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Sample records for prokaryotic phenylalanine ammonia-lyase

  1. Phenylalanine ammonia-lyase (PAL) gene activity in response to ...

    African Journals Online (AJOL)

    Phenylalanine ammonia-lyase (PAL) catalyzes the biosynthesis of rosmarinic acid (RA), tyrosine and phenylalanine are the precursors of RA, while proline drives metabolite precursors toward Shikimate and phenylpropanoid pathway ending with the production of RA. The aim of this study was to investigate the PAL gene ...

  2. Circadian Rhythmicity in the Activities of Phenylalanine Ammonia-Lyase from Lemna perpusilla and Spirodela polyrhiza 1

    Science.gov (United States)

    Gordon, William R.; Koukkari, Willard L.

    1978-01-01

    The oscillations in phenylalanine ammonia-lyase activity from Spirodela polyrhiza and phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities from Lemna perpusilla displayed a circadian rhythm under continuous light. Rhythmicity in enzymic activity could not be detected in continuous darkness since under this condition phenylalanine ammonia-lyase activity remains at a fairly constantly low level. Results from our studies of the oscillatory pattern of the respective activities of phenylalanine and tyrosine ammonia-lyase support their “inseparability.” PMID:16660569

  3. Phenylalanine ammonia-lyase through evolution: A bioinformatic approach

    Directory of Open Access Journals (Sweden)

    Shiva Hemmati

    2015-03-01

    Full Text Available Phenylalanine ammonia-lyase (PAL is the first entry enzyme of the phenylpropanoid pathway that converts phenylalanine to cinnamic acid which is the precursor of various secondary metabolites. PAL is recently formulated for phenylketonuric patients in pegylated forms; therefore, screening a PAL with the highest affinity to the substrate is of a great importance. PAL exists in all higher plants and some fungi and few bacteria. Ancestors of land plants have been adopted by evolving metabolic pathways. A multi-gene family encodes PAL by gene duplication events in most plants. In this study, the taxonomic distribution and phylogeny of pal gene found in land plants, fungi and bacteria have been analyzed. It seems that the ancestor of plants acquired a pal gene via horizontal gene transfer in symbioses with bacteria and fungi. Gymnosperms have kept a diverse set of pal genes that arose from gene duplication events. In angiosperms, after the divergence of dicotyledons from monocots, pal genes were duplicated many times. The close paralogues of pal genes in some species indicate expansion of gene families after the divergence in plant pal gene evolution. Interestingly, some of the plant pals clustered by species in a way that pals within one species are more closely related to each other than to homologs in the other species which indicates this duplication event occurred more recently.

  4. Genomic Characterization of Phenylalanine Ammonia Lyase Gene in Buckwheat.

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    Karthikeyan Thiyagarajan

    Full Text Available Phenylalanine Ammonia Lyase (PAL gene which plays a key role in bio-synthesis of medicinally important compounds, Rutin/quercetin was sequence characterized for its efficient genomics application. These compounds possessing anti-diabetic and anti-cancer properties and are predominantly produced by Fagopyrum spp. In the present study, PAL gene was sequenced from three Fagopyrum spp. (F. tataricum, F. esculentum and F. dibotrys and showed the presence of three SNPs and four insertion/deletions at intra and inter specific level. Among them, the potential SNP (position 949th bp G>C with Parsimony Informative Site was selected and successfully utilised to individuate the zygosity/allelic variation of 16 F. tataricum varieties. Insertion mutations were identified in coding region, which resulted the change of a stretch of 39 amino acids on the putative protein. Our Study revealed that autogamous species (F. tataricum has lower frequency of observed SNPs as compared to allogamous species (F. dibotrys and F. esculentum. The identified SNPs in F. tataricum didn't result to amino acid change, while in other two species it caused both conservative and non-conservative variations. Consistent pattern of SNPs across the species revealed their phylogenetic importance. We found two groups of F. tataricum and one of them was closely related with F. dibotrys. Sequence characterization information of PAL gene reported in present investigation can be utilized in genetic improvement of buckwheat in reference to its medicinal value.

  5. Phenylalanine ammonia lyase catalyzed synthesis of amino acids by an MIO-cofactor independent pathway.

    Science.gov (United States)

    Lovelock, Sarah L; Lloyd, Richard C; Turner, Nicholas J

    2014-04-25

    Phenylalanine ammonia lyases (PALs) belong to a family of 4-methylideneimidazole-5-one (MIO) cofactor dependent enzymes which are responsible for the conversion of L-phenylalanine into trans-cinnamic acid in eukaryotic and prokaryotic organisms. Under conditions of high ammonia concentration, this deamination reaction is reversible and hence there is considerable interest in the development of PALs as biocatalysts for the enantioselective synthesis of non-natural amino acids. Herein the discovery of a previously unobserved competing MIO-independent reaction pathway, which proceeds in a non-stereoselective manner and results in the generation of both L- and D-phenylalanine derivatives, is described. The mechanism of the MIO-independent pathway is explored through isotopic-labeling studies and mutagenesis of key active-site residues. The results obtained are consistent with amino acid deamination occurring by a stepwise E1 cB elimination mechanism. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Synthesis of d‐ and l‐Phenylalanine Derivatives by Phenylalanine Ammonia Lyases: A Multienzymatic Cascade Process†

    Science.gov (United States)

    Parmeggiani, Fabio; Lovelock, Sarah L.; Weise, Nicholas J.; Ahmed, Syed T.

    2015-01-01

    Abstract The synthesis of substituted d‐phenylalanines in high yield and excellent optical purity, starting from inexpensive cinnamic acids, has been achieved with a novel one‐pot approach by coupling phenylalanine ammonia lyase (PAL) amination with a chemoenzymatic deracemization (based on stereoselective oxidation and nonselective reduction). A simple high‐throughput solid‐phase screening method has also been developed to identify PALs with higher rates of formation of non‐natural d‐phenylalanines. The best variants were exploited in the chemoenzymatic cascade, thus increasing the yield and ee value of the d‐configured product. Furthermore, the system was extended to the preparation of those l‐phenylalanines which are obtained with a low ee value using PAL amination. PMID:27478261

  7. Synthesis of d- and l-Phenylalanine Derivatives by Phenylalanine Ammonia Lyases: A Multienzymatic Cascade Process**

    Science.gov (United States)

    Parmeggiani, Fabio; Lovelock, Sarah L; Weise, Nicholas J; Ahmed, Syed T; Turner, Nicholas J

    2015-01-01

    The synthesis of substituted d-phenylalanines in high yield and excellent optical purity, starting from inexpensive cinnamic acids, has been achieved with a novel one-pot approach by coupling phenylalanine ammonia lyase (PAL) amination with a chemoenzymatic deracemization (based on stereoselective oxidation and nonselective reduction). A simple high-throughput solid-phase screening method has also been developed to identify PALs with higher rates of formation of non-natural d-phenylalanines. The best variants were exploited in the chemoenzymatic cascade, thus increasing the yield and ee value of the d-configured product. Furthermore, the system was extended to the preparation of those l-phenylalanines which are obtained with a low ee value using PAL amination. PMID:25728350

  8. Synthesis of specifically labelled L-phenylalanines using phenylalanine ammonia lyase activity

    International Nuclear Information System (INIS)

    Haedener, A.; Tamm, Ch.

    1987-01-01

    Specifically labelled L-phenylalanines have been prepared using a variety of classical synthetic methods in combination with phenylalanine ammonia lyase (PAL) enzyme activity of the yeast Rhodosporidium toruloides ATCC 10788 or Rhodotorula glutinis IFO 0559, respectively. Thus, L-[2- 2 H]phenyl-[2- 2 H]alanine was formed from (E) -[2,2'- 2 H 2 ]cinnamic acid and ammonia in 46% yield, whereas L-phenyl-[2- 13 C, 15 N]alanine was obtained from (E)-[2- 13 C]cinnamic acid in 45% overall yield. Generally, labelled cinnamic acids were recovered in pure form from the reaction mixture, with a loss of 6-8%. Likewise, unchanged 15 NH 3 was reisolated as 15 NH 4 Cl after steam distillation with overall losses of less than 4%. Labelled cinnamic acids were prepared by Knoevenagel condensations between appropriately labelled benzaldehydes and malonic acids. [2- 2 H]Benzaldehyde was obtained from 2-bromotoluene by decomposition of the corresponding Grignard reagent with 2 H 2 O and subsequent oxidation. Since simple molecules, most of them commercially available in labelled form or otherwise easily accessible, may serve as starting material, and due to its defined stereochemistry, the reaction catalysed by PAL opens a short and attractive route to specifically labelled L-phenylalanines. (author)

  9. Synthesis of specifically labelled L-phenylalanines using phenylalanine ammonia lyase activity

    Energy Technology Data Exchange (ETDEWEB)

    Haedener, A.; Tamm, Ch.

    1987-11-01

    Specifically labelled L-phenylalanines have been prepared using a variety of classical synthetic methods in combination with phenylalanine ammonia lyase (PAL) enzyme activity of the yeast Rhodosporidium toruloides ATCC 10788 or Rhodotorula glutinis IFO 0559, respectively. Thus, L-(2-/sup 2/H)phenyl-(2-/sup 2/H)alanine was formed from (E) -(2,2'-/sup 2/H/sub 2/)cinnamic acid and ammonia in 46% yield, whereas L-phenyl-(2-/sup 13/C, /sup 15/N)alanine was obtained from (E)-(2-/sup 13/C)cinnamic acid in 45% overall yield. Generally, labelled cinnamic acids were recovered in pure form from the reaction mixture, with a loss of 6-8%. Likewise, unchanged /sup 15/NH/sub 3/ was reisolated as /sup 15/NH/sub 4/Cl after steam distillation with overall losses of less than 4%. Labelled cinnamic acids were prepared by Knoevenagel condensations between appropriately labelled benzaldehydes and malonic acids. (2-/sup 2/H)Benzaldehyde was obtained from 2-bromotoluene by decomposition of the corresponding Grignard reagent with /sup 2/H/sub 2/O and subsequent oxidation. Since simple molecules, most of them commercially available in labelled form or otherwise easily accessible, may serve as starting material, and due to its defined stereochemistry, the reaction catalysed by PAL opens a short and attractive route to specifically labelled L-phenylalanines.

  10. Reduced phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities and lignin synthesis in wheat grown under low pressure sodium lamps

    Science.gov (United States)

    Guerra, D.; Anderson, A. J.; Salisbury, F. B.

    1985-01-01

    Wheat (Triticum aestivum L. cv Fremont) grown in hydroponic culture under 24-hour continuous irradiation at 560 to 580 micromoles per square meter per second from either metalhalide (MH), high pressure sodium (HPS), or low pressure sodium (LPS) lamps reached maturity in 70 days. Grain yields were similar under all three lamps, although LPS-grown plants lodged at maturity. Phenylalanine ammonia-lyase (PAL) and a tyrosine ammonia lyase (TAL) with lesser activity were detected in all extracts of leaf, inflorescence, and stem. Ammonia-lyase activities increased with age of the plant, and plants grown under the LPS lamp displayed PAL and TAL activities lower than wheat cultured under MH and HPS radiation. Greenhouse solar-grown wheat had the highest PAL and TAL activities. Lignin content of LPS-grown wheat was also significantly reduced from that of plants grown under MH or HPS lamps or in the greenhouse, showing a correlation with the reduced PAL and TAL activities. Ratios of far red-absorbing phytochrome to total phytochrome were similar for all three lamps, but the data do not yet warrant a conclusion about specific wavelengths missing from the LPS lamps that might have induced PAL and TAL activities in plants under the other lamps.

  11. Expression and properties of the highly alkalophilic phenylalanine ammonia-lyase of thermophilic Rubrobacter xylanophilus.

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    Klaudia Kovács

    Full Text Available The sequence of a phenylalanine ammonia-lyase (PAL; EC: 4.3.1.24 of the thermophilic and radiotolerant bacterium Rubrobacter xylanophilus (RxPAL was identified by screening the genomes of bacteria for members of the phenylalanine ammonia-lyase family. A synthetic gene encoding the RxPAL protein was cloned and overexpressed in Escherichia coli TOP 10 in a soluble form with an N-terminal His6-tag and the recombinant RxPAL protein was purified by Ni-NTA affinity chromatography. The activity assay of RxPAL with l-phenylalanine at various pH values exhibited a local maximum at pH 8.5 and a global maximum at pH 11.5. Circular dichroism (CD studies showed that RxPAL is associated with an extensive α-helical character (far UV CD and two distinctive near-UV CD peaks. These structural characteristics were well preserved up to pH 11.0. The extremely high pH optimum of RxPAL can be rationalized by a three-dimensional homology model indicating possible disulfide bridges, extensive salt-bridge formation and an excess of negative electrostatic potential on the surface. Due to these properties, RxPAL may be a candidate as biocatalyst in synthetic biotransformations leading to unnatural l- or d-amino acids or as therapeutic enzyme in treatment of phenylketonuria or leukemia.

  12. Cooperative functioning between phenylalanine ammonia lyase and isochorishmate synthase activities contributes to salicylic acid biosynthesis in soybean

    Science.gov (United States)

    Salicylic acid (SA), an essential regulator of plant defense, is derived from chorismate via either the phenylalanine ammonia lyase (PAL), or the isochorishmate synthase (ICS) catalyzed steps. The ICS pathway is thought to be the primary contributor of defense-related SA, at least in Arabidopsis. We...

  13. Possible regulatory role of phenylalanine ammonia-lyase in the production of anthocyanins in asparagus (Asparagus officinalis L)

    NARCIS (Netherlands)

    Flores, F.B.; Oosterhaven, J.; Martinez-Madrid, M.C.; Romojaro, F.

    2005-01-01

    The regulatory role of phenylalanine ammonia-lyase (PAL) in the light-induced accumulation of anthocyanins in the epidermis of asparagus spears has been analysed. A correlation between the stimulation of PAL activity and the rise in total anthocyanin content has been observed. Light radiation

  14. Cloning, expression and characterization of phenylalanine ammonia-lyase from Rhodotorula glutinis.

    Science.gov (United States)

    Zhu, Longbao; Cui, Wenjing; Fang, Yueqin; Liu, Yi; Gao, Xinxing; Zhou, Zhemin

    2013-05-01

    The industrial-scale production of phenylalanine ammonia-lyase (PAL) mainly uses strains of Rhodotorula. However, the PAL gene from Rhodotorula has not been cloned. Here, the full-length gene of PAL from Rhodotorula glutinis was isolated. It was 2,121 bp, encoding a polypeptide with 706 amino acids and a calculated MW of 75.5 kDa. Though R. glutinis is an anamorph of Rhodosporium toruloides, the amino acid sequences of PALs them are not the same (about 74 % identity). PAL was expressed in E. coli and characterized. Its specific activity was 4.2 U mg(-1) and the k cat/K m was 1.9 × 10(4) mM(-1) s(-1), exhibiting the highest catalytic ability among the reported PALs. The genetic and biochemical information reported here should facilitate future application in industry.

  15. Stabilization of Phenylalanine Ammonia Lyase from Rhodotorula glutinis by Encapsulation in Polyethyleneimine-Mediated Biomimetic Silica.

    Science.gov (United States)

    Cui, Jiandong; Liang, Longhao; Han, Cong; Lin Liu, Rong

    2015-06-01

    Phenylalanine ammonia lyase (PAL) from Rhodotorula glutinis was encapsulated within polyethyleneimine-mediated biomimetic silica. The main factors in the preparation of biomimetic silica were optimized by response surface methodology (RSM). Compared to free PAL (about 2 U), the encapsulated PAL retained more than 43 % of their initial activity after 1 h of incubation time at 60 °C, whereas free PAL lost most of activity in the same conditions. It was clearly indicated that the thermal stability of PAL was improved by encapsulation. Moreover, the encapsulated PAL exhibited the excellent stability of the enzyme against denaturants and storage stability, and pH stability was improved by encapsulation. Operational stability of 7 reaction cycles showed that the encapsulated PAL was stable. Nevertheless, the K m value of encapsulated PAL in biomimetic silica was higher than that of the free PAL due to lower total surface area and increased mass transfer resistance.

  16. Optimization of oligomeric enzyme activity in ionic liquids using Rhodotorula glutinis yeast phenylalanine ammonia lyase.

    Science.gov (United States)

    Barron, Christiaan C; Sponagle, Brandon J D; Arivalagan, Pugazhendhi; D'Cunha, Godwin B

    2017-01-01

    Phenylalanine ammonia lyase (E.C.4.3.1.24, PAL) activity of Rhodotorula glutinis yeast has been demonstrated in four commonly used ionic liquids. PAL forward reaction was carried out in 1-butyl-3-methylimidazolium methyl sulfate ([BMIM][MeSO 4 ]), 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF 4 ]), 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF 6 ]) and 1-butyl-3-methylimidazolium lactate ([BMIM][lactate]). Our experiments have revealed that PAL is catalytically active in ionic liquids and the enzyme activity in ([BMIM][PF 6 ]) is comparable to that obtained in aqueous buffer medium. Different conditions were optimized for maximal PAL forward activity including time of incubation (30.0min) L -phenylalanine substrate concentration (30.0mM), nature of buffer (50.0mM Tris-HCl), pH (9.0), temperature (37°C), and speed of agitation (100 rev min -1 ). Under these optimized conditions, about 83% conversion of substrate to product was obtained for the PAL forward reaction that was determined using UV spectroscopy at 290nm. PAL reverse reaction in ([BMIM][PF 6 ]) was determined spectrophotometrically at 520nm; and about 59% substrate conversion was obtained. This data provides further knowledge in enzyme biocatalysis in non-aqueous media, and may be of importance when studying the function of other oligomeric/multimeric proteins and enzymes in ionic liquids. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Single-dose, subcutaneous recombinant phenylalanine ammonia lyase conjugated with polyethylene glycol in adult patients with phenylketonuria: an open-label, multicentre, phase 1 dose-escalation trial.

    Science.gov (United States)

    Longo, Nicola; Harding, Cary O; Burton, Barbara K; Grange, Dorothy K; Vockley, Jerry; Wasserstein, Melissa; Rice, Gregory M; Dorenbaum, Alejandro; Neuenburg, Jutta K; Musson, Donald G; Gu, Zhonghua; Sile, Saba

    2014-07-05

    Phenylketonuria is an inherited disease caused by impaired activity of phenylalanine hydroxylase, the enzyme that converts phenylalanine to tyrosine, leading to accumulation of phenylalanine and subsequent neurocognitive dysfunction. Phenylalanine ammonia lyase is a prokaryotic enzyme that converts phenylalanine to ammonia and trans-cinnamic acid. We aimed to assess the safety, tolerability, pharmacokinetic characteristics, and efficacy of recombinant Anabaena variabilis phenylalanine ammonia lyase (produced in Escherichia coli) conjugated with polyethylene glycol (rAvPAL-PEG) in reducing phenylalanine concentrations in adult patients with phenylketonuria. In this open-label, phase 1, multicentre trial, single subcutaneous injections of rAvPAL-PEG were given in escalating doses (0·001, 0·003, 0·010, 0·030, and 0·100 mg/kg) to adults with phenylketonuria. Participants aged 18 years or older with blood phenylalanine concentrations of 600 μmol/L or higher were recruited from among patients attending metabolic disease clinics in the USA. The primary endpoints were safety and tolerability of rAvPAL-PEG. Secondary endpoints were the pharmacokinetic characteristics of the drug and its effect on concentrations of phenylalanine. Participants and investigators were not masked to assigned dose group. This study is registered with ClinicalTrials.gov, number NCT00925054. 25 participants were recruited from seven centres between May 6, 2008, and April 15, 2009, with five participants assigned to each escalating dose group. All participants were included in the safety population. The most frequently reported adverse events were injection-site reactions and dizziness, which were self-limited and without sequelae. Two participants had serious adverse reactions to intramuscular medroxyprogesterone acetate, a drug that contains polyethylene glycol as an excipient. Three of five participants given the highest dose of rAvPAL-PEG (0·100 mg/kg) developed a generalised skin rash

  18. Formulation and PEGylation optimization of the therapeutic PEGylated phenylalanine ammonia lyase for the treatment of phenylketonuria.

    Science.gov (United States)

    Bell, Sean M; Wendt, Dan J; Zhang, Yanhong; Taylor, Timothy W; Long, Shinong; Tsuruda, Laurie; Zhao, Bin; Laipis, Phillip; Fitzpatrick, Paul A

    2017-01-01

    Phenylketonuria (PKU) is a genetic metabolic disease in which the decrease or loss of phenylalanine hydroxylase (PAH) activity results in elevated, neurotoxic levels of phenylalanine (Phe). Due to many obstacles, PAH enzyme replacement therapy is not currently an option. Treatment of PKU with an alternative enzyme, phenylalanine ammonia lyase (PAL), was first proposed in the 1970s. However, issues regarding immunogenicity, enzyme production and mode of delivery needed to be overcome. Through the evaluation of PAL enzymes from multiple species, three potential PAL enzymes from yeast and cyanobacteria were chosen for evaluation of their therapeutic potential. The addition of polyethylene glycol (PEG, MW = 20,000), at a particular ratio to modify the protein surface, attenuated immunogenicity in an animal model of PKU. All three PEGylated PAL candidates showed efficacy in a mouse model of PKU (BTBR Pahenu2) upon subcutaneous injection. However, only PEGylated Anabaena variabilis (Av) PAL-treated mice demonstrated sustained low Phe levels with weekly injection and was the only PAL evaluated that maintained full enzymatic activity upon PEGylation. A PEGylated recombinant double mutant version of AvPAL (Cys503Ser/Cys565Ser), rAvPAL-PEG, was selected for drug development based on its positive pharmacodynamic profile and favorable expression titers. PEGylation was shown to be critical for rAvPAL-PEG efficacy as under PEGylated rAvPAL had a lower pharmacodynamic effect. rAvPAL and rAvPAL-PEG had poor stability at 4°C. L-Phe and trans-cinnamate were identified as activity stabilizing excipients. rAvPAL-PEG is currently in Phase 3 clinical trials to assess efficacy in PKU patients.

  19. Formulation and PEGylation optimization of the therapeutic PEGylated phenylalanine ammonia lyase for the treatment of phenylketonuria.

    Directory of Open Access Journals (Sweden)

    Sean M Bell

    Full Text Available Phenylketonuria (PKU is a genetic metabolic disease in which the decrease or loss of phenylalanine hydroxylase (PAH activity results in elevated, neurotoxic levels of phenylalanine (Phe. Due to many obstacles, PAH enzyme replacement therapy is not currently an option. Treatment of PKU with an alternative enzyme, phenylalanine ammonia lyase (PAL, was first proposed in the 1970s. However, issues regarding immunogenicity, enzyme production and mode of delivery needed to be overcome. Through the evaluation of PAL enzymes from multiple species, three potential PAL enzymes from yeast and cyanobacteria were chosen for evaluation of their therapeutic potential. The addition of polyethylene glycol (PEG, MW = 20,000, at a particular ratio to modify the protein surface, attenuated immunogenicity in an animal model of PKU. All three PEGylated PAL candidates showed efficacy in a mouse model of PKU (BTBR Pahenu2 upon subcutaneous injection. However, only PEGylated Anabaena variabilis (Av PAL-treated mice demonstrated sustained low Phe levels with weekly injection and was the only PAL evaluated that maintained full enzymatic activity upon PEGylation. A PEGylated recombinant double mutant version of AvPAL (Cys503Ser/Cys565Ser, rAvPAL-PEG, was selected for drug development based on its positive pharmacodynamic profile and favorable expression titers. PEGylation was shown to be critical for rAvPAL-PEG efficacy as under PEGylated rAvPAL had a lower pharmacodynamic effect. rAvPAL and rAvPAL-PEG had poor stability at 4°C. L-Phe and trans-cinnamate were identified as activity stabilizing excipients. rAvPAL-PEG is currently in Phase 3 clinical trials to assess efficacy in PKU patients.

  20. Control of phenylalanine ammonia-lyase gene promoters from pea by UV radiation

    International Nuclear Information System (INIS)

    Pluskota, W.E.; Michalczyk, D.J.; Gorecki, R.J.

    2005-01-01

    The gene fusion system was used to study UV light-control of PS PAL1 and PS PAL2 genes encoding phenylalanine ammonia-lyase of pea. The induction of pea PAL promoters was analysed in transgenic tobacco plants. Binary plasmids (derivatives of pBI101.2 vector) containing 5' regulatory fragments of PS PAL1 and PS PAL2 linked to reporter genes (GUS, LUC) were constructed. The analyses were performed with the use of single constructs (containing one variant of PS PAL promoter and one reporter gene) and dual constructs (containing both PS PAL1 and PS PAL2 promoters connected with different reporter genes). The use of dual constructs enabled the evaluation of both PS PAL promoters activity in the same plant. The analyses of in vitro grown plants have shown that both PAL promoters are strongly induced in leaves subjected to UV radiation. In some cases, the UV-stimulated expression exceeded the exposed areas. This phenomenon was observed more often in the leaves of plants containing the PS PAL1::GUS than PS PAL2::GUS construct. Removal of boxes 2, 4, 5 from PS PAL1 promoter and deletion of its 5' end region (-339 to -1394) decreases the level of gene expression but does not eliminate its responsiveness to UV

  1. Molecular Cloning and Sequence Analysis of a Phenylalanine Ammonia-Lyase Gene from Dendrobium

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    Cai, Yongping; Lin, Yi

    2013-01-01

    In this study, a phenylalanine ammonia-lyase (PAL) gene was cloned from Dendrobium candidum using homology cloning and RACE. The full-length sequence and catalytic active sites that appear in PAL proteins of Arabidopsis thaliana and Nicotiana tabacum are also found: PAL cDNA of D. candidum (designated Dc-PAL1, GenBank No. JQ765748) has 2,458 bps and contains a complete open reading frame (ORF) of 2,142 bps, which encodes 713 amino acid residues. The amino acid sequence of DcPAL1 has more than 80% sequence identity with the PAL genes of other plants, as indicated by multiple alignments. The dominant sites and catalytic active sites, which are similar to that showing in PAL proteins of Arabidopsis thaliana and Nicotiana tabacum, are also found in DcPAL1. Phylogenetic tree analysis revealed that DcPAL is more closely related to PALs from orchidaceae plants than to those of other plants. The differential expression patterns of PAL in protocorm-like body, leaf, stem, and root, suggest that the PAL gene performs multiple physiological functions in Dendrobium candidum. PMID:23638048

  2. Molecular cloning and sequence analysis of a phenylalanine ammonia-lyase gene from dendrobium.

    Directory of Open Access Journals (Sweden)

    Qing Jin

    Full Text Available In this study, a phenylalanine ammonia-lyase (PAL gene was cloned from Dendrobium candidum using homology cloning and RACE. The full-length sequence and catalytic active sites that appear in PAL proteins of Arabidopsis thaliana and Nicotiana tabacum are also found: PAL cDNA of D. candidum (designated Dc-PAL1, GenBank No. JQ765748 has 2,458 bps and contains a complete open reading frame (ORF of 2,142 bps, which encodes 713 amino acid residues. The amino acid sequence of DcPAL1 has more than 80% sequence identity with the PAL genes of other plants, as indicated by multiple alignments. The dominant sites and catalytic active sites, which are similar to that showing in PAL proteins of Arabidopsis thaliana and Nicotiana tabacum, are also found in DcPAL1. Phylogenetic tree analysis revealed that DcPAL is more closely related to PALs from orchidaceae plants than to those of other plants. The differential expression patterns of PAL in protocorm-like body, leaf, stem, and root, suggest that the PAL gene performs multiple physiological functions in Dendrobium candidum.

  3. Genome-wide characterization of phenylalanine ammonia-lyase gene family in watermelon (Citrullus lanatus).

    Science.gov (United States)

    Dong, Chun-Juan; Shang, Qing-Mao

    2013-07-01

    Phenylalanine ammonia-lyase (PAL), the first enzyme in the phenylpropanoid pathway, plays a critical role in plant growth, development, and adaptation. PAL enzymes are encoded by a gene family in plants. Here, we report a genome-wide search for PAL genes in watermelon. A total of 12 PAL genes, designated ClPAL1-12, are identified . Nine are arranged in tandem in two duplication blocks located on chromosomes 4 and 7, and the other three ClPAL genes are distributed as single copies on chromosomes 2, 3, and 8. Both the cDNA and protein sequences of ClPALs share an overall high identity with each other. A phylogenetic analysis places 11 of the ClPALs into a separate cucurbit subclade, whereas ClPAL2, which belongs to neither monocots nor dicots, may serve as an ancestral PAL in plants. In the cucurbit subclade, seven ClPALs form homologous pairs with their counterparts from cucumber. Expression profiling reveals that 11 of the ClPAL genes are expressed and show preferential expression in the stems and male and female flowers. Six of the 12 ClPALs are moderately or strongly expressed in the fruits, particularly in the pulp, suggesting the potential roles of PAL in the development of fruit color and flavor. A promoter motif analysis of the ClPAL genes implies redundant but distinctive cis-regulatory structures for stress responsiveness. Finally, duplication events during the evolution and expansion of the ClPAL gene family are discussed, and the relationships between the ClPAL genes and their cucumber orthologs are estimated.

  4. Structural And Biochemical Characterization of the Therapeutic A. Variabilis Phenylalanine Ammonia Lyase

    Energy Technology Data Exchange (ETDEWEB)

    Wang, L.; Gamez, A.; Archer, H.; Abola, E.E.; Sarkissian, C.N.; Fitzpatrick, P.; Wendt, D.; Zhang, Y.; Vellard, M.; Bliesath, J.; Bell, S.; Lemont, J.; Scriver, C.R.; Stevens, R.C.

    2009-05-26

    We have recently observed promising success in a mouse model for treating the metabolic disorder phenylketonuria with phenylalanine ammonia lyase (PAL) from Rhodosporidium toruloides and Anabaena variabilis. Both molecules, however, required further optimization in order to overcome problems with protease susceptibility, thermal stability, and aggregation. Previously, we optimized PAL from R. toruloides, and in this case we reduced aggregation of the A. variabilis PAL by mutating two surface cysteine residues (C503 and C565) to serines. Additionally, we report the structural and biochemical characterization of the A. variabilis PAL C503S/C565S double mutant and carefully compare this molecule with the R. toruloides engineered PAL molecule. Unlike previously published PAL structures, significant electron density is observed for the two active-site loops in the A. variabilis C503S/C565S double mutant, yielding a complete view of the active site. Docking studies and N-hydroxysuccinimide-biotin binding studies support a proposed mechanism in which the amino group of the phenylalanine substrate is attacked directly by the 4-methylidene-imidazole-5-one prosthetic group. We propose a helix-to-loop conformational switch in the helices flanking the inner active-site loop that regulates accessibility of the active site. Differences in loop stability among PAL homologs may explain the observed variation in enzyme efficiency, despite the highly conserved structure of the active site. A. variabilis C503S/C565S PAL is shown to be both more thermally stable and more resistant to proteolytic cleavage than R. toruloides PAL. Additional increases in thermal stability and protease resistance upon ligand binding may be due to enhanced interactions among the residues of the active site, possibly locking the active-site structure in place and stabilizing the tetramer. Examination of the A. variabilis C503S/C565S PAL structure, combined with analysis of its physical properties, provides

  5. The effect of cadmium on phenylalanine ammonia lyase activity and lipid peroxidation in pepper (Capsicum annuum L. seedlings

    Directory of Open Access Journals (Sweden)

    Esra Koç

    2015-04-01

    Full Text Available In this study, the effect on differrent concentrations (20, 40, 80µM ve 100 µM CdCl2 of cadmium (CdCl2 on the activity of phenylalanine ammonia-lyase (PAL and lipid peroxidation amount in leaf and stem of Kahramanmaraş- Hot (Capsicum annum L. pepper seedlings were researched. Activity of phenylalanine ammonia-lyase (PAL, the first enzyme in the phenylpropanoid biosynthetic pathway, was increased at 2 and 4 days in KM-Hot plants exposed to CdCl2 stress. The highest PAL activity was detected in 20 μM CdCl2 application, on the four day after the application in the leaves of KM-Hot pepper. Moreover, it was observed that treatment of pepper with Cd led to an increased the rate of lipid peroxidation (which is indicated by increasing MDA content in the leaf and stem tissues. The highest MDA content was detected in 80 μM CdCl2 application, on the four day after the application in the leaf tissues. These results suggest that the activation of PAL may be associated with increased production of MDA

  6. Biochemical Evaluation of Phenylalanine Ammonia Lyase from Endemic Plant Cyathobasis fruticulosa (Bunge Aellen. for the Dietary Treatment of Phenylketonuria

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    Seda Şirin

    2016-01-01

    Full Text Available Enzyme substitution therapy with the phenylalanine ammonia lyase (PAL is a new approach to the treatment of patients with phenylketonuria (PKU. This enzyme is responsible for the conversion of phenylalanine to trans-cinnamic acid. We assessed the PAL enzyme of the endemic plant Cyathobasis fruticulosa (Bunge Aellen. for its possible role in the dietary treatment of PKU. The enzyme was found to have a high activity of (64.9±0.1 U/mg, with the optimum pH, temperature and buffer (Tris–HCl and L-phenylalanine concentration levels of pH=8.8, 37 °C and 100 mM, respectively. Optimum enzyme activity was achieved at pH=4.0 and 7.5, corresponding to pH levels of gastric and intestinal juice, and NaCl concentration of 200 mM. The purifi cation of the enzyme by 1.87-fold yielded an activity of 98.6 U/mg. PAL activities determined by HPLC analyses before and after purification were similar. Two protein bands, one at 70 and the other at 23 kDa, were determined by Western blot analysis of the enzyme. This enzyme is a potential candidate for serial production of dietary food and biotechnological products.

  7. Efficient preparation of enantiopure D-phenylalanine through asymmetric resolution using immobilized phenylalanine ammonia-lyase from Rhodotorula glutinis JN-1 in a recirculating packed-bed reactor.

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    Longbao Zhu

    Full Text Available An efficient enzymatic process was developed to produce optically pure D-phenylalanine through asymmetric resolution of the racemic DL-phenylalanine using immobilized phenylalanine ammonia-lyase (RgPAL from Rhodotorula glutinis JN-1. RgPAL was immobilized on a modified mesoporous silica support (MCM-41-NH-GA. The resulting MCM-41-NH-GA-RgPAL showed high activity and stability. The resolution efficiency using MCM-41-NH-GA-RgPAL in a recirculating packed-bed reactor (RPBR was higher than that in a stirred-tank reactor. Under optimal operational conditions, the volumetric conversion rate of L-phenylalanine and the productivity of D-phenylalanine reached 96.7 mM h⁻¹ and 0.32 g L⁻¹ h⁻¹, respectively. The optical purity (eeD of D-phenylalanine exceeded 99%. The RPBR ran continuously for 16 batches, the conversion ratio did not decrease. The reactor was scaled up 25-fold, and the productivity of D-phenylalanine (eeD>99% in the scaled-up reactor reached 7.2 g L⁻¹ h⁻¹. These results suggest that the resolution process is an alternative method to produce highly pure D-phenylalanine.

  8. Efficient preparation of enantiopure D-phenylalanine through asymmetric resolution using immobilized phenylalanine ammonia-lyase from Rhodotorula glutinis JN-1 in a recirculating packed-bed reactor.

    Science.gov (United States)

    Zhu, Longbao; Zhou, Li; Huang, Nan; Cui, Wenjing; Liu, Zhongmei; Xiao, Ke; Zhou, Zhemin

    2014-01-01

    An efficient enzymatic process was developed to produce optically pure D-phenylalanine through asymmetric resolution of the racemic DL-phenylalanine using immobilized phenylalanine ammonia-lyase (RgPAL) from Rhodotorula glutinis JN-1. RgPAL was immobilized on a modified mesoporous silica support (MCM-41-NH-GA). The resulting MCM-41-NH-GA-RgPAL showed high activity and stability. The resolution efficiency using MCM-41-NH-GA-RgPAL in a recirculating packed-bed reactor (RPBR) was higher than that in a stirred-tank reactor. Under optimal operational conditions, the volumetric conversion rate of L-phenylalanine and the productivity of D-phenylalanine reached 96.7 mM h⁻¹ and 0.32 g L⁻¹ h⁻¹, respectively. The optical purity (eeD) of D-phenylalanine exceeded 99%. The RPBR ran continuously for 16 batches, the conversion ratio did not decrease. The reactor was scaled up 25-fold, and the productivity of D-phenylalanine (eeD>99%) in the scaled-up reactor reached 7.2 g L⁻¹ h⁻¹. These results suggest that the resolution process is an alternative method to produce highly pure D-phenylalanine.

  9. One-Pot Enzymatic Synthesis of D-Arylalanines Using Phenylalanine Ammonia Lyase and L-Amino Acid Deaminase.

    Science.gov (United States)

    Zhu, Longbao; Feng, Guoqiang; Ge, Fei; Song, Ping; Wang, Taotao; Liu, Yi; Tao, Yugui; Zhou, Zhemin

    2018-06-08

    The phenylalanine ammonia-lyase (AvPAL) from Anabaena variabilis catalyzes the amination of substituent trans-cinnamic acid (t-CA) to produce racemic D,L-enantiomer arylalanine mixture owing to its low stereoselectivity. To produce high optically pure D-arylalanine, a modified AvPAL with high D-selectivity is expected. Based on the analyses of catalytic mechanism and structure, the Asn347 residue in the active site was proposed to control stereoselectivity. Therefore, Asn347 was mutated to construct mutant AvPAL-N347A, the stereoselectivity of AvPAL-N347A for D-enantiomer arylalanine was 2.3-fold higher than that of wild-type AvPAL (WtPAL). Furthermore, the residual L-enantiomer product in reaction solution could be converted into the D-enantiomer product through stereoselective oxidation by PmLAAD and nonselective reduction by reducing agent NH 3 BH 3 . At optimal conditions, the conversion rate of t-CA and optical purity (enantiomeric excess (ee D )) of D-phenylalanine reached 82% and exceeded 99%, respectively. The two enzymes displayed activity toward a broad range of substrate and could be used to efficiently synthesize D-arylalanine with different groups on the phenyl ring. Among these D-arylalanines, the yield of m-nitro-D-phenylalanine was highest and reached 96%, and the ee D exceeded 99%. This one-pot synthesis using AvPAL and PmLAAD has prospects for industrial application.

  10. Phenylalanine ammonia-lyase (pal) and peroxidase activity in brown rust infected tissues of pakistani wheat cultivars

    International Nuclear Information System (INIS)

    Riaz, A.; Tahir, M.I.

    2014-01-01

    Besides other factors resistance and susceptibility is the outcome of biochemical processes such as activities of defense-related enzymes. So in this study, Phenylalanine ammonia-lyase (PAL) and Peroxidase activity of resistant (Inqilab-91) and susceptible (Kirin-95) wheat cultivars were determined through spectrophotometer to address the biochemical aspect related to the disease after 8 hours, 24 hours, 48 hours and 72 hours of leaf rust inoculation. The results have shown that these enzymes were present in both the resistant and susceptible cultivars but the activity was more pronounced in the resistant one. The effect of PAL and peroxidase activity was also investigated among inoculated and uninoculated plants within the same cultivar. The activity of both PAL and peroxidase were more significant in inoculated ones. The results have shown that the after 72 hours of inoculation Inqilab-91 had more PAL activity i.e., 5.47 IU/ml/min than in Kirin-95 i.e., 2.08 IU/ml/min at 270 nm. While peroxidase activity in Inqilab-91 was 6.41 IU/ml/min and in Kirin-95, 3.66 IU/ml/min after 72 hours of inoculation, observed under 470 nm wavelength. Increase in one's activity increases the other enzyme's activity. The activity was more prominent after 72 hours of infection as pathogen had successfully established itself in the host plant tissue. The activities of these enzymes act as plants active defense mechanism against the attack of pathogen. (author)

  11. Cadmium-induced changes in pigments, total phenolics, and phenylalanine ammonia-lyase activity in fronds of Azolla imbricata.

    Science.gov (United States)

    Dai, Ling-Peng; Xiong, Zhi-Ting; Huang, Yu; Li, Min-Jing

    2006-10-01

    This study was designed to examine the effects of cadmium on several color-related parameters (including chlorophyll, carotenoid, and anthocyanin), total phenolics, and phenylalanine ammonia-lyase (PAL) activity in an aquatic fern species Azolla imbricate (A. imbricata). Cd accumulation and effects in the fronds were closely related with Cd concentration in the growth medium. The fronds under 0.5 mg/L Cd treatment turned red on the 3rd day, and this color change also appeared under 0.05 and 0.1 mg/L Cd treatment on the 5th day. Correlated with the color change, the contents of chlorophyll and carotenoid in the fronds significantly decreased in the presence of high Cd concentrations, while the anthocyanin content increased during the experiment. Significant increase in total phenolics content and PAL activity were also detected during Cd treatment. The results suggested that the Cd-induced change in color of fronds might be due to the decrease in chlorophyll and carotenoid and the increase in anthocyanin. Anthocyanin, total phenolics and their biosynthesis-related PAL might play a role in detoxification of Cd in A. imbricata.

  12. [The relationship between activity and gene expression of phenylalanine ammonia-lyase and peel pitting in 'Fengjie' navel orange fruits].

    Science.gov (United States)

    Li, Zheng-Guo; Gao, Xue; Fan, Jing; Yang, Ying-Wu; Li, Dao-Gao; Kanellis, Angelos K

    2006-06-01

    Citrus fruit is prone to develop peel pitting during development and storage, which greatly decreases its fresh market value because of the deterioration of the peel. In the present study, we have examined the effect of different temperatures (15 degrees C and 4 degrees C), waxing and mechanical damage on the changes in the activity of phenylalanine ammonia-lyase (PAL) and the incidence of peel pitting in 'Fengjie' navel orange (Citrus sinensis Osbeck) fruits. The expression levels of PAL2, PAL6 genes in the peel during the development of peel pitting have been investigated through semi-quantitative PCR method. The incidence of peel pitting was greatly enhanced by waxing and mechanical damage and was decreased in lower temperature storage (4 degrees C) (Fig.1). Waxing and mechanical damage might be the important factors inducing peel pitting and suitable low temperature could decrease the incidence of this disease. The PAL activity increased during the whole storage period in accordance with the development of this pitting (Fig.2). The expression levels of PAL2 and PAL6 genes in damaged peel were higher than those in healthy peel and the expression of PAL2 is much more higher than that of PAL6 (Figs.4 and 5). The results suggested that the enzyme activity of PAL, along with the expression of PAL2 gene is highly related to this peel pitting occurred on 'Fengjie' navel orange fruits.

  13. Molecular and analysis of a phenylalanine ammonia-lyase gene (LrPAL2) from Lycoris radiata.

    Science.gov (United States)

    Jiang, Yumei; Xia, Bing; Liang, Lijian; Li, Xiaodan; Xu, Sheng; Peng, Feng; Wang, Ren

    2013-03-01

    Phenylalanine ammonia-lyase (PAL), the first enzyme of phenylpropanoid biosynthesis, participates in the biosynthesis of flavonoids, lignins, stilbenes and many other compounds. In this study, we cloned a 2,326 bp full-length PAL2 gene from Lycoris radiata by using degenerate oligonucleotide primer PCR (DOP-PCR) and the rapid amplification of cDNA ends method. The cDNA contains a 2,124 bp coding region encoding 707 amino acids. The LrPAL2 shares about 77.0 % nucleic acid identity and 83 % amino acid identity with LrPAL1. Furthermore, genome sequence analysis demonstrated that LrPAL2 gene contains one intron and two exons. The 5' flanking sequence of LrPAL2 was also cloned by self-formed adaptor PCR (SEFA-PCR), and a group of putative cis-acting elements such as TATA box, CAAT box, G box, TC-rich repeats, CGTCA motif and TCA-element were identified. The LrPAL2 was detected in all tissues examined, with high abundance in bulbs at leaf sprouting stage and in petals at blooming stage. Besides, LrPAL2 drastically responded to MJ, SNP and UV, moderately responded to GA and SA, and a little increased under wounding. Comparison of LrPAL2 expression and LrPAL1 expression demonstrated that LrPAL2 can be more significantly induced than LrPAL1 under the above treatments, and LrPAL2 transcripts accumulated prominently at blooming stage, especially in petals, while LrPAL1 transcripts did not accumulated significantly at blooming stage. All these results suggested that LrPAL2 might play distinct roles in different branches of the phenylpropanoid pathway.

  14. Suppressed phenylalanine ammonia-lyase activity after heat shock in transgenic Nicotiana plumbaginifolia containing an Arabidopsis HSP18.2-parsley PAL2 chimera gene.

    Science.gov (United States)

    Moriwaki, M; Yamakawa, T; Washino, T; Kodama, T; Igarashi, Y

    1999-01-01

    The activity of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) after heat shock (HS) in leaves and buds of transgenic Nicotiana plumbaginifolia containing an Arabidopsis HSP18.2 promoter-parsley phenylalanine ammonia-lyase 2 (HSP18.2-PAL2) chimera gene was examined. Immediately after HS treatment at 44 degrees C for 5 h, the PAL activity in both transgenic and normal (untransformed) plants was 35-38% lower than that before HS. At normal temperature (25-26 degrees C), the PAL activity recovered within 5 h of ending the HS treatment in normal plants, but not until 12-24 h in transgenic plants containing the HSP18.2-PAL2 gene. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed the presence of parsley PAL2 mRNA in transgenic plants, which remained for 8-12 h following 5-h HS at 44 degrees C; the mRNA was not observed before HS. The content of chlorogenic acid (CGA; 3-caffeoylquinic acid) decreased drastically 8-12 h after HS in transgenic plants, but only slightly in normal plants. Thus, the decrease in PAL activity accompanied by expression of the parsley PAL2 gene after HS treatment corresponded to the decrease in CGA synthesis. These results might be attributed to post-transcriptional degradation of endogenous PAL mRNA triggered by transcription of the transgene.

  15. Ammonia lyases and aminomutases as biocatalysts for the synthesis of α-amino and β-amino acids.

    Science.gov (United States)

    Turner, Nicholas J

    2011-04-01

    Ammonia lyases catalyse the reversible addition of ammonia to cinnamic acid (1: R=H) and p-hydroxycinnamic (1: R=OH) to generate L-phenylalanine (2: R=H) and L-tyrosine (2: R=OH) respectively (Figure 1a). Both phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL) are widely distributed in plants, fungi and prokaryotes. Recently there has been interest in the use of these enzymes for the synthesis of a broader range of L-arylalanines. Aminomutases catalyse a related reaction, namely the interconversion of α-amino acids to β-amino acids (Figure 1b). In the case of L-phenylalanine, this reaction is catalysed by phenylalanine aminomutase (PAM) and proceeds stereospecifically via the intermediate cinnamic acid to generate β-Phe 3. Ammonia lyases and aminomutases are related in sequence and structure and share the same active site cofactor 4-methylideneimidazole-5-one (MIO). There is currently interest in the possibility of using these biocatalysts to prepare a wide range of enantiomerically pure l-configured α-amino and β-amino acids. Recent reviews have focused on the mechanism of these MIO containing enzymes. The aim of this review is to review recent progress in the application of ammonia lyase and aminomutase enzymes to prepare enantiomerically pure α-amino and β-amino acids. Copyright © 2010 Elsevier Ltd. All rights reserved.

  16. Change in the Content of Salicylic Acid and in the Activities of Phenylalanine Ammonia-Lyase and Catalase in Wheat Seedling Roots Under the Effect of Azospirillum Lectins

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    Alen'kina S.A.

    2012-05-01

    Full Text Available We investigated the time course of changes in the endogenous content of salicylic acid, the ratio between the acid's free and bound forms, and changes in the activities of phenylalanine ammonia-lyase and catalase in wheat seedling roots under the effect of the lectins of two strains of the associative nitrogen-fixing bacterium Azospirillum: A. brasilense Sp7 and its mutant defective in lectin activity, A. brasilense Sp7.2.3. Differences in plant response to the action of the lectins from these two strains were established. On the basis of the obtained data, a model was proposed for lectin-assisted induction of resistance, according to which the lectin effect on the roots of seedlings results in accumulation of free salicylic acid, which inhibits catalase activity, ultimately leading to accumulation of hydrogen peroxide and to formation of induced resistance.

  17. Structure and characterization of a cDNA clone for phenylalanine ammonia-lyase from cut-injured roots of sweet potato

    International Nuclear Information System (INIS)

    Tanaka, Yoshiyuki; Matsuoka, Makoto; Yamanoto, Naoki; Ohashi, Yuko; Kano-Murakami, Yuriko; Ozeki, Yoshihiro

    1989-01-01

    A cDNA clone for phenylalanine ammonia-lyase (PAL) induced in wounded sweet potato (Ipomoea batatas Lam.) root was obtained by immunoscreening a cDNA library. The protein produced in Escherichia coli cells containing the plasmid pPAL02 was indistinguishable from sweet potato PAL as judged by Ouchterlony double diffusion assays. The M r of its subunit was 77,000. The cells converted [ 14 C]-L-phenylalanine into [ 14 C]-t-cinnamic acid and PAL activity was detected in the homogenate of the cells. The activity was dependent on the presence of the pPAL02 plasmid DNA. The nucleotide sequence of the cDNA contained a 2,121-base pair (bp) open-reading frame capable of coding for a polypeptide with 707 amino acids (M r 77,137), a 22-bp 5'-noncoding region and a 207-bp 3'-noncoding region. The results suggest that the insert DNA fully encoded the amino acid sequence for sweet potato PAL that is induced by wounding. Comparison of the deduced amino acid sequence with that of a PAL cDNA fragment from Phaseolus vulgaris revealed 78.9% homology. The sequence from amino acid residues 258 to 494 was highly conserved, showing 90.7% homology

  18. Regulation of a phenylalanine ammonia lyase (BbPAL) by calmodulin in response to environmental changes in the entomopathogenic fungus Beauveria bassiana.

    Science.gov (United States)

    Kim, Jiyoung; Park, Hyesung; Han, Jae-Gu; Oh, Junsang; Choi, Hyung-Kyoon; Kim, Seong Hwan; Sung, Gi-Ho

    2015-11-01

    Phenylalanine ammonia lyase (PAL, E.C. 4.3.1.5) catalyses the deamination of L -phenylalanine to trans-cinnamic acid and ammonia, facilitating a critical step in the phenylpropanoid pathway that produces a variety of secondary metabolites. In this study, we isolated BbPAL gene in the entomopathogenic fungus Beauveria bassiana. According to multiple sequence alignment, homology modelling and in vitro PAL activity, we demonstrated that BbPAL acts as a typical PAL enzyme in B. bassiana. BbPAL interacted with calmodulin (CaM) in vitro and in vivo, indicating that BbPAL is a novel CaM-binding protein. The functional role of CaM in BbPAL action was to negatively regulate the BbPAL activity in B. bassiana. High-performance liquid chromatography analysis revealed that L -phenylalanine was reduced and trans-cinnamic acid was increased in response to the CaM inhibitor W-7. Dark conditions suppressed BbPAL activity in B. bassiana, compared with light. In addition, heat and cold stresses inhibited BbPAL activity in B. bassiana. Interestingly, these negative effects of BbPAL activity by dark, heat and cold conditions were recovered by W-7 treatment, suggesting that the inhibitory mechanism is mediated through stimulation of CaM activity. Therefore, this work suggests that BbPAL plays a role in the phenylpropanoid pathway mediated by environmental stimuli via the CaM signalling pathway. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

  19. Cloning and Expression Analysis of Phenylalanine Ammonia-Lyase Gene in the Mycelium and Fruit Body of the Edible Mushroom Flammulina velutipes

    Science.gov (United States)

    Yun, Yeo Hong; Koo, Ja Sun

    2015-01-01

    Phenylalanine ammonia-lyase (PAL) gene is known to be expressed in plants, and is involved in the differentiation, growth and synthesis of secondary metabolites. However, its expression in fungi remains to be explored. To understand its expression in mushroom fungi, the PAL gene of the edible mushroom Flammulina velutipes (Fvpal) was cloned and characterized. The cloned Fvpal consists of 2,175 bp, coding for a polypeptide containing 724 amino acids and having 11 introns. The translated amino acid sequence of Fvpal shares a high identity (66%) with that of ectomycorrhizal fungus Tricholoma matsutake. Distinctively, the Fvpal expression in the mycelium was higher in minimal medium supplemented with L-tyrosine than with other aromatic amino acids. During cultivation of the mushroom on sawdust medium, Fvpal expression in the fruit body correspondingly increased as the mushroom grew. In the fruiting body, Fvpal was expressed more in the stipe than in the pileus. These results suggest that F. velutipes PAL activity differs in the different organs of the mushroom. Overall, this is first report to show that the PAL gene expression is associated with mushroom growth in fungi. PMID:26539050

  20. DNA Methylation Influences Chlorogenic Acid Biosynthesis in Lonicera japonica by Mediating LjbZIP8 to Regulate Phenylalanine Ammonia-Lyase 2 Expression

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    Liangping Zha

    2017-07-01

    Full Text Available The content of active compounds differ in buds and flowers of Lonicera japonica (FLJ and L. japonica var. chinensis (rFLJ. Chlorogenic acid (CGAs were major active compounds of L. japonica and regarded as measurements for quality evaluation. However, little is known concerning the formation of active compounds at the molecular level. We quantified the major CGAs in FLJ and rFLJ, and found the concentrations of CGAs were higher in the buds of rFLJ than those of FLJ. Further analysis of CpG methylation of CGAs biosynthesis genes showed differences between FLJ and rFLJ in the 5′-UTR of phenylalanine ammonia-lyase 2 (PAL2. We identified 11 LjbZIP proteins and 24 rLjbZIP proteins with conserved basic leucine zipper domains, subcellular localization, and electrophoretic mobility shift assay showed that the transcription factor LjbZIP8 is a nuclear-localized protein that specifically binds to the G-box element of the LjPAL2 5′-UTR. Additionally, a transactivation assay and LjbZIP8 overexpression in transgenic tobacco indicated that LjbZIP8 could function as a repressor of transcription. Finally, treatment with 5-azacytidine decreased the transcription level of LjPAL2 and CGAs content in FLJ leaves. These results raise the possibility that DNA methylation might influence the recruitment of LjbZIP8, regulating PAL2 expression level and CGAs content in L. japonica.

  1. Rhodotorulaglutinis phenylalanine/tyrosine ammonia lyase enzyme catalyzed synthesis of the methyl ester of para-hydroxycinnamic acid and its potential antibacterial activity

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    Marybeth C MacDonald

    2016-03-01

    Full Text Available Biotransformation of L-tyrosine methyl ester (L-TM to the methyl ester of para- hydroxycinnamic acid (p-HCAM using Rhodotorula glutinis yeast phenylalanine/tyrosine ammonia lyase (PTAL; EC 4.3.1.26 enzyme was successfully demonstrated for the first time; progress of the reaction was followed by spectrophotometric determination at 315 nm. The following conditions were optimized for maximal formation of p-HCAM: pH (8.5, temperature (37 C, speed of agitation (50 rpm, enzyme concentration (0.080 µM, and substrate concentration (0.50 mM. Under these conditions, the yield of the reaction was ~15% in 1 h incubation period and ~63% after an overnight (~18 h incubation period. The product (p-HCAM of the reaction of PTAL with L-TM was confirmed using Nuclear Magnetic Resonance spectroscopy (NMR. Fourier Transform Infra-Red spectroscopy (FTIR was carried out to rule out potential hydrolysis of p-HCAM during overnight incubation. Potential antibacterial activity of p-HCAM was tested against several strains of Gram positive and Gram negative bacteria. This study describes a synthetically useful transformation, and could have future clinical and industrial applications.

  2. Developmental role of phenylalanine-ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H) genes during adventitious rooting of Juglans regia L. microshoots.

    Science.gov (United States)

    Cheniany, Monireh; Ganjeali, Ali

    2016-12-01

    Phenylalanine-ammonia-lyase and cinnamate-4-hydroxylase play important role in the phenylpropanoid pathway, which produces many biologically important secondary metabolites participating in normal plant development. Flavonol quercetin is the main representant of these compounds that has been identified in numerous Juglans spp. In this survey, the developmental expression patterns of PAL and C4H genes during in vitro rooting of two walnut cultivars 'Sunland' and 'Howard' was examined by RT-PCR. To understand the potential role in rooting, the changing pattern of endogenous content of quercetin was also analyzed by HPLC. The 'Sunland' with better capacity to root had more quercetin content during the "inductive phase" of rooting than 'Howard'. In each cultivar, the level of PAL transcripts showed the same behavior with the changing patterns of quercetin during root formation of microshoots. The positive correlation between the changes of quercetin and PAL-mRNA indicated that PAL gene may have an immediate effect on flavonoid pathway metabolites including quercetin. Although the behavioral change of C4H expression was similar in both cultivars during root formation (with significantly more level for 'Howard'), it was not coincide with the changes of quercerin concentrations. Our results showed that C4H function is important for the normal development, but its transcriptional regulation does not correlate with quercetin as an efficient phenolic compound for walnut rhizogenesis.

  3. Expression and enzymatic activity of phenylalanine ammonia-lyase and p-coumarate 3-hydroxylase in mango (Mangifera indica 'Ataulfo') during ripening.

    Science.gov (United States)

    Palafox-Carlos, H; Contreras-Vergara, C A; Muhlia-Almazán, A; Islas-Osuna, M A; González-Aguilar, G A

    2014-05-16

    Phenylalanine ammonia lyase (PAL) and p-coumarate 3-hydroxylase (C3H) are key enzymes in the phenylpropanoid pathway. The relative expression of PAL and C3H was evaluated in mango fruit cultivar 'Ataulfo' in four ripening stages (RS1, RS2, RS3, and RS4) by quantitative polymerase chain reaction. In addition, enzyme activity of PAL and C3H was determined in mango fruits during ripening. The PAL levels were downregulated at the RS2 and RS3 stages, while C3H levels were upregulated in fruits only at RS3. The enzyme activity of PAL followed a pattern that was different from that of the PAL expression, thus suggesting regulation at several levels. For C3H, a regulation at the transcriptional level is suggested because a similar pattern was revealed by its activity and transcript level. In this study, the complexity of secondary metabolite biosynthesis regulation is emphasized because PAL and C3H enzymes are involved in the biosynthesis of several secondary metabolites that are active during all mango ripening stages.

  4. A phenylalanine ammonia-lyase ortholog (PkPAL1) from Picrorhiza kurrooa Royle ex. Benth: molecular cloning, promoter analysis and response to biotic and abiotic elicitors.

    Science.gov (United States)

    Bhat, Wajid Waheed; Razdan, Sumeer; Rana, Satiander; Dhar, Niha; Wani, Tariq Ahmad; Qazi, Parvaiz; Vishwakarma, Ram; Lattoo, Surrinder K

    2014-09-01

    Picrorhiza kurrooa Royle ex Benth. is a highly reputed medicinal herb utilised in the preparation of a number of herbal drug formulations, principally due to the presence of novel monoterpene iridoid glycosides kenned as picrosides. Phenylalanine ammonia-lyase catalyses an important rate-limiting step in phenylpropanoid pathway and supplies precursors like cinnamic acid, vanillic acid, ferulic acid, etc., to a variety of secondary metabolites including picrosides. The imperilled status of P. kurrooa coupled with lack of information regarding biogenesis of picrosides necessitates deciphering the biosynthetic pathway for picrosides. In the present study, a PAL gene, designated PkPAL1 was isolated from P. kurrooa. The cDNA is 2312 bp in length, consisting of an ORF of 2142 bp encoding for a 713 amino acid protein having a predicted molecular weight of 77.66 kDa and an isoelectric point of pH 6.82. qRT-PCR analysis of various tissues of P. kurrooa showed that PkPAL1 transcript levels were highest in the leaves, consistent with picroside accumulation pattern. Using Genome walking, a 718 bp promoter region was also isolated resulting in identification of distinct cis-regulatory elements including TGA-element, TGACG-motif, CGTCA-motif, etc. qRT-PCR indicated up-regulation of PkPAL1 by methyl jasmonate, salicylic acid, 2,4-dicholorophenoxy acetic acid and UV-B elicitations that corroborated positively with the identified cis-elements within the promoter region. Moreover, altitude was found to have a positive effect on the PkPAL1 transcript levels, driving the expression of PkPAL1 abundantly. Based on docking analysis, we identified eight residues as potentially essential for substrate binding in PkPAL1. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Isolation and Functional Characterization of a Phenylalanine Ammonia-Lyase Gene (SsPAL1 from Coleus (Solenostemon scutellarioides (L. Codd

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    Qinlong Zhu

    2015-09-01

    Full Text Available Phenylalanine ammonia-lyase (PAL is the first enzyme involved in the phenylpropanoid pathway and plays important roles in the secondary metabolisms, development and defense of plants. To study the molecular function of PAL in anthocyanin synthesis of Coleus (Solenostemon scutellarioides (L. Codd, a Coleus PAL gene designated as SsPAL1 was cloned and characterized using a degenerate oligonucleotide primer PCR and RACE method. The full-length SsPAL1 was 2450 bp in size and consisted of one intron and two exons encoding a polypeptide of 711 amino acids. The deduced SsPAL1 protein showed high identities and structural similarities with other functional plant PAL proteins. A series of putative cis-acting elements involved in transcriptional regulation, light and stress responsiveness were found in the upstream regulatory sequence of SsPAL1. Transcription pattern analysis indicated that SsPAL1 was constitutively expressed in all tissues examined and was enhanced by light and different abiotic factors. The recombinant SsPAL1 protein exhibited high PAL activity, at optimal conditions of 60 °C and pH 8.2. Although the levels of total PAL activity and total anthocyanin concentration have a similar variation trend in different Coleus cultivars, there was no significant correlation between them (r = 0.7529, p > 0.1, suggesting that PAL was not the rate-limiting enzyme for the downstream anthocyanin biosynthetic branch in Coleus. This study enables us to further understand the role of SsPAL1 in the phenylpropanoid (flavonoids, anthocyanins biosynthesis in Coleus at the molecular level.

  6. Electromagnetic Field Effects on Production of Salicylic Acid and Phenylalanine ammonia lyase in Tobacco Infected with Potato Virus X (PVX

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    amin radmard titkanlo

    2018-02-01

    Full Text Available Introduction: Potato virus x (PVX is one of the most devastating viruses in the fields of tobacco and no effective method to control the virus has been provided yet. Magnetism is known as one of the environmental stresses that can directly or indirectly affect the plants, especially plants under stress. The magnetic fields can have beneficial affects plants. Materials and Methods: In the present study to investigate the effects of electromagnetic fields on PVX control, an experiment based on completely randomized design with eleven treatments (10, 50 and 90 mT in a period of 60, 90 and 120 minutes on tobacco seedlings infected with the virus and two control, and the other one is free from analog infected with the virus in tobacco seedlings carried on six repeats.. After inoculation at the four-leaf stage and after 14 days, the amount of turbidity virus infection rate is calculated and then ensure equality of tobacco seedlings, where the electromagnetic device. 30 days after applying an electromagnetic field analysis of the results of ELISA test two samples were tested with antisera Chndhmsanh of PVX. The amount of salicylic acid and the enzyme phenylalanine ammonia in systemic acquired resistance and plant defense mechanisms are involved, the treatments were measured, for evaluation of HPLC as salicylic acid and phenylalanine ammonia-based assessment of trans-cinnamic acid was used. Results and Discussion: Statistical analysis ELISA test results revealed that there is a significant difference between treatments 0/05. So that the treatment was a no pollution treatment and 11 who were infected but had not been exposed to the electromagnetic field had a significant difference compared to other treatments, among the treatments that were exposed to the electromagnetic field treatment 8 (100 mT, 120 minutes showed the lowest OD in ELISA and later treatment 7 (50 mT and 60 minutes was the lowest OD, the two treatments were observed in less than two OD

  7. Hydrogen Peroxide Treatment and the Phenylpropanoid Pathway Precursors Feeding Improve Phenolics and Antioxidant Capacity of Quinoa Sprouts via an Induction of L-Tyrosine and L-Phenylalanine Ammonia-Lyases Activities

    Directory of Open Access Journals (Sweden)

    Michał Świeca

    2016-01-01

    Full Text Available Hydrogen peroxide treatment and the phenylpropanoid pathway precursors feeding affected the antioxidant capacity of quinoa sprouts. Compared to the control, total phenolics content was significantly increased by treatment of control sprouts with 50 mM and 200 mM H2O2—an elevation of about 24% and 28%, respectively. The highest increase of flavonoids content was found for the sprouts treated with 200 mM H2O2 obtained from seeds fed with shikimic acid. All the studied modifications increased the antioxidant potential of sprouts (at least by 50% compared to control. The highest reducing power was found for the sprouts treated with 200 mM H2O2 obtained by phenylalanine feeding (5.03 mg TE/g DW and those obtained from the seeds fed with tyrosine (5.26 mg TE/g DW. The activities of L-tyrosine (TAL and L-phenylalanine (PAL ammonia-lyases were strongly affected by germination time as well as the applied modification of sprouting. On the 3rd day the highest PAL activity was determined for both untreated and induced with 50 mM H2O2 sprouts obtained by phenylalanine feeding. H2O2 induced TAL activity; the highest TAL activity was determined for 3-day-old sprouts induced with 200 mM H2O2 obtained from seeds fed with phenylalanine.

  8. Wound-induced ethylene synthesis and expression and formation of 1-aminocyclopropane-1-carboxylate (ACC) synthase, ACC oxidase, phenylalanine ammonia-lyase, and peroxidase in wounded mesocarp tissue of Cucurbita maxima.

    Science.gov (United States)

    Kato, M; Hayakawa, Y; Hyodo, H; Ikoma, Y; Yano, M

    2000-04-01

    1-Aminocyclopropane-1-carboxylate (ACC) synthase was rapidly induced in mesocarp tissue of Cucurbita maxima after wounding in the cut surface layer in 1 mm thickness (ca. 9 cells) (first layer) in both the enzyme activity and the levels of transcript. This led to a rapid accumulation of ACC and hence ethylene production. In the inside tissue (1-2 mm) (second layer), no significant induction of ACC synthase was observed, which resulted in a low level of ACC, although ethylene was evolved at a much lower rate than the first one. In contrast to ACC synthase, ACC oxidase was induced markedly in both the first and second layers and the development of its activity and the levels of mRNA remained high until later stages. It was considered that wound ethylene was closely associated with the development of ACC oxidase, since 2,5-norbornadiene (NBD), an inhibitor of ethylene action, substantially suppressed it. Phenylalanine ammonia-lyase (PAL) greatly increased in activity after wounding similarly to that of ACC synthase, in which increase in PAL activity occurred predominantly in the first layer. Induction of peroxidase activity after wounding had a close correlation in profile with that of ACC oxidase in that marked increases in the activity were observed in both the first and second layers and were strongly suppressed by NBD application. Four peroxidase isozymes were found by PAGE, among which a fraction was newly detected after wounding.

  9. Highly Active and Specific Tyrosine Ammonia-Lyases from Diverse Origins Enable Enhanced Production of Aromatic Compounds in Bacteria and Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Jendresen, Christian Bille; Stahlhut, Steen Gustav; Li, Mingji

    2015-01-01

    Phenylalanine and tyrosine ammonia-lyases form cinnamic acid and p-coumaric acid, which are precursors of a wide range of aromatic compounds of biotechnological interest. Lack of highly active and specific tyrosine ammonia-lyases has previously been a limitation in metabolic engineering approaches...

  10. Phenylalanine ammonia lyase from Arabidopsis thaliana (AtPAL2): A potent MIO-enzyme for the synthesis of non-canonical aromatic alpha-amino acids: Part I: Comparative characterization to the enzymes from Petroselinum crispum (PcPAL1) and Rhodosporidium toruloides (RtPAL).

    Science.gov (United States)

    Dreßen, Alana; Hilberath, Thomas; Mackfeld, Ursula; Billmeier, Arne; Rudat, Jens; Pohl, Martina

    2017-09-20

    Phenylalanine ammonia lyase (PAL) from Arabidopsis thaliana (AtPAL2) was comparatively characterized to the well-studied enzyme from parsley (PcPAL1) and Rhodosporidium toruloides (RtPAL) with respect to kinetic parameters for the deamination and the amination reaction, pH- and temperature optima and the substrate range of the amination reaction. Whereas both plant enzymes are specific for phenylalanine, the bifunctional enzyme from Rhodosporidium toruloides shows K M -values for L-Phe and L-Tyr in the same order of magnitude and, compared to both plant enzymes, a 10-15-fold higher activity. At 30°C all enzymes were sufficiently stable with half-lives of 3.4days (PcPAL1), 4.6days (AtPAL2) and 9.7days (RtPAL/TAL). Very good results for the amination of various trans-cinnamic acid derivatives were obtained using E. coli cells as whole cell biocatalysts in ammonium carbonate buffer. Investigation of the substrate ranges gave interesting results for the newly tested enzymes from A. thaliana and R. toruloides. Only the latter accepts besides 4-hydroxy-CA also 3-methoxy-4-hydroxy-CA as a substrate, which is an interesting intermediate for the formation of pharmaceutically relevant L-Dopa. AtPAL2 is a very good catalyst for the formation of (S)-3-F-Phe, (S)-4-F-Phe and (S)-2-Cl-Phe. Such non-canonical amino acids are valuable building blocks for the formation of various drug molecules. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Priming ammonia lyases and aminomutases for industrial and therapeutic applications

    NARCIS (Netherlands)

    Heberling, Matthew M.; Wu, Bian; Bartsch, Sebastian; Janssen, Dick B.

    Ammonia lyases (AL) and aminomutases (AM) are emerging in green synthetic routes to chiral amines and an AL is being explored as an enzyme therapeutic for treating phenylketonuria and cancer. Although the restricted substrate range of the wild-type enzymes limits their widespread application, the

  12. Processes for the production of hydroxycinnamic acids using polypeptides having tyrosine ammonia lyase activity

    DEFF Research Database (Denmark)

    2016-01-01

    The present invention generally relates to the field of biotechnology as it applies to the production of hydroxycinnamic acids using polypeptides having tyrosine ammonia lyase activity. More particularly, the present invention pertains to polypeptides having tyrosine ammonia lyase activity and high...... substrate specificity towards tyrosine, which makes them particularly suitable in the production of p-coumaric acid and other hydroxycinnamic acids. The present invention thus provides processes for the production of p-coumaric acid and other hydroxycinnamic acids employing these polypeptides as well...

  13. Catalytic Mechanisms and Biocatalytic Applications of Aspartate and Methylaspartate Ammonia Lyases

    NARCIS (Netherlands)

    de Villiers, Marianne; Veetil, Vinod Puthan; Raj, Hans; de Villiers, Jandre; Poelarends, Gerrit J.

    2012-01-01

    Ammonia lyases catalyze the formation of alpha-beta-unsaturated bonds by the elimination of ammonia from their substrates. This conceptually straightforward reaction has been the emphasis of many studies, with the main focus on the catalytic mechanism of these enzymes and/or the use of these enzymes

  14. Biocatalytic Enantioselective Synthesis of N-Substituted Aspartic Acids by Aspartate Ammonia Lyase

    NARCIS (Netherlands)

    Weiner, Barbara; Poelarends, Gerrit J.; Janssen, Dick B.; Feringa, Ben L.

    2008-01-01

    The gene encoding aspartate ammonia lyase (aspB) from Bacillus sp. YM55-1 has been cloned and overexpressed, and the recombinant enzyme containing a C-terminal His6 tag has been purified to homogeneity and subjected to kinetic characterization. Kinetic studies have shown that the His6 tag does not

  15. Engineering methylaspartate ammonia lyase for the asymmetric synthesis of unnatural amino acids

    NARCIS (Netherlands)

    Raj, Hans; Szymanski, Wiktor; de Villiers, Jandre; Rozeboom, Henriëtte J.; Puthan Veetil, Vinod; Reis, Carlos R.; Villiers, Marianne de; Dekker, Frank J.; Wildeman, Stefaan de; Quax, Wim J.; Thunnissen, Andy-Mark W.H.; Feringa, Ben L.; Janssen, Dick B.; Poelarends, Gerrit J.

    The redesign of enzymes to produce catalysts for a predefined transformation remains a major challenge in protein engineering. Here, we describe the structure-based engineering of methylaspartate ammonia lyase (which in nature catalyses the conversion of 3-methylaspartate to ammonia and

  16. Kinetic Resolution and Stereoselective Synthesis of 3-Substituted Aspartic Acids by Using Engineered Methylaspartate Ammonia Lyases

    NARCIS (Netherlands)

    Raj, Hans; Szymanski, Wiktor; Villiers, Jandré de; Puthan Veetil, Vinod; Quax, Wim J.; Shimamoto, Keiko; Janssen, Dick B.; Feringa, Ben L.; Poelarends, Gerrit J.

    2013-01-01

    Kinetic resolution and asymmetric synthesis of various valuable 3-substituted aspartic acids, which were obtained in fair to good yields with diastereomeric ratio values of up to >98:2 and enantiomeric excess values of up to >99 %, by using engineered methylaspartate ammonia lyases are described.

  17. Molecular cloning, characterization and expression of phenylalanine ...

    African Journals Online (AJOL)

    A full-length cDNA and genomic DNA of phenylalanine ammonia-lyase gene, which catalyzes the first step in the flavonoid biosynthetic pathway, were isolated from Ginkgo biloba for the first time (designated as GbPAL, GenBank Accession No. EU071050). The cDNA and genomic DNA sequences of GbPAL were the same, ...

  18. Crystal structure and characterization of a novel L-serine ammonia-lyase from Rhizomucor miehei

    Energy Technology Data Exchange (ETDEWEB)

    Qin, Zhen [College of Food Science and Nutritional Engineering, Beijing Advanced Innovation Center of Food Nutrition and Human Health, China Agricultural University, Beijing 100083 (China); Yan, Qiaojuan [College of Engineering, China Agricultural University, Beijing 100083 (China); Ma, Qingjun [Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071 (China); Jiang, Zhengqiang, E-mail: zhqjiang@cau.edu.cn [College of Food Science and Nutritional Engineering, Beijing Advanced Innovation Center of Food Nutrition and Human Health, China Agricultural University, Beijing 100083 (China)

    2015-10-23

    L-serine ammonia-lyase, as a member of the β-family of pyridoxal-5′-phosphate (PLP) dependent enzymes, catalyzes the conversion of L-serine (L-threonine) to pyruvate (α-ketobutyrate) and ammonia. The crystal structure of L-serine ammonia-lyase from Rhizomucor miehei (RmSDH) was solved at 1.76 Å resolution by X-ray diffraction method. The overall structure of RmSDH had the characteristic β-family PLP dependent enzyme fold. It consisted of two distinct domains, both of which show the typical open twisted α/β structure. A PLP cofactor was located in the crevice between the two domains, which was attached to Lys52 by a Schiff-base linkage. Unique residue substitutions (Gly78, Pro79, Ser146, Ser147 and Thr312) were discovered at the catalytic site of RmSDH by comparison of structures of RmSDH and other reported eukaryotic L-serine ammonia-lyases. Optimal pH and temperature of the purified RmSDH were 7.5 and 40 °C, respectively. It was stable in the pH range of 7.0–9.0 and at temperatures below 40 °C. This is the first crystal structure of a fungal L-serine ammonia-lyase. It will be useful to study the catalytic mechanism of β-elimination enzymes and will provide a basis for further enzyme engineering. - Highlights: • The crystal structure of a fungal L-serine ammonia-lyase (RmSDH) was solved. • Five unique residue substitutions are found at the catalytic site of RmSDH. • RmSDH was expressed in Pichia. pastoris and biochemically characterized. • RmSDH has potential application in splitting D/L-serine.

  19. Bioproduction of L-Aspartic Acid and Cinnamic Acid by L-Aspartate Ammonia Lyase from Pseudomonas aeruginosa PAO1.

    Science.gov (United States)

    Patel, Arti T; Akhani, Rekha C; Patel, Manisha J; Dedania, Samir R; Patel, Darshan H

    2017-06-01

    Aspartase (L-aspartate ammonia lyase, EC 4.3.1.1) catalyses the reversible amination and deamination of L-aspartic acid to fumaric acid which can be used to produce important biochemical. In this study, we have explored the characteristics of aspartase from Pseudomonas aeruginosa PAO1 (PA-AspA). To overproduce PA-AspA, the 1425-bp gene was introduced in Escherichia coli BL21 and purified. A 51.0-kDa protein was observed as a homogenous purified protein on SDS-PAGE. The enzyme was optimally active at pH 8.0 and 35 °C. PA-AspA has retained 56% activity after 7 days of incubation at 35 °C, which displays the hyperthermostablility characteristics of the enzyme. PA-AspA is activated in the presence of metal ions and Mg2+ is found to be most effective. Among the substrates tested for specificity of PA-AspA, L-phenylalanine (38.35 ± 2.68) showed the highest specific activity followed by L-aspartic acid (31.21 ± 3.31) and fumarate (5.42 ± 2.94). K m values for L-phenylalanine, L-aspartic acid and fumarate were 1.71 mM, 0.346 μM and 2 M, respectively. The catalytic efficiency (k cat /K m ) for L-aspartic acid (14.18 s -1  mM -1 ) was higher than that for L-phenylalanine (4.65 s -1  mM -1 ). For bioconversion, from an initial concentration of 1000 mM of fumarate and 30 mM of L-phenylalanine, PA-AspA was found to convert 395.31 μM L-aspartic acid and 3.47 mM cinnamic acid, respectively.

  20. Phenylalanine Ammonia Lyase Activity in Stem of Pepper (Capsicum annuum L.) Infected by Phytophthora capsici L.

    OpenAIRE

    KOC, Esra; USTUN, Ayşen Sulun

    2012-01-01

    In this study, PAL activity in stems of pepper (Capsicum annuum L.) plants infected by the root rot pathogen Phytophthora capsici-22 in a resistant (PM-702) and two susceptible (Kahramanmaraş-Hot (KM-Hot) and Demre-8) cultivar were studied. The response of the PAL activity in the resistant cultivar was faster and higher than in the susceptible cultivars (p < 0.01). The increase in production of  PAL upon Phytophthora capsici-22 were higher in the infected plants compared to the non-in...

  1. Kinetic resolution and stereoselective synthesis of 3-substituted aspartic acids by using engineered methylaspartate ammonia lyases.

    Science.gov (United States)

    Raj, Hans; Szymanski, Wiktor; de Villiers, Jandré; Puthan Veetil, Vinod; Quax, Wim J; Shimamoto, Keiko; Janssen, Dick B; Feringa, Ben L; Poelarends, Gerrit J

    2013-08-19

    Enzymatic amino acid synthesis: Kinetic resolution and asymmetric synthesis of various valuable 3-substituted aspartic acids, which were obtained in fair to good yields with diastereomeric ratio values of up to >98:2 and enantiomeric excess values of up to >99 %, by using engineered methylaspartate ammonia lyases are described. These biocatalytic methodologies for the selective preparation of aspartic acid derivatives appear to be attractive alternatives for existing chemical methods. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Probing Reversible Chemistry in Coenzyme B12-Dependent Ethanolamine Ammonia Lyase with Kinetic Isotope Effects

    Science.gov (United States)

    Jones, Alex R; Rentergent, Julius; Scrutton, Nigel S; Hay, Sam

    2015-01-01

    Coenzyme B12-dependent enzymes such as ethanolamine ammonia lyase have remarkable catalytic power and some unique properties that enable detailed analysis of the reaction chemistry and associated dynamics. By selectively deuterating the substrate (ethanolamine) and/or the β-carbon of the 5′-deoxyadenosyl moiety of the intrinsic coenzyme B12, it was possible to experimentally probe both the forward and reverse hydrogen atom transfers between the 5′-deoxyadenosyl radical and substrate during single-turnover stopped-flow measurements. These data are interpreted within the context of a kinetic model where the 5′-deoxyadenosyl radical intermediate may be quasi-stable and rearrangement of the substrate radical is essentially irreversible. Global fitting of these data allows estimation of the intrinsic rate constants associated with CoC homolysis and initial H-abstraction steps. In contrast to previous stopped-flow studies, the apparent kinetic isotope effects are found to be relatively small. PMID:25950663

  3. Identification of the substrate radical intermediate derived from ethanolamine during catalysis by ethanolamine ammonia-lyase.

    Science.gov (United States)

    Bender, Güneş; Poyner, Russell R; Reed, George H

    2008-10-28

    Rapid-mix freeze-quench (RMFQ) methods and electron paramagnetic resonance (EPR) spectroscopy have been used to characterize the steady-state radical in the deamination of ethanolamine catalyzed by adenosylcobalamin (AdoCbl)-dependent ethanolamine ammonia-lyase (EAL). EPR spectra of the radical intermediates formed with the substrates, [1-13C]ethanolamine, [2-13C]ethanolamine, and unlabeled ethanolamine were acquired using RMFQ trapping methods from 10 ms to completion of the reaction. Resolved 13C hyperfine splitting in EPR spectra of samples prepared with [1-13C]ethanolamine and the absence of such splitting in spectra of samples prepared with [2-13C]ethanolamine show that the unpaired electron is localized on C1 (the carbinol carbon) of the substrate. The 13C splitting from C1 persists from 10 ms throughout the time course of substrate turnover, and there was no evidence of a detectable amount of a product like radical having unpaired spin on C2. These results correct an earlier assignment for this radical intermediate [Warncke, K., et al. (1999) J. Am. Chem. Soc. 121, 10522-10528]. The EPR signals of the substrate radical intermediate are altered by electron spin coupling to the other paramagnetic species, cob(II)alamin, in the active site. The dipole-dipole and exchange interactions as well as the 1-13C hyperfine splitting tensor were analyzed via spectral simulations. The sign of the isotropic exchange interaction indicates a weak ferromagnetic coupling of the two unpaired electrons. A Co2+-radical distance of 8.7 A was obtained from the magnitude of the dipole-dipole interaction. The orientation of the principal axes of the 13C hyperfine splitting tensor shows that the long axis of the spin-bearing p orbital on C1 of the substrate radical makes an angle of approximately 98 degrees with the unique axis of the d(z2) orbital of Co2+.

  4. Virus-induced gene silencing of WRKY53 and an inducible phenylalanine ammonia-lyase in wheat reduces aphid resistance

    Science.gov (United States)

    Although several wheat genes differentially expressed during the Russian wheat aphid resistance response have recently been identified, their requirement for and specific role in resistance remain unclear. Progress in wheat-aphid interaction research is hampered by inadequate collections of mutant g...

  5. Relationship between browning and the activities of polyphenol oxidase and phenylalanine ammonia lyase in banana peel during low temperature storage

    NARCIS (Netherlands)

    Nguyen, T.B.T.; Ketsa, S.; Doorn, van W.G.

    2003-01-01

    Kluai Khai (Musa AA Group) and Kluai Hom Thong (Musa AAA Group) bananas were stored at 6 and 10 °C. Visible chilling injury (CI) in the peel, mainly browning, occurred at both temperatures, but more so at 6 °C, and without significant differences between the cultivars. At the time of harvest, total

  6. Preliminary structural investigations of the Eut-L shell protein of the ethanolamine ammonia-lyase metabolosome of Escherichia coli

    International Nuclear Information System (INIS)

    Nikolakakis, Kiel; Ohtaki, Akashi; Newton, Keith; Chworos, Arkadiusz; Sagermann, Martin

    2009-01-01

    Preliminary X-ray analysis of crystals of the bacterial microcompartment shell protein Eut-L from Escherichia coli is reported. The ethanolamine ammonia-lyase microcompartment is composed of five different shell proteins that have been proposed to assemble into symmetrically shaped polyhedral particles of varying sizes. Here, preliminary X-ray analysis of crystals of the bacterial microcompartment shell protein Eut-L from Escherichia coli is reported. Cloning, overexpression and purification resulted in highly pure protein that crystallized readily under many different conditions. In all cases the protein forms thin hexagonal plate-shaped crystals belonging to space group P3 that are of unusually high stability against different solvent conditions. The crystals diffracted to a resolution of 2.0 Å using synchrotron radiation but proved to be radiation-sensitive. Preparations of heavy-atom-derivatized crystals for use in determining the three-dimensional structure are under way

  7. Phenolics and Flavonoids Compounds, Phenylanine Ammonia Lyase and Antioxidant Activity Responses to Elevated CO2 in Labisia pumila (Myrisinaceae

    Directory of Open Access Journals (Sweden)

    Hawa Z.E. Jaafar

    2012-05-01

    Full Text Available A split plot 3 × 3 experiment was designed to examine the impact of three concentrations of CO2 (400, 800 and 1,200 µmol·mol−1 on the phenolic and flavonoid compound profiles, phenylalanine ammonia lyase (PAL and antioxidant activity in three varieties of Labisia pumila Benth. (var. alata, pumila and lanceolata after 15 weeks of exposure. HPLC analysis revealed a strong influence of increased CO2 concentration on the modification of phenolic and flavonoid profiles, whose intensity depended on the interaction between CO2 levels and L. pumila varieties. Gallic acid and quercetin were the most abundant phenolics and flavonoids commonly present in all the varieties. With elevated CO2 (1,200 µmol·mol−1 exposure, gallic acid increased tremendously, especially in var. alata and pumila (101–111%, whilst a large quercetin increase was noted in var. lanceolata (260%, followed closely by alata (201%. Kaempferol, although detected under ambient CO2 conditions, was undetected in all varieties after exposure. Instead, caffeic acid was enhanced tremendously in var. alata (338~1,100% and pumila (298~433%. Meanwhile, pyragallol and rutin were only seen in var. alata (810 µg·g−1 DW and pumila (25 µg·g−1 DW, respectively, under ambient conditions; but the former compound went undetected in all varieties while rutin continued to increase by 262% after CO2 enrichment. Interestingly, naringenin that was present in all varieties under ambient conditions went undetected under enrichment, except for var. pumila where it was enhanced by 1,100%. PAL activity, DPPH and FRAP also increased with increasing CO2 levels implying the possible improvement of health-promoting quality of Malaysian L. pumila

  8. Differential metabolism of L-phenylalanine in the formation of aromatic volatiles in melon (Cucumis melo L.) fruit.

    Science.gov (United States)

    Gonda, Itay; Davidovich-Rikanati, Rachel; Bar, Einat; Lev, Shery; Jhirad, Pliaa; Meshulam, Yuval; Wissotsky, Guy; Portnoy, Vitaly; Burger, Joseph; Schaffer, Arthur A; Tadmor, Yaakov; Giovannoni, James J; Fei, Zhangjun; Fait, Aaron; Katzir, Nurit; Lewinsohn, Efraim

    2018-04-01

    Studies on the active pathways and the genes involved in the biosynthesis of L-phenylalanine-derived volatiles in fleshy fruits are sparse. Melon fruit rinds converted stable-isotope labeled L-phe into more than 20 volatiles. Phenylpropanes, phenylpropenes and benzenoids are apparently produced via the well-known phenylpropanoid pathway involving phenylalanine ammonia lyase (PAL) and being (E)-cinnamic acid a key intermediate. Phenethyl derivatives seemed to be derived from L-phe via a separate biosynthetic route not involving (E)-cinnamic acid and PAL. To explore for a biosynthetic route to (E)-cinnamaldehyde in melon rinds, soluble protein cell-free extracts were assayed with (E)-cinnamic acid, CoA, ATP, NADPH and MgSO 4 , producing (E)-cinnamaldehyde in vitro. In this context, we characterized CmCNL, a gene encoding for (E)-cinnamic acid:coenzyme A ligase, inferred to be involved in the biosynthesis of (E)-cinnamaldehyde. Additionally we describe CmBAMT, a SABATH gene family member encoding a benzoic acid:S-adenosyl-L-methionine carboxyl methyltransferase having a role in the accumulation of methyl benzoate. Our approach leads to a more comprehensive understanding of L-phe metabolism into aromatic volatiles in melon fruit. Copyright © 2018 Elsevier Ltd. All rights reserved.

  9. Prokaryote metabolism activity

    OpenAIRE

    Biederman, Lori

    2017-01-01

    I wrote this activity to emphasize that prokaryotic organisms can carry out 6 different types of metabolisms (as presented in Freeman’s Biological Science textbook) and this contrasts to eukaryotes, which can only use 2 metabolism pathways (photoautotroph and heterotroph).    For in class materials I remove the  red box (upper right corner) and print slides 3-10, place them back-to-back and laminate them.  The students get a key (slide 2) and a two-sided organism sheet...

  10. Thiol biochemistry of prokaryotes

    Science.gov (United States)

    Fahey, Robert C.

    1986-01-01

    The present studies have shown that GSH metabolism arose in the purple bacteria and cyanobacteria where it functions to protect against oxygen toxicity. Evidence was obtained indicating that GSH metabolism was incorporated into eucaryotes via the endosymbiosis giving rise to mitochrondria and chloroplasts. Aerobic bacteria lacking GSH utilize other thiols for apparently similar functions, the thiol being coenzyme A in Gram positive bacteria and chi-glutamylcysteine in the halobacteria. The thiol biochemistry of prokaryotes is thus seen to be much more highly diversified than that of eucaryotes and much remains to be learned about this subject.

  11. Browse Title Index

    African Journals Online (AJOL)

    Items 101 - 150 of 1010 ... Vol 26, No 1 (2018), Characterisation of Phaseolus coccineus interspecific germplasm accessions for disease resistance, grain market class and ... Vol 20, No 2 (2012), Comparative activities of phenylalanine ammonia-lyase and tyrosine ammonia-lyase and phenolic compounds accumulated in ...

  12. Halophilic & halotolerant prokaryotes in humans.

    Science.gov (United States)

    Seck, El Hadji; Dufour, Jean-Charles; Raoult, Didier; Lagier, Jean-Christophe

    2018-05-04

    Halophilic prokaryotes are described as microorganisms living in hypersaline environments. Here, we list the halotolerant and halophilic bacteria which have been isolated in humans. Of the 52 halophilic prokaryotes, 32 (61.54%) were moderately halophilic, 17 (32.69%) were slightly halophilic and three (5.76%) were extremely halophilic prokaryotes. At the phylum level, 29 (54.72%) belong to Firmicutes, 15 (28.84%) to Proteobacteria, four (7.69%) to Actinobacteria, three (5.78%) to Euryarchaeota and one (1.92%) belongs to Bacteroidetes. Halophilic prokaryotes are rarely pathogenic: of these 52 halophilic prokaryotes only two (3.92%) species were classified in Risk Group 2 (Vibrio cholerae, Vibrio parahaemolyticus) and one (1.96%), species in Risk Group 3 (Bacillus anthracis).

  13. Evolution of small prokaryotic genomes

    Directory of Open Access Journals (Sweden)

    David José Martínez-Cano

    2015-01-01

    Full Text Available As revealed by genome sequencing, the biology of prokaryotes with reduced genomes is strikingly diverse. These include free-living prokaryotes with ~800 genes as well as endosymbiotic bacteria with as few as ~140 genes. Comparative genomics is revealing the evolutionary mechanisms that led to these small genomes. In the case of free-living prokaryotes, natural selection directly favored genome reduction, while in the case of endosymbiotic prokaryotes neutral processes played a more prominent role. However, new experimental data suggest that selective processes may be at operation as well for endosymbiotic prokaryotes at least during the first stages of genome reduction. Endosymbiotic prokaryotes have evolved diverse strategies for living with reduced gene sets inside a host-defined medium. These include utilization of host-encoded functions (some of them coded by genes acquired by gene transfer from the endosymbiont and/or other bacteria; metabolic complementation between co-symbionts; and forming consortiums with other bacteria within the host. Recent genome sequencing projects of intracellular mutualistic bacteria showed that previously believed universal evolutionary trends like reduced G+C content and conservation of genome synteny are not always present in highly reduced genomes. Finally, the simplified molecular machinery of some of these organisms with small genomes may be used to aid in the design of artificial minimal cells. Here we review recent genomic discoveries of the biology of prokaryotes endowed with small gene sets and discuss the evolutionary mechanisms that have been proposed to explain their peculiar nature.

  14. Novel thiols of prokaryotes.

    Science.gov (United States)

    Fahey, R C

    2001-01-01

    Glutathione metabolism is associated with oxygenic cyanobacteria and the oxygen-utilizing purple bacteria, but is absent in many other prokaryotes. This review focuses on novel thiols found in those bacteria lacking glutathione. Included are glutathione amide and its perthiol, produced by phototrophic purple sulfur bacteria and apparently involved in their sulfide metabolism. Among archaebacteria, coenzyme M (2-mercaptoethanesulfonic acid) and coenzyme B (7-mercaptoheptanoylthreonine phosphate) play central roles in the anaerobic production of CH4 and associated energy conversion by methanogens, whereas the major thiol in the aerobic phototrophic halobacteria is gamma-glutamylcysteine. The highly aerobic actinomycetes produce mycothiol, a conjugate of N-acetylcysteine with a pseudodisaccharide of glucosamine and myo-inositol, AcCys-GlcNalpha(1 --> 1)Ins, which appears to play an antioxidant role similar to glutathione. Ergothioneine, also produced by actinomycetes, remains a mystery despite many years of study. Available data on the biosynthesis and metabolism of these and other novel thiols is summarized and key areas for additional study are identified.

  15. Capturing prokaryotic dark matter genomes.

    Science.gov (United States)

    Gasc, Cyrielle; Ribière, Céline; Parisot, Nicolas; Beugnot, Réjane; Defois, Clémence; Petit-Biderre, Corinne; Boucher, Delphine; Peyretaillade, Eric; Peyret, Pierre

    2015-12-01

    Prokaryotes are the most diverse and abundant cellular life forms on Earth. Most of them, identified by indirect molecular approaches, belong to microbial dark matter. The advent of metagenomic and single-cell genomic approaches has highlighted the metabolic capabilities of numerous members of this dark matter through genome reconstruction. Thus, linking functions back to the species has revolutionized our understanding of how ecosystem function is sustained by the microbial world. This review will present discoveries acquired through the illumination of prokaryotic dark matter genomes by these innovative approaches. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  16. Effects of germination on the activities of amylases and phenolic enzymes in sorghum varieties grouped according to food end-use properties

    NARCIS (Netherlands)

    Dicko, M.H.; Gruppen, H.; Zouzouho, O.C.; Traore, A.S.; Berkel, van W.J.H.; Voragen, A.G.J.

    2006-01-01

    Fifty sorghum varieties were screened to determine the effects of germination on levels of starch, -amylase, -amylase, phenylalanine ammonia lyase (PAL), peroxidase (POX) and polyphenol oxidase (PPO). Germination decreased starch content, with amylose being more degraded than amylopectin. In

  17. Phenolic compounds and related enzymes as determinants of sorghum for food use

    NARCIS (Netherlands)

    Dicko, M.H.; Gruppen, H.; Traore, A.S.; Voragen, A.G.J.; Berkel, van W.J.H.

    2006-01-01

    Phenolic compounds and related enzymes such as phenol biosynthesizing enzymes (phenylalanine ammonia lyase) and phenol catabolizing enzymes (polyphenol oxidase and peroxidase) are determinants for sorghum utilization as human food because they influence product properties during and after sorghum

  18. Biochemical studies on the Combination of Bemul-wax and Calcium ...

    African Journals Online (AJOL)

    Afolabi S

    2011-04-04

    Apr 4, 2011 ... coating combined with calcium chloride treatment ... storage methods for sweet potatoes are by leaving the ..... Phenylalanine ammonia lyase activity of treated sweet potato at ambient temperature ..... Theme: Innovative.

  19. Download this PDF file

    African Journals Online (AJOL)

    amino acids analysis (Especially for the free amino acids), this formula was then analyzed for .... Methods: Skim milk hydrolysates were pre-. 1. pared by two enzymes: purified. Protease ...... recombinant phenylalanine ammonia lyase. Proc.

  20. Phenylketonuria: Direct and indirect effects of phenylalanine.

    Science.gov (United States)

    Schlegel, Gudrun; Scholz, Ralf; Ullrich, Kurt; Santer, René; Rune, Gabriele M

    2016-07-01

    High phenylalanine concentrations in the brain due to dysfunctional phenylalanine hydroxylase (Pah) are considered to account for mental retardation in phenylketonuria (PKU). In this study, we treated hippocampal cultures with the amino acid in order to determine the role of elevated levels of phenylalanine in PKU-related mental retardation. Synapse density and dendritic length were dramatically reduced in hippocampal cultures treated with phenylalanine. Changes in cofilin expression and phosphorylation status, which were restored by NMDA, as well as reduced activation of the small GTPase Rac1, likely underlie these structural alterations. In the Pah(enu2) mouse, which carries a mutated Pah gene, we previously found higher synaptic density due to delayed synaptic pruning in response to insufficient microglia function. Microglia activity and C3 complement expression, both of which were reduced in the Pah(enu2) mouse, however, were unaffected in hippocampal cultures treated with phenylalanine. The lack of a direct effect of phenylalanine on microglia is the key to the opposite effects regarding synapse stability in vitro and in the Pah(enu2) mouse. Judging from our data, it appears that another player is required for the inactivation of microglia in the Pah(enu2) mouse, rather than high concentrations of phenylalanine alone. Altogether, the data underscore the necessity of a lifelong phenylalanine-restricted diet. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Prokaryotic photosynthesis and phototrophy illuminated

    DEFF Research Database (Denmark)

    Bryant, Donald A; Frigaard, Niels-Ulrik

    2006-01-01

    Genome sequencing projects are revealing new information about the distribution and evolution of photosynthesis and phototrophy. Although coverage of the five phyla containing photosynthetic prokaryotes (Chlorobi, Chloroflexi, Cyanobacteria, Proteobacteria and Firmicutes) is limited and uneven...... components that have not yet been described. Metagenomics has already shown how the relatively simple phototrophy based upon rhodopsins has spread laterally throughout Archaea, Bacteria and eukaryotes. In this review, we present examples that reflect recent advances in phototroph biology as a result...

  2. The Epigenomic Landscape of Prokaryotes.

    Directory of Open Access Journals (Sweden)

    Matthew J Blow

    2016-02-01

    Full Text Available DNA methylation acts in concert with restriction enzymes to protect the integrity of prokaryotic genomes. Studies in a limited number of organisms suggest that methylation also contributes to prokaryotic genome regulation, but the prevalence and properties of such non-restriction-associated methylation systems remain poorly understood. Here, we used single molecule, real-time sequencing to map DNA modifications including m6A, m4C, and m5C across the genomes of 230 diverse bacterial and archaeal species. We observed DNA methylation in nearly all (93% organisms examined, and identified a total of 834 distinct reproducibly methylated motifs. This data enabled annotation of the DNA binding specificities of 620 DNA Methyltransferases (MTases, doubling known specificities for previously hard to study Type I, IIG and III MTases, and revealing their extraordinary diversity. Strikingly, 48% of organisms harbor active Type II MTases with no apparent cognate restriction enzyme. These active 'orphan' MTases are present in diverse bacterial and archaeal phyla and show motif specificities and methylation patterns consistent with functions in gene regulation and DNA replication. Our results reveal the pervasive presence of DNA methylation throughout the prokaryotic kingdoms, as well as the diversity of sequence specificities and potential functions of DNA methylation systems.

  3. Phenylalanine kinetics in human adipose tissue.

    OpenAIRE

    Coppack, S W; Persson, M; Miles, J M

    1996-01-01

    Very little is known about the regulation of protein metabolism in adipose tissue. In this study systemic, adipose tissue, and forearm phenylalanine kinetics were determined in healthy postabsorptive volunteers before and during a 2-h glucose infusion (7 mg.kg-1.min-1). [3H]Phenylalanine was infused and blood was sampled from a radial artery, a subcutaneous abdominal vein, and a deep forearm vein. Adipose tissue and forearm blood flow were measured with 133Xe and plethysmography, respectively...

  4. Therapeutic implication of L-phenylalanine aggregation mechanism and its modulation by D-phenylalanine in phenylketonuria

    Science.gov (United States)

    Singh, Virender; Rai, Ratan Kumar; Arora, Ashish; Sinha, Neeraj; Thakur, Ashwani Kumar

    2014-01-01

    Self-assembly of phenylalanine is linked to amyloid formation toxicity in phenylketonuria disease. We are demonstrating that L-phenylalanine self-assembles to amyloid fibrils at varying experimental conditions and transforms to a gel state at saturated concentration. Biophysical methods including nuclear magnetic resonance, resistance by alpha-phenylglycine to fibril formation and preference of protected phenylalanine to self-assemble show that this behaviour of L-phenylalanine is governed mainly by hydrophobic interactions. Interestingly, D-phenylalanine arrests the fibre formation by L-phenylalanine and gives rise to flakes. These flakes do not propagate further and prevent fibre formation by L-phenylalanine. This suggests the use of D-phenylalanine as modulator of L-phenylalanine amyloid formation and may qualify as a therapeutic molecule in phenylketonuria. PMID:24464217

  5. Binding of radiation-induced phenylalanine radicals to DNA

    International Nuclear Information System (INIS)

    Schans, G.P. van der; Rijn, C.J.S. van; Bleichrodt, J.F.

    1975-11-01

    When an aqueous solution of double-stranded DNA of bacteriophage PM2 containing phenylalanine and saturated with N 2 O is irradiated with γ-rays, radiation-induced phenylalanine radicals are bound covalently. Under the conditions used about 25 phenylalanine molecules may be bound per lethal hit. Also for single-stranded PM2 DNA, most of the phenylalanine radicals bound are non-lethal. Evidence is presented that in double-stranded DNA an appreciable fraction of the single-strand breaks is induced by phenylalanine radicals. Radiation products of phenylalanine and the phenylalanine bound to the DNA decrease the sensitivity of the DNA to the induction of single-strand breaks. There are indications that the high efficiency of protection by radiation products of phenylalanine is due to their positive charge, which will result in a relatively high concentration of these compounds in the vicinity of the negatively charged DNA molecules

  6. Glycomacropeptide in children with phenylketonuria: does its phenylalanine content affect blood phenylalanine control?

    Science.gov (United States)

    Daly, A; Evans, S; Chahal, S; Santra, S; MacDonald, A

    2017-08-01

    In phenylketonuria (PKU), there are no data available for children with respect to evaluating casein glycomacropeptide (CGMP) as an alternative to phenylalanine-free protein substitutes [Phe-free L-amino acid (AA)]. CGMP contains a residual amount of phenylalanine, which may alter blood phenylalanine control. In a prospective 6-month pilot study, we investigated the effect on blood phenylalanine control of CGMP-amino acid (CGMP-AA) protein substitute in 22 PKU subjects (13 boys, nine girls), median age (range) 11 years (6-16 years). Twelve received CGMP-AA and nine received Phe-free L-AA, (1 CGMP-AA withdrawal). Subjects partially or wholly replaced Phe-free L-AA with CGMP-AA. If blood phenylalanine exceeded the target range, the CGMP-AA dose was reduced and replaced with Phe-free L-amino acids. The control group remained on Phe-free L-AAs. Phenylalanine, tyrosine and Phe : Tyr ratio concentrations were compared with the results for the previous year. In the CGMP-AA group, there was a significant increase in blood phenylalanine concentrations (pre-study, 275 μmol L -1 ; CGMP-AA, 317 μmol L -1 ; P = 0.02), a decrease in tyrosine concentrations (pre-study, 50 μmol L -1 ; CGMP-AA, 40 μmol L -1 ; P = 0.03) and an increase in Phe : Tyr ratios (pre-study, Phe : Tyr 4.9:1; CGMP-AA, Phe : Tyr 8:1; P = 0.02). In the control group there was a non-significant fall in phenylalanine concentrations (pre-study 325μmol/L: study 280μmol/L [p = 0.9], and no significant changes for tyrosine or phe/tyr ratios [p = 0.9]. Children taking the CGMP-AA found it more acceptable to L-AA. Blood phenylalanine control declined with CGMP-AA but, by titrating the dose of CGMP-AA, blood phenylalanine control remained within target range. The additional intake of phenylalanine may have contributed to the change in blood phenylalanine concentration. CGMP-AA use requires careful monitoring in children. © 2017 The British Dietetic Association Ltd.

  7. Production of manganese peroxidase by white rot fungi from potato ...

    African Journals Online (AJOL)

    phenylalanine and L-tyrosine blocked the MnP biosynthetic pathway. Ammonium ion released from the L-phenylalanine by the L-phenylalanine ammonia-lyase participated in the repression of the MnP biosynthetic pathway of the strain L-25.

  8. Assessment of brain phenylalanine dynamics in phenylketonuria patients

    International Nuclear Information System (INIS)

    Bik-Multanowski, M.; Pietrzyk, J. J.; Pasowicz, M.; Banys, R.P.

    2006-01-01

    Phenylketonuria (PKU) is the most common inborn error of metabolism in man. Brain phenylalanine kinetics can determine neurological treatment outcome in phenylketonuria. The aim of our study wa sto test a simplified magnetic resonance spectroscopy method for assessment of brain phenylalanine dynamics in PKU patients. Brain phenylalanine concentration (measured by means of magnetic resonance spectroscopy) and blood phenylalanine concentrations changes occurring within 24 hours after oral phenylalanine loading were analyzed in 5 PKU patients. The brain/blood phenylalanine ratio in 3 persons with normal intelligence was lower than in 2 with borderline intelligence or mild mental retardation. In our opinion the proposed method could be useful for assessment of brain phenylalanine dynamics in PKU patients. (author)

  9. Fluorinated Phenylalanine Precursor Resistance in Yeast

    Directory of Open Access Journals (Sweden)

    Ian S. Murdoch

    2018-06-01

    Full Text Available Development of a counter-selection method for phenylalanine auxotrophy could be a useful tool in the repertoire of yeast genetics. Fluorinated and sulfurated precursors of phenylalanine were tested for toxicity in Saccharomyces cerevisiae. One such precursor, 4-fluorophenylpyruvate (FPP, was found to be toxic to several strains from the Saccharomyces and Candida genera. Toxicity was partially dependent on ARO8 and ARO9, and correlated with a strain’s ability to convert FPP into 4-fluorophenylalanine (FPA. Thus, strains with deletions in ARO8 and ARO9, having a mild phenylalanine auxotrophy, could be separated from a culture of wild-type strains using FPP. Tetrad analysis suggests FPP resistance in one strain is due to two genes. Strains resistant to FPA have previously been shown to exhibit increased phenylethanol production. However, FPP resistant isolates did not follow this trend. These results suggest that FPP could effectively be used for counter-selection but not for enhanced phenylethanol production.

  10. Genome resource utilization during prokaryotic development

    Czech Academy of Sciences Publication Activity Database

    Vohradský, Jiří; Ramsden, J. J.

    2001-01-01

    Roč. 15, - (2001), s. 2054-2056 ISSN 0892-6638 R&D Projects: GA ČR GA204/00/1253 Institutional research plan: CEZ:AV0Z5020903 Keywords : prokaryotic development Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 8.817, year: 2001

  11. How crowded is the prokaryotic cytoplasm?

    NARCIS (Netherlands)

    Spitzer, Jan; Poolman, Bert; Ferguson, Stuart

    2013-01-01

    We consider biomacromolecular crowding within the cytoplasm of prokaryotic cells as a two-phase system of 'supercrowded' cytogel and 'dilute' cytosol; we simplify and quantify this model for a coccoid cell over a wide range of biomacromolecular crowding. The key result shows that the supercrowded

  12. Plasmid and chromosome segregation in prokaryotes

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Bugge Jensen, Rasmus; Gerdes, Kenn

    2000-01-01

    Recent major advances in the understanding of prokaryotic DNA segregation have been achieved by using fluorescence microscopy to visualize the localization of cellular components. Plasmids and bacterial chromosomes are partitioned in a highly dynamic fashion, suggesting the presence of a mitotic...

  13. Diversity and activity in marine prokaryotes

    NARCIS (Netherlands)

    Arrieta López de Uralde, Jesús Maria

    2005-01-01

    Life on Earth epends on the endless recycling of elements as matter and energy are required to sustain life. The prokaryotes (Bacteria and Archaea) are the masters of the trade of life. After all, they were already responsible for the major biogeochemical cycles 3.000 million years ago, long before

  14. Getting out : protein traffic in prokaryotes

    NARCIS (Netherlands)

    Pugsley, A.P; Francetic, O; Driessen, A.J.M.; de Lorenzo, V.

    Protein secretion systems in prokaryotes are increasingly shifting from being considered as experimental models for 'more complex' processes (i.e. eukaryotes) to being a major source of key biological questions in their own right. The pathways by which proteins move between compartments or insert

  15. [Kinetic study on inhibition effects of dansyl-L-phenylalanine and L-phenylalanine on calf intestinal alkaline phosphatase].

    Science.gov (United States)

    Li, Li-Na; Wu, Yu-Qing; Buchet, René

    2009-10-01

    To evaluate the inhibition effect of dansyl-L-phenylalanine on calf intestinal alkaline phosphatase (CIAP), UV-Vis spectrophotometric method was employed. It was found that dansyl-L-phenylalanine can selectively inhibit CIAP. The kinetic inhibition processes of dansyl-L-phenylalanine and L-phenylalanine were comparatively studied. The authors' finding elucidates that at the optimized alkaline pH of alkaline phosphatase (pH 10.4) and 37 degrees C, dansyl-L-phenylalanine can inhibit alkaline phosphatase activity of CIAP efficiently and specifically, similar as L-phenylalanine. Both inhibition types were uncompetitive inhibition resulting from the double reciprocal curve fitting of upsilon versus substrate concentrations, and the inhibition constants Ki of both inhibitors were determined to be 2.3 and 1.1 mmol L(-1) respectively, both of which were at millimolar level. The investigation of the inhibition effect of dansyl modified L-phenylalanine on calf intestinal alkaline phosphatase not only helped get insight into the detailed inhibition mechanism of L-phenylalanine on tissue specific alkaline phosphatase, such as in the case of intestinal alkaline phosphatase, but also provided the possibility to employ fluorescence spectroscopy by labeling the specific inhibitors of alkaline phosphatase with chromophoric groups.

  16. Prokaryotes versus Eukaryotes: Who is hosting whom?

    Directory of Open Access Journals (Sweden)

    Guillermo eTellez

    2014-10-01

    Full Text Available Microorganisms represent the largest component of biodiversity in our world. For millions of years, prokaryotic microorganisms have functioned as a major selective force shaping eukaryotic evolution. Microbes that live inside and on animals outnumber the animals’ actual somatic and germ cells by an estimated 10-fold. Collectively, the intestinal microbiome represents a ‘forgotten organ’, functioning as an organ inside another that can execute many physiological responsibilities. The nature of primitive eukaryotes was drastically changed due to the association with symbiotic prokaryotes facilitating mutual coevolution of host and microbe. Phytophagous insects have long been used to test theories of evolutionary diversification; moreover, the diversification of a number of phytophagous insect lineages has been linked to mutualisms with microbes. From termites and honey bees to ruminants and mammals, depending on novel biochemistries provided by the prokaryotic microbiome, the association helps to metabolize several nutrients that the host cannot digest and converting these into useful end products (such as short chain fatty acids, a process which has huge impact on the biology and homeostasis of metazoans. More importantly, in a direct and/or indirect way, the intestinal microbiota influences the assembly of gut-associated lymphoid tissue, helps to educate immune system, affects the integrity of the intestinal mucosal barrier, modulates proliferation and differentiation of its epithelial lineages, regulates angiogenesis, and modifies the activity of enteric as well as the central nervous system,. Despite these important effects, the mechanisms by which the gut microbial community influences the host’s biology remains almost entirely unknown. Our aim here is to encourage empirical inquiry into the relationship between mutualism and evolutionary diversification between prokaryotes and eukaryotes which encourage us to postulate: Who is

  17. Phenylketonuria : tyrosine supplementation in phenylalanine-restricted diets

    NARCIS (Netherlands)

    van Spronsen, FJ; van Rijn, M; Bekhof, J; Koch, R; Smit, PGA

    Treatment of phenylketonuria (PKU) consists of restriction of natural protein and provision of a protein substitute that lacks phenylalanine but is enriched in tyrosine. Large and unexplained differences exist, however, in the tyrosine enrichment of the protein substitutes. Furthermore, some

  18. Analog kefir production with a low phenylalanine for Phenylketonuria

    OpenAIRE

    Amir Yari; Yousef Ramezan

    2017-01-01

    Phenylketonuria (PKU) is one of the most prevalent types of hereditary metabolic disorders which is caused due to an absence or reduction of the activity of the Phenylalanine hydroxylase enzyme in the liver which in turn, inhibits the transformation of phenylalanine (Phe) to tyrosine. In clinical terms, this disorder is displayed with severe, permanent and irreversible mental retardation. This research was aimed at development of a highly nutrient and acceptable suitable analogue Kefir drink ...

  19. Emerging spatial patterns in Antarctic prokaryotes

    Directory of Open Access Journals (Sweden)

    Chun Wie eChong

    2015-09-01

    Full Text Available Recent advances in knowledge of patterns of biogeography in terrestrial eukaryotic organisms have led to a fundamental paradigm shift in understanding of the controls and history of life on land in Antarctica, and its interactions over the long term with the glaciological and geological processes that have shaped the continent. However, while it has long been recognized that the terrestrial ecosystems of Antarctica are dominated by microbes and their processes, knowledge of microbial diversity and distributions has lagged far behind that of the macroscopic eukaryote organisms. Increasing human contact with and activity in the continent is leading to risks of biological contamination and change in a region whose isolation has protected it for millions of years at least; these risks may be particularly acute for microbial communities which have, as yet, received scant recognition and attention. Even a matter apparently as straightforward as Protected Area designation in Antarctica requires robust biodiversity data which, in most parts of the continent, remain almost completely unavailable. A range of important contributing factors mean that it is now timely to reconsider the state of knowledge of Antarctic terrestrial prokaryotes. Rapid advances in molecular biological approaches are increasingly demonstrating that bacterial diversity in Antarctica may be far greater than previously thought, and that there is overlap in the environmental controls affecting both Antarctic prokaryotic and eukaryotic communities. Bacterial dispersal mechanisms and colonization patterns remain largely unaddressed, although evidence for regional evolutionary differentiation is rapidly accruing and, with this, there is increasing appreciation of patterns in regional bacterial biogeography in this large part of the globe. In this review, we set out to describe the state of knowledge of Antarctic prokaryote diversity patterns, drawing analogy with those of eukaryote

  20. Emerging spatial patterns in Antarctic prokaryotes.

    Science.gov (United States)

    Chong, Chun-Wie; Pearce, David A; Convey, Peter

    2015-01-01

    Recent advances in knowledge of patterns of biogeography in terrestrial eukaryotic organisms have led to a fundamental paradigm shift in understanding of the controls and history of life on land in Antarctica, and its interactions over the long term with the glaciological and geological processes that have shaped the continent. However, while it has long been recognized that the terrestrial ecosystems of Antarctica are dominated by microbes and their processes, knowledge of microbial diversity and distributions has lagged far behind that of the macroscopic eukaryote organisms. Increasing human contact with and activity in the continent is leading to risks of biological contamination and change in a region whose isolation has protected it for millions of years at least; these risks may be particularly acute for microbial communities which have, as yet, received scant recognition and attention. Even a matter apparently as straightforward as Protected Area designation in Antarctica requires robust biodiversity data which, in most parts of the continent, remain almost completely unavailable. A range of important contributing factors mean that it is now timely to reconsider the state of knowledge of Antarctic terrestrial prokaryotes. Rapid advances in molecular biological approaches are increasingly demonstrating that bacterial diversity in Antarctica may be far greater than previously thought, and that there is overlap in the environmental controls affecting both Antarctic prokaryotic and eukaryotic communities. Bacterial dispersal mechanisms and colonization patterns remain largely unaddressed, although evidence for regional evolutionary differentiation is rapidly accruing and, with this, there is increasing appreciation of patterns in regional bacterial biogeography in this large part of the globe. In this review, we set out to describe the state of knowledge of Antarctic prokaryote diversity patterns, drawing analogy with those of eukaryote groups where appropriate

  1. Surveying the expanding prokaryotic Rubisco multiverse.

    Science.gov (United States)

    Liu, Di; Ramya, Ramaswamy Chettiyan Seetharaman; Mueller-Cajar, Oliver

    2017-09-01

    The universal, but catalytically modest, CO2-fixing enzyme Rubisco is currently experiencing intense interest by researchers aiming to enhance crop photosynthesis. These efforts are mostly focused on the highly conserved hexadecameric enzyme found in land plants. In comparison, prokaryotic organisms harbor a far greater diversity in Rubisco forms. Recent work towards improving our appreciation of microbial Rubisco properties and harnessing their potential is surveyed. New structural models are providing informative glimpses into catalytic subtleties and diverse oligomeric states. Ongoing characterization is informing us about the conservation of constraints, such as sugar phosphate inhibition and the associated dependence on Rubisco activase helper proteins. Prokaryotic Rubiscos operate under a far wider range of metabolic contexts than the photosynthetic function of higher plant enzymes. Relaxed selection pressures may have resulted in the exploration of a larger volume of sequence space than permitted in organisms performing oxygenic photosynthesis. To tap into the potential of microbial Rubiscos, in vivo selection systems are being used to discover functional metagenomic Rubiscos. Various directed evolution systems to optimize their function have been developed. It is anticipated that this approach will provide access to biotechnologically valuable enzymes that cannot be encountered in the higher plant Rubisco space. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. Fluctuations in phenylalanine concentrations in phenylketonuria: a review of possible relationships with outcomes.

    Science.gov (United States)

    Cleary, Maureen; Trefz, Friedrich; Muntau, Ania C; Feillet, François; van Spronsen, Francjan J; Burlina, Alberto; Bélanger-Quintana, Amaya; Giżewska, Maria; Gasteyger, Christoph; Bettiol, Esther; Blau, Nenad; MacDonald, Anita

    2013-12-01

    Fluctuations in blood phenylalanine concentrations may be an important determinant of intellectual outcome in patients with early and continuously treated phenylketonuria (PKU). This review evaluates the studies on phenylalanine fluctuations, factors affecting fluctuations, and if stabilizing phenylalanine concentrations affects outcomes, particularly neurocognitive outcome. Electronic literature searches of Embase and PubMed were performed for English-language publications, and the bibliographies of identified publications were also searched. In patients with PKU, phenylalanine concentrations are highest in the morning. Factors that can affect phenylalanine fluctuations include age, diet, timing and dosing of protein substitute and energy intake, dietary adherence, phenylalanine hydroxylase genotype, changes in dietary phenylalanine intake and protein metabolism, illness, and growth rate. Even distribution of phenylalanine-free protein substitute intake throughout 24h may reduce blood phenylalanine fluctuations. Patients responsive to and treated with 6R-tetrahydrobiopterin seem to have less fluctuation in their blood phenylalanine concentrations than controls. An increase in blood phenylalanine concentration may result in increased brain and cerebrospinal fluid phenylalanine concentrations within hours. Although some evidence suggests that stabilization of blood phenylalanine concentrations may have benefits in patients with PKU, more studies are needed to distinguish the effects of blood phenylalanine fluctuations from those of poor metabolic control. © 2013.

  3. A framework for classification of prokaryotic protein kinases.

    Directory of Open Access Journals (Sweden)

    Nidhi Tyagi

    Full Text Available BACKGROUND: Overwhelming majority of the Serine/Threonine protein kinases identified by gleaning archaeal and eubacterial genomes could not be classified into any of the well known Hanks and Hunter subfamilies of protein kinases. This is owing to the development of Hanks and Hunter classification scheme based on eukaryotic protein kinases which are highly divergent from their prokaryotic homologues. A large dataset of prokaryotic Serine/Threonine protein kinases recognized from genomes of prokaryotes have been used to develop a classification framework for prokaryotic Ser/Thr protein kinases. METHODOLOGY/PRINCIPAL FINDINGS: We have used traditional sequence alignment and phylogenetic approaches and clustered the prokaryotic kinases which represent 72 subfamilies with at least 4 members in each. Such a clustering enables classification of prokaryotic Ser/Thr kinases and it can be used as a framework to classify newly identified prokaryotic Ser/Thr kinases. After series of searches in a comprehensive sequence database we recognized that 38 subfamilies of prokaryotic protein kinases are associated to a specific taxonomic level. For example 4, 6 and 3 subfamilies have been identified that are currently specific to phylum proteobacteria, cyanobacteria and actinobacteria respectively. Similarly subfamilies which are specific to an order, sub-order, class, family and genus have also been identified. In addition to these, we also identify organism-diverse subfamilies. Members of these clusters are from organisms of different taxonomic levels, such as archaea, bacteria, eukaryotes and viruses. CONCLUSION/SIGNIFICANCE: Interestingly, occurrence of several taxonomic level specific subfamilies of prokaryotic kinases contrasts with classification of eukaryotic protein kinases in which most of the popular subfamilies of eukaryotic protein kinases occur diversely in several eukaryotes. Many prokaryotic Ser/Thr kinases exhibit a wide variety of modular

  4. Phenylalanine-coupled solid lipid nanoparticles for brain tumor targeting

    Energy Technology Data Exchange (ETDEWEB)

    Kharya, Parul; Jain, Ashish; Gulbake, Arvind; Shilpi, Satish; Jain, Ankit; Hurkat, Pooja [Dr. Hari Singh Gour University, Pharmaceutical Research Projects Laboratory, Department of Pharmaceutical Sciences (India); Majumdar, Subrata [Bose Institute, Division of Molecular Medicine (India); Jain, Sanjay K., E-mail: drskjainin@yahoo.com [Dr. Hari Singh Gour University, Pharmaceutical Research Projects Laboratory, Department of Pharmaceutical Sciences (India)

    2013-11-15

    The purpose of this study is to investigate the targeting potential of amino acid (phenylalanine)-coupled solid lipid nanoparticles (SLN) loaded with ionically complexed doxorubicin HCl (Dox). Ionic complexation was used to enhance the loading efficiency and release characteristics of water soluble form of Dox. l-Type amino acid transporters (LAT1) are highly expressed on blood brain barrier as well as on many brain cancer cells, thus targeting LAT1 using phenylalanine improved anticancer activity of prepared nanocarrier. The phenylalanine-coupled SLN were characterized by fourier transform infrared spectroscopy, scanning electron microscope, transmission electron microscopy, particle size, zeta potential, entrapment efficiency and in vitro release. The particle size of the resulting SLN was found to be in the range of 163.3 ± 5.2 to 113.0 ± 2.6 nm, with a slightly negative surface charge. In ex vivo study on C6 glioma cell lines, the cellular cytotoxicity of the SLN was highly increased when coupled with phenylalanine. In addition, stealthing sheath of PEG present on the surface of the SLN enhanced the cellular uptake of the SLN on C6 glioma cell line. Results of biodistribution and fluorescence studies clearly revealed that phenylalanine-coupled SLN could deliver high amount of drug into the brain tumor cells and showed the brain-targeting potential.

  5. The rate of incorporation and degradation of phenylalanine-14C by tissue slices of roosters fed a phenylalanine-free diet

    International Nuclear Information System (INIS)

    Ishibashi, Teru; Kametaka, Masao

    1975-01-01

    To investigate the reason why adult roosters maintain nitrogen equilibrium on a phenylalanine-free diet for long periods, the rates of incorporation and of degradation of phenylalanine, tyrosine- and isoleucine- 14 C by tissue slices of roosters on the control, phenylalanine-free or isoleucine-free diets were measured. The degradation rate of isoleucine by liver and muscle slices decreased significantly for the isoleucine-free diet. However, the degradation rate of phenylalanine- 14 C by liver slices for the phenylalanine-free diet was not significantly lower than that for the control diet in contrast to the previous observation in vivo. The incorporation rates of 14 C into liver and muscle proteins were not affected by the dietary conditions in this experiment except in the case of phenylalanine- 14 C by muscle slices for the phenylalanine-free diet. (auth.)

  6. Bioinformatics analysis of disordered proteins in prokaryotes

    Directory of Open Access Journals (Sweden)

    Malkov Saša N

    2011-03-01

    Full Text Available Abstract Background A significant number of proteins have been shown to be intrinsically disordered, meaning that they lack a fixed 3 D structure or contain regions that do not posses a well defined 3 D structure. It has also been proven that a protein's disorder content is related to its function. We have performed an exhaustive analysis and comparison of the disorder content of proteins from prokaryotic organisms (i.e., superkingdoms Archaea and Bacteria with respect to functional categories they belong to, i.e., Clusters of Orthologous Groups of proteins (COGs and groups of COGs-Cellular processes (Cp, Information storage and processing (Isp, Metabolism (Me and Poorly characterized (Pc. We also analyzed the disorder content of proteins with respect to various genomic, metabolic and ecological characteristics of the organism they belong to. We used correlations and association rule mining in order to identify the most confident associations between specific modalities of the characteristics considered and disorder content. Results Bacteria are shown to have a somewhat higher level of protein disorder than archaea, except for proteins in the Me functional group. It is demonstrated that the Isp and Cp functional groups in particular (L-repair function and N-cell motility and secretion COGs of proteins in specific possess the highest disorder content, while Me proteins, in general, posses the lowest. Disorder fractions have been confirmed to have the lowest level for the so-called order-promoting amino acids and the highest level for the so-called disorder promoters. For each pair of organism characteristics, specific modalities are identified with the maximum disorder proteins in the corresponding organisms, e.g., high genome size-high GC content organisms, facultative anaerobic-low GC content organisms, aerobic-high genome size organisms, etc. Maximum disorder in archaea is observed for high GC content-low genome size organisms, high GC content

  7. Translational selection is ubiquitous in prokaryotes.

    Directory of Open Access Journals (Sweden)

    Fran Supek

    2010-06-01

    Full Text Available Codon usage bias in prokaryotic genomes is largely a consequence of background substitution patterns in DNA, but highly expressed genes may show a preference towards codons that enable more efficient and/or accurate translation. We introduce a novel approach based on supervised machine learning that detects effects of translational selection on genes, while controlling for local variation in nucleotide substitution patterns represented as sequence composition of intergenic DNA. A cornerstone of our method is a Random Forest classifier that outperformed previous distance measure-based approaches, such as the codon adaptation index, in the task of discerning the (highly expressed ribosomal protein genes by their codon frequencies. Unlike previous reports, we show evidence that translational selection in prokaryotes is practically universal: in 460 of 461 examined microbial genomes, we find that a subset of genes shows a higher codon usage similarity to the ribosomal proteins than would be expected from the local sequence composition. These genes constitute a substantial part of the genome--between 5% and 33%, depending on genome size--while also exhibiting higher experimentally measured mRNA abundances and tending toward codons that match tRNA anticodons by canonical base pairing. Certain gene functional categories are generally enriched with, or depleted of codon-optimized genes, the trends of enrichment/depletion being conserved between Archaea and Bacteria. Prominent exceptions from these trends might indicate genes with alternative physiological roles; we speculate on specific examples related to detoxication of oxygen radicals and ammonia and to possible misannotations of asparaginyl-tRNA synthetases. Since the presence of codon optimizations on genes is a valid proxy for expression levels in fully sequenced genomes, we provide an example of an "adaptome" by highlighting gene functions with expression levels elevated specifically in

  8. Detecting uber-operons in prokaryotic genomes.

    Science.gov (United States)

    Che, Dongsheng; Li, Guojun; Mao, Fenglou; Wu, Hongwei; Xu, Ying

    2006-01-01

    We present a study on computational identification of uber-operons in a prokaryotic genome, each of which represents a group of operons that are evolutionarily or functionally associated through operons in other (reference) genomes. Uber-operons represent a rich set of footprints of operon evolution, whose full utilization could lead to new and more powerful tools for elucidation of biological pathways and networks than what operons have provided, and a better understanding of prokaryotic genome structures and evolution. Our prediction algorithm predicts uber-operons through identifying groups of functionally or transcriptionally related operons, whose gene sets are conserved across the target and multiple reference genomes. Using this algorithm, we have predicted uber-operons for each of a group of 91 genomes, using the other 90 genomes as references. In particular, we predicted 158 uber-operons in Escherichia coli K12 covering 1830 genes, and found that many of the uber-operons correspond to parts of known regulons or biological pathways or are involved in highly related biological processes based on their Gene Ontology (GO) assignments. For some of the predicted uber-operons that are not parts of known regulons or pathways, our analyses indicate that their genes are highly likely to work together in the same biological processes, suggesting the possibility of new regulons and pathways. We believe that our uber-operon prediction provides a highly useful capability and a rich information source for elucidation of complex biological processes, such as pathways in microbes. All the prediction results are available at our Uber-Operon Database: http://csbl.bmb.uga.edu/uber, the first of its kind.

  9. Interactions Between Prokaryotes and Dissolved Organic Matter in Marine Waters

    DEFF Research Database (Denmark)

    Traving, Sachia Jo

    organic bound carbon equal in size to atmospheric carbon dioxide. Prokaryotes mediate the fate of a large part of marine DOM, which is their principal source of energy and substrate. However, a large fraction is also left behind in the water column persisting for millennia, and prokaryotes may hold...... the key to understanding the mechanisms controlling the cycling of DOM within marine waters. In the thesis presented here, the aim was to investigate the activity and composition of prokaryotes to determine their functional role in DOM utilization. The thesis incorporates a range of study systems...

  10. The effect of streptozotocin-induced diabetes on phenylalanine hydroxylase expression in rat liver.

    OpenAIRE

    Taylor, D S; Dahl, H H; Mercer, J F; Green, A K; Fisher, M J

    1989-01-01

    The impact of experimentally induced diabetes on the expression of rat liver phenylalanine hydroxylase has been investigated. A significant elevation in maximal enzymic activity was observed in diabetes. This was associated with significant increases in the amount of enzyme, the phenylalanine hydroxylase-specific translational activity of hepatic RNA and the abundance of phenylalanine hydroxylase-specific mRNA. These changes in phenylalanine hydroxylase expression were not observed when diabe...

  11. Physiological mechanism of resistance to anthracnose of different ...

    African Journals Online (AJOL)

    However, enzyme activity of resistant cultivars improved markedly after pathogen inoculation, while those of susceptible cultivars did not change. This study broadens the understanding of the mechanisms of disease resistance in Camellia. Keywords: Anthracnose, Camellia oleifera, phenylalanine ammonia lyase, ...

  12. Antioxidant characterization and sensory evaluation during storage of ultraviolet-B light exposed baby carrots (abstract)

    Science.gov (United States)

    Baby carrot processing induces wounding stress activation of phenylalanine ammonia-lyase (PAL), enhancing its nutrient content by increasing synthesis of secondary metabolites. Ultraviolet-B (UV-B) exposure further promotes the formation of soluble phenolic compounds, significantly increasing antiox...

  13. Determination of specific growth stages of plant cell suspension cultures by monitoring conductivity changes in the medium.

    Science.gov (United States)

    Hahlbrock, K; Ebel, J; Oaks, A; Auden, J; Liersch, M

    1974-03-01

    Conductivity changes in the medium of cultured soybean (Glycine max L.) cells were shown to be strictly correlated with nitrate uptake and growth of the cultures. A continuous record of the conductivity was used as a simple and reliable method of determining specific growth stages and concomitant peaks in the activities of nitrate reductase and phenylalanine ammonia-lyase.

  14. Elevated levels of plasma phenylalanine in schizophrenia: a guanosine triphosphate cyclohydrolase-1 metabolic pathway abnormality?

    Directory of Open Access Journals (Sweden)

    Olaoluwa Okusaga

    Full Text Available BACKGROUND: Phenylalanine and tyrosine are precursor amino acids required for the synthesis of dopamine, the main neurotransmitter implicated in the neurobiology of schizophrenia. Inflammation, increasingly implicated in schizophrenia, can impair the function of the enzyme Phenylalanine hydroxylase (PAH; which catalyzes the conversion of phenylalanine to tyrosine and thus lead to elevated phenylalanine levels and reduced tyrosine levels. This study aimed to compare phenylalanine, tyrosine, and their ratio (a proxy for PAH function in a relatively large sample of schizophrenia patients and healthy controls. METHODS: We measured non-fasting plasma phenylalanine and tyrosine in 950 schizophrenia patients and 1000 healthy controls. We carried out multivariate analyses to compare log transformed phenylalanine, tyrosine, and phenylalanine:tyrosine ratio between patients and controls. RESULTS: Compared to controls, schizophrenia patients had higher phenylalanine (p<0.0001 and phenylalanine: tyrosine ratio (p<0.0001 but tyrosine did not differ between the two groups (p = 0.596. CONCLUSIONS: Elevated phenylalanine and phenylalanine:tyrosine ratio in the blood of schizophrenia patients have to be replicated in longitudinal studies. The results may relate to an abnormal PAH function in schizophrenia that could become a target for novel preventative and interventional approaches.

  15. Phase 1 Trial of Subcutaneous rAvPAL-PEG in Subjects with Phenylketonuria

    Science.gov (United States)

    Longo, Nicola; Harding, Cary O.; Burton, Barbara K.; Grange, Dorothy K.; Vockley, Jerry; Wasserstein, Melissa; Rice, Gregory M.; Musson, Donald G.; Gu, Zhonghua; Sile, Saba

    2014-01-01

    Objective Phenylketonuria is an inherited disease caused by impaired activity of phenylalanine hydroxylase, the enzyme that converts phenylalanine to tyrosine, leading to accumulation of phenylalanine and subsequent neurocognitive dysfunction. A phenylalanine-restricted diet initiated early in life can ameliorate the toxic effects of phenylalanine. However, the diet is onerous and compliance is extremely difficult. Phenylalanine ammonia lyase (PAL) is a prokaryotic enzyme that converts phenylalanine to ammonia and trans-cinnamic acid. This Phase 1, multicenter clinical trial evaluated the safety, tolerability, pharmacokinetics and efficacy of rAvPAL-PEG (recombinant Anabaena variabilis PAL produced in E. coli conjugated with polyethylene glycol [PEG] to reduce immunogenicity) in reducing phenylalanine levels in subjects with phenylketonuria. Methods Single subcutaneous injections of rAvPAL-PEG in escalating doses (0·001, 0·003, 0·01, 0·03, and 0·1 mg/kg) were administered to 25 adults with phenylketonuria recruited from those attending metabolic clinics in North America whose blood phenylalanine concentrations were ≥600 μmol/L. Results The most frequently reported adverse events were injection-site reactions and dizziness. Reactions were self-limited without sequelae. During the trial, two subjects had adverse reactions to intramuscular (IM) medroxyprogesterone acetate, a drug containing polyethylene glycol as an excipient. Three subjects developed a generalized skin rash at the highest rAvPAL-PEG dose (0·1 mg/kg). Drug levels peaked ∼5 days after the injection. Treatment was effective in reducing blood phenylalanine in all five subjects receiving the highest dose (0·1 mg/kg, mean percent change of -58 from baseline), with a nadir ∼6 days after injection and inverse correlation between drug and phenylalanine concentrations in plasma. Phenylalanine concentrations returned to near-baseline levels ∼20 days after the single injection. Conclusions

  16. Polymorphism and modulation of para-substituted L-Phenylalanine

    NARCIS (Netherlands)

    Sögütoglu, Leyla-cann; Lutz, Martin; Meekes, Hugo; De Gelder, Rene; Vlieg, Elias

    2017-01-01

    The crystal structure of para-methyl-L-phenylalanine at 230 K resembles that of the para-fluorinated analogue from the literaturebut is commensurately modulated with seven molecules in the asymmetric unit (Z′ = 7). At 100 K, the superstructure loses its modulation, leading to a unit cell with Z′ =

  17. Enzymatic Preparation of Low-Phenylalanine Formula Derived from ...

    African Journals Online (AJOL)

    Background: Phenylketonuria (PKU) is one of the most common inborn errors of amino acids metabolism. WHO guidelines introduced in 1979 and revised 1988 for breast-feeding infants with PKU included a formula containing low amounts of phenylalanine as a part of dietary prescription. Mental retardation can be ...

  18. Eosin-sensitized photooxidation of substituted phenylalanines and tyrosines

    Energy Technology Data Exchange (ETDEWEB)

    Rizzuto, F.; Spikes, J.D.

    1977-01-01

    The cosin-sensitized photooxidation of tyrosine and a number of compounds related to tyrosine (substituted phenylalanines) was studied by steady-state kinetic and flash photolysis techniques. In particular, the role of the phenolic group and the amino and carboxyl groups of the alanyl side chain in the photooxidation mechanism was investigated in detail. Several relationships between substrate structure and susceptibility to photooxidation as well as effects of substrate structure on photooxidation mechanisms were found. For example, phenylalanine is not photooxidizable, but substitution of electron-donating (activating) groups such as -OH (as in tyrosine) or -NH/sub 2/ (as in p-aminophenylalanine) results in rapidly photooxidized derivatives. However, substituting deactivating groups such as -Cl (as in p-chlorophenylalanine) or weakly activating groups such as -OCH/sub 3/ (as in 4-methoxyphenylalanine) result in non-photooxidizable derivatives. Substitution of additional activating groups to the ring of hydroxy-substituted phenylalanines results in increased rates of photooxidation, whereas additional deactivating groups result in decreased photooxidation rates. The rate-determining step in the photooxidation mechanism is shown to be dependent on the presence and position of an electron-donating substituent on the benzenoid ring. Only minor involvement of the side chain amino and carboxyl groups was found. Both singlet oxygen and hydrogen abstraction mechanisms are involved in the eosin-sensitized photooxidation of hydroxy-substituted phenylalanines (e.g., tyrosine). The hydrogen abstraction mechanism probably predominates at both pH 8 and 11.

  19. Two Tales of Prokaryotic Genomic Diversity: Escherichia coli and Halophiles

    Directory of Open Access Journals (Sweden)

    Lejla Pašić

    2014-01-01

    Full Text Available Prokaryotes are generally characterized by vast genomic diversity that has been shaped by mutations, horizontal gene transfer, bacteriocins and phage predation. Enormous genetic diversity has developed as a result of stresses imposed in harsh environments and the ability of microorganisms to adapt. Two examples of prokaryotic diversity are presented: on intraspecies level, exemplified by Escherichia coli, and the diversity of the hypersaline environment, with the discussion of food-related health issues and biotechnological potential.

  20. Engineering prokaryotic transcriptional activators as metabolite biosensors in yeast

    DEFF Research Database (Denmark)

    Skjødt, Mette Louise; Snoek, Tim; Kildegaard, Kanchana Rueksomtawin

    2016-01-01

    ,cis-muconic acid at different levels, and found that reporter gene output correlated with production. The transplantation of prokaryotic transcriptional activators into the eukaryotic chassis illustrates the potential of a hitherto untapped biosensor resource useful for biotechnological applications....... real-time monitoring of production has attracted attention. Here we applied systematic engineering of multiple parameters to search for a general biosensor design in the budding yeast Saccharomyces cerevisiae based on small-molecule binding transcriptional activators from the prokaryote superfamily...

  1. Effects of viruses and predators on prokaryotic community composition.

    Science.gov (United States)

    Jardillier, Ludwig; Bettarel, Yvan; Richardot, Mathilde; Bardot, Corinne; Amblard, Christian; Sime-Ngando, Télesphore; Debroas, Didier

    2005-11-01

    Dialysis bags were used to examine the impact of predation and viral lysis on prokaryotic community composition (PCC) over a 5-day experiment in the oligomesotrophic Lake Pavin (France). The impact of the different predator communities (protists and metazoans) of prokaryotes was estimated by water fractionation (protists, which also affected PCC, whereas viruses seemed to be essentially responsible for profound changes in PCC via direct and indirect actions.

  2. Functional characterization of a prokaryotic Kir channel.

    Science.gov (United States)

    Enkvetchakul, Decha; Bhattacharyya, Jaya; Jeliazkova, Iana; Groesbeck, Darcy K; Cukras, Catherine A; Nichols, Colin G

    2004-11-05

    The Kir gene family encodes inward rectifying K+ (Kir) channels that are widespread and critical regulators of excitability in eukaryotic cells. A related gene family (KirBac) has recently been identified in prokaryotes. While a crystal structure of one member, Kir-Bac1.1, has been solved, there has been no functional characterization of any KirBac gene products. Here we present functional characterization of KirBac1.1 reconstituted in liposomes. Utilizing a 86Rb+ uptake assay, we demonstrate that KirBac1.1 generates a K+ -selective permeation path that is inhibited by extraliposomal Ba2+ and Ca2+ ions. In contrast to KcsA (an acid-activated bacterial potassium channel), KirBac1.1 is inhibited by extraliposomal acid (pKa approximately 6). This characterization of KirBac1.1 activity now paves the way for further correlation of structure and function in this model Kir channel.

  3. Prokaryotic Argonautes - variations on the RNA interference theme

    Science.gov (United States)

    van der Oost, John; Swarts, Daan C.; Jore, Matthijs M.

    2014-01-01

    The discovery of RNA interference (RNAi) has been a major scientific breakthrough. This RNA-guided RNA interference system plays a crucial role in a wide range of regulatory and defense mechanisms in eukaryotes. The key enzyme of the RNAi system is Argonaute (Ago), an endo-ribonuclease that uses a small RNA guide molecule to specifically target a complementary RNA transcript. Two functional classes of eukaryotic Ago have been described: catalytically active Ago that cleaves RNA targets complementary to its guide, and inactive Ago that uses its guide to bind target RNA to down-regulate translation efficiency. A recent comparative genomics study has revealed that Argonaute-like proteins are also encoded by prokaryotic genomes. Interestingly, there is a lot of variation among these prokaryotic Argonaute (pAgo) proteins with respect to domain architecture: some resemble the eukaryotic Ago (long pAgo) containing a complete or disrupted catalytic site, while others are truncated versions (short pAgo) that generally contain an incomplete catalytic site. Prokaryotic Agos with an incomplete catalytic site often co-occur with (predicted) nucleases. Based on this diversity, and on the fact that homologs of other RNAi-related protein components (such as Dicer nucleases) have never been identified in prokaryotes, it has been predicted that variations on the eukaryotic RNAi theme may occur in prokaryotes. PMID:28357239

  4. Differential response of marine flagellate communities to prokaryotic food quality

    Science.gov (United States)

    De Corte, D.; Paredes, G.; Sintes, E.; Herndl, G. J.

    2016-02-01

    Marine prokaryotes play a major role in the biogeochemical cycles. The main predators of prokaryotes are heterotrophic nanoflagellates (HNF). HNF are thus a major link connecting dissolved organic material through prokaryotic grazing to the higher trophic levels. However, little is known about the grazing specificity of HNF on specific prokaryotic taxa. Bacterial and archaeal microbes may have different nutritive values for the HNF communities, thus affecting growth rates and community composition of HNFs. In this study we investigated the influence of prey food quality on Cafeteria roenbergensis and on a natural HNF community isolated in the northern Adriatic Sea. Two Nitrosopumilus maritimus-related strains isolated from the northern Adriatic Sea (Nitrosopumilus adriaticus, Nitrosopumilus piranensis), two Nitrosococcus strains and two fast growing marine Bacteria (Pseudomonas marina and Marinobacter algicola) were fed to the HNFs. The two fast growing bacterial strains resulted in high growth rates of Cafeteria roenbergensis and the mixed HNF community, while the two Nitrosococcus strains did not. Cafeteria roenbergensis fed on N. adriaticus but it did not graze N. piranensis, suggesting that the subtle metabolic and physiological differences between these two closely related thaumarchaeal strains affect the grazing pressure to which they are exposed. Our study also indicates that prokaryotic community composition influences the composition of the HNF community.

  5. Biosynthesis and Metabolic Fate of Phenylalanine in Conifers

    OpenAIRE

    Pascual, María B.; El-Azaz, Jorge; de la Torre, Fernando N.; Cañas, Rafael A.; Avila, Concepción; Cánovas, Francisco M.

    2016-01-01

    The amino acid phenylalanine (Phe) is a critical metabolic node that plays an essential role in the interconnection between primary and secondary metabolism in plants. Phe is used as a protein building block but it is also as a precursor for numerous plant compounds that are crucial for plant reproduction, growth, development, and defense against different types of stresses. The metabolism of Phe plays a central role in the channeling of carbon from photosynthesis to the biosynthesis of pheny...

  6. Simple sequence proteins in prokaryotic proteomes

    Directory of Open Access Journals (Sweden)

    Ramachandran Srinivasan

    2006-06-01

    Full Text Available Abstract Background The structural and functional features associated with Simple Sequence Proteins (SSPs are non-globularity, disease states, signaling and post-translational modification. SSPs are also an important source of genetic and possibly phenotypic variation. Analysis of 249 prokaryotic proteomes offers a new opportunity to examine the genomic properties of SSPs. Results SSPs are a minority but they grow with proteome size. This relationship is exhibited across species varying in genomic GC, mutational bias, life style, and pathogenicity. Their proportion in each proteome is strongly influenced by genomic base compositional bias. In most species simple duplications is favoured, but in a few cases such as Mycobacteria, large families of duplications occur. Amino acid preference in SSPs exhibits a trend towards low cost of biosynthesis. In SSPs and in non-SSPs, Alanine, Glycine, Leucine, and Valine are abundant in species widely varying in genomic GC whereas Isoleucine and Lysine are rich only in organisms with low genomic GC. Arginine is abundant in SSPs of two species and in the non-SSPs of Xanthomonas oryzae. Asparagine is abundant only in SSPs of low GC species. Aspartic acid is abundant only in the non-SSPs of Halobacterium sp NRC1. The abundance of Serine in SSPs of 62 species extends over a broader range compared to that of non-SSPs. Threonine(T is abundant only in SSPs of a couple of species. SSPs exhibit preferential association with Cell surface, Cell membrane and Transport functions and a negative association with Metabolism. Mesophiles and Thermophiles display similar ranges in the content of SSPs. Conclusion Although SSPs are a minority, the genomic forces of base compositional bias and duplications influence their growth and pattern in each species. The preferences and abundance of amino acids are governed by low biosynthetic cost, evolutionary age and base composition of codons. Abundance of charged amino acids Arginine

  7. Analog kefir production with a low phenylalanine for Phenylketonuria

    Directory of Open Access Journals (Sweden)

    Amir Yari

    2017-06-01

    Full Text Available Phenylketonuria (PKU is one of the most prevalent types of hereditary metabolic disorders which is caused due to an absence or reduction of the activity of the Phenylalanine hydroxylase enzyme in the liver which in turn, inhibits the transformation of phenylalanine (Phe to tyrosine. In clinical terms, this disorder is displayed with severe, permanent and irreversible mental retardation. This research was aimed at development of a highly nutrient and acceptable suitable analogue Kefir drink for these patients. The mentioned drink is based on milk permeate, cream powder and includes glycomacropeptide (GMP as a source of protein, starter as a fermentation source, the trance glutamines (TG enzyme, dough stabilizer and modified corn starch as tissue maker, salt and water. The GMP used in this analogue drink is intended for enrichment of the product and therefore it was added by 3% to one formula. The aforementioned sample had a lower calculated amount of pH and alcohol percentage in comparison with the 16 samples which did not have GMP. The results of this study showed that the analog kefir has a low level of phenylalanine (30.40 mg/100g and in that regard, it can be considered to be useful for patients with PKU.

  8. The Role of Prokaryotes in Sediment Carbon Cycling

    DEFF Research Database (Denmark)

    Piil, Kristoffer

    in the sediment. In particular, the work has focused on estimating how rapidly amino acids derived from plankton are degraded and replaced by amino acids from prokaryotes and how extensive this reworking of amino acids is in surface sediments. Another part of my work has focused on establishing reliable estimates...... of cell specific amino acid and muramic acid concentrations in sediment bacteria. Such estimates are important tools when studying the reworking of amino acids by bacteria and the preservation of bacterial cell walls. In addition, it has been an aim of the work to investigate how abundant endospores...... are in marine sediment and how dynamic the endospore population is, as very little is known about this compartment of the prokaryotic community. Prokaryotic reworking of amino acids was investigated by two independent methods. The first approach involved estimating the amount of amino acids produced...

  9. Prokaryotic DNA segregation by an actin-like filament

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Bugge Jensen, Rasmus; Löwe, Jan

    2002-01-01

    The mechanisms responsible for prokaryotic DNA segregation are largely unknown. The partitioning locus (par) encoded by the Escherichia coli plasmid R1 actively segregates its replicon to daughter cells. We show here that the ParM ATPase encoded by par forms dynamic actin-like filaments with prop...... point for ParM polymerization. Hence, we provide evidence for a simple prokaryotic analogue of the eukaryotic mitotic spindle apparatus.......The mechanisms responsible for prokaryotic DNA segregation are largely unknown. The partitioning locus (par) encoded by the Escherichia coli plasmid R1 actively segregates its replicon to daughter cells. We show here that the ParM ATPase encoded by par forms dynamic actin-like filaments...

  10. Viral Diversity Threshold for Adaptive Immunity in Prokaryotes

    Science.gov (United States)

    Weinberger, Ariel D.; Wolf, Yuri I.; Lobkovsky, Alexander E.; Gilmore, Michael S.; Koonin, Eugene V.

    2012-01-01

    ABSTRACT Bacteria and archaea face continual onslaughts of rapidly diversifying viruses and plasmids. Many prokaryotes maintain adaptive immune systems known as clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (Cas). CRISPR-Cas systems are genomic sensors that serially acquire viral and plasmid DNA fragments (spacers) that are utilized to target and cleave matching viral and plasmid DNA in subsequent genomic invasions, offering critical immunological memory. Only 50% of sequenced bacteria possess CRISPR-Cas immunity, in contrast to over 90% of sequenced archaea. To probe why half of bacteria lack CRISPR-Cas immunity, we combined comparative genomics and mathematical modeling. Analysis of hundreds of diverse prokaryotic genomes shows that CRISPR-Cas systems are substantially more prevalent in thermophiles than in mesophiles. With sequenced bacteria disproportionately mesophilic and sequenced archaea mostly thermophilic, the presence of CRISPR-Cas appears to depend more on environmental temperature than on bacterial-archaeal taxonomy. Mutation rates are typically severalfold higher in mesophilic prokaryotes than in thermophilic prokaryotes. To quantitatively test whether accelerated viral mutation leads microbes to lose CRISPR-Cas systems, we developed a stochastic model of virus-CRISPR coevolution. The model competes CRISPR-Cas-positive (CRISPR-Cas+) prokaryotes against CRISPR-Cas-negative (CRISPR-Cas−) prokaryotes, continually weighing the antiviral benefits conferred by CRISPR-Cas immunity against its fitness costs. Tracking this cost-benefit analysis across parameter space reveals viral mutation rate thresholds beyond which CRISPR-Cas cannot provide sufficient immunity and is purged from host populations. These results offer a simple, testable viral diversity hypothesis to explain why mesophilic bacteria disproportionately lack CRISPR-Cas immunity. More generally, fundamental limits on the adaptability of biological

  11. Minimally invasive (13)C-breath test to examine phenylalanine metabolism in children with phenylketonuria.

    Science.gov (United States)

    Turki, Abrar; Murthy, Gayathri; Ueda, Keiko; Cheng, Barbara; Giezen, Alette; Stockler-Ipsiroglu, Sylvia; Elango, Rajavel

    2015-01-01

    Phenylketonuria (PKU) is an autosomal recessive disorder caused by deficiency of hepatic phenylalanine hydroxylase (PAH) leading to increased levels of phenylalanine in the plasma. Phenylalanine levels and phenylalanine hydroxylase (PAH) activity monitoring are currently limited to conventional blood dot testing. 1-(13)C-phenylalanine, a stable isotope can be used to examine phenylalanine metabolism, as the conversion of phenylalanine to tyrosine occurs in vivo via PAH and subsequently releases the carboxyl labeled (13)C as (13)CO2 in breath. Our objective was to examine phenylalanine metabolism in children with PKU using a minimally-invasive 1-(13)C-phenylalanine breath test ((13)C-PBT). Nine children (7 M: 2 F, mean age 12.5 ± 2.87 y) with PKU participated in the study twice: once before and once after sapropterin supplementation. Children were provided 6 mg/kg oral dose of 1-(13)C-phenylalanine and breath samples were collected at 20 min intervals for a period of 2h. Rate of CO2 production was measured at 60 min post-oral dose using indirect calorimetry. The percentage of 1-(13)C-phenylalanine exhaled as (13)CO2 was measured over a 2h period. Prior to studying children with PKU, we tested the study protocol in healthy children (n = 6; 4M: 2F, mean age 10.2 ± 2.48 y) as proof of principle. Production of a peak enrichment (Cmax) of (13)CO2 (% of dose) in all healthy children occurred at 20 min ranging from 17-29% of dose, with a subsequent return to ~5% by the end of 2h. Production of (13)CO2 from 1-(13)C-phenylalanine in all children with PKU prior to sapropterin treatment remained low. Following sapropterin supplementation for a week, production of (13)CO2 significantly increased in five children with a subsequent decline in blood phenylalanine levels, suggesting improved PAH activity. Sapropterin treatment was not effective in three children whose (13)CO2 production remained unchanged, and did not show a reduction in blood phenylalanine levels and improvement

  12. PKU: high plasma phenylalanine concentrations are associated with increased prevalence of mood swings.

    Science.gov (United States)

    Anjema, Karen; van Rijn, Margreet; Verkerk, Paul H; Burgerhof, Johannes G M; Heiner-Fokkema, M Rebecca; van Spronsen, Francjan J

    2011-11-01

    In phenylketonuria, knowledge about the relation between behavior and plasma phenylalanine is scarce. The aim of this study was to determine whether high phenylalanine is associated with disturbed behavior noticed by the patient and or close environment (parents or partners). 48 early treated PKU patients (median age 8.5, range 0-35 years) participated (median phenylalanine concentration in total sample 277 (range 89-1171) μmol/l; and in patients introvert or extravert behavior. The interviewer as well as the respondents were blinded with regard to the phenylalanine concentration. Patients reported less deviant behavior compared to close environment. Mood swings were positively associated with phenylalanine concentrations in the total group (P=0.039) and patients introvert and extravert behavior were not statistically significant. there is a positive association between phenylalanine concentrations and mood swings. Copyright © 2011 Elsevier Inc. All rights reserved.

  13. Phenylalanine metabolism in isolated rat liver cells. Effects of glucagon and diabetes.

    OpenAIRE

    Carr, F P; Pogson, C I

    1981-01-01

    1. Methods are described for monitoring the metabolic flux through phenylalanine hydroxylase, the tyrosine catabolic pathway and phenylalanine: pyruvate transaminase in isolated liver cell incubations. 2. The relationship between hydroxylase flux and phenylalanine concentration is sigmoidal. 3. Glucagon increases hydroxylase activity at low, near-physiological, substrate concentrations only. The hormone does not affect the rate of formation of phenylpyruvate. 4. Experimental diabetes (for 10 ...

  14. Construction and prokaryotic expression of the fusion gene PRRSV ...

    African Journals Online (AJOL)

    ajl4

    2013-07-24

    Jul 24, 2013 ... The fusion expressing plasmid pET32-GP5-Hsp70 was constructed and expressed in ... 2004). Hsps, expressed by prokaryotes and eukaryotes and their action as molecular ..... Facts, thoughts, and dreams. Shock. 12(4): ...

  15. Top-Down Control of Diesel-Degrading Prokaryotic Communities.

    Science.gov (United States)

    Sauret, Caroline; Böttjer, Daniela; Talarmin, Agathe; Guigue, Catherine; Conan, Pascal; Pujo-Pay, Mireille; Ghiglione, Jean-François

    2015-08-01

    Biostimulation through the addition of inorganic nutrients has been the most widely practiced bioremediation strategy in oil-polluted marine waters. However, little attention has so far been paid to the microbial food web and the impact of top-down control that directly or indirectly influences the success of the bioremediation. We designed a mesocosm experiment using pre-filtered (diesel fuel. Prokaryotes, HNF and VLP abundances showed a predator-prey succession, with a co-development of HNF and VLP. In the polluted system, we observed a stronger impact of viral lysis on prokaryotic abundances than in the control. Analysis of the diversity revealed that a bloom of Vibrio sp. occurred in the polluted mesocosm. That bloom was rapidly followed by a less abundant and more even community of predation-resistant bacteria, including known hydrocarbon degraders such as Oleispira spp. and Methylophaga spp. and opportunistic bacteria such as Percisivirga spp., Roseobacter spp. and Phaeobacter spp. The shift in prokaryotic dominance in response to viral lysis provided clear evidence of the 'killing the winner' model. Nevertheless, despite clear effects on prokaryotic abundance, activity and diversity, the diesel degradation was not impacted by top-down control. The present study investigates for the first time the functioning of a complex microbial network (including VLP) using a nutrient-based biostimulation strategy and highlights some key processes useful for tailoring bioremediation.

  16. Small CRISPR RNAs guide antiviral defense in prokaryotes

    NARCIS (Netherlands)

    Brouns, S.J.J.; Jore, M.M.; Lundgren, M.; Westra, E.R.; Slijkhuis, R.J.; Snijders, A.P.; Dickman, M.J.; Makarova, K.S.; Koonin, E.V.; Oost, van der J.

    2008-01-01

    Prokaryotes acquire virus resistance by integrating short fragments of viral nucleic acid into clusters of regularly interspaced short palindromic repeats (CRISPRs). Here we show how virus-derived sequences contained in CRISPRs are used by CRISPR-associated (Cas) proteins from the host to mediate an

  17. Do marine natural products interfere with prokaryotic AHL regulatory systems?

    DEFF Research Database (Denmark)

    Kjelleberg, S.; Steinberg, P.; Givskov, Michael Christian

    1997-01-01

    Recent studies indicate that a taxonomically diverse range of marine eukaryotes produce metabolites which inhibit phenotypic traits in bacteria, with no or minimal effects on growth. In this review, we present evidence for the existence of such eukaryotic interference with a conserved prokaryotic...

  18. RNA in defense: CRISPRs protect prokaryotes against mobile genetic elements

    NARCIS (Netherlands)

    Jore, M.M.; Brouns, S.J.J.; Oost, van der J.

    2012-01-01

    The CRISPR/Cas system in prokaryotes provides resistance against invading viruses and plasmids. Three distinct stages in the mechanism can be recognized. Initially, fragments of invader DNA are integrated as new spacers into the repetitive CRISPR locus. Subsequently, the CRISPR is transcribed and

  19. Global diversity and biogeography of deep-sea pelagic prokaryotes

    KAUST Repository

    Salazar, Guillem

    2015-08-07

    The deep-sea is the largest biome of the biosphere, and contains more than half of the whole ocean\\'s microbes. Uncovering their general patterns of diversity and community structure at a global scale remains a great challenge, as only fragmentary information of deep-sea microbial diversity exists based on regional-scale studies. Here we report the first globally comprehensive survey of the prokaryotic communities inhabiting the bathypelagic ocean using high-throughput sequencing of the 16S rRNA gene. This work identifies the dominant prokaryotes in the pelagic deep ocean and reveals that 50% of the operational taxonomic units (OTUs) belong to previously unknown prokaryotic taxa, most of which are rare and appear in just a few samples. We show that whereas the local richness of communities is comparable to that observed in previous regional studies, the global pool of prokaryotic taxa detected is modest (∼3600 OTUs), as a high proportion of OTUs are shared among samples. The water masses appear to act as clear drivers of the geographical distribution of both particle-attached and free-living prokaryotes. In addition, we show that the deep-oceanic basins in which the bathypelagic realm is divided contain different particle-attached (but not free-living) microbial communities. The combination of the aging of the water masses and a lack of complete dispersal are identified as the main drivers for this biogeographical pattern. All together, we identify the potential of the deep ocean as a reservoir of still unknown biological diversity with a higher degree of spatial complexity than hitherto considered.

  20. Global diversity and biogeography of deep-sea pelagic prokaryotes

    KAUST Repository

    Salazar, Guillem; Cornejo-Castillo, Francisco M.; Bení tez-Barrios, Veró nica; Fraile-Nuez, Eugenio; Á lvarez-Salgado, X. Antó n; Duarte, Carlos M.; Gasol, Josep M.; Acinas, Silvia G.

    2015-01-01

    The deep-sea is the largest biome of the biosphere, and contains more than half of the whole ocean's microbes. Uncovering their general patterns of diversity and community structure at a global scale remains a great challenge, as only fragmentary information of deep-sea microbial diversity exists based on regional-scale studies. Here we report the first globally comprehensive survey of the prokaryotic communities inhabiting the bathypelagic ocean using high-throughput sequencing of the 16S rRNA gene. This work identifies the dominant prokaryotes in the pelagic deep ocean and reveals that 50% of the operational taxonomic units (OTUs) belong to previously unknown prokaryotic taxa, most of which are rare and appear in just a few samples. We show that whereas the local richness of communities is comparable to that observed in previous regional studies, the global pool of prokaryotic taxa detected is modest (∼3600 OTUs), as a high proportion of OTUs are shared among samples. The water masses appear to act as clear drivers of the geographical distribution of both particle-attached and free-living prokaryotes. In addition, we show that the deep-oceanic basins in which the bathypelagic realm is divided contain different particle-attached (but not free-living) microbial communities. The combination of the aging of the water masses and a lack of complete dispersal are identified as the main drivers for this biogeographical pattern. All together, we identify the potential of the deep ocean as a reservoir of still unknown biological diversity with a higher degree of spatial complexity than hitherto considered.

  1. Gene duplications in prokaryotes can be associated with environmental adaptation.

    Science.gov (United States)

    Bratlie, Marit S; Johansen, Jostein; Sherman, Brad T; Huang, Da Wei; Lempicki, Richard A; Drabløs, Finn

    2010-10-20

    Gene duplication is a normal evolutionary process. If there is no selective advantage in keeping the duplicated gene, it is usually reduced to a pseudogene and disappears from the genome. However, some paralogs are retained. These gene products are likely to be beneficial to the organism, e.g. in adaptation to new environmental conditions. The aim of our analysis is to investigate the properties of paralog-forming genes in prokaryotes, and to analyse the role of these retained paralogs by relating gene properties to life style of the corresponding prokaryotes. Paralogs were identified in a number of prokaryotes, and these paralogs were compared to singletons of persistent orthologs based on functional classification. This showed that the paralogs were associated with for example energy production, cell motility, ion transport, and defence mechanisms. A statistical overrepresentation analysis of gene and protein annotations was based on paralogs of the 200 prokaryotes with the highest fraction of paralog-forming genes. Biclustering of overrepresented gene ontology terms versus species was used to identify clusters of properties associated with clusters of species. The clusters were classified using similarity scores on properties and species to identify interesting clusters, and a subset of clusters were analysed by comparison to literature data. This analysis showed that paralogs often are associated with properties that are important for survival and proliferation of the specific organisms. This includes processes like ion transport, locomotion, chemotaxis and photosynthesis. However, the analysis also showed that the gene ontology terms sometimes were too general, imprecise or even misleading for automatic analysis. Properties described by gene ontology terms identified in the overrepresentation analysis are often consistent with individual prokaryote lifestyles and are likely to give a competitive advantage to the organism. Paralogs and singletons dominate

  2. Gene duplications in prokaryotes can be associated with environmental adaptation

    Directory of Open Access Journals (Sweden)

    Lempicki Richard A

    2010-10-01

    Full Text Available Abstract Background Gene duplication is a normal evolutionary process. If there is no selective advantage in keeping the duplicated gene, it is usually reduced to a pseudogene and disappears from the genome. However, some paralogs are retained. These gene products are likely to be beneficial to the organism, e.g. in adaptation to new environmental conditions. The aim of our analysis is to investigate the properties of paralog-forming genes in prokaryotes, and to analyse the role of these retained paralogs by relating gene properties to life style of the corresponding prokaryotes. Results Paralogs were identified in a number of prokaryotes, and these paralogs were compared to singletons of persistent orthologs based on functional classification. This showed that the paralogs were associated with for example energy production, cell motility, ion transport, and defence mechanisms. A statistical overrepresentation analysis of gene and protein annotations was based on paralogs of the 200 prokaryotes with the highest fraction of paralog-forming genes. Biclustering of overrepresented gene ontology terms versus species was used to identify clusters of properties associated with clusters of species. The clusters were classified using similarity scores on properties and species to identify interesting clusters, and a subset of clusters were analysed by comparison to literature data. This analysis showed that paralogs often are associated with properties that are important for survival and proliferation of the specific organisms. This includes processes like ion transport, locomotion, chemotaxis and photosynthesis. However, the analysis also showed that the gene ontology terms sometimes were too general, imprecise or even misleading for automatic analysis. Conclusions Properties described by gene ontology terms identified in the overrepresentation analysis are often consistent with individual prokaryote lifestyles and are likely to give a competitive

  3. Mechanistic, Mutational, and Structural Evaluation of a Taxus Phenylalanine Aminomutase

    Energy Technology Data Exchange (ETDEWEB)

    Feng, Lei; Wanninayake, Udayanga; Strom, Susan; Geiger, James; Walker, Kevin D. (MSU)

    2014-10-02

    The structure of a phenylalanine aminomutase (TcPAM) from Taxus canadensis has been determined at 2.4 {angstrom} resolution. The active site of the TcPAM contains the signature 4-methylidene-1H-imidazol-5(4H)-one prosthesis, observed in all catalysts of the class I lyase-like family. This catalyst isomerizes (S)-{alpha}-phenylalanine to the (R)-{beta}-isomer by exchange of the NH{sub 2}/H pair. The stereochemistry of the TcPAM reaction product is opposite of the (S)-{beta}-tyrosine made by the mechanistically related tyrosine aminomutase (SgTAM) from Streptomyces globisporus. Since TcPAM and SgTAM share similar tertiary- and quaternary-structures and have several highly conserved aliphatic residues positioned analogously in their active sites for substrate recognition, the divergent product stereochemistries of these catalysts likely cannot be explained by differences in active site architecture. The active site of the TcPAM structure also is in complex with (E)-cinnamate; the latter functions as both a substrate and an intermediate. To account for the distinct (3R)-{beta}-amino acid stereochemistry catalyzed by TcPAM, the cinnamate skeleton must rotate the C{sub 1}-C{sub {alpha}} and C{sub ipso}-C{sub {beta}} bonds 180{sup o} in the active site prior to exchange and rebinding of the NH{sub 2}/H pair to the cinnamate, an event that is not required for the corresponding acrylate intermediate in the SgTAM reaction. Moreover, the aromatic ring of the intermediate makes only one direct hydrophobic interaction with Leu-104. A L104A mutant of TcPAM demonstrated an 1.5-fold increase in k{sub cat} and a decrease in K{sub M} values for sterically demanding 3'-methyl-{alpha}-phenylalanine and styryl-{alpha}-alanine substrates, compared to the kinetic parameters for TcPAM. These parameters did not change significantly for the mutant with 4'-methyl-{alpha}-phenylalanine compared to those for TcPAM.

  4. Biosynthesis and metabolic fate of phenylalanine in conifers

    Directory of Open Access Journals (Sweden)

    María Belén Pascual

    2016-07-01

    Full Text Available The amino acid phenylalanine (Phe is a critical metabolic node that plays an essential role in the interconnection between primary and secondary metabolism in plants. Phe is used as a protein building block but it is also as a precursor for numerous plant compounds that are crucial for plant reproduction, growth, development and defense against different types of stresses. The metabolism of Phe plays a central role in the channeling of carbon from photosynthesis to the biosynthesis of phenylpropanoids. The study of this metabolic pathway is particularly relevant in trees, which divert large amounts of carbon into the biosynthesis of Phe-derived compounds, particularly lignin, an important constituent of wood. The trunks of trees are metabolic sinks that consume a considerable percentage of carbon and energy from photosynthesis, and carbon is finally immobilized in wood. This paper reviews recent advances in the biosynthesis and metabolic utilization of Phe in conifer trees. Two alternative routes have been identified: the ancient phenylpyruvate pathway that is present in microorganisms, and the arogenate pathway that possibly evolved later during plant evolution. Additionally, an efficient nitrogen recycling mechanism is required to maintain sustained growth during xylem formation. The relevance of phenylalanine metabolic pathways in wood formation, the biotic interactions and ultraviolet protection is discussed. The genetic manipulation and transcriptional regulation of the pathways are also outlined.

  5. Biosynthesis and Metabolic Fate of Phenylalanine in Conifers.

    Science.gov (United States)

    Pascual, María B; El-Azaz, Jorge; de la Torre, Fernando N; Cañas, Rafael A; Avila, Concepción; Cánovas, Francisco M

    2016-01-01

    The amino acid phenylalanine (Phe) is a critical metabolic node that plays an essential role in the interconnection between primary and secondary metabolism in plants. Phe is used as a protein building block but it is also as a precursor for numerous plant compounds that are crucial for plant reproduction, growth, development, and defense against different types of stresses. The metabolism of Phe plays a central role in the channeling of carbon from photosynthesis to the biosynthesis of phenylpropanoids. The study of this metabolic pathway is particularly relevant in trees, which divert large amounts of carbon into the biosynthesis of Phe-derived compounds, particularly lignin, an important constituent of wood. The trunks of trees are metabolic sinks that consume a considerable percentage of carbon and energy from photosynthesis, and carbon is finally immobilized in wood. This paper reviews recent advances in the biosynthesis and metabolic utilization of Phe in conifer trees. Two alternative routes have been identified: the ancient phenylpyruvate pathway that is present in microorganisms, and the arogenate pathway that possibly evolved later during plant evolution. Additionally, an efficient nitrogen recycling mechanism is required to maintain sustained growth during xylem formation. The relevance of phenylalanine metabolic pathways in wood formation, the biotic interactions, and ultraviolet protection is discussed. The genetic manipulation and transcriptional regulation of the pathways are also outlined.

  6. Adsorptive molecularly imprinted composite membranes for chiral separation of phenylalanine

    Directory of Open Access Journals (Sweden)

    Shah Nasrullah

    2016-09-01

    Full Text Available Two types of composite imprinted membranes, i.e., composite membrane comprised of D-Phe imprinted beads and D-Phe imprinted membrane or DCM and composite membrane comprised of L-Phe imprinted beads and L-Phe imprinted membranes or LCM, were synthesized by phase inversion technique after a uniform dispersion of beads within the polymeric solutions using simple physico-mechanical process. The assemblies of the prepared DCM, LCM and control membranes were employed in ultrafiltration for chiral separation of D, L-Phenylalanine racemate solution. DCM and LCM showed an improved adsorption capacity (0.334 mg g-1 and 0.365 mg g-1 respectively, and adsorption selectivity (2.72 and 2.98 respectively. However, the percent rejection of the template and counter enantiomer were lower than that of control membranes. Compared to control membrane, the DCM and LCM showed inverse permselectivity. These composite membranes having better adsorption and separation ability for Phenylalanine racemate solution will be suitable in the future for various other applications.

  7. Characterization of mutations at the mouse phenylalanine hydroxylase locus

    Energy Technology Data Exchange (ETDEWEB)

    McDonald, J.D.; Charlton, C.K. [Wichita State Univ., KS (United States)

    1997-02-01

    Two genetic mouse models for human phenylketonuria have been characterized by DNA sequence analysis. For each, a distinct mutation was identified within the protein coding sequence of the phenylalanine hydroxylase gene. This establishes that the mutated locus is the same as that causing human phenylketonuria and allows a comparison between these mouse phenylketonuria models and the human disease. A genotype/phenotype relationship that is strikingly similar to the human disease emerges, underscoring the similarity of phenylketonuria in mouse and man. In PAH{sup ENU1}, the phenotype is mild. The Pah{sup enu1} mutation predicts a conservative valine to alanine amino acid substitution and is located in exon 3, a gene region where serious mutations are rare in humans. In PAH{sup ENU2} the phenotype is severe. The Pah{sup enu2} mutation predicts a radical phenylalanine to serine substitution and is located in exon 7, a gene region where serious mutations are common in humans. In PAH{sup ENU2}, the sequence information was used to devise a direct genotyping system based on the creation of a new Alw26I restriction endonuclease site. 26 refs., 2 figs., 1 tab.

  8. Differential impact of lytic viruses on prokaryotic morphopopulations in a tropical estuarine system (Cochin estuary, India).

    Science.gov (United States)

    Jasna, Vijayan; Pradeep Ram, Angia Sriram; Parvathi, Ammini; Sime-Ngando, Telesphore

    2018-01-01

    Our understanding on the importance of viral lysis in the functioning of tropical estuarine ecosystem is limited. This study examines viral infection of prokaryotes and subsequent lysis of cells belonging to different morphotypes across a salinity gradient in monsoon driven estuarine ecosystem (Cochin estuary, India). High standing stock of viruses and prokaryotes accompanied by lytic infection rates in the euryhaline/mesohaline region of the estuary suggests salinity to have an influential role in driving interactions between prokaryotes and viruses. High prokaryotic mortality rates, up to 42% of prokaryote population in the pre-monsoon season is further substantiated by a high virus to prokaryote ratio (VPR), suggesting that maintenance of a high number of viruses is dependent on the most active fraction of bacterioplankton. Although myoviruses were the dominant viral morphotype (mean = 43%) throughout the study period, there was significant variation among prokaryotic morphotypes susceptible to viral infection. Among them, the viral infected short rod prokaryote morphotype with lower burst estimates (mean = 18 viruses prokaryote-1) was dominant (35%) in the dry seasons whereas a substantial increase in cocci forms (30%) infected by viruses with high burst size (mean = 31 viruses prokaryote-1) was evident during the monsoon season. Such preferential infections of prokaryotic morphopopulations with respect to seasons can have a strong and variable impact on the carbon and energy flow in this tropical ecosystem.

  9. PKU : high plasma phenylalanine concentrations are associated with increased prevalence of mood swings

    NARCIS (Netherlands)

    Anjema, Karen; van Rijn, Margreet; Verkerk, Paul H; Burgerhof, Johannes G M; van Spronsen, Francjan J; Fokkema, Margaretha

    2011-01-01

    UNLABELLED: In phenylketonuria, knowledge about the relation between behavior and plasma phenylalanine is scarce. The aim of this study was to determine whether high phenylalanine is associated with disturbed behavior noticed by the patient and or close environment (parents or partners). 48 early

  10. PKU: High plasma phenylalanine concentrations are associated with increased prevalence of mood swings

    NARCIS (Netherlands)

    Anjema, K.; Rijn, M. van; Verkerk, P.H.; Burgerhof, J.G.M.; Heiner-Fokkema, M.R.; Spronsen, F.J. van

    2011-01-01

    In phenylketonuria, knowledge about the relation between behavior and plasma phenylalanine is scarce. The aim of this study was to determine whether high phenylalanine is associated with disturbed behavior noticed by the patient and or close environment (parents or partners). 48 early treated PKU

  11. PKU : high plasma phenylalanine concentrations are associated with increased prevalence of mood swings

    NARCIS (Netherlands)

    Anjema, Karen; van Rijn, Margreet; Verkerk, Paul H; Burgerhof, Johannes G M; van Spronsen, Francjan J; Fokkema, Margaretha

    UNLABELLED: In phenylketonuria, knowledge about the relation between behavior and plasma phenylalanine is scarce. The aim of this study was to determine whether high phenylalanine is associated with disturbed behavior noticed by the patient and or close environment (parents or partners). 48 early

  12. Geochemical Interactions and Viral-Prokaryote Relationships in Freshwater Environments

    Science.gov (United States)

    Kyle, J. E.; Ferris, G.

    2009-05-01

    Viral and prokaryotic abundances were surveyed throughout southern Ontario aquatic habitats to determine relationships with geochemical parameters in the natural environment. Surface water samples were collected from acid mine drainage in summer of 2007 and 2008 and from circum-neutral pH environments in October to November 2008. Site determination was based on collecting samples from various aquatic habitats (acid mine drainage, lakes, rivers, tributaries, wetlands) with differing bedrock geology (limestone and shale dominated vs granitic Canadian Shield) to obtain a range of geochemical conditions. At each site, measurements of temperature, pH, and Eh were conducted. Samples collected for microbial counts and electron imaging were preserved to a final concentration of 2.5 % (v/v) glutaraldehyde. Additional sample were filtered into 60 mL nalgene bottles and amber EPA certified 40 mL glass vials to determine chemical constituents and dissolved organic carbon (DOC), respectively. Water was also collected to determine additional physiochemical parameters (dissolved total iron, ferric iron, nitrate, sulfate, phosphate, alkalinity, and turbidity). All samples were stored at 4 °C until analysis. Viral and prokaryotic abundance was determined by staining samples with SYBR Green I and examining with a epifluorescence microscope under blue excitation. Multiple regression analysis using stepwise backwards regression and general linear models revealed that viral abundance was the most influential predictor of prokaryotic abundance. Additional predictors include pH, sulfate, phosphate, and magnesium. The strength of the model was very strong with 90 % of the variability explained (R2 = 0.90, p < 0.007). This is the first report, to our knowledge, of viruses exhibiting such strong controls over prokaryotic abundance in the natural environment. All relationships are positively correlated with the exception of Mg, which is negatively correlated. Iron was also noted as a

  13. Surface plasmon resonance spectroscopic study of UV-addressable phenylalanine sensing based on a self-assembled spirooxazine derivative monolayer

    International Nuclear Information System (INIS)

    Suk, Shinae; Suh, Hee-Jung; Gun An, Won; Kim, Jae-Ho; Jin, Sung-Ho; Kim, Sung-Hoon; Gal, Yeong-Soon; Koh, Kwangnak

    2004-01-01

    Light-addressable compounds are very interesting due to the possibilities of their practical use such as optical switches and memories or variable transmission materials. For example, transportation of phenylalanine across liposomal bilayers mediated by a photoresponsive carrier like spirooxazine through electrostatic interaction between phenylalanine and spirooxazine derivative. Thus, the spirooxazine is expected to form a UV-addressable phenylalanine sensing interface. In this study, we prepared phenylalanine sensing interface of a spirooxazine derivative by self-assembly technique and evaluated interaction between a spirooxazine moiety and phenylalanine with a surface plasmon resonance (SPR). The refractive index change of monolayer caused by interaction between a spirooxazine derivative and phenylalanine led to the SPR angle shifts upon UV irradiation. The SPR angle shift increased with increasing the concentration of phenylalanine solution. These results indicated that the spirooxazine derivative self-assembled monolayer (SAM) has an application potential for UV-addressable phenylalanine sensing

  14. Determination of Phenylalanine and Tyrosine by High Performance Liquid Chromatography-Tandem Mass Spectrometry.

    Science.gov (United States)

    Peat, Judy; Garg, Uttam

    2016-01-01

    Hyperphenylalaninemia/phenylketonuria (PKU) is one of the most common inborn errors of amino acid metabolism affecting about 1:15,000 infants in the United States. PKU is an autosomal recessive disorder that if untreated results in mental retardation. The most common cause of PKU is deficiency of the enzyme phenylalanine hydroxylase that converts phenylalanine to tyrosine. Tyrosine deficiency results in impaired synthesis of catecholamines and thyroxine. Less commonly, it can result from defects in the synthesis or regeneration of tetrahydrobiopterin (BH4), an essential cofactor for the enzyme phenylalanine hydroxylase. Increased phenylalanine and decreased tyrosine in blood are used in the diagnosis and follow-up of patients with PKU. LC/MS/MS method is described for the quantification of phenylalanine and tyrosine.

  15. Rule Mining Techniques to Predict Prokaryotic Metabolic Pathways

    KAUST Repository

    Saidi, Rabie

    2017-08-28

    It is becoming more evident that computational methods are needed for the identification and the mapping of pathways in new genomes. We introduce an automatic annotation system (ARBA4Path Association Rule-Based Annotator for Pathways) that utilizes rule mining techniques to predict metabolic pathways across wide range of prokaryotes. It was demonstrated that specific combinations of protein domains (recorded in our rules) strongly determine pathways in which proteins are involved and thus provide information that let us very accurately assign pathway membership (with precision of 0.999 and recall of 0.966) to proteins of a given prokaryotic taxon. Our system can be used to enhance the quality of automatically generated annotations as well as annotating proteins with unknown function. The prediction models are represented in the form of human-readable rules, and they can be used effectively to add absent pathway information to many proteins in UniProtKB/TrEMBL database.

  16. Rule Mining Techniques to Predict Prokaryotic Metabolic Pathways

    KAUST Repository

    Saidi, Rabie; Boudellioua, Imene; Martin, Maria J.; Solovyev, Victor

    2017-01-01

    It is becoming more evident that computational methods are needed for the identification and the mapping of pathways in new genomes. We introduce an automatic annotation system (ARBA4Path Association Rule-Based Annotator for Pathways) that utilizes rule mining techniques to predict metabolic pathways across wide range of prokaryotes. It was demonstrated that specific combinations of protein domains (recorded in our rules) strongly determine pathways in which proteins are involved and thus provide information that let us very accurately assign pathway membership (with precision of 0.999 and recall of 0.966) to proteins of a given prokaryotic taxon. Our system can be used to enhance the quality of automatically generated annotations as well as annotating proteins with unknown function. The prediction models are represented in the form of human-readable rules, and they can be used effectively to add absent pathway information to many proteins in UniProtKB/TrEMBL database.

  17. Patterns of prokaryotic lateral gene transfers affecting parasitic microbial eukaryotes

    DEFF Research Database (Denmark)

    Alsmark, Cecilia; Foster, Peter G; Sicheritz-Pontén, Thomas

    2013-01-01

    BACKGROUND: The influence of lateral gene transfer on gene origins and biology in eukaryotes is poorly understood compared with those of prokaryotes. A number of independent investigations focusing on specific genes, individual genomes, or specific functional categories from various eukaryotes have...... approach to systematically investigate lateral gene transfer affecting the proteomes of thirteen, mainly parasitic, microbial eukaryotes, representing four of the six eukaryotic super-groups. All of the genomes investigated have been significantly affected by prokaryote-to-eukaryote lateral gene transfers...... indicated that lateral gene transfer does indeed affect eukaryotic genomes. However, the lack of common methodology and criteria in these studies makes it difficult to assess the general importance and influence of lateral gene transfer on eukaryotic genome evolution. RESULTS: We used a phylogenomic...

  18. Trends and barriers to lateral gene transfer in prokaryotes.

    Science.gov (United States)

    Popa, Ovidiu; Dagan, Tal

    2011-10-01

    Gene acquisition by lateral gene transfer (LGT) is an important mechanism for natural variation among prokaryotes. Laboratory experiments show that protein-coding genes can be laterally transferred extremely fast among microbial cells, inherited to most of their descendants, and adapt to a new regulatory regime within a short time. Recent advance in the phylogenetic analysis of microbial genomes using networks approach reveals a substantial impact of LGT during microbial genome evolution. Phylogenomic networks of LGT among prokaryotes reconstructed from completely sequenced genomes uncover barriers to LGT in multiple levels. Here we discuss the kinds of barriers to gene acquisition in nature including physical barriers for gene transfer between cells, genomic barriers for the integration of acquired DNA, and functional barriers for the acquisition of new genes. Copyright © 2011 Elsevier Ltd. All rights reserved.

  19. Proteolytic enzymes in seawater: contribution of prokaryotes and protists

    Science.gov (United States)

    Obayashi, Y.; Suzuki, S.

    2016-02-01

    Proteolytic enzyme is one of the major catalysts of microbial processing of organic matter in biogeochemical cycle. Here we summarize some of our studies about proteases in seawater, including 1) distribution of protease activities in coastal and oceanic seawater, 2) responses of microbial community and protease activities in seawater to organic matter amending, and 3) possible contribution of heterotrophic protists besides prokaryotes to proteases in seawater, to clarify cleared facts and remaining questions. Activities of aminopeptidases, trypsin-type and chymotrypsin-type proteases were detected from both coastal and oceanic seawater by using MCA-substrate assay. Significant activities were detected from not only particulate (cell-associated) fraction but also dissolved fraction of seawater, especially for trypsin-type and chymotrypsin-type proteases. Hydrolytic enzymes in seawater have been commonly thought to be mainly derived from heterotrophic prokaryotes; however, it was difficult to determine actual source organisms of dissolved enzymes in natural seawater. Our experiment with addition of dissolved protein to subtropical oligotrophic Pacific water showed drastically enhancement of the protease activities especially aminopeptidases in seawater, and the prokaryotic community structure simultaneously changed to be dominant of Bacteroidetes, indicating that heterotrophic bacteria were actually one of the sources of proteases in seawater. Another microcosm experiment with free-living marine heterotrophic ciliate Paranophrys marina together with an associated bacterium showed that extracellular trypsin-type activity was mainly attributed to the ciliate. The protist seemed to work in organic matter digestion in addition to be a grazer. From the results, we propose a system of organic matter digestion by prokaryotes and protists in aquatic environments, although their actual contribution in natural environments should be estimated in future studies.

  20. A quantitative account of genomic island acquisitions in prokaryotes

    Directory of Open Access Journals (Sweden)

    Roos Tom E

    2011-08-01

    Full Text Available Abstract Background Microbial genomes do not merely evolve through the slow accumulation of mutations, but also, and often more dramatically, by taking up new DNA in a process called horizontal gene transfer. These innovation leaps in the acquisition of new traits can take place via the introgression of single genes, but also through the acquisition of large gene clusters, which are termed Genomic Islands. Since only a small proportion of all the DNA diversity has been sequenced, it can be hard to find the appropriate donors for acquired genes via sequence alignments from databases. In contrast, relative oligonucleotide frequencies represent a remarkably stable genomic signature in prokaryotes, which facilitates compositional comparisons as an alignment-free alternative for phylogenetic relatedness. In this project, we test whether Genomic Islands identified in individual bacterial genomes have a similar genomic signature, in terms of relative dinucleotide frequencies, and can therefore be expected to originate from a common donor species. Results When multiple Genomic Islands are present within a single genome, we find that up to 28% of these are compositionally very similar to each other, indicative of frequent recurring acquisitions from the same donor to the same acceptor. Conclusions This represents the first quantitative assessment of common directional transfer events in prokaryotic evolutionary history. We suggest that many of the resident Genomic Islands per prokaryotic genome originated from the same source, which may have implications with respect to their regulatory interactions, and for the elucidation of the common origins of these acquired gene clusters.

  1. [Experimental interaction of halophilic prokaryotes and opportunistic bacteria in brine].

    Science.gov (United States)

    Selivanova, E A; Nemtseva, N V

    2013-01-01

    Study the effect of extremely halophilic archaea and moderately halophilic bacteria on preservation of opportunistic bacteria in brine. 17 strains of moderately halophilic bacteria and 2 strains of extremely halophilic archaea were isolated from continental hypersaline lake Razval of Sol-Iletsk area of Orenburg Region. Identification of pure cultures of prokaryotes was carried out taking into account their phenotype properties and based on determination of 16S RNA gene sequence. The effect of halophilic prokaryote on elimination of Escherichia coli from brine was evaluated during co-cultivation. Antagonistic activity of cell extracts of the studied microorganisms was evaluated by photometric method. A more prolonged preservation of an E. coli strain in brine in the presence of live cells of extremely halophilic archaea Halorubrum tebenquichense and moderately halophilic bacteria Marinococcus halophilus was established. Extracts of cells of extremely halophilic archaea and moderately halophilic bacteria on the contrary displayed antagonistic activity. The protective effect of live cells of halophilic prokaryotes and antagonistic activity of their cell extracts change the period of conservation of opportunistic bacteria in brine that regulates inter-microbial interactions and changes the period of self-purification that reflects the sanitary condition of a hypersaline water body.

  2. Prokaryotic cells: structural organisation of the cytoskeleton and organelles

    Directory of Open Access Journals (Sweden)

    Wanderley de Souza

    2012-05-01

    Full Text Available For many years, prokaryotic cells were distinguished from eukaryotic cells based on the simplicity of their cytoplasm, in which the presence of organelles and cytoskeletal structures had not been discovered. Based on current knowledge, this review describes the complex components of the prokaryotic cell cytoskeleton, including (i tubulin homologues composed of FtsZ, BtuA, BtuB and several associated proteins, which play a fundamental role in cell division, (ii actin-like homologues, such as MreB and Mb1, which are involved in controlling cell width and cell length, and (iii intermediate filament homologues, including crescentin and CfpA, which localise on the concave side of a bacterium and along its inner curvature and associate with its membrane. Some prokaryotes exhibit specialised membrane-bound organelles in the cytoplasm, such as magnetosomes and acidocalcisomes, as well as protein complexes, such as carboxysomes. This review also examines recent data on the presence of nanotubes, which are structures that are well characterised in mammalian cells that allow direct contact and communication between cells.

  3. The prokaryote-eukaryote dichotomy: meanings and mythology.

    Science.gov (United States)

    Sapp, Jan

    2005-06-01

    Drawing on documents both published and archival, this paper explains how the prokaryote-eukaryote dichotomy of the 1960s was constructed, the purposes it served, and what it implied in terms of classification and phylogeny. In doing so, I first show how the concept was attributed to Edouard Chatton and the context in which he introduced the terms. Following, I examine the context in which the terms were reintroduced into biology in 1962 by Roger Stanier and C. B. van Niel. I study the discourse over the subsequent decade to understand how the organizational dichotomy took on the form of a natural classification as the kingdom Monera or superkingdom Procaryotae. Stanier and van Niel admitted that, in regard to constructing a natural classification of bacteria, structural characteristics were no more useful than physiological properties. They repeatedly denied that bacterial phylogenetics was possible. I thus examine the great historical irony that the "prokaryote," in both its organizational and phylogenetic senses, was defined (negatively) on the basis of structure. Finally, we see how phylogenetic research based on 16S rRNA led by Carl Woese and his collaborators confronted the prokaryote concept while moving microbiology to the center of evolutionary biology.

  4. A computational genomics pipeline for prokaryotic sequencing projects.

    Science.gov (United States)

    Kislyuk, Andrey O; Katz, Lee S; Agrawal, Sonia; Hagen, Matthew S; Conley, Andrew B; Jayaraman, Pushkala; Nelakuditi, Viswateja; Humphrey, Jay C; Sammons, Scott A; Govil, Dhwani; Mair, Raydel D; Tatti, Kathleen M; Tondella, Maria L; Harcourt, Brian H; Mayer, Leonard W; Jordan, I King

    2010-08-01

    New sequencing technologies have accelerated research on prokaryotic genomes and have made genome sequencing operations outside major genome sequencing centers routine. However, no off-the-shelf solution exists for the combined assembly, gene prediction, genome annotation and data presentation necessary to interpret sequencing data. The resulting requirement to invest significant resources into custom informatics support for genome sequencing projects remains a major impediment to the accessibility of high-throughput sequence data. We present a self-contained, automated high-throughput open source genome sequencing and computational genomics pipeline suitable for prokaryotic sequencing projects. The pipeline has been used at the Georgia Institute of Technology and the Centers for Disease Control and Prevention for the analysis of Neisseria meningitidis and Bordetella bronchiseptica genomes. The pipeline is capable of enhanced or manually assisted reference-based assembly using multiple assemblers and modes; gene predictor combining; and functional annotation of genes and gene products. Because every component of the pipeline is executed on a local machine with no need to access resources over the Internet, the pipeline is suitable for projects of a sensitive nature. Annotation of virulence-related features makes the pipeline particularly useful for projects working with pathogenic prokaryotes. The pipeline is licensed under the open-source GNU General Public License and available at the Georgia Tech Neisseria Base (http://nbase.biology.gatech.edu/). The pipeline is implemented with a combination of Perl, Bourne Shell and MySQL and is compatible with Linux and other Unix systems.

  5. Depth Dependent Relationships between Temperature and Ocean Heterotrophic Prokaryotic Production

    KAUST Repository

    Lønborg, Christian

    2016-06-07

    Marine prokaryotes play a key role in cycling of organic matter and nutrients in the ocean. Using a unique dataset (>14,500 samples), we applied a space-for-time substitution analysis to assess the temperature dependence of prokaryotic heterotrophic production (PHP) in epi- (0-200 m), meso- (201-1000 m) and bathypelagic waters (1001-4000 m) of the global ocean. Here, we show that the temperature dependence of PHP is fundamentally different between these major oceanic depth layers, with an estimated ecosystem-level activation energy (E) of 36 ± 7 kJ mol for the epipelagic, 72 ± 15 kJ mol for the mesopelagic and 274 ± 65 kJ mol for the bathypelagic realm. We suggest that the increasing temperature dependence with depth is related to the parallel vertical gradient in the proportion of recalcitrant organic compounds. These Ea predict an increased PHP of about 5, 12, and 55% in the epi-, meso-, and bathypelagic ocean, respectively, in response to a water temperature increase by 1°C. Hence, there is indication that a major thus far underestimated feedback mechanism exists between future bathypelagic ocean warming and heterotrophic prokaryotic activity.

  6. Depth Dependent Relationships between Temperature and Ocean Heterotrophic Prokaryotic Production

    KAUST Repository

    Lø nborg, Christian; Cuevas, L. Antonio; Reinthaler, Thomas; Herndl, Gerhard J.; Gasol, Josep M.; Moran, Xose Anxelu G.; Bates, Nicholas R.; á lvarez-Salgado, Xosé A.

    2016-01-01

    Marine prokaryotes play a key role in cycling of organic matter and nutrients in the ocean. Using a unique dataset (>14,500 samples), we applied a space-for-time substitution analysis to assess the temperature dependence of prokaryotic heterotrophic production (PHP) in epi- (0-200 m), meso- (201-1000 m) and bathypelagic waters (1001-4000 m) of the global ocean. Here, we show that the temperature dependence of PHP is fundamentally different between these major oceanic depth layers, with an estimated ecosystem-level activation energy (E) of 36 ± 7 kJ mol for the epipelagic, 72 ± 15 kJ mol for the mesopelagic and 274 ± 65 kJ mol for the bathypelagic realm. We suggest that the increasing temperature dependence with depth is related to the parallel vertical gradient in the proportion of recalcitrant organic compounds. These Ea predict an increased PHP of about 5, 12, and 55% in the epi-, meso-, and bathypelagic ocean, respectively, in response to a water temperature increase by 1°C. Hence, there is indication that a major thus far underestimated feedback mechanism exists between future bathypelagic ocean warming and heterotrophic prokaryotic activity.

  7. The conversion of phenylalanine to tyrosine in man. Direct measurement by continuous intravenous tracer infusions of L-[ring-2H5]phenylalanine and L-[1-13C] tyrosine in the postabsorptive state

    International Nuclear Information System (INIS)

    Clarke, J.T.; Bier, D.M.

    1982-01-01

    Steady state phenylalanine and tyrosine turnover and the rate of conversion of phenylalanine of tyrosine in vivo were determined in 6 healthy postabsorptive adult volunteers. Continuous infusions of tracer amounts of L-[ring- 2 H5]phenylalanine were determined intravenously for 13-14 hr. After 9-10 hr, a priming dose followed by a continuous infusion of L-[1- 13 C]tyrosine was added and maintained, along with the [ 2 H5]phenylalanine infusion, for 4 hr. Venous plasma samples were obtained before the initiation of each infusion and every 30 min during the course of the combined [ 2 H5]phenylalanine and [ 13 C]tyrosine infusion for determination of isotopic enrichments of [ 2 H5]phenylalanine, [ 13 C]tyrosine, and [ 2 H4]tyrosine by gas chromatograph-mass spectrometric analysis of the N-trifluoroacetyl-, methyl ester derivatives of the amino acids. Calculated from the observed enrichments, free phenylalanine and tyrosine turnover rates were 36.1 +/- 5.1 mumole . kg-1 . h-1 and 39.8 +/- 3.5 mumole . kg-1 . h-1, respectively. Phenylalanine was converted to tyrosine at the rate of 5.83 +/- 0.59 mumole . kg-1 . h-1, accounting for approximately 16% of either the phenylalanine or the tyrosine flux. The results indicate that the normal basal steady state phenylalanine hydroxylase activity in vivo in man is lower than that obtained from phenylalanine loading studies. This supports the existence of some type of substance activation of the enzyme as reflected in the previously reported exponential relationship between phenylalanine concentration and phenylalanine hydroxylase activity in vitro. The use of continuous simultaneous infusions of tracer amounts of stable isotope-labeled phenylalanine and tyrosine provides a direct means for studying physiological regulation of phenylalanine hydroxylase activity in vivo

  8. Age–related psychophysiological vulnerability to phenylalanine in phenylketonuria

    Directory of Open Access Journals (Sweden)

    Vincenzo eLeuzzi

    2014-06-01

    Full Text Available Background. Phenylketonuria (PKU is caused by the inherited defect of the phenylalanine hydroxylase enzyme, which converts phenylalanine (Phe into tyrosine (Tyr. Neonatal screening programs and early treatment have radically changed the natural history of PKU. Nevertheless, an increased risk of neurocognitive and psychiatric problems in adulthood remains a challenging aspect of the disease. In order to assess the vulnerability of complex skills to Phe, we explored: a the effect of a rapid increase in blood Phe levels on event-related potentials (ERP in PKU subjects during their second decade of life; b the association (if existing between psychophysiological and neurocognitive features.Methods. Seventeen early-treated PKU subjects, aged 10 to 20, underwent ERP (Mismatch Negativity, auditory P300, Contingent Negative Variation (CNV, and Intensity Dependence of Auditory Evoked Potentials recording before and 2 hours after an oral loading of Phe. Neurocognitive functioning, historical and concurrent biochemical values of blood Phe, Tyr, and Phe/Tyr ratio, were all included in the statistical analysis.Results. ERP components were normally detected in all the subjects. In subjects younger than 13 CNV amplitude, W2-CNV area, P3b latency, and Reaction Times in motor responses were negatively influenced by Phe loading. Independently from the psychophysiological vulnerability, some neurocognitive skills were more impaired in younger patients. No correlation was found between biochemical alterations and neurocognitive and psychophysiological findings. Conclusion. The vulnerability of the emerging neurocognitive functions to Phe suggests a strict metabolic control in adolescents affected by PKU and a neurodevelopmental approach in the study of neurocognitive outcome in PKU.

  9. Use of deuterated tyrosine and phenylalanine in the study of catecholamine and aromatic acid metabolism

    International Nuclear Information System (INIS)

    Curtius, H.C.; Redweik, U.; Steinmann, B.; Leimbacher, W.; Wegmann, H.

    1975-01-01

    Deuterated tyrosine and phenylalanine have been used for the study of their respective metabolism in patients with phenylketonuria (PKU) and in healthy persons. Urinary excretion of dopamine and its metabolites was studied by GC-MS after oral administration of deuterated L-tyrosine in 2 patients with PKU and in normal controls at low and high plasma phenylalanine levels. From these studies it seemed that the in vivo tyrosine 3-hydroxylase activity and thus the formation of L-dopa depend on the phenylalanine concentration in plasma and also in tissues. After loading 3 mentally retarded patients with 3,5-[ 2 H 2 ]-4-hydroxyphenylalanine, we found, among others, excretion of deuterated m-hydroxyphenyl-hydracrylic acid, p-hydroxymandelic acid, p-hydroxybenzoic acid, p-hydroxyhippuric acid, benzoic acid and hippuric acid. An intramolecular rearrangement is postulated. Deuterated phenylalanine was used to investigate phenylalanine and dopa metabolism in PKU. In addition, one untreated person with PKU of normal intelligence and normal excretion of catecholamines at high plasma phenylalanine concentration was investigated in order to see whether there exists an alternative metabolic pathway from phenylalanine to dopa formation

  10. Highest Plasma Phenylalanine Levels in (Very Premature Infants on Intravenous Feeding; A Need for Concern.

    Directory of Open Access Journals (Sweden)

    Ernesto Cortés-Castell

    Full Text Available To analyse the association in newborns between blood levels of phenylalanine and feeding method and gestational age.This observational, cross-sectional study included a sample of 11,829 infants between 2008 and 2013 in a Spanish region. Data were recorded on phenylalanine values, feeding method [breast, formula, mixed (breast plus formula, or partial or fully intravenous feeding], gestational age in weeks (<32, 32-37, ≥37, gender and days since birth at the moment of blood collection. Outcomes were [phenylalanine] and [phenylalanine] ≥95th percentile. Associations were analysed using multivariate models [linear (means difference and logistic regression (adjusted odds ratios].Higher phenylalanine values were associated with lower gestational age (p<0.001 and with intravenous feeding (p<0.001.The degree of prematurity and intravenous feeding influenced the plasma concentration of phenylalanine in the newborn. Caution should be taken in [phenylalanine] for newborns with intravenous feeding, monitoring them carefully. Very preterm infants given the recommended amount of amino acids should also be strictly monitored. These findings should be taken into consideration and call for adapting the amounts to the needs of the infant.

  11. Phenylalanine hydroxylase deficiency caused by a single base substitution in an exon of the human phenylalanine hydroxylase gene

    International Nuclear Information System (INIS)

    Lichter-Konecki, U.; Konecki, D.S.; DiLella, A.G.; Brayton, K.; Marvit, J.; Hahn, T.M.; Trefz, E.K.; Woo, S.L.C.

    1988-01-01

    A novel restriction fragment length polymorphism in the phenylalanine hydroxylase (PAH) locus generated by the restriction endonuclease MspI was observed in a German phenylketonuria (PKU) patient. Molecular cloning and DNA sequence analyses revealed that the MspI polymorphism was created by a T to C transition in exon 9 of the human PAH gene, which also resulted in the conversion of a leucine codon to proline codon. The effect of the amino acid substitution was investigated by creating a corresponding mutation in a full-length human PAD cDNA by site-directed mutagenesis followed by expression analysis in cultured mammalian cells. Results demonstrate that the mutation in the gene causes the synthesis of an unstable protein in the cell corresponding to a CRM - phenotype. Together with the other mutations recently reported in the PAH gene,the data support previous biochemical and clinical observations that PKU is a heterogeneous disorder at the gene level

  12. Phenylalanine hydroxylase deficiency caused by a single base substitution in an exon of the human phenylalanine hydroxylase gene

    Energy Technology Data Exchange (ETDEWEB)

    Lichter-Konecki, U.; Konecki, D.S.; DiLella, A.G.; Brayton, K.; Marvit, J.; Hahn, T.M.; Trefz, E.K.; Woo, S.L.C.

    1988-04-19

    A novel restriction fragment length polymorphism in the phenylalanine hydroxylase (PAH) locus generated by the restriction endonuclease MspI was observed in a German phenylketonuria (PKU) patient. Molecular cloning and DNA sequence analyses revealed that the MspI polymorphism was created by a T to C transition in exon 9 of the human PAH gene, which also resulted in the conversion of a leucine codon to proline codon. The effect of the amino acid substitution was investigated by creating a corresponding mutation in a full-length human PAD cDNA by site-directed mutagenesis followed by expression analysis in cultured mammalian cells. Results demonstrate that the mutation in the gene causes the synthesis of an unstable protein in the cell corresponding to a CRM/sup -/ phenotype. Together with the other mutations recently reported in the PAH gene,the data support previous biochemical and clinical observations that PKU is a heterogeneous disorder at the gene level.

  13. Genomes in turmoil: quantification of genome dynamics in prokaryote supergenomes.

    Science.gov (United States)

    Puigbò, Pere; Lobkovsky, Alexander E; Kristensen, David M; Wolf, Yuri I; Koonin, Eugene V

    2014-08-21

    Genomes of bacteria and archaea (collectively, prokaryotes) appear to exist in incessant flux, expanding via horizontal gene transfer and gene duplication, and contracting via gene loss. However, the actual rates of genome dynamics and relative contributions of different types of event across the diversity of prokaryotes are largely unknown, as are the sizes of microbial supergenomes, i.e. pools of genes that are accessible to the given microbial species. We performed a comprehensive analysis of the genome dynamics in 35 groups (34 bacterial and one archaeal) of closely related microbial genomes using a phylogenetic birth-and-death maximum likelihood model to quantify the rates of gene family gain and loss, as well as expansion and reduction. The results show that loss of gene families dominates the evolution of prokaryotes, occurring at approximately three times the rate of gain. The rates of gene family expansion and reduction are typically seven and twenty times less than the gain and loss rates, respectively. Thus, the prevailing mode of evolution in bacteria and archaea is genome contraction, which is partially compensated by the gain of new gene families via horizontal gene transfer. However, the rates of gene family gain, loss, expansion and reduction vary within wide ranges, with the most stable genomes showing rates about 25 times lower than the most dynamic genomes. For many groups, the supergenome estimated from the fraction of repetitive gene family gains includes about tenfold more gene families than the typical genome in the group although some groups appear to have vast, 'open' supergenomes. Reconstruction of evolution for groups of closely related bacteria and archaea reveals an extremely rapid and highly variable flux of genes in evolving microbial genomes, demonstrates that extensive gene loss and horizontal gene transfer leading to innovation are the two dominant evolutionary processes, and yields robust estimates of the supergenome size.

  14. Surface modification of hydroxyapatite nanoparticles by poly(L-phenylalanine) via ROP of L-phenylalanine N-carboxyanhydride (Pha-NCA)

    International Nuclear Information System (INIS)

    Dai Yanfeng; Xu Min; Wei Junchao; Zhang Haobin; Chen Yiwang

    2012-01-01

    The surface of hydroxyapatite nanoparticles was modified by poly(L-phenylalanine) via the ring opening polymerization (ROP) of L-phenylalanine N-carboxyanhydride. The preparation procedure was monitored by Fourier transform infrared spectroscopy (FTIR), and the modified hydroxyapatite was characterized by thermal gravimetric analysis (TGA) and X-ray diffraction (XRD). The results showed that the surface grafting amounts of poly(L-phenylalanine) on HA ranging from 20.26% to 38.92% can be achieved by tuning the reaction condition. The XRD patterns demonstrated that the crystalline structure of the modified hydroxyapatite was nearly the same with that of HA, implying that the ROP was an efficient surface modification method. The MTT assay proved that the biocompatibility of modified HA was very good, which showed the potential application of modified HA in bone tissue engineering.

  15. Characterization of prokaryotic and eukaryotic promoters usinghidden Markov models

    DEFF Research Database (Denmark)

    Pedersen, Anders Gorm; Baldi, Pierre; Brunak, Søren

    1996-01-01

    In this paper we utilize hidden Markov models (HMMs) and information theory to analyze prokaryotic and eukaryotic promoters. We perform this analysis with special emphasis on the fact that promoters are divided into a number of different classes, depending on which polymerase-associated factors...... that bind to them. We find that HMMs trained on such subclasses of Escherichia coli promoters (specifically, the so-called sigma-70 and sigma-54 classes) give an excellent classification of unknown promoters with respect to sigma-class. HMMs trained on eukaryotic sequences from human genes also model nicely...

  16. Characterization of prokaryotic and eukaryotic promoters using hidden Markov models

    DEFF Research Database (Denmark)

    Pedersen, Anders Gorm; Baldi, P.; Chauvin, Y.

    1996-01-01

    In this paper we utilize hidden Markov models (HMMs) and information theory to analyze prokaryotic and eukaryotic promoters. We perform this analysis with special emphasis on the fact that promoters are divided into a number of different classes, depending on which polymerase-associated factors...... that bind to them. We find that HMMs trained on such subclasses of Escherichia coli promoters (specifically, the so-called sigma 70 and sigma 54 classes) give an excellent classification of unknown promoters with respect to sigma-class. HMMs trained on eukaryotic sequences from human genes also model nicely...

  17. BLAST Ring Image Generator (BRIG: simple prokaryote genome comparisons

    Directory of Open Access Journals (Sweden)

    Beatson Scott A

    2011-08-01

    Full Text Available Abstract Background Visualisation of genome comparisons is invaluable for helping to determine genotypic differences between closely related prokaryotes. New visualisation and abstraction methods are required in order to improve the validation, interpretation and communication of genome sequence information; especially with the increasing amount of data arising from next-generation sequencing projects. Visualising a prokaryote genome as a circular image has become a powerful means of displaying informative comparisons of one genome to a number of others. Several programs, imaging libraries and internet resources already exist for this purpose, however, most are either limited in the number of comparisons they can show, are unable to adequately utilise draft genome sequence data, or require a knowledge of command-line scripting for implementation. Currently, there is no freely available desktop application that enables users to rapidly visualise comparisons between hundreds of draft or complete genomes in a single image. Results BLAST Ring Image Generator (BRIG can generate images that show multiple prokaryote genome comparisons, without an arbitrary limit on the number of genomes compared. The output image shows similarity between a central reference sequence and other sequences as a set of concentric rings, where BLAST matches are coloured on a sliding scale indicating a defined percentage identity. Images can also include draft genome assembly information to show read coverage, assembly breakpoints and collapsed repeats. In addition, BRIG supports the mapping of unassembled sequencing reads against one or more central reference sequences. Many types of custom data and annotations can be shown using BRIG, making it a versatile approach for visualising a range of genomic comparison data. BRIG is readily accessible to any user, as it assumes no specialist computational knowledge and will perform all required file parsing and BLAST comparisons

  18. BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons.

    Science.gov (United States)

    Alikhan, Nabil-Fareed; Petty, Nicola K; Ben Zakour, Nouri L; Beatson, Scott A

    2011-08-08

    Visualisation of genome comparisons is invaluable for helping to determine genotypic differences between closely related prokaryotes. New visualisation and abstraction methods are required in order to improve the validation, interpretation and communication of genome sequence information; especially with the increasing amount of data arising from next-generation sequencing projects. Visualising a prokaryote genome as a circular image has become a powerful means of displaying informative comparisons of one genome to a number of others. Several programs, imaging libraries and internet resources already exist for this purpose, however, most are either limited in the number of comparisons they can show, are unable to adequately utilise draft genome sequence data, or require a knowledge of command-line scripting for implementation. Currently, there is no freely available desktop application that enables users to rapidly visualise comparisons between hundreds of draft or complete genomes in a single image. BLAST Ring Image Generator (BRIG) can generate images that show multiple prokaryote genome comparisons, without an arbitrary limit on the number of genomes compared. The output image shows similarity between a central reference sequence and other sequences as a set of concentric rings, where BLAST matches are coloured on a sliding scale indicating a defined percentage identity. Images can also include draft genome assembly information to show read coverage, assembly breakpoints and collapsed repeats. In addition, BRIG supports the mapping of unassembled sequencing reads against one or more central reference sequences. Many types of custom data and annotations can be shown using BRIG, making it a versatile approach for visualising a range of genomic comparison data. BRIG is readily accessible to any user, as it assumes no specialist computational knowledge and will perform all required file parsing and BLAST comparisons automatically. There is a clear need for a user

  19. Amino acid tolerance test using L-β-phenylalanine-125I

    International Nuclear Information System (INIS)

    Hafiez, A.A.; Megahed, Y.M.; Ismail, A.A.; Abdel-Wahab, M.F.; Khater, R.A.

    1978-01-01

    An amino acid tolerance test is described. L-β-phenylalanine- 125 I was used as representative of L-amino acids. The change in radioactivity of the blood after giving a test dose of tagged L-β-phenylalanine was also investigated. L-β-phenylalanine- 125 I tolerance curves were found to be irreproducible when the test dose was given without a carrier. The addition of 2.5 g untagged phenylalanine as a carrier to the test dose allowed a reproducible and precise type of tolerance curves. Metformin in a dose of 0.5 g t.d.s. for three days induced an inhibitory effect on amino acid absorption in normal persons. (author)

  20. The effect of experimental diabetes on phenylalanine metabolism in isolated liver cells.

    OpenAIRE

    Santana, M A; Fisher, M J; Bate, A J; Pogson, C I

    1985-01-01

    Chronic (10-day) diabetes was associated with increased metabolic flux through phenylalanine hydroxylase in isolated liver cells. This flux was stimulated by 0.1 microM-glucagon, but not by 10 microM-noradrenaline; 0.1 microM-insulin affected neither basal nor glucagon-stimulated flux. The increased rate of phenylalanine hydroxylation in diabetes was accompanied by parallel increases in enzyme activity (as measured with artificial cofactor) and immunoreactive-enzyme-protein content. In contra...

  1. Retroviral-mediated gene transfer and expression of human phenylalanine hydroxylase in primary mouse hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Peng, H.; Armentano, D.; Mackenzie-Graham, L.; Shen, R.F.; Darlington, G.; Ledley, F.D.; Woo, S.L.C. (Baylor College of Medicine, Houston, TX (USA))

    1988-11-01

    Genetic therapy for phenylketonuria (severe phenylalanine hydroxylase deficiency) may require introduction of a normal phenylalanine hydroxylase gene into hepatic cells of patients. The authors report development of a recombinant retrovirus based on the N2 vector for gene transfer and expression of human phenylalanine hydroxylase cDNA in primary mouse hepatocytes. This construct contains an internal promoter of the human {alpha}{sub 1}-antitrypsin gene driving transcription of the phenylalanine hydroxylase cDNA. Primary mouse hepatocytes were isolated from newborn mice, infected with the recombinant virus, and selected for expression of the neomycin-resistance gene. Hepatocytes transformed with the recombinant virus contained high levels of human phenylalanine hydroxylase mRNA transcripts originating from the retroviral and internal promoters. These results demonstrate that the transcriptional regulatory elements of the {alpha}{sub 1} antitrypsin gene retain their tissue-specific function in the recombinant provirus and establish a method for efficient transfer and high-level expression of human phenylalanine hydroxylase in primary hepatocytes.

  2. Retroviral-mediated gene transfer and expression of human phenylalanine hydroxylase in primary mouse hepatocytes

    International Nuclear Information System (INIS)

    Peng, H.; Armentano, D.; Mackenzie-Graham, L.; Shen, R.F.; Darlington, G.; Ledley, F.D.; Woo, S.L.C.

    1988-01-01

    Genetic therapy for phenylketonuria (severe phenylalanine hydroxylase deficiency) may require introduction of a normal phenylalanine hydroxylase gene into hepatic cells of patients. The authors report development of a recombinant retrovirus based on the N2 vector for gene transfer and expression of human phenylalanine hydroxylase cDNA in primary mouse hepatocytes. This construct contains an internal promoter of the human α 1 -antitrypsin gene driving transcription of the phenylalanine hydroxylase cDNA. Primary mouse hepatocytes were isolated from newborn mice, infected with the recombinant virus, and selected for expression of the neomycin-resistance gene. Hepatocytes transformed with the recombinant virus contained high levels of human phenylalanine hydroxylase mRNA transcripts originating from the retroviral and internal promoters. These results demonstrate that the transcriptional regulatory elements of the α 1 antitrypsin gene retain their tissue-specific function in the recombinant provirus and establish a method for efficient transfer and high-level expression of human phenylalanine hydroxylase in primary hepatocytes

  3. Lengths of Orthologous Prokaryotic Proteins Are Affected by Evolutionary Factors

    Directory of Open Access Journals (Sweden)

    Tatiana Tatarinova

    2015-01-01

    Full Text Available Proteins of the same functional family (for example, kinases may have significantly different lengths. It is an open question whether such variation in length is random or it appears as a response to some unknown evolutionary driving factors. The main purpose of this paper is to demonstrate existence of factors affecting prokaryotic gene lengths. We believe that the ranking of genomes according to lengths of their genes, followed by the calculation of coefficients of association between genome rank and genome property, is a reasonable approach in revealing such evolutionary driving factors. As we demonstrated earlier, our chosen approach, Bubble-sort, combines stability, accuracy, and computational efficiency as compared to other ranking methods. Application of Bubble Sort to the set of 1390 prokaryotic genomes confirmed that genes of Archaeal species are generally shorter than Bacterial ones. We observed that gene lengths are affected by various factors: within each domain, different phyla have preferences for short or long genes; thermophiles tend to have shorter genes than the soil-dwellers; halophiles tend to have longer genes. We also found that species with overrepresentation of cytosines and guanines in the third position of the codon (GC3 content tend to have longer genes than species with low GC3 content.

  4. Lengths of Orthologous Prokaryotic Proteins Are Affected by Evolutionary Factors.

    Science.gov (United States)

    Tatarinova, Tatiana; Salih, Bilal; Dien Bard, Jennifer; Cohen, Irit; Bolshoy, Alexander

    2015-01-01

    Proteins of the same functional family (for example, kinases) may have significantly different lengths. It is an open question whether such variation in length is random or it appears as a response to some unknown evolutionary driving factors. The main purpose of this paper is to demonstrate existence of factors affecting prokaryotic gene lengths. We believe that the ranking of genomes according to lengths of their genes, followed by the calculation of coefficients of association between genome rank and genome property, is a reasonable approach in revealing such evolutionary driving factors. As we demonstrated earlier, our chosen approach, Bubble-sort, combines stability, accuracy, and computational efficiency as compared to other ranking methods. Application of Bubble Sort to the set of 1390 prokaryotic genomes confirmed that genes of Archaeal species are generally shorter than Bacterial ones. We observed that gene lengths are affected by various factors: within each domain, different phyla have preferences for short or long genes; thermophiles tend to have shorter genes than the soil-dwellers; halophiles tend to have longer genes. We also found that species with overrepresentation of cytosines and guanines in the third position of the codon (GC3 content) tend to have longer genes than species with low GC3 content.

  5. Primer design for a prokaryotic differential display RT-PCR.

    Science.gov (United States)

    Fislage, R; Berceanu, M; Humboldt, Y; Wendt, M; Oberender, H

    1997-05-01

    We have developed a primer set for a prokaryotic differential display of mRNA in the Enterobacteriaceae group. Each combination of ten 10mer and ten 11mer primers generates up to 85 bands from total Escherichia coli RNA, thus covering expressed sequences of a complete bacterial genome. Due to the lack of polyadenylation in prokaryotic RNA the type T11VN anchored oligonucleotides for the reverse transcriptase reaction had to be replaced with respect to the original method described by Liang and Pardee [ Science , 257, 967-971 (1992)]. Therefore, the sequences of both the 10mer and the new 11mer oligonucleotides were determined by a statistical evaluation of species-specific coding regions extracted from the EMBL database. The 11mer primers used for reverse transcription were selected for localization in the 3'-region of the bacterial RNA. The 10mer primers preferentially bind to the 5'-end of the RNA. None of the primers show homology to rRNA or other abundant small RNA species. Randomly sampled cDNA bands were checked for their bacterial origin either by re-amplification, cloning and sequencing or by re-amplification and direct sequencing with 10mer and 11mer primers after asymmetric PCR.

  6. Insights into structural variations and genome rearrangements in prokaryotic genomes.

    Science.gov (United States)

    Periwal, Vinita; Scaria, Vinod

    2015-01-01

    Structural variations (SVs) are genomic rearrangements that affect fairly large fragments of DNA. Most of the SVs such as inversions, deletions and translocations have been largely studied in context of genetic diseases in eukaryotes. However, recent studies demonstrate that genome rearrangements can also have profound impact on prokaryotic genomes, leading to altered cell phenotype. In contrast to single-nucleotide variations, SVs provide a much deeper insight into organization of bacterial genomes at a much better resolution. SVs can confer change in gene copy number, creation of new genes, altered gene expression and many other functional consequences. High-throughput technologies have now made it possible to explore SVs at a much refined resolution in bacterial genomes. Through this review, we aim to highlight the importance of the less explored field of SVs in prokaryotic genomes and their impact. We also discuss its potential applicability in the emerging fields of synthetic biology and genome engineering where targeted SVs could serve to create sophisticated and accurate genome editing. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  7. Transcriptome dynamics-based operon prediction in prokaryotes.

    Science.gov (United States)

    Fortino, Vittorio; Smolander, Olli-Pekka; Auvinen, Petri; Tagliaferri, Roberto; Greco, Dario

    2014-05-16

    Inferring operon maps is crucial to understanding the regulatory networks of prokaryotic genomes. Recently, RNA-seq based transcriptome studies revealed that in many bacterial species the operon structure vary with the change of environmental conditions. Therefore, new computational solutions that use both static and dynamic data are necessary to create condition specific operon predictions. In this work, we propose a novel classification method that integrates RNA-seq based transcriptome profiles with genomic sequence features to accurately identify the operons that are expressed under a measured condition. The classifiers are trained on a small set of confirmed operons and then used to classify the remaining gene pairs of the organism studied. Finally, by linking consecutive gene pairs classified as operons, our computational approach produces condition-dependent operon maps. We evaluated our approach on various RNA-seq expression profiles of the bacteria Haemophilus somni, Porphyromonas gingivalis, Escherichia coli and Salmonella enterica. Our results demonstrate that, using features depending on both transcriptome dynamics and genome sequence characteristics, we can identify operon pairs with high accuracy. Moreover, the combination of DNA sequence and expression data results in more accurate predictions than each one alone. We present a computational strategy for the comprehensive analysis of condition-dependent operon maps in prokaryotes. Our method can be used to generate condition specific operon maps of many bacterial organisms for which high-resolution transcriptome data is available.

  8. ProOpDB: Prokaryotic Operon DataBase.

    Science.gov (United States)

    Taboada, Blanca; Ciria, Ricardo; Martinez-Guerrero, Cristian E; Merino, Enrique

    2012-01-01

    The Prokaryotic Operon DataBase (ProOpDB, http://operons.ibt.unam.mx/OperonPredictor) constitutes one of the most precise and complete repositories of operon predictions now available. Using our novel and highly accurate operon identification algorithm, we have predicted the operon structures of more than 1200 prokaryotic genomes. ProOpDB offers diverse alternatives by which a set of operon predictions can be retrieved including: (i) organism name, (ii) metabolic pathways, as defined by the KEGG database, (iii) gene orthology, as defined by the COG database, (iv) conserved protein domains, as defined by the Pfam database, (v) reference gene and (vi) reference operon, among others. In order to limit the operon output to non-redundant organisms, ProOpDB offers an efficient method to select the most representative organisms based on a precompiled phylogenetic distances matrix. In addition, the ProOpDB operon predictions are used directly as the input data of our Gene Context Tool to visualize their genomic context and retrieve the sequence of their corresponding 5' regulatory regions, as well as the nucleotide or amino acid sequences of their genes.

  9. Prokaryote genome fluidity: toward a system approach of the mobilome.

    Science.gov (United States)

    Toussaint, Ariane; Chandler, Mick

    2012-01-01

    The importance of horizontal/lateral gene transfer (LGT) in shaping the genomes of prokaryotic organisms has been recognized in recent years as a result of analysis of the increasing number of available genome sequences. LGT is largely due to the transfer and recombination activities of mobile genetic elements (MGEs). Bacterial and archaeal genomes are mosaics of vertically and horizontally transmitted DNA segments. This generates reticulate relationships between members of the prokaryotic world that are better represented by networks than by "classical" phylogenetic trees. In this review we summarize the nature and activities of MGEs, and the problems that presently limit their analysis on a large scale. We propose routes to improve their annotation in the flow of genomic and metagenomic sequences that currently exist and those that become available. We describe network analysis of evolutionary relationships among some MGE categories and sketch out possible developments of this type of approach to get more insight into the role of the mobilome in bacterial adaptation and evolution.

  10. Plant hormone interaction and phenolic metabolism in the regulation of russet spotting in iceberg lettuce.

    Science.gov (United States)

    Ke, D; Saltveit, M E

    1988-12-01

    Russet spotting (RS) is a physiological disorder induced in iceberg lettuce (Lactuca sativa L.) by exposure to parts per million levels of ethylene at 5 +/- 2 degrees C. Ethylene induced phenylalanine ammonia-lyase and ionically bound peroxidase activities that correlated with development of RS symptoms. The ethylene-treated tissue had significantly higher lignin content than air control tissue with lignification localized in walls of RS-affected cells. Ethylene also caused the accumulation of the flavonoids (+)catechin and (-)epicatechin and the chlorogenic acid derivatives 3-caffeoyl-quinic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid. These soluble phenolic compounds were readily oxidized to brown substances by polyphenol oxidase isolated from RS tissue. Ethylene substantially increased ionically bound indole-3-acetic acid (IAA) oxidase activity, while IAA application greatly reduced ethylene-induced phenylalanine ammonia-lyase, peroxidase, and IAA oxidase activities, soluble phenolic content, and RS development.

  11. Links between viruses and prokaryotes throughout the water column along a North Atlantic latitudinal transect

    NARCIS (Netherlands)

    De Corte, Daniele; Sintes, Eva; Yokokawa, Taichi; Reinthaler, Thomas; Herndl, Gerhard J.

    Viruses are an abundant, diverse and dynamic component of marine ecosystems and have a key role in the biogeochemical processes of the ocean by controlling prokaryotic and phytoplankton abundance and diversity. However, most of the studies on virus-prokaryote interactions in marine environments have

  12. A proposed genus boundary for the prokaryotes based on genomic insights.

    Science.gov (United States)

    Qin, Qi-Long; Xie, Bin-Bin; Zhang, Xi-Ying; Chen, Xiu-Lan; Zhou, Bai-Cheng; Zhou, Jizhong; Oren, Aharon; Zhang, Yu-Zhong

    2014-06-01

    Genomic information has already been applied to prokaryotic species definition and classification. However, the contribution of the genome sequence to prokaryotic genus delimitation has been less studied. To gain insights into genus definition for the prokaryotes, we attempted to reveal the genus-level genomic differences in the current prokaryotic classification system and to delineate the boundary of a genus on the basis of genomic information. The average nucleotide sequence identity between two genomes can be used for prokaryotic species delineation, but it is not suitable for genus demarcation. We used the percentage of conserved proteins (POCP) between two strains to estimate their evolutionary and phenotypic distance. A comprehensive genomic survey indicated that the POCP can serve as a robust genomic index for establishing the genus boundary for prokaryotic groups. Basically, two species belonging to the same genus would share at least half of their proteins. In a specific lineage, the genus and family/order ranks showed slight or no overlap in terms of POCP values. A prokaryotic genus can be defined as a group of species with all pairwise POCP values higher than 50%. Integration of whole-genome data into the current taxonomy system can provide comprehensive information for prokaryotic genus definition and delimitation. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  13. Heterogeneous distribution of prokaryotes and viruses at the microscale in a tidal sediment

    DEFF Research Database (Denmark)

    Carreira, Cátia; Larsen, Morten; Glud, Ronnie

    2013-01-01

    In this study we show for the first time the microscale (mm) 2- and 3-dimensional spatial distribution and abundance of prokaryotes, viruses, and oxygen in a tidal sediment. Prokaryotes and viruses were highly heterogeneously distributed with patches of elevated abundances surrounded by areas of ...

  14. Temperature regulation of marine heterotrophic prokaryotes increases latitudinally as a breach between bottom-up and top-down controls

    KAUST Repository

    Moran, Xose Anxelu G.; Gasol, Josep M.; Pernice, Massimo C.; Mangot, Jean-Franç ois; Massana, Ramon; Lara, Elena; Vaqué , Dolors; Duarte, Carlos M.

    2017-01-01

    Planktonic heterotrophic prokaryotes make up the largest living biomass and process most organic matter in the ocean. Determining when and where the biomass and activity of heterotrophic prokaryotes are controlled by resource availability (bottom

  15. Evaluation of pal activity, phenolic and flavonoid contents in three pistachio ( Pistacia vera L. ) cultivars grafted onto three different rootstocks

    OpenAIRE

    Nadernejad, N.; Ahmadimoghadam, A.; Hossyinifard, J.; Poorseyedi, S.

    2013-01-01

    Phenylalanine ammonia lyase (PAL) is a biochemical marker of the environmental stress and plays a pivotal role in phenolic synthesis. The lower ROS level and oxidative damage was observed in grafted plants and the rootstocks have a profound influence on the biochemical composition, especially phenolic compounds. Regarding the importance of the effect rootstocks have on scion in pistachio trees, this study was carried out to assess and compare three pistachio cultivars ("Ahmadaghaii", "Ohadi" ...

  16. Gene-enzyme relationships in somatic cells and their organismal derivatives in higher plants. Progress report

    International Nuclear Information System (INIS)

    Jensen, R.A.

    1983-01-01

    Several enzymes involved in the biosynthesis of aromatic amino acids have been isolated from Nicotiana silvestris. Isozymes of chlorismate mutase were isolated, partially purified and subjected to enzyme kinetic analysis. In addition, studies investigating the role of 5-enolpyruvyl-shikimate-3-phosphate synthetase, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase, shikimate dehydrogenase, prephenate aminotransferase, arogenate dehydrogenase and phenylalanine ammonia-lyase in regulation of aromatic amino acids levels in tobacco are reported

  17. Local and systemic hormonal responses in pepper leaves during compatible and incompatible pepper-tobamovirus interactions

    Czech Academy of Sciences Publication Activity Database

    Dziurka, M.; Janeczko, A.; Juhasz, C.; Gullner, G.; Oklešťková, Jana; Novák, Ondřej; Saja, D.; Skoczowski, A.; Tobias, I.; Barna, B.

    2016-01-01

    Roč. 109, DEC (2016), s. 355-364 ISSN 0981-9428 R&D Projects: GA ČR GA14-34792S Institutional support: RVO:61389030 Keywords : tobacco-mosaic-virus * pathogenesis-related proteins * salicylic-acid * abscisic-acid * acquired- resistance * disease resistance * nicotiana-benthamiana * arabidopsis-thaliana * defense response * immune-responses * Brassinosteroids * Ethylene * Hormone * Pepper * Phenylalanine ammonia lyase * Progesterone * Salicylic acid * Tobamovirus Subject RIV: EF - Botanics Impact factor: 2.724, year: 2016

  18. Physiological and biochemical characteristics of tobacco transgenic plants expressing bacterial dioxygenase

    Czech Academy of Sciences Publication Activity Database

    Piruzian, S.; Goldenkova, V.; Lenets, A.; Cvikrová, Milena; Macháčková, Ivana; Kobets, N.; Mett, V.

    2002-01-01

    Roč. 49, č. 6 (2002), s. 817-822 ISSN 1021-4437 R&D Projects: GA MŠk LN00A081; GA ČR GA206/00/1354 Institutional research plan: CEZ:AV0Z5038910 Keywords : phenylalanine ammonia lyase * polyphenol oxidases * biosynthesis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.102, year: 2002

  19. Diversity of sulfur isotope fractionations by sulfate-reducing prokaryotes

    DEFF Research Database (Denmark)

    Detmers, Jan; Brüchert, Volker; Habicht, K S

    2001-01-01

    Batch culture experiments were performed with 32 different sulfate-reducing prokaryotes to explore the diversity in sulfur isotope fractionation during dissimilatory sulfate reduction by pure cultures. The selected strains reflect the phylogenetic and physiologic diversity of presently known...... sulfate reducers and cover a broad range of natural marine and freshwater habitats. Experimental conditions were designed to achieve optimum growth conditions with respect to electron donors, salinity, temperature, and pH. Under these optimized conditions, experimental fractionation factors ranged from 2.......0 to 42.0 per thousand. Salinity, incubation temperature, pH, and phylogeny had no systematic effect on the sulfur isotope fractionation. There was no correlation between isotope fractionation and sulfate reduction rate. The type of dissimilatory bisulfite reductase also had no effect on fractionation...

  20. Functional Implications of Domain Organization Within Prokaryotic Rhomboid Proteases.

    Science.gov (United States)

    Panigrahi, Rashmi; Lemieux, M Joanne

    2015-01-01

    Intramembrane proteases are membrane embedded enzymes that cleave transmembrane substrates. This interesting class of enzyme and its water mediated substrate cleavage mechanism occurring within the hydrophobic lipid bilayer has drawn the attention of researchers. Rhomboids are a family of ubiquitous serine intramembrane proteases. Bacterial forms of rhomboid proteases are mainly composed of six transmembrane helices that are preceded by a soluble N-terminal domain. Several crystal structures of the membrane domain of the E. coli rhomboid protease ecGlpG have been solved. Independently, the ecGlpG N-terminal cytoplasmic domain structure was solved using both NMR and protein crystallography. Despite these structures, we still do not know the structure of the full-length protein, nor do we know the functional role of these domains in the cell. This chapter will review the structural and functional roles of the different domains associated with prokaryotic rhomboid proteases. Lastly, we will address questions remaining in the field.

  1. Cloning and prokaryotic expression of the porcine lipasin gene.

    Science.gov (United States)

    Li, M M; Geng, J; Guo, Y J; Jiao, X Q; Lu, W F; Zhu, H S; Wang, Y Y; Yang, G Y

    2015-11-23

    Lipasin has recently been demonstrated to be involved in lipid metabolism. In this study, two specific primers were used to amplify the lipasin open reading frame from porcine liver tissue. The polymerase chain reaction product was cloned to a pGEM®-T Easy Vector, digested by SalI and NotI, and sequenced. The lipasin fragment was then cloned to a pET21(b) vector and digested by the same restriction enzyme. The recombinant plasmid was transferred to Escherichia coli (BL21), and the lipasin protein was induced with isopropyl-β-D-thiogalactopyranoside. The protein obtained was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting. A pET-lipasin prokaryotic recombinant expression vector was successfully constructed, and a 25.2-kDa protein was obtained. This study provides a basis for further research on the biological function of porcine lipasin.

  2. Metabolic Compensation and Circadian Resilience in Prokaryotic Cyanobacteria

    Science.gov (United States)

    Johnson, Carl Hirschie; Egli, Martin

    2014-01-01

    For a biological oscillator to function as a circadian pacemaker that confers a fitness advantage, its timing functions must be stable in response to environmental and metabolic fluctuations. One such stability enhancer, temperature compensation, has long been a defining characteristic of these timekeepers. However, an accurate biological timekeeper must also resist changes in metabolism, and this review suggests that temperature compensation is actually a subset of a larger phenomenon, namely metabolic compensation, which maintains the frequency of circadian oscillators in response to a host of factors that impinge on metabolism and would otherwise destabilize these clocks. The circadian system of prokaryotic cyanobacteria is an illustrative model because it is composed of transcriptional and nontranscriptional oscillators that are coupled to promote resilience. Moreover, the cyanobacterial circadian program regulates gene activity and metabolic pathways, and it can be manipulated to improve the expression of bioproducts that have practical value. PMID:24905782

  3. Known knowns, known unknowns and unknown unknowns in prokaryotic transposition.

    Science.gov (United States)

    Siguier, Patricia; Gourbeyre, Edith; Chandler, Michael

    2017-08-01

    Although the phenomenon of transposition has been known for over 60 years, its overarching importance in modifying and streamlining genomes took some time to recognize. In spite of a robust understanding of transposition of some TE, there remain a number of important TE groups with potential high genome impact and unknown transposition mechanisms and yet others, only recently identified by bioinformatics, yet to be formally confirmed as mobile. Here, we point to some areas of limited understanding concerning well established important TE groups with DDE Tpases, to address central gaps in our knowledge of characterised Tn with other types of Tpases and finally, to highlight new potentially mobile DNA species. It is not exhaustive. Examples have been chosen to provide encouragement in the continued exploration of the considerable prokaryotic mobilome especially in light of the current threat to public health posed by the spread of multiple Ab R . Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Prokaryotic communities differ along a geothermal soil photic gradient.

    Science.gov (United States)

    Meadow, James F; Zabinski, Catherine A

    2013-01-01

    Geothermal influenced soils exert unique physical and chemical limitations on resident microbial communities but have received little attention in microbial ecology research. These environments offer a model system in which to investigate microbial community heterogeneity and a range of soil ecological concepts. We conducted a 16S bar-coded pyrosequencing survey of the prokaryotic communities in a diatomaceous geothermal soil system and compared communities across soil types and along a conspicuous photic depth gradient. We found significant differences between the communities of the two different soils and also predictable differences between samples taken at different depths. Additionally, we targeted three ecologically relevant bacterial phyla, Cyanobacteria, Planctomycetes, and Verrucomicrobia, for clade-wise comparisons with these variables and found strong differences in their abundances, consistent with the autecology of these groups.

  5. Reconstruction of phylogenetic trees of prokaryotes using maximal common intervals.

    Science.gov (United States)

    Heydari, Mahdi; Marashi, Sayed-Amir; Tusserkani, Ruzbeh; Sadeghi, Mehdi

    2014-10-01

    One of the fundamental problems in bioinformatics is phylogenetic tree reconstruction, which can be used for classifying living organisms into different taxonomic clades. The classical approach to this problem is based on a marker such as 16S ribosomal RNA. Since evolutionary events like genomic rearrangements are not included in reconstructions of phylogenetic trees based on single genes, much effort has been made to find other characteristics for phylogenetic reconstruction in recent years. With the increasing availability of completely sequenced genomes, gene order can be considered as a new solution for this problem. In the present work, we applied maximal common intervals (MCIs) in two or more genomes to infer their distance and to reconstruct their evolutionary relationship. Additionally, measures based on uncommon segments (UCS's), i.e., those genomic segments which are not detected as part of any of the MCIs, are also used for phylogenetic tree reconstruction. We applied these two types of measures for reconstructing the phylogenetic tree of 63 prokaryotes with known COG (clusters of orthologous groups) families. Similarity between the MCI-based (resp. UCS-based) reconstructed phylogenetic trees and the phylogenetic tree obtained from NCBI taxonomy browser is as high as 93.1% (resp. 94.9%). We show that in the case of this diverse dataset of prokaryotes, tree reconstruction based on MCI and UCS outperforms most of the currently available methods based on gene orders, including breakpoint distance and DCJ. We additionally tested our new measures on a dataset of 13 closely-related bacteria from the genus Prochlorococcus. In this case, distances like rearrangement distance, breakpoint distance and DCJ proved to be useful, while our new measures are still appropriate for phylogenetic reconstruction. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  6. Phenylalanine Removal from Water by Fe3O4 Nanoparticles Functionalized with Two Different Surfactants

    Directory of Open Access Journals (Sweden)

    Ameneh Heidari

    2016-07-01

    Full Text Available In the present study, the application for the removal of phenylalanine by using two nano sorbents, namely, cetyltrimethylammonium bromide –Coated and BKC (benzal-conium chloride-Coated Fe3O4 nanoparticles was investigated. Solid-phase extraction (SPE and ultra violet–visible spectroscopy were used for studying the removal ability of each nano-sorbent in this study. Scanning Electron Microscopy, X-ray diffraction and Fourier infrared were used to characterize the synthesized magnetite nanoparticles. Batch adsorption studies were carried out to study the effect of various parameters, such as contact time, solution pH and concentration of phenylalanine. The equilibrium adsorption data of phenylalanine onto Fe3O4 nanoparticles (non-functionalized sample, cetyltrimethylammonium bromide -Coated and BKC -Coated were analyzed using Freundlich and Langmuir adsorption isotherms. The results indicated that adsorption of phenylalanine increased with increasing solution pH and maximum removal of phenylalanine was obtained at pH=9.0. Correlation coefficient were determined by analyzing each isotherm. It was found that the Freundlich equation showed better correlation with the experimental data than the Langmuir.

  7. A Survey of Aspartate Phenylalanine and Glutamate Phenylalanine Interactions in the Protein Data Bank: Searching for Anion Pairs

    Energy Technology Data Exchange (ETDEWEB)

    Philip, Vivek M [ORNL; Harris, Jason B [ORNL; Adams, Rachel M [ORNL; Nguyen, Don [University of Tennessee, Knoxville (UTK); Spires, Jeremy [University of Tennessee, Knoxville (UTK); Howell, Elizabeth E. [University of Tennessee, Knoxville (UTK); Hinde, Robert J [ORNL

    2011-01-01

    Protein structures are stabilized using noncovalent interactions. In addition to the traditional noncovalent interactions, newer types of interactions are thought to be present in proteins. One such interaction, an anion pair, in which the positively charged edge of an aromatic ring interacts with an anion, forming a favorable anion quadrupole interaction, has been previously proposed [Jackson, M. R., et al. (2007) J. Phys. Chem. B111, 8242 8249]. To study the role of anion interactions in stabilizing protein structure, we analyzed pairwise interactions between phenylalanine (Phe) and the anionic amino acids, aspartate (Asp) and glutamate (Glu). Particular emphasis was focused on identification of Phe Asp or Glu pairs separated by less than 7 in the high-resolution, nonredundant Protein Data Bank. Simplifying Phe to benzene and Asp or Glu to formate molecules facilitated in silico analysis of the pairs. Kitaura Morokuma energy calculations were performed on roughly 19000 benzene formate pairs and the resulting energies analyzed as a function of distance and angle. Edgewise interactions typically produced strongly stabilizing interaction energies (2 to 7.3 kcal/mol), while interactions involving the ring face resulted in weakly stabilizing to repulsive interaction energies. The strongest, most stabilizing interactions were identified as preferentially occurring in buried residues. Anion pairs are found throughout protein structures, in helices as well as strands. Numerous pairs also had nearby cation interactions as well as potential stacking. While more than 1000 structures did not contain an anion pair, the 3134 remaining structures contained approximately 2.6 anion pairs per protein, suggesting it is a reasonably common motif that could contribute to the overall structural stability of a protein.

  8. Biodegradation of Emiliania huxleyi Aggregates by natural Prokaryotic Communities under Increasing Hydrostatic Pressure.

    Science.gov (United States)

    Riou, V.; Para, J.; Garel, M.; Guigue, C.; Al Ali, B.; Santinelli, C.; Lefèvre, D.; Gattuso, J. P.; Goutx, M.; Panagiotopoulos, C.; Beaufort, L.; Jacquet, S.; Le Moigne, F. A. C.; Tachikawa, K.; Tamburini, C.

    2016-02-01

    Fluxes of particulate organic carbon (POC) and minerals are positively correlated, suggesting that minerals could enhance the flux of POC into the deep ocean. The so called "ballast effect" posits that minerals could increase sinking particle densities and/or protect the organic matter from heterotrophic degradation. Laboratory controlled experiments on coccolithophorid aggregates under atmospheric pressure show that biogenic calcite both increases particle settling velocities and preserves the organic matter. However, such experiments have yet to include genuine prokaryote rates indicators as well as the effect of increasing pressure. Here, we used the PArticle Sinking Simulator (PASS) to investigate the effect of the increasing pressure on the degradation of Emiliania huxleyi (calcifiers) aggregates. Extra care was taken to obtain culture aggregates with low prokaryotic abundance prior to exposure to natural mesopelagic prokaryotic communities. Particulate organic and inorganic carbon and dissolved organic carbon concentrations were monitored along with the lipid and carbohydrate compositions, as well as prokaryotic community abundance and specific diversity. A control experiment, without natural prokaryotic community addition, indicates that the pressure increase did not have any effect on calcite dissolution observed after ten days. In contrast, the addition of natural prokaryotic community accelerates calcite dissolution under conditions of increasing pressure. Prokaryotic community development and the lipid fraction of E. huxleyi particulate organic carbon are enhanced under increasing pressure. These results suggest that hydrostatic pressure denatures the structural integrity of the carbonate skeleton that protects the cellular organic matter.

  9. Effects of sodium azide on the abundance of prokaryotes and viruses in marine samples.

    Directory of Open Access Journals (Sweden)

    Christian Winter

    Full Text Available Flow cytometry is set to become the standard method for enumerating prokaryotes and viruses in marine samples. However, the samples need to be flash-frozen in liquid nitrogen directly after aldehyde fixation. Because liquid nitrogen may not always be available, we tested the potential of sodium azide as a preservative for prokaryotes and viruses in marine samples as a possible alternative. For that we conducted incubation experiments with untreated and sodium azide treated marine water samples at 4°C and room temperature. The data indicate that sodium azide cannot be used to maintain marine samples used for the enumeration of prokaryotes and viruses.

  10. Incorporation of exogenous L-phenylalanine into C-glycosylflavones in buckwheat cotyledons

    International Nuclear Information System (INIS)

    Margna, U.; Margna, E.

    1978-01-01

    By tracer experiments it was demonstrated that in excised buckwheat cotyledons L-phenylalanine fed exogeneously was incorporated predominantly into the luteolinic C-glycosylflavones orientin and iso-orientin, but not into their simpler apigeninic analogues vitexin and isovitexin, as it could be expected theoretically. Evidence is presented that L-phenylalanine supplied exogenously does not mix with the endogenous pool of that precursor, and that ratios of L-phenylalanine distribution between pathways of apigeninic and luteolinic C-glycosylflavones are different depending on wether the flavonoids are synthesized from endogenous or exogenous material. This phenomenon is suggested to be caused by the differences between enzyme complexes responsible for the biosynthesis of separate C-glycosylflavones in their capability of consuming common endogenous precursors. (author)

  11. The metabolism of C14-labeled phenylalanine and tyrosine in malaria-infected Culex-females

    International Nuclear Information System (INIS)

    Maier, W.A.; Nassif-Makki, H.

    1975-01-01

    Culex females are fed on C14-phenylalanine or C14-tyrosine in sugar solution. Autoradiographic studies on homogenated females 1 or 4 days after feeding, show that the labeled amino acids are metabolized on the first day and are not detectable on the fourth day. After increase of the amino acid concentration by saturation of the sugar solution with the unlabeled amino acid, the labeled acid and its metabolites are visible over a longer period of time. Phenylalanine is metabolized to tyrosine and at least four other substances. Radioactivity on the starting point of the chromatogram can be interpreted as incorporation of tyrosine into proteins. After infection with Plasmodium cathemerium, and feeding of C14-phenylalanine C14-tyrosine is demonstrable over a longer period. (orig.) [de

  12. Mapping the functional landscape of frequent phenylalanine hydroxylase (PAH) genotypes promotes personalised medicine in phenylketonuria.

    Science.gov (United States)

    Danecka, Marta K; Woidy, Mathias; Zschocke, Johannes; Feillet, François; Muntau, Ania C; Gersting, Søren W

    2015-03-01

    In phenylketonuria, genetic heterogeneity, frequent compound heterozygosity, and the lack of functional data for phenylalanine hydroxylase genotypes hamper reliable phenotype prediction and individualised treatment. A literature search revealed 690 different phenylalanine hydroxylase genotypes in 3066 phenylketonuria patients from Europe and the Middle East. We determined phenylalanine hydroxylase function of 30 frequent homozygous and compound heterozygous genotypes covering 55% of the study population, generated activity landscapes, and assessed the phenylalanine hydroxylase working range in the metabolic (phenylalanine) and therapeutic (tetrahydrobiopterin) space. Shared patterns in genotype-specific functional landscapes were linked to biochemical and pharmacological phenotypes, where (1) residual activity below 3.5% was associated with classical phenylketonuria unresponsive to pharmacological treatment; (2) lack of defined peak activity induced loss of response to tetrahydrobiopterin; (3) a higher cofactor need was linked to inconsistent clinical phenotypes and low rates of tetrahydrobiopterin response; and (4) residual activity above 5%, a defined peak of activity, and a normal cofactor need were associated with pharmacologically treatable mild phenotypes. In addition, we provide a web application for retrieving country-specific information on genotypes and genotype-specific phenylalanine hydroxylase function that warrants continuous extension, updates, and research on demand. The combination of genotype-specific functional analyses with biochemical, clinical, and therapeutic data of individual patients may serve as a powerful tool to enable phenotype prediction and to establish personalised medicine strategies for dietary regimens and pharmacological treatment in phenylketonuria. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  13. Nuclear magnetic resonance studies of phenylalanine analog interactions with normal and sicklen hemoglobin

    International Nuclear Information System (INIS)

    Lee, Y.H.

    1985-01-01

    Several phenylalanine derivatives have been found to inhibit the gelation of deoxygenated sickle hemoglobin (deoxy HbS). Proton and 19 F-NMR techniques were used to monitor the interaction of selected phenylalanine derivatives with the Hb molecule by using fluorine containing phenylalanine derivatives, Hb labeled at the β93 position with N-(2,2,2-trifluoroethyl) iodoacetamide (IA-F 3 ), and by monitoring the relaxation rates of the C2 and C4 histidine protons. The results show that the 19 F spin-spin relaxation times of L-phenylalanin-4-fluorobenzylamide (PheNBz1-F), which has a deoxy HbS antigelling activity comparable to that of the amino acid, tryptophan, are affected much more strongly by interaction with Hb than are those of glycin-4-fluorobenzylamide (GlyNBz1-F). In contrast, it is shown that N-(2,2,5,5-tetramethylpyrrolidin-1-oxy-3-carboxyl)-L-phenylalanine t-butyl ester (SL-Phe) exhibits specific binding to Hb, and an antigelling activity more than two orders of magnitude greater than that of phenylalanine. These results indicate that the fluorine nuclei strongly influenced by the presence of spin label nitroxide are located in a conformation within a few angstroms of the SL-Phe binding site. Proton NMR relaxation measurements of the C2 and C4 proton resonances from the β2, 4b143 and β146 histidine residues show significant and selective effects from the binding of SL-Phe to Hb, indicating that the SL-Phe binding site must be close to the side chains of these three residues. The strong antigelation activity of SL-Phe suggests that this binding site may be one of the intermolecular contact sites of importance to the deoxy HbS aggregation process

  14. Racemization of aspartic acid and phenylalanine in the sweetener aspartame at 100 degrees C.

    Science.gov (United States)

    Boehm, M F; Bada, J L

    1984-01-01

    The racemization half-lives (i.e., the time required to reach a D/L = 0.33) at pH 6.8 for aspartic acid and phenylalanine in the sweetener aspartame (L-aspartyl-L-phenylalanine methyl ester) were determined to be 13 and 23 hours, respectively, at 100 degrees C. Racemization at this pH does not occur in aspartame but rather in its diketopiperazine decomposition product. Our results indicate that the use of aspartame to sweeten neutral pH foods and beverages that are then heated at elevated temperature could generate D-aspartic acid and D-phenylalanine. The nutritive consequences of these D-amino acids in the human diet are not well established, and thus aspartame should probably not be used as a sweetener when the exposure of neutral pH foods and beverages to elevated temperatures is required. At pH 4, a typical pH of most foods and beverages that might be sweetened with aspartame, the half-lives are 47 hours for aspartic acid and 1200 hours for phenylalanine at 100 degrees C. Racemization at pH 4 takes place in aspartame itself. Although the racemization rates at pH 4 are slow and no appreciable racemization of aspartic acid and phenylalanine should occur during the normal use of aspartame, some food and beverage components could conceivably act as catalysts. Additional studies are required to evaluate whether the use of aspartame as a sugar substitute might not in turn result in an increased human consumption of D-aspartic acid and D-phenylalanine. PMID:6591191

  15. Fifteen-year follow-up of pulmonary function in individuals heterozygous for the cystic fibrosis phenylalanine-508 deletion

    DEFF Research Database (Denmark)

    Dahl, Morten; Nordestgaard, B G; Lange, P

    2001-01-01

    In a cross-sectional study, we previously showed that cystic fibrosis phenylalanine-508 deletion (DeltaF508) heterozygosity may be overrepresented among individuals with asthma.......In a cross-sectional study, we previously showed that cystic fibrosis phenylalanine-508 deletion (DeltaF508) heterozygosity may be overrepresented among individuals with asthma....

  16. Phenylalanine tolerance can already reliably be assessed at the age of 2 years in patients with PKU

    NARCIS (Netherlands)

    Spronsen, F.J. van; Rijn, M.; Dorgelo, B.; Hoeksma, M.; Bosch, A.M.; Mulder, M.F.; Klerk, J.B.C. de; Koning, T. de; Rubio-Gozalbo, M.E.; Vries, M. de; Verkerk, P.H.

    2009-01-01

    Background: The clinical severity of phenylalanine hydroxylase deficiency is usually defined by either pre-treatment phenylalanine (Phe) concentration or Phe tolerance at 5 years of age. So far, little is known about the course of Phe tolerance or the ability of both pre-treatment Phe and Phe

  17. Phenylalanine tolerance can already reliably be assessed at the age of 2 years in patients with PKU

    NARCIS (Netherlands)

    van Spronsen, F. J.; van Rijn, M.; Dorgelo, B.; Hoeksma, M.; Bosch, A. M.; Mulder, M. F.; de Klerk, J. B. C.; de Koning, T.; Rubio-Gozalbo, M. Estela; de Vries, M.; Verkerk, P. H.

    The clinical severity of phenylalanine hydroxylase deficiency is usually defined by either pre-treatment phenylalanine (Phe) concentration or Phe tolerance at 5 years of age. So far, little is known about the course of Phe tolerance or the ability of both pre-treatment Phe and Phe tolerance at early

  18. Phenylalanine tolerance can already reliably be assessed at the age of 2 years in patients with PKU

    NARCIS (Netherlands)

    van Spronsen, F. J.; van Rijn, M. [=Margreet; Dorgelo, B.; Hoeksma, M.; Bosch, A. M.; Mulder, M. F.; de Klerk, J. B. C.; de Koning, T.; Rubio-Gozalbo, M. Estela; de Vries, M.; Verkerk, P. H.

    2009-01-01

    The clinical severity of phenylalanine hydroxylase deficiency is usually defined by either pre-treatment phenylalanine (Phe) concentration or Phe tolerance at 5 years of age. So far, little is known about the course of Phe tolerance or the ability of both pre-treatment Phe and Phe tolerance at early

  19. Chiral separation of substituted phenylalanine analogues using chiral palladium phosphine complexes with enantioselective liquid-liquid extraction

    NARCIS (Netherlands)

    Verkuijl, B.J.V.; Schuur, B.; Minnaard, A.J.; Vries, de J.G.; Feringa, B.L.

    2010-01-01

    Chiral palladium phosphine complexes have been employed in the chiral separation of amino acids and phenylalanine analogues in particular. The use of (S)-xylyl-BINAP as a ligand for the palladium complex in enantioselective liquid–liquid extraction allowed the separation of the phenylalanine

  20. What Is the Best Blood Sampling Time for Metabolic Control of Phenylalanine and Tyrosine Concentrations in Tyrosinemia Type 1 Patients?

    NARCIS (Netherlands)

    van Dam, Esther; Daly, Anne; Venema-Liefaard, Gineke; van Rijn, Margreet; Derks, Terry G J; McKiernan, Patrick J; Heiner-Fokkema, Rebecca; MacDonald, Anita; van Spronsen, Francjan J

    2017-01-01

    BACKGROUND: Treatment of hereditary tyrosinemia type 1 with nitisinone and phenylalanine and tyrosine restricted diet has largely improved outcome, but the best blood sampling time for assessment of metabolic control is not known. AIM: To study diurnal and day-to-day variation of phenylalanine and

  1. Effect of nitrate addition on prokaryotic diversity and the activity of sulfate-reducing prokaryotes in high-temperature oil production systems

    DEFF Research Database (Denmark)

    Gittel, Antje; Wieczorek, Adam; Sørensen, Ketil

    Adding nitrate to injection water is a possible strategy to control the activity of sulfate-reducing prokaryotes (SRP) in oil production system. To assess the effects of nitrate addition, prokaryotic diversity (Bacteria, Archaea, SRP) and SRP activity were studied in the production waters......-treated site was additionally supported by demonstrating their potential activity at 58°C, indicating that the troublesome SRP were pipeline-derived. Consistent with the low frequency of SRP in the clone libraries, no activity could be shown for samples from the nitrate-treated system suggesting that SRP were...... inhibited by nitrate addition. Visualization and quantification of the identified troublesome prokaryotes and potential competitors using the CARD-FISH technique will be performed on production water from both sites....

  2. Emerging experimental and computational technologies for purpose designed engineering of photosynthetic prokaryotes

    KAUST Repository

    Lindblad, Peter

    2016-01-01

    With recent advances in synthetic molecular tools to be used in photosynthetic prokaryotes, like cyanobacteria, it is possible to custom design and construct microbial cells for specific metabolic functions. This cross-disciplinary area of research

  3. Connectivity between surface and deep waters determines prokaryotic diversity in the North Atlantic Deep Water.

    Science.gov (United States)

    Frank, Alexander H; Garcia, Juan A L; Herndl, Gerhard J; Reinthaler, Thomas

    2016-06-01

    To decipher the influence of depth stratification and surface provincialism on the dark ocean prokaryotic community composition, we sampled the major deep-water masses in the eastern North Atlantic covering three biogeographic provinces. Their diversity was evaluated using ordination and canonical analysis of 454 pyrotag sequences. Variance partitioning suggested that 16% of the variation in the bacterial community composition was based on depth stratification while 9% of the variation was due to geographic location. General linear mixed effect models showed that the community of the subsurface waters was connected to the dark ocean prokaryotic communities in different biogeographic provinces. Cluster analysis indicated that some prokaryotic taxa are specific to distinct regions in bathypelagic water masses. Taken together, our data suggest that the dark ocean prokaryotic community composition of the eastern North Atlantic is primed by the formation and the horizontal transport of water masses. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

  4. Microscopic Identification of Prokaryotes in Modern and Ancient Halite, Saline Valley and Death Valley, California

    Science.gov (United States)

    Schubert, Brian A.; Lowenstein, Tim K.; Timofeeff, Michael N.

    2009-06-01

    Primary fluid inclusions in halite crystallized in Saline Valley, California, in 1980, 2004-2005, and 2007, contain rod- and coccoid-shaped microparticles the same size and morphology as archaea and bacteria living in modern brines. Primary fluid inclusions from a well-dated (0-100,000 years), 90 m long salt core from Badwater Basin, Death Valley, California, also contain microparticles, here interpreted as halophilic and halotolerant prokaryotes. Prokaryotes are distinguished from crystals on the basis of morphology, optical properties (birefringence), and uniformity of size. Electron micrographs of microparticles from filtered modern brine (Saline Valley), dissolved modern halite crystals (Saline Valley), and dissolved ancient halite crystals (Death Valley) support in situ microscopic observations that prokaryotes are present in fluid inclusions in ancient halite. In the Death Valley salt core, prokaryotes in fluid inclusions occur almost exclusively in halite precipitated in perennial saline lakes 10,000 to 35,000 years ago. This suggests that trapping and preservation of prokaryotes in fluid inclusions is influenced by the surface environment in which the halite originally precipitated. In all cases, prokaryotes in fluid inclusions in halite from the Death Valley salt core are miniaturized (<1 μm diameter cocci, <2.5 μm long, very rare rod shapes), which supports interpretations that the prokaryotes are indigenous to the halite and starvation survival may be the normal response of some prokaryotes to entrapment in fluid inclusions for millennia. These results reinforce the view that fluid inclusions in halite and possibly other evaporites are important repositories of microbial life and should be carefully examined in the search for ancient microorganisms on Earth, Mars, and elsewhere in the Solar System.

  5. Modeling the winter-to-summer transition of prokaryotic and viral abundance in the Arctic Ocean.

    Science.gov (United States)

    Winter, Christian; Payet, Jérôme P; Suttle, Curtis A

    2012-01-01

    One of the challenges in oceanography is to understand the influence of environmental factors on the abundances of prokaryotes and viruses. Generally, conventional statistical methods resolve trends well, but more complex relationships are difficult to explore. In such cases, Artificial Neural Networks (ANNs) offer an alternative way for data analysis. Here, we developed ANN-based models of prokaryotic and viral abundances in the Arctic Ocean. The models were used to identify the best predictors for prokaryotic and viral abundances including cytometrically-distinguishable populations of prokaryotes (high and low nucleic acid cells) and viruses (high- and low-fluorescent viruses) among salinity, temperature, depth, day length, and the concentration of Chlorophyll-a. The best performing ANNs to model the abundances of high and low nucleic acid cells used temperature and Chl-a as input parameters, while the abundances of high- and low-fluorescent viruses used depth, Chl-a, and day length as input parameters. Decreasing viral abundance with increasing depth and decreasing system productivity was captured well by the ANNs. Despite identifying the same predictors for the two populations of prokaryotes and viruses, respectively, the structure of the best performing ANNs differed between high and low nucleic acid cells and between high- and low-fluorescent viruses. Also, the two prokaryotic and viral groups responded differently to changes in the predictor parameters; hence, the cytometric distinction between these populations is ecologically relevant. The models imply that temperature is the main factor explaining most of the variation in the abundances of high nucleic acid cells and total prokaryotes and that the mechanisms governing the reaction to changes in the environment are distinctly different among the prokaryotic and viral populations.

  6. Modeling the Winter–to–Summer Transition of Prokaryotic and Viral Abundance in the Arctic Ocean

    Science.gov (United States)

    Winter, Christian; Payet, Jérôme P.; Suttle, Curtis A.

    2012-01-01

    One of the challenges in oceanography is to understand the influence of environmental factors on the abundances of prokaryotes and viruses. Generally, conventional statistical methods resolve trends well, but more complex relationships are difficult to explore. In such cases, Artificial Neural Networks (ANNs) offer an alternative way for data analysis. Here, we developed ANN-based models of prokaryotic and viral abundances in the Arctic Ocean. The models were used to identify the best predictors for prokaryotic and viral abundances including cytometrically-distinguishable populations of prokaryotes (high and low nucleic acid cells) and viruses (high- and low-fluorescent viruses) among salinity, temperature, depth, day length, and the concentration of Chlorophyll-a. The best performing ANNs to model the abundances of high and low nucleic acid cells used temperature and Chl-a as input parameters, while the abundances of high- and low-fluorescent viruses used depth, Chl-a, and day length as input parameters. Decreasing viral abundance with increasing depth and decreasing system productivity was captured well by the ANNs. Despite identifying the same predictors for the two populations of prokaryotes and viruses, respectively, the structure of the best performing ANNs differed between high and low nucleic acid cells and between high- and low-fluorescent viruses. Also, the two prokaryotic and viral groups responded differently to changes in the predictor parameters; hence, the cytometric distinction between these populations is ecologically relevant. The models imply that temperature is the main factor explaining most of the variation in the abundances of high nucleic acid cells and total prokaryotes and that the mechanisms governing the reaction to changes in the environment are distinctly different among the prokaryotic and viral populations. PMID:23285186

  7. Viral infections stimulate the metabolism and shape prokaryotic assemblages in submarine mud volcanoes.

    Science.gov (United States)

    Corinaldesi, Cinzia; Dell'Anno, Antonio; Danovaro, Roberto

    2012-06-01

    Mud volcanoes are geological structures in the oceans that have key roles in the functioning of the global ecosystem. Information on the dynamics of benthic viruses and their interactions with prokaryotes in mud volcano ecosystems is still completely lacking. We investigated the impact of viral infection on the mortality and assemblage structure of benthic prokaryotes of five mud volcanoes in the Mediterranean Sea. Mud volcano sediments promote high rates of viral production (1.65-7.89 × 10(9) viruses g(-1) d(-1)), viral-induced prokaryotic mortality (VIPM) (33% cells killed per day) and heterotrophic prokaryotic production (3.0-8.3 μgC g(-1) d(-1)) when compared with sediments outside the mud volcano area. The viral shunt (that is, the microbial biomass converted into dissolved organic matter as a result of viral infection, and thus diverted away from higher trophic levels) provides 49 mgC m(-2) d(-1), thus fuelling the metabolism of uninfected prokaryotes and contributing to the total C budget. Bacteria are the dominant components of prokaryotic assemblages in surface sediments of mud volcanoes, whereas archaea dominate the subsurface sediment layers. Multivariate multiple regression analyses show that prokaryotic assemblage composition is not only dependant on the geochemical features and processes of mud volcano ecosystems but also on synergistic interactions between bottom-up (that is, trophic resources) and top-down (that is, VIPM) controlling factors. Overall, these findings highlight the significant role of the viral shunt in sustaining the metabolism of prokaryotes and shaping their assemblage structure in mud volcano sediments, and they provide new clues for our understanding of the functioning of cold-seep ecosystems.

  8. NMR comparison of prokaryotic and eukaryotic cytochromes c

    International Nuclear Information System (INIS)

    Chau, Meihing; Cai, Meng Li; Timkovich, R.

    1990-01-01

    1 H NMR spectroscopy has been used to examine ferrocytochrome c-551 from Pseudomonas aeruginosa (ATCC 19429) over the pH range 3.5-10.6 and the temperature range 4-60 degree C. Resonance assignments are proposed for main-chain and side-chain protons. Comparison of results for cytochrome c-551 to recently assigned spectra for horse cytochrome c and mutants of yeast iso-1 cytochrome reveals some unique resonances with unusual chemical shifts in all cytochromes that may serve as markers for the heme region. Results for cytochrome c-551 indicate that in the smaller prokaryotic cytochrome, all benzoid side chains are rapidly flipping on the NMR time scale. In contrast, in eukaryotic cytochromes there are some rings flipping slowly on the NMR time scale. The ferrocytochrome c-551 undergoes a transition linked to pH with a pK around 7. The pH behavior of assigned resonances provides evidence that the site of protonation is the inner or buried 17-propionic acid heme substituent (IUPAC-IUB porphyrin nomenclature). Conformational heterogeneity has been observed for segments near the inner heme propionate substituent

  9. Structural similarities between prokaryotic and eukaryotic 5S ribosomal RNAs

    International Nuclear Information System (INIS)

    Welfle, H.; Boehm, S.; Damaschun, G.; Fabian, H.; Gast, K.; Misselwitz, R.; Mueller, J.J.; Zirwer, D.; Filimonov, V.V.; Venyaminov, S.Yu.; Zalkova, T.N.

    1986-01-01

    5S RNAs from rat liver and E. coli have been studied by diffuse X-ray and dynamic light scattering and by infrared and Raman spectroscopy. Identical structures at a resolution of 1 nm can be deduced from the comparison of the experimental X-ray scattering curves and electron distance distribution functions and from the agreement of the shape parameters. A flat shape model with a compact central region and two protruding arms was derived. Double helical stems are eleven-fold helices with a mean base pair distance of 0.28 nm. The number of base pairs (26 GC, 9 AU for E. coli; 27 GC, 9 AU for rat liver) and the degree of base stacking are the same within the experimental error. A very high regularity in the ribophosphate backbone is indicated for both 5S RNAs. The observed structural similarity and the consensus secondary structure pattern derived from comparative sequence analyses suggest the conclusion that prokaryotic and eukaryotic 5S RNAs are in general very similar with respect to their fundamental structural features. (author)

  10. Current Developments in Prokaryotic Single Cell Whole Genome Amplification

    Energy Technology Data Exchange (ETDEWEB)

    Goudeau, Danielle; Nath, Nandita; Ciobanu, Doina; Cheng, Jan-Fang; Malmstrom, Rex

    2014-03-14

    Our approach to prokaryotic single-cell Whole Genome Amplification at the JGI continues to evolve. To increase both the quality and number of single-cell genomes produced, we explore all aspects of the process from cell sorting to sequencing. For example, we now utilize specialized reagents, acoustic liquid handling, and reduced reaction volumes eliminate non-target DNA contamination in WGA reactions. More specifically, we use a cleaner commercial WGA kit from Qiagen that employs a UV decontamination procedure initially developed at the JGI, and we use the Labcyte Echo for tip-less liquid transfer to set up 2uL reactions. Acoustic liquid handling also dramatically reduces reagent costs. In addition, we are exploring new cell lysis methods including treatment with Proteinase K, lysozyme, and other detergents, in order to complement standard alkaline lysis and allow for more efficient disruption of a wider range of cells. Incomplete lysis represents a major hurdle for WGA on some environmental samples, especially rhizosphere, peatland, and other soils. Finding effective lysis strategies that are also compatible with WGA is challenging, and we are currently assessing the impact of various strategies on genome recovery.

  11. Prokaryotic Heme Biosynthesis: Multiple Pathways to a Common Essential Product.

    Science.gov (United States)

    Dailey, Harry A; Dailey, Tamara A; Gerdes, Svetlana; Jahn, Dieter; Jahn, Martina; O'Brian, Mark R; Warren, Martin J

    2017-03-01

    The advent of heme during evolution allowed organisms possessing this compound to safely and efficiently carry out a variety of chemical reactions that otherwise were difficult or impossible. While it was long assumed that a single heme biosynthetic pathway existed in nature, over the past decade, it has become clear that there are three distinct pathways among prokaryotes, although all three pathways utilize a common initial core of three enzymes to produce the intermediate uroporphyrinogen III. The most ancient pathway and the only one found in the Archaea converts siroheme to protoheme via an oxygen-independent four-enzyme-step process. Bacteria utilize the initial core pathway but then add one additional common step to produce coproporphyrinogen III. Following this step, Gram-positive organisms oxidize coproporphyrinogen III to coproporphyrin III, insert iron to make coproheme, and finally decarboxylate coproheme to protoheme, whereas Gram-negative bacteria first decarboxylate coproporphyrinogen III to protoporphyrinogen IX and then oxidize this to protoporphyrin IX prior to metal insertion to make protoheme. In order to adapt to oxygen-deficient conditions, two steps in the bacterial pathways have multiple forms to accommodate oxidative reactions in an anaerobic environment. The regulation of these pathways reflects the diversity of bacterial metabolism. This diversity, along with the late recognition that three pathways exist, has significantly slowed advances in this field such that no single organism's heme synthesis pathway regulation is currently completely characterized. Copyright © 2017 American Society for Microbiology.

  12. Prokaryotic phylogenetic diversity of Hungarian deep subsurface geothermal well waters.

    Science.gov (United States)

    Németh, Andrea; Szirányi, Barbara; Krett, Gergely; Janurik, Endre; Kosáros, Tünde; Pekár, Ferenc; Márialigeti, Károly; Borsodi, Andrea K

    2014-09-01

    Geothermal wells characterized by thermal waters warmer than 30°C can be found in more than 65% of the area of Hungary. The examined thermal wells located nearby Szarvas are used for heating industrial and agricultural facilities because of their relatively high hydrocarbon content. The aim of this study was to reveal the prokaryotic community structure of the water of SZR18, K87 and SZR21 geothermal wells using molecular cloning methods and Denaturing Gradient Gel Electrophoresis (DGGE). Water samples from the outflow pipes were collected in 2012 and 2013. The phylogenetic distribution of archaeal molecular clones was very similar in each sample, the most abundant groups belonged to the genera Methanosaeta, Methanothermobacter and Thermofilum. In contrast, the distribution of bacterial molecular clones was very diverse. Many of them showed the closest sequence similarities to uncultured clone sequences from similar thermal environments. From the water of the SZR18 well, phylotypes closely related to genera Fictibacillus and Alicyclobacillus (Firmicutes) were only revealed, while the bacterial diversity of the K87 well water was much higher. Here, the members of the phyla Thermodesulfobacteria, Proteobacteria, Nitrospira, Chlorobi, OP1 and OPB7 were also detected besides Firmicutes.

  13. Light-controlled motility in prokaryotes and the problem of directional light perception.

    Science.gov (United States)

    Wilde, Annegret; Mullineaux, Conrad W

    2017-11-01

    The natural light environment is important to many prokaryotes. Most obviously, phototrophic prokaryotes need to acclimate their photosynthetic apparatus to the prevailing light conditions, and such acclimation is frequently complemented by motility to enable cells to relocate in search of more favorable illumination conditions. Non-phototrophic prokaryotes may also seek to avoid light at damaging intensities and wavelengths, and many prokaryotes with diverse lifestyles could potentially exploit light signals as a rich source of information about their surroundings and a cue for acclimation and behavior. Here we discuss our current understanding of the ways in which bacteria can perceive the intensity, wavelength and direction of illumination, and the signal transduction networks that link light perception to the control of motile behavior. We discuss the problems of light perception at the prokaryotic scale, and the challenge of directional light perception in small bacterial cells. We explain the peculiarities and the common features of light-controlled motility systems in prokaryotes as diverse as cyanobacteria, purple photosynthetic bacteria, chemoheterotrophic bacteria and haloarchaea. © FEMS 2017.

  14. Serum phenylalanine in preterm newborns fed different diets of human milk

    Directory of Open Access Journals (Sweden)

    Débora M. Thomaz

    2014-09-01

    Conclusion: The observed results demonstrated that human milk with fortifiers derived from human milk acted as a good substratum for preterm infant feeding both in the evaporated or the lyophilized form, without significant increases in plasma phenylalanine levels in comparison to human milk with commercial fortifier.

  15. Doxycycline hinders phenylalanine fibril assemblies revealing a potential novel therapeutic approach in phenylketonuria.

    Science.gov (United States)

    De Luigi, Ada; Mariani, Alessandro; De Paola, Massimiliano; Re Depaolini, Andrea; Colombo, Laura; Russo, Luca; Rondelli, Valeria; Brocca, Paola; Adler-Abramovich, Lihi; Gazit, Ehud; Del Favero, Elena; Cantù, Laura; Salmona, Mario

    2015-10-29

    A new paradigm for the aetiopathology of phenylketonuria suggests the presence of amyloid-like assemblies in the brains of transgenic mouse models and patients with phenylketonuria, possibly shedding light on the selective cognitive deficit associated with this disease. Paralleling the amyloidogenic route that identifies different stages of peptide aggregation, corresponding to different levels of toxicity, we experimentally address for the first time, the physico-chemical properties of phenylalanine aggregates via Small Angle, Wide Angle X-ray Scattering and Atomic Force Microscopy. Results are consistent with the presence of well-structured, aligned fibres generated by milliMolar concentrations of phenylalanine. Moreover, the amyloid-modulating doxycycline agent affects the local structure of phenylalanine aggregates, preventing the formation of well-ordered crystalline structures. Phenylalanine assemblies prove toxic in vitro to immortalized cell lines and primary neuronal cells. Furthermore, these assemblies also cause dendritic sprouting alterations and synaptic protein impairment in neurons. Doxycycline counteracts these toxic effects, suggesting an approach for the development of future innovative non-dietary preventive therapies.

  16. Formation of tyrosine isomers in aqueous phenylalanine solutions by gamma irradiation

    International Nuclear Information System (INIS)

    Aflaki, F.; Salahinejad, M.; Roozbehani, A.

    2009-01-01

    Ortho-tyrosine detection method can be used for detection of irradiated protein rich foods. Tyrosine isomers produced by gamma radiation of aqueous phenylalanine solutions at wide dose levels (0.1-50 k Gy) were examined to obtain basic information for o-tyrosine detection method of irradiated foods. Determination of tyrosines produced in aqueous phenylalanine solutions were carried out by high performance liquid chromatography and fluorescence detection. The detection limit of o-tyrosine was 0.01 ppm and the linear range of calibration and the relative standard deviation of analysis was 50 ng and 4-13%, respectively. The amounts of the tyrosines increased with the irradiation level up to 10 k Gy and no further tyrosine formation was observed when the dose level was increased. At a constant dose level, the yield of tyrosines initially increased with the phenylalanine concentration, while with further increase of phenylalanine concentration no effect on increase of tyrosine yield was observed. When the dose rate was varying from 2.3 k Gy/h to 1.2 k Gy/h with a total amount of 10 k Gy in each case, there was no significant effect on tyrosine isomers formation was observed. Also the results showed that tyrosine yield was affected by temperature, p H and the presence of oxygen

  17. Mid-IR spectra of different conformers of phenylalanine in the gas phase

    NARCIS (Netherlands)

    von Helden, G.; Compagnon, I.; Blom, M. N.; Frankowski, M.; Erlekam, U.; Oomens, J.; Brauer, B.; Gerber, R. B.; Meijer, G.

    2008-01-01

    The experimental mid- and far-IR spectra of six conformers of phenylalanine in the gas phase are presented. The experimental spectra are compared to spectra calculated at the B3LYP and at the MP2 level. The differences between B3LYP and MP2 IR spectra are found to be small. The agreement between

  18. Cationized phenylalanine conformations characterized by IRMPD and computation for singly and doubly charged ions

    NARCIS (Netherlands)

    Dunbar, R.C.; Steill, J.D.; Oomens, J.

    2010-01-01

    Electrospray ionization produces phenylalanine (Phe) complexes of the alkali metal ion series, plus Ag+ and Ba2+. Infrared multiple photon dissociation (IRMPD) spectroscopy using the FELIX free electron laser light source is used to characterize the conformations of the ions, in conjunction with

  19. Resolving Phenylalanine Metabolism Sheds Light on Natural Synthesis of Penicillin G in Penicillium chrysogenum

    NARCIS (Netherlands)

    Veiga, T.; Solis-Escalante, D.; Romagnoli, G.; Ten Pierick, A.; Hanemaaijer, M.; Deshmuhk, A.; Wahl, A.; Pronk, J.T.; Daran, J.M.

    2011-01-01

    The industrial production of penicillin G by Penicillium chrysogenum requires the supplementation of the growth medium with the side chain precursor phenylacetate. The growth of P. chrysogenum with phenylalanine as the sole nitrogen source resulted in the extracellular production of phenylacetate

  20. 1 Synthesis of Selected Phenylalanine Esters C1 to C4 and their ...

    African Journals Online (AJOL)

    Figure 1 Synthesis of Taxol via the Wieland Miescher Ketone. Important natural products .... ml) at 0°C. Thionyl chloride (1ml, 13.8 mmol) was added dropwise to the reaction mixture ... L-Phenylalanine propyl ester hydrochloride. (Yield: 93%).

  1. Synthesis of bradykinin labelled with tritium on the phenylalanine residue in position 5

    International Nuclear Information System (INIS)

    Seproedi, J.; Teplan, I.; Medzihradszky, K.

    1976-01-01

    The polypeptide hormone bradykinine, labelled with tritium in the phenylalanine chain in position 5, has been prepared. The preparation method is described; the tritium atom is incorporated in the last step of the synthesis. The hormone can therefore be labelled immediately before use, which avoids autoradiolysis during storage

  2. Blood phenylalanine control in phenylketonuria : a survey of 10 European centres

    NARCIS (Netherlands)

    Ahring, K.; Belanger-Quintana, A.; Dokoupil, K.; Gokmen-Ozel, H.; Lammardo, A. M.; MacDonald, A.; Motzfeldt, K.; Nowacka, M.; van Rijn, M.; Robert, M.

    Background: Only limited data are available on the blood phenylalanine (Phe) concentrations achieved in European patients with phenylketonuria (PKU) on a low-Phe diet. Objective: A survey was conducted to compare blood Phe control achieved in diet-treated patients with PKU of different age groups in

  3. Study of interaction between tryptophan, tyrosine, and phenylalanine separately with silver nanoparticles by fluorescence quenching method

    International Nuclear Information System (INIS)

    Roy, S.; Das, T.K.

    2015-01-01

    Using the spectroscopic method, the individual interaction of the three biochemically important amino acids, which are constituents of protein, namely, tryptophan, tyrosine, and phenylalanine with biologically synthesized silver nanoparticles has been investigated. The obtained UV-Vis spectra show the formation of ground-state complexes between tryptophan, tyrosine, and phenylalanine with silver nanoparticles. Silver nanoparticles possess the ability to quench the intrinsic fluorescence of the aforesaid amino acids by a dynamic quenching process. The binding constant, number of binding sites, and corresponding thermodynamic parameters (ΔH, ΔS, and ΔG) based on the interaction system were calculated for 293, 303, and 313 K. In the case of tryptophan and phenylalanine, with increase in temperature, the binding constant K was found to decrease; conversely, it was found to increase with increase in temperature in the case of tyrosine. The thermodynamic results revealed that the binding process was spontaneous; hydrogen bonding and van der Waals interaction were the predominant forces responsible for the complex stabilization in the case of tryptophan and phenylalanine, respectively, whereas in the case of tyrosine, hydrophobic interaction was the sole force conferring stability. Moreover, the Förster non-radiation energy transfer theory has been applied to calculate the average binding distance among the above amino acids and silver nanoparticles. The results show a binding distance of <7 nm, which ensures that energy transfer does occur between the said amino acids and silver nanoparticles. (authors)

  4. ATGC: a database of orthologous genes from closely related prokaryotic genomes and a research platform for microevolution of prokaryotes

    Energy Technology Data Exchange (ETDEWEB)

    Novichkov, Pavel S.; Ratnere, Igor; Wolf, Yuri I.; Koonin, Eugene V.; Dubchak, Inna

    2009-07-23

    The database of Alignable Tight Genomic Clusters (ATGCs) consists of closely related genomes of archaea and bacteria, and is a resource for research into prokaryotic microevolution. Construction of a data set with appropriate characteristics is a major hurdle for this type of studies. With the current rate of genome sequencing, it is difficult to follow the progress of the field and to determine which of the available genome sets meet the requirements of a given research project, in particular, with respect to the minimum and maximum levels of similarity between the included genomes. Additionally, extraction of specific content, such as genomic alignments or families of orthologs, from a selected set of genomes is a complicated and time-consuming process. The database addresses these problems by providing an intuitive and efficient web interface to browse precomputed ATGCs, select appropriate ones and access ATGC-derived data such as multiple alignments of orthologous proteins, matrices of pairwise intergenomic distances based on genome-wide analysis of synonymous and nonsynonymous substitution rates and others. The ATGC database will be regularly updated following new releases of the NCBI RefSeq. The database is hosted by the Genomics Division at Lawrence Berkeley National laboratory and is publicly available at http://atgc.lbl.gov.

  5. Oxidation of inorganic sulfur compounds in acidophilic prokaryotes

    Energy Technology Data Exchange (ETDEWEB)

    Rohwerder, T.; Sand, W. [Universitaet Duisburg-Essen, Biofilm Centre, Aquatic Biotechnology, Duisburg (Germany)

    2007-07-15

    The oxidation of reduced inorganic sulfur compounds to sulfuric acid is of great importance for biohydrometallurgical technologies as well as the formation of acidic (below pH 3) and often heavy metal-contaminated environments. The use of elemental sulfur as an electron donor is the predominant energy-yielding process in acidic natural sulfur-rich biotopes but also at mining sites containing sulfidic ores. Contrary to its significant role in the global sulfur cycle and its biotechnological importance, the microbial fundamentals of acidophilic sulfur oxidation are only incompletely understood. Besides giving an overview of sulfur-oxidizing acidophiles, this review describes the so far known enzymatic reactions related to elemental sulfur oxidation in acidophilic bacteria and archaea. Although generally similar reactions are employed in both prokaryotic groups, the stoichiometry of the key enzymes is different. Bacteria oxidize elemental sulfur by a sulfur dioxygenase to sulfite whereas in archaea, a sulfur oxygenase reductase is used forming equal amounts of sulfide and sulfite. In both cases, the activation mechanism of elemental sulfur is not known but highly reactive linear sulfur forms are assumed to be the actual substrate. Inhibition as well as promotion of these biochemical steps is highly relevant in bioleaching operations. An efficient oxidation can prevent the formation of passivating sulfur layers. In other cases, a specific inhibition of sulfur biooxidation may be beneficial for reducing cooling and neutralization costs. In conclusion, the demand for a better knowledge of the biochemistry of sulfur-oxidizing acidophiles is underlined. (Abstract Copyright [2007], Wiley Periodicals, Inc.)

  6. Metabolic profiles of prokaryotic and eukaryotic communities in deep-sea sponge Neamphius huxleyi indicated by metagenomics

    Science.gov (United States)

    Li, Zhi-Yong; Wang, Yue-Zhu; He, Li-Ming; Zheng, Hua-Jun

    2014-01-01

    The whole metabolism of a sponge holobiont and the respective contributions of prokaryotic and eukaryotic symbionts and their associations with the sponge host remain largely unclear. Meanwhile, compared with shallow water sponges, deep-sea sponges are rarely understood. Here we report the metagenomic exploration of deep-sea sponge Neamphius huxleyi at the whole community level. Metagenomic data showed phylogenetically diverse prokaryotes and eukaryotes in Neamphius huxleyi. MEGAN and gene enrichment analyses indicated different metabolic potentials of prokaryotic symbionts from eukaryotic symbionts, especially in nitrogen and carbon metabolisms, and their molecular interactions with the sponge host. These results supported the hypothesis that prokaryotic and eukaryotic symbionts have different ecological roles and relationships with sponge host. Moreover, vigorous denitrification, and CO2 fixation by chemoautotrophic prokaryotes were suggested for this deep-sea sponge. The study provided novel insights into the respective potentials of prokaryotic and eukaryotic symbionts and their associations with deep-sea sponge Neamphius huxleyi. PMID:24463735

  7. Metabolic profiles of prokaryotic and eukaryotic communities in deep-sea sponge Neamphius huxleyi [corrected]. indicated by metagenomics.

    Science.gov (United States)

    Li, Zhi-Yong; Wang, Yue-Zhu; He, Li-Ming; Zheng, Hua-Jun

    2014-01-27

    The whole metabolism of a sponge holobiont and the respective contributions of prokaryotic and eukaryotic symbionts and their associations with the sponge host remain largely unclear. Meanwhile, compared with shallow water sponges, deep-sea sponges are rarely understood. Here we report the metagenomic exploration of deep-sea sponge Neamphius huxleyi [corrected] . at the whole community level. Metagenomic data showed phylogenetically diverse prokaryotes and eukaryotes in Neamphius huxleyi [corrected]. MEGAN and gene enrichment analyses indicated different metabolic potentials of prokaryotic symbionts from eukaryotic symbionts, especially in nitrogen and carbon metabolisms, and their molecular interactions with the sponge host. These results supported the hypothesis that prokaryotic and eukaryotic symbionts have different ecological roles and relationships with sponge host. Moreover, vigorous denitrification, and CO2 fixation by chemoautotrophic prokaryotes were suggested for this deep-sea sponge. The study provided novel insights into the respective potentials of prokaryotic and eukaryotic symbionts and their associations with deep-sea sponge Neamphius huxleyi [corrected].

  8. Metabolic profiles of prokaryotic and eukaryotic communities in deep-sea sponge Lamellomorpha sp. indicated by metagenomics

    Science.gov (United States)

    Li, Zhi-Yong; Wang, Yue-Zhu; He, Li-Ming; Zheng, Hua-Jun

    2014-01-01

    The whole metabolism of a sponge holobiont and the respective contributions of prokaryotic and eukaryotic symbionts and their associations with the sponge host remain largely unclear. Meanwhile, compared with shallow water sponges, deep-sea sponges are rarely understood. Here we report the metagenomic exploration of deep-sea sponge Lamellomorpha sp. at the whole community level. Metagenomic data showed phylogenetically diverse prokaryotes and eukaryotes in Lamellomorpha sp.. MEGAN and gene enrichment analyses indicated different metabolic potentials of prokaryotic symbionts from eukaryotic symbionts, especially in nitrogen and carbon metabolisms, and their molecular interactions with the sponge host. These results supported the hypothesis that prokaryotic and eukaryotic symbionts have different ecological roles and relationships with sponge host. Moreover, vigorous denitrification, and CO2 fixation by chemoautotrophic prokaryotes were suggested for this deep-sea sponge. The study provided novel insights into the respective potentials of prokaryotic and eukaryotic symbionts and their associations with deep-sea sponge Lamellomorpha sp..

  9. PSSRdb: a relational database of polymorphic simple sequence repeats extracted from prokaryotic genomes.

    Science.gov (United States)

    Kumar, Pankaj; Chaitanya, Pasumarthy S; Nagarajaram, Hampapathalu A

    2011-01-01

    PSSRdb (Polymorphic Simple Sequence Repeats database) (http://www.cdfd.org.in/PSSRdb/) is a relational database of polymorphic simple sequence repeats (PSSRs) extracted from 85 different species of prokaryotes. Simple sequence repeats (SSRs) are the tandem repeats of nucleotide motifs of the sizes 1-6 bp and are highly polymorphic. SSR mutations in and around coding regions affect transcription and translation of genes. Such changes underpin phase variations and antigenic variations seen in some bacteria. Although SSR-mediated phase variation and antigenic variations have been well-studied in some bacteria there seems a lot of other species of prokaryotes yet to be investigated for SSR mediated adaptive and other evolutionary advantages. As a part of our on-going studies on SSR polymorphism in prokaryotes we compared the genome sequences of various strains and isolates available for 85 different species of prokaryotes and extracted a number of SSRs showing length variations and created a relational database called PSSRdb. This database gives useful information such as location of PSSRs in genomes, length variation across genomes, the regions harboring PSSRs, etc. The information provided in this database is very useful for further research and analysis of SSRs in prokaryotes.

  10. Effects of Predation by Protists on Prokaryotic Community Function, Structure, and Diversity in Anaerobic Granular Sludge

    Science.gov (United States)

    Hirakata, Yuga; Oshiki, Mamoru; Kuroda, Kyohei; Hatamoto, Masashi; Kubota, Kengo; Yamaguchi, Takashi; Harada, Hideki; Araki, Nobuo

    2016-01-01

    Predation by protists is top-down pressure that regulates prokaryotic abundance, community function, structure, and diversity in natural and artificial ecosystems. Although the effects of predation by protists have been studied in aerobic ecosystems, they are poorly understood in anoxic environments. We herein studied the influence of predation by Metopus and Caenomorpha ciliates—ciliates frequently found in anoxic ecosystems—on prokaryotic community function, structure, and diversity. Metopus and Caenomorpha ciliates were cocultivated with prokaryotic assemblages (i.e., anaerobic granular sludge) in an up-flow anaerobic sludge blanket (UASB) reactor for 171 d. Predation by these ciliates increased the methanogenic activities of granular sludge, which constituted 155% of those found in a UASB reactor without the ciliates (i.e., control reactor). Sequencing of 16S rRNA gene amplicons using Illumina MiSeq revealed that the prokaryotic community in the UASB reactor with the ciliates was more diverse than that in the control reactor; 2,885–3,190 and 2,387–2,426 operational taxonomic units (>97% sequence similarities), respectively. The effects of predation by protists in anaerobic engineered systems have mostly been overlooked, and our results show that the influence of predation by protists needs to be examined and considered in the future for a better understanding of prokaryotic community structure and function. PMID:27431197

  11. Evolution of glutamate dehydrogenase genes: evidence for lateral gene transfer within and between prokaryotes and eukaryotes

    Directory of Open Access Journals (Sweden)

    Roger Andrew J

    2003-06-01

    Full Text Available Abstract Background Lateral gene transfer can introduce genes with novel functions into genomes or replace genes with functionally similar orthologs or paralogs. Here we present a study of the occurrence of the latter gene replacement phenomenon in the four gene families encoding different classes of glutamate dehydrogenase (GDH, to evaluate and compare the patterns and rates of lateral gene transfer (LGT in prokaryotes and eukaryotes. Results We extend the taxon sampling of gdh genes with nine new eukaryotic sequences and examine the phylogenetic distribution pattern of the various GDH classes in combination with maximum likelihood phylogenetic analyses. The distribution pattern analyses indicate that LGT has played a significant role in the evolution of the four gdh gene families. Indeed, a number of gene transfer events are identified by phylogenetic analyses, including numerous prokaryotic intra-domain transfers, some prokaryotic inter-domain transfers and several inter-domain transfers between prokaryotes and microbial eukaryotes (protists. Conclusion LGT has apparently affected eukaryotes and prokaryotes to a similar extent within the gdh gene families. In the absence of indications that the evolution of the gdh gene families is radically different from other families, these results suggest that gene transfer might be an important evolutionary mechanism in microbial eukaryote genome evolution.

  12. Effects of Predation by Protists on Prokaryotic Community Function, Structure, and Diversity in Anaerobic Granular Sludge.

    Science.gov (United States)

    Hirakata, Yuga; Oshiki, Mamoru; Kuroda, Kyohei; Hatamoto, Masashi; Kubota, Kengo; Yamaguchi, Takashi; Harada, Hideki; Araki, Nobuo

    2016-09-29

    Predation by protists is top-down pressure that regulates prokaryotic abundance, community function, structure, and diversity in natural and artificial ecosystems. Although the effects of predation by protists have been studied in aerobic ecosystems, they are poorly understood in anoxic environments. We herein studied the influence of predation by Metopus and Caenomorpha ciliates-ciliates frequently found in anoxic ecosystems-on prokaryotic community function, structure, and diversity. Metopus and Caenomorpha ciliates were cocultivated with prokaryotic assemblages (i.e., anaerobic granular sludge) in an up-flow anaerobic sludge blanket (UASB) reactor for 171 d. Predation by these ciliates increased the methanogenic activities of granular sludge, which constituted 155% of those found in a UASB reactor without the ciliates (i.e., control reactor). Sequencing of 16S rRNA gene amplicons using Illumina MiSeq revealed that the prokaryotic community in the UASB reactor with the ciliates was more diverse than that in the control reactor; 2,885-3,190 and 2,387-2,426 operational taxonomic units (>97% sequence similarities), respectively. The effects of predation by protists in anaerobic engineered systems have mostly been overlooked, and our results show that the influence of predation by protists needs to be examined and considered in the future for a better understanding of prokaryotic community structure and function.

  13. PSP: rapid identification of orthologous coding genes under positive selection across multiple closely related prokaryotic genomes.

    Science.gov (United States)

    Su, Fei; Ou, Hong-Yu; Tao, Fei; Tang, Hongzhi; Xu, Ping

    2013-12-27

    With genomic sequences of many closely related bacterial strains made available by deep sequencing, it is now possible to investigate trends in prokaryotic microevolution. Positive selection is a sub-process of microevolution, in which a particular mutation is favored, causing the allele frequency to continuously shift in one direction. Wide scanning of prokaryotic genomes has shown that positive selection at the molecular level is much more frequent than expected. Genes with significant positive selection may play key roles in bacterial adaption to different environmental pressures. However, selection pressure analyses are computationally intensive and awkward to configure. Here we describe an open access web server, which is designated as PSP (Positive Selection analysis for Prokaryotic genomes) for performing evolutionary analysis on orthologous coding genes, specially designed for rapid comparison of dozens of closely related prokaryotic genomes. Remarkably, PSP facilitates functional exploration at the multiple levels by assignments and enrichments of KO, GO or COG terms. To illustrate this user-friendly tool, we analyzed Escherichia coli and Bacillus cereus genomes and found that several genes, which play key roles in human infection and antibiotic resistance, show significant evidence of positive selection. PSP is freely available to all users without any login requirement at: http://db-mml.sjtu.edu.cn/PSP/. PSP ultimately allows researchers to do genome-scale analysis for evolutionary selection across multiple prokaryotic genomes rapidly and easily, and identify the genes undergoing positive selection, which may play key roles in the interactions of host-pathogen and/or environmental adaptation.

  14. Sodium sulfite pH-buffering effect for improved xylose-phenylalanine conversion to N-(1-deoxy-d-xylulos-1-yl)-phenylalanine during an aqueous Maillard reaction.

    Science.gov (United States)

    Cui, Heping; Duhoranimana, Emmanuel; Karangwa, Eric; Jia, Chengsheng; Zhang, Xiaoming

    2018-04-25

    The yield of the Maillard reaction intermediate (MRI), prepared in aqueous medium, is usually unsatisfactory. However, the addition of sodium sulfite could improve the conversion of xylose-phenylalanine (Xyl-Phe) to the MRI (N-(1-deoxy-d-xylulos-1-yl)-phenylalanine) in aqueous medium. Sodium sulfite (Na 2 SO 3 ) showed a significant pH-buffering effect during the Maillard reaction, which accounted for its facilitation of the N-(1-deoxy-d-xylulos-1-yl)-phenylalanine yield. The results revealed that the pH could be maintained at a relatively high level (above 7.0) for an optimized pH-buffering effect when Na 2 SO 3 (4.0%) was added before the reaction of Xyl-Phe. Thus, the conversion of Xyl-Phe to N-(1-deoxy-d-xylulos-1-yl)-phenylalanine increased from 47.23% to 74.86%. Furthermore, the addition moment of Na 2 SO 3 and corresponding solution pH were crucial factors in regulating the pH-buffering effect of Na 2 SO 3 on N-(1-deoxy-d-xylulos-1-yl)-phenylalanine yield. Based on the pH-buffering effect of Na 2 SO 3 and maintaining the optimal pH 7.4 relatively stable, the conversion of Xyl-Phe to N-(1-deoxy-d-xylulos-1-yl)-phenylalanine was successfully improved. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Shape Memory Properties and Enzymatic Degradability of Poly(ε-caprolactone)-Based Polyurethane Urea Containing Phenylalanine-Derived Chain Extender.

    Science.gov (United States)

    Wang, Rong; Zhang, Fanjun; Lin, Weiwei; Liu, Wenkai; Li, Jiehua; Luo, Feng; Wang, Yaning; Tan, Hong

    2018-04-24

    Biodegradable shape memory polymers are promising biomaterials for minimally invasive surgical procedures. Herein, a series of linear biodegradable shape memory poly(ε-caprolactone) (PCL)-based polyurethane ureas (PUUs) containing a novel phenylalanine-derived chain extender is synthesized. The phenylalanine-derived chain extender, phenylalanine-hexamethylenediamine-phenylalanine (PHP), contains two chymotrypsin cleaving sites to enhance the enzymatic degradation of PUUs. The degradation rate, the crystallinity, and mechanical properties of PUUs are tailored by the content of PHP. Meanwhile, semicrystalline PCL is not only hydrolytically degradable but also vital for shape memory. Good shape memory ability under body temperature is achieved for PUUs due to the strong interactions in hard segments for permanent crosslinking and the crystallization-melt transition of PCL to switch temporary shape. The PUUs would have a great potential in application as implanting stent. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. New LASER fluorometric HPLC detection for ortho-tyrosine in gamma-irradiated phenylalanine solution and pork

    International Nuclear Information System (INIS)

    Miyahara, Makoto; Toyoda, Masatake; Saito, Yukio; Nagasawa, Taeko; Izumi, Keiko; Kitamura, Mayumi

    1999-01-01

    New analytical procedure for o-tyrosine was studied to investigate effects of gamma irradiation on aqueous phenylalanine solution and pork. The process includes extraction and hydrolysis of protein, derivatization of the free amino acid by fluororeagent, and finally separation and detection by LASER fluorometric HPLC. The detection limit was 25ng. To study how the procedure works, irradiated phenylalanine solution and pork were analyzed. The samples were irradiated at doses up to 10 kGy at room temperature. Three tyrosine isomers were detected in phenylalanine solution, and 2 isomers (o-and p-tyrosine) were found in pork. Dose response was found in the formation of the isomers both in phenylalanine solution and in pork. O-tyrosine peak obtained from irradiated pork was separated from interference successfully. Those findings illustrate the procedure may be applicable to detection of irradiated food. (author)

  17. Effect of inhaled formyl-methionyl-leucyl-phenylalanine on airway function.

    OpenAIRE

    Berend, N; Peters, M J; Armour, C L; Black, J L; Ward, H E

    1988-01-01

    Formyl-methionyl-leucyl-phenylalanine (FMLP), a synthetic, acylated tripeptide analogous to bacterial chemotactic factors, has been shown to cause bronchoconstriction in guinea pig, rabbit, and human airways in vitro. To determine whether FMLP causes bronchoconstriction in man in vivo, a preliminary study was undertaken in which five non-smokers (mean age 35 years, FEV1 94% (SEM 5%) predicted) and five smokers (mean age 34 years, FEV1 93% (6%) predicted) inhaled aerosols of FMLP. None of the ...

  18. No Tryptophan, Tyrosine and Phenylalanine Abnormalities in Children with Attention-Deficit/Hyperactivity Disorder

    OpenAIRE

    Bergwerff, C.E.; Luman, M.; Blom, H.J.; Oosterlaan, J.

    2016-01-01

    Background The aim of the current study was to explore the role of aromatic amino acids (AAAs) in blood in relation to attention-deficit/hyperactivity disorder (ADHD). Given their impact on the synthesis of serotonin and dopamine, decreased concentrations of the AAAs tryptophan, tyrosine and phenylalanine in blood may contribute to the expression of ADHD symptoms. Decreased AAA blood concentrations, in turn, may be related to lowered dietary protein intake or to abnormal AAA catabolism, as ev...

  19. Influence of sample quality on phenylalanine and 17-hydroxyprogesterone levels in neonatal screening

    OpenAIRE

    del Río Fabre, Lesley; González, Ernesto C; Frómeta, Amarilys; Castells, Elisa M; Tejeda, Yileidis

    2010-01-01

    Quality of the sample and phenylalanine and 17-hydroxyprogesterone levels in neonatal screening. In the programs for neonatal screening many different analytes are quantified from dried blood on filter paper cards. Several factors affect the quality of the samples invalidating their employment in the laboratory: inadequate collection procedures, quality of the filter paper, the drying, storage and transportation under extreme environmental conditions. This article aims to show how the quality...

  20. Prokaryotic caspase homologs: phylogenetic patterns and functional characteristics reveal considerable diversity.

    Directory of Open Access Journals (Sweden)

    Johannes Asplund-Samuelsson

    Full Text Available Caspases accomplish initiation and execution of apoptosis, a programmed cell death process specific to metazoans. The existence of prokaryotic caspase homologs, termed metacaspases, has been known for slightly more than a decade. Despite their potential connection to the evolution of programmed cell death in eukaryotes, the phylogenetic distribution and functions of these prokaryotic metacaspase sequences are largely uncharted, while a few experiments imply involvement in programmed cell death. Aiming at providing a more detailed picture of prokaryotic caspase homologs, we applied a computational approach based on Hidden Markov Model search profiles to identify and functionally characterize putative metacaspases in bacterial and archaeal genomes. Out of the total of 1463 analyzed genomes, merely 267 (18% were identified to contain putative metacaspases, but their taxonomic distribution included most prokaryotic phyla and a few archaea (Euryarchaeota. Metacaspases were particularly abundant in Alphaproteobacteria, Deltaproteobacteria and Cyanobacteria, which harbor many morphologically and developmentally complex organisms, and a distinct correlation was found between abundance and phenotypic complexity in Cyanobacteria. Notably, Bacillus subtilis and Escherichia coli, known to undergo genetically regulated autolysis, lacked metacaspases. Pfam domain architecture analysis combined with operon identification revealed rich and varied configurations among the metacaspase sequences. These imply roles in programmed cell death, but also e.g. in signaling, various enzymatic activities and protein modification. Together our data show a wide and scattered distribution of caspase homologs in prokaryotes with structurally and functionally diverse sub-groups, and with a potentially intriguing evolutionary role. These features will help delineate future characterizations of death pathways in prokaryotes.

  1. Evolution and Diversity of the Ras Superfamily of Small GTPases in Prokaryotes

    Science.gov (United States)

    Wuichet, Kristin; Søgaard-Andersen, Lotte

    2015-01-01

    The Ras superfamily of small GTPases are single domain nucleotide-dependent molecular switches that act as highly tuned regulators of complex signal transduction pathways. Originally identified in eukaryotes for their roles in fundamental cellular processes including proliferation, motility, polarity, nuclear transport, and vesicle transport, recent studies have revealed that single domain GTPases also control complex functions such as cell polarity, motility, predation, development and antibiotic resistance in bacteria. Here, we used a computational genomics approach to understand the abundance, diversity, and evolution of small GTPases in prokaryotes. We collected 520 small GTPase sequences present in 17% of 1,611 prokaryotic genomes analyzed that cover diverse lineages. We identified two discrete families of small GTPases in prokaryotes that show evidence of three distinct catalytic mechanisms. The MglA family includes MglA homologs, which are typically associated with the MglB GTPase activating protein, whereas members of the Rup (Ras superfamily GTPase of unknown function in prokaryotes) family are not predicted to interact with MglB homologs. System classification and genome context analyses support the involvement of small GTPases in diverse prokaryotic signal transduction pathways including two component systems, laying the foundation for future experimental characterization of these proteins. Phylogenetic analysis of prokaryotic and eukaryotic GTPases supports that the last universal common ancestor contained ancestral MglA and Rup family members. We propose that the MglA family was lost from the ancestral eukaryote and that the Ras superfamily members in extant eukaryotes are the result of vertical and horizontal gene transfer events of ancestral Rup GTPases. PMID:25480683

  2. Phenylalanine-a biogenic ligand with flexible η6- and η6:κ1-coordination at ruthenium(ii) centres

    KAUST Repository

    Reiner, Thomas; Jantke, Dominik; Miao, Xiaohe; Marziale, Alexander N.; Kiefer, Florian J.; Eppinger, Jö rg

    2013-01-01

    The reaction of (S)-2,5-dihydrophenylalanine 1 with ruthenium(iii) chloride yields the μ-chloro-bridged dimeric η6-phenylalanine ethyl ester complex 3, which can be converted into the monomeric analogue, η6:κ1-phenylalanine ethyl ester complex 12, under basic conditions. Studies were carried out to determine the stability and reactivity of complexes bearing η6- and η6: κ1-chelating phenylalanine ligands under various conditions. Reaction of 3 with ethylenediamine derivatives N-p-tosylethylenediamine or 1,4-di-N-p-tosylethylenediamine results in the formation of monomeric η6:κ1-phenylalanine ethyl ester complexes 14 and 15, which could be saponified yielding complexes 16 and 17 without changing the inner coordination sphere of the metal centre. The structure of η6:κ1-phenylalanine complex 17 and an N-κ1-phenylalanine complex 13 resulting from the reaction of 3 with an excess of pyridine were confirmed by X-ray crystallography. © 2013 The Royal Society of Chemistry.

  3. Copolyacrylates with phenylalanine and anthracene entities prepared by ATRP and microwave irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Buruiana, E.C., E-mail: emilbur@icmpp.r [Romanian Academy, Petru Poni Institute of Macromolecular Chemistry, Grigore Ghica Voda Alley 41A, 700487 Iasi (Romania); Murariu, Mioara; Buruiana, Tinca [Romanian Academy, Petru Poni Institute of Macromolecular Chemistry, Grigore Ghica Voda Alley 41A, 700487 Iasi (Romania)

    2010-10-15

    In this study, two amino acid copolymers containing anthracene incorporated either on the one end, poly(N-acryloyl-L-phenylalanine-co-methyl methacrylate)-1 or as pendant groups, poly-(N-acryloyl-L-phenylalanine-co-methyl methacrylate)-2 were prepared directly from N-acryloyl-L-phenylalanine (APhe) and methyl methacrylate (MMA) through atom transfer radical polymerization (ATRP) and microwave-assisted synthesis. In the first case, 9-(chloromethyl)anthracene was used as an ATRP-initiator to obtain a copolymer that contains amino acid sequences and anthracene end-capped units (0.03 molar fraction). Rapid synthesis of copolymer under microwave irradiation (250 W) in the presence of 1,1'-azobis(cyclohexanecarbonitrile) used as an initiator was followed of a functionalization of the formed copolymer with an anthracene derivative yielding copolyacrylate with pendant anthracene (0.02 molar fraction). The structure of the copolymers was verified by {sup 1}H NMR, UV-Vis and FTIR spectroscopy, gel permeation chromatography (GPC), and fluorescence spectroscopy. The fluorescence quenching process of anthracene which exists in copolymers by FeCl{sub 3}, cobalt acetate, nitrobenzene, maleic anhydride, diethylaniline and nitromethane in DMF solutions shows that this involves an electron transfer between the excited state anthracene and the present transitional metal cations, more efficiently being FeCl{sub 3} for poly-(APhe-co-MMA)-1 and cobalt acetate for the latter copolymer.

  4. Effect of template on chiral separation of phenylalanine using molecularly imprinted membrane in aqueous medium

    International Nuclear Information System (INIS)

    Haq, N.U.

    2014-01-01

    Wet phase inversion method was used to prepare L-Phenylalanine (L-Phe) and D-Phenylalanine (D-Phe) imprinted poly ((acrylonitrile)-co-(acrylic acid)) membranes for chiral separation. Ultrafiltration experiments were conducted to evaluate the chiral separation ability of the prepared membrane towards racemate aqueous solution of Phenylalanine. The continuous permselectivity was observed by novel membrane. The chiral resolution ability of L-Phe imprinted membrane was much better than that of D-Phe. It was observed that both membranes simultaneously, selectively reject, selectively adsorbed and selectively permeate solute. The achieved adsorption selectivities of L-Phe imprinted membrane (AlphaAds)L and D-Phe imprinted membrane (AlphaAds)D were 2.6 and 2.40 respectively. Permselectivity of L-Phe imprinted membrane (AlphaPerm)L was 2.56 while D-Phe imprinted membrane permselectivity (AlphaPerm)D was 2.03. The rejection selectivities of L-Phe and D-Phe imprinted membranes were (AlphaRej)L=0.32 and (AlphaRej)D =0.28 respectively. (author)

  5. PpNAC1, a main regulator of phenylalanine biosynthesis and utilization in maritime pine.

    Science.gov (United States)

    Pascual, María Belén; Llebrés, María-Teresa; Craven-Bartle, Blanca; Cañas, Rafael A; Cánovas, Francisco M; Ávila, Concepción

    2018-05-01

    The transcriptional regulation of phenylalanine metabolism is particularly important in conifers, long-lived species that use large amounts of carbon in wood. Here, we show that the Pinus pinaster transcription factor, PpNAC1, is a main regulator of phenylalanine biosynthesis and utilization. A phylogenetic analysis classified PpNAC1 in the NST proteins group and was selected for functional characterization. PpNAC1 is predominantly expressed in the secondary xylem and compression wood of adult trees. Silencing of PpNAC1 in P. pinaster results in the alteration of stem vascular radial patterning and the down-regulation of several genes associated with cell wall biogenesis and secondary metabolism. Furthermore, transactivation and EMSA analyses showed that PpNAC1 is able to activate its own expression and PpMyb4 promoter, while PpMyb4 is able to activate PpMyb8, a transcriptional regulator of phenylalanine and lignin biosynthesis in maritime pine. Together, these results suggest that PpNAC1 is a functional ortholog of the ArabidopsisSND1 and NST1 genes and support the idea that key regulators governing secondary cell wall formation could be conserved between gymnosperms and angiosperms. Understanding the molecular switches controlling wood formation is of paramount importance for fundamental tree biology and paves the way for applications in conifer biotechnology. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  6. Production of tyrosine through phenylalanine hydroxylation bypasses the intrinsic feedback inhibition in Escherichia coli.

    Science.gov (United States)

    Huang, Jin; Lin, Yuheng; Yuan, Qipeng; Yan, Yajun

    2015-04-01

    Tyrosine is a proteinogenic aromatic amino acid that is often used as a supplement of food and animal feed, as well as a (bio-)synthetic precursor to various pharmaceutically or industrially important molecules. Extensive metabolic engineering efforts have been made towards the efficient and cost-effective microbial production of tyrosine. Conventional strategies usually focus on eliminating intrinsic feedback inhibition and redirecting carbon flux into the shikimate pathway. In this study, we found that continuous conversion of phenylalanine into tyrosine by the action of tetrahydromonapterin (MH4)-utilizing phenylalanine 4-hydroxylase (P4H) can bypass the feedback inhibition in Escherichia coli, leading to tyrosine accumulation in the cultures. First, expression of the P4H from Xanthomonas campestris in combination with an MH4 recycling system in wild-type E. coli allowed the strain to accumulate tyrosine at 262 mg/L. On this basis, enhanced expression of the key enzymes associated with the shikimate pathway and the MH4 biosynthetic pathway resulted in the elevation of tyrosine production up to 401 mg/L in shake flasks. This work demonstrated a novel approach to tyrosine production and verified the possibility to alleviate feedback inhibition by creating a phenylalanine sink.

  7. Design, Engineering, and Characterization of Prokaryotic Ligand-Binding Transcriptional Activators as Biosensors in Yeast

    DEFF Research Database (Denmark)

    Ambri, Francesca; Snoek, Tim; Skjødt, Mette Louise

    2018-01-01

    process. In the yeast Saccharomyces cerevisiae, implementation of allosterically regulated transcription factors from prokaryotes as metabolite biosensors has proven a valuable strategy to alleviate this screening bottleneck. Here, we present a protocol to select and incorporate prokaryotic...... transcriptional activators as metabolite biosensors in S. cerevisiae. As an example, we outline the engineering and characterization of the LysR-type transcriptional regulator (LTTR) family member BenM from Acetinobacter sp. ADP1 for monitoring accumulation of cis,cis-muconic acid, a bioplast precursor, in yeast...

  8. Once in a lifetime: strategies for preventing re-replication in prokaryotic and eukaryotic cells

    DEFF Research Database (Denmark)

    Nielsen, Olaf; Løbner-Olesen, Anders

    2008-01-01

    DNA replication is an extremely accurate process and cells have evolved intricate control mechanisms to ensure that each region of their genome is replicated only once during S phase. Here, we compare what is known about the processes that prevent re-replication in prokaryotic and eukaryotic cells...... prokaryotes and eukaryotes are inactivated until the next cell cycle. Furthermore, in both systems the beta-clamp of the replicative polymerase associates with enzymatic activities that contribute to the inactivation of the helicase loaders. Finally, recent studies suggest that the control mechanism...

  9. Antihypertension and anti-cardiovascular remodeling by phenylalanine in spontaneously hypertensive rats: effectiveness and mechanisms.

    Science.gov (United States)

    Zhao, G; Li, Z; Gu, T

    2001-03-01

    To investigate mechanisms of anti-hypertension and anti-cardiovascular remodeling by phenylalanine (phe) in spontaneously hypertensive rats (SHRs). The comparison of blood pressure (BP) increment with the ages and cardiovascular changes of SHRs was made between the 3% phe-intervented group (SHR-phe) and the control SHRs group. Detection of the structural changes with the VIDAS digital vedio-frequency processing technique and light and electron microscopy were made. The cell growth and proliferation of cultured smooth muscle cells (CSMCs) of the thoracic aortas or myocardial fibroblasts were evaluated by measuring the 3H-thymidine counts per minute (cpm) incorporated into the new synthesized desoxyribonucleic acid (DNA) and determining the cell number with the crystal violet stain technique. The Ca2+ influx was measured in counts/min of 45CaCl2 after incubating it with 5 different concentrations of phenylalanine and the intracellular [Ca2+]i by Fura-II/Am indicator. The total messenger ribonucleic acid (mRNA) of the myocardium was extracted and Northern blot analysis was performed with the probe collagen alpha 2 (I) cDNA. The tyrosine hydroxylase (TH) activity was measured by high-performance liquid chromatography (HPLC) with electrochemical detector after having reacted with its substrate tyrosine and other reagents. The catecholamine contents in brain homogenat were detected by HPLC method. The comparison of pharmacokinetics of phenylalanine among SHR-phe, SHRs and control Wistar Kyoto (WKY) rats was made after intravenous injection of 3H-L-phe (1 ml/kg) by PK-GRAPH Program for kinetic calculation. The 3H-L-phe uptake by CSMCs after incubating for definite intervals was also detected and compared. Phenylalanine could prevent the increase of BP with ages and the heart weight (heart/body weight index). The aortic media thickness and the collagen content in the myocardium were decreased significantly in SHR-phe. Whereas the dearranged cardiovascular structure was

  10. Read length and repeat resolution: exploring prokaryote genomes using next-generation sequencing technologies.

    Directory of Open Access Journals (Sweden)

    Matt J Cahill

    Full Text Available BACKGROUND: There are a growing number of next-generation sequencing technologies. At present, the most cost-effective options also produce the shortest reads. However, even for prokaryotes, there is uncertainty concerning the utility of these technologies for the de novo assembly of complete genomes. This reflects an expectation that short reads will be unable to resolve small, but presumably abundant, repeats. METHODOLOGY/PRINCIPAL FINDINGS: Using a simple model of repeat assembly, we develop and test a technique that, for any read length, can estimate the occurrence of unresolvable repeats in a genome, and thus predict the number of gaps that would need to be closed to produce a complete sequence. We apply this technique to 818 prokaryote genome sequences. This provides a quantitative assessment of the relative performance of various lengths. Notably, unpaired reads of only 150nt can reconstruct approximately 50% of the analysed genomes with fewer than 96 repeat-induced gaps. Nonetheless, there is considerable variation amongst prokaryotes. Some genomes can be assembled to near contiguity using very short reads while others require much longer reads. CONCLUSIONS: Given the diversity of prokaryote genomes, a sequencing strategy should be tailored to the organism under study. Our results will provide researchers with a practical resource to guide the selection of the appropriate read length.

  11. Exploring cultivable Bacteria from the prokaryotic community associated with the carnivorous sponge Asbestopluma hypogea.

    Science.gov (United States)

    Dupont, Samuel; Carre-Mlouka, Alyssa; Domart-Coulon, Isabelle; Vacelet, Jean; Bourguet-Kondracki, Marie-Lise

    2014-04-01

    Combining culture-dependent and independent approaches, we investigated for the first time the cultivable fraction of the prokaryotic community associated with the carnivorous sponge Asbestopluma hypogea. The heterotrophic prokaryotes isolated from this tiny sponge were compared between specimens freshly collected from cave and maintained in aquarium. Overall, 67 isolates obtained in pure culture were phylogenetically affiliated to the bacterial phyla Proteobacteria, Bacteroidetes, Actinobacteria, and Firmicutes. This cultivable diversity was lower than the prokaryotic diversity obtained by previous pyrosequencing study and comparable to that of another Mediterranean demosponge, the filter-feeding Phorbas tenacior. Furthermore, using fluorescence in situ hybridization, we visualized bacterial and archaeal cells, confirming the presence of both prokaryotes in A. hypogea tissue. Approximately 16% of the bacterial isolates tested positive for chitinolytic activity, suggesting potential microbial involvement in the digestion processes of crustacean prey by this carnivorous sponge. Additionally, 6% and 16% of bacterial isolates revealed antimicrobial and antioxidant activities, respectively. One Streptomyces sp. S1CA strain was identified as a promising candidate for the production of antimicrobial and antioxidant secondary metabolites as well as chitinolytic enzymes. Implications in the context of the sponge biology and prey-feeding strategy are discussed. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  12. Horizontal transfer, not duplication, drives the expansion of protein families in prokaryotes.

    Directory of Open Access Journals (Sweden)

    Todd J Treangen

    2011-01-01

    Full Text Available Gene duplication followed by neo- or sub-functionalization deeply impacts the evolution of protein families and is regarded as the main source of adaptive functional novelty in eukaryotes. While there is ample evidence of adaptive gene duplication in prokaryotes, it is not clear whether duplication outweighs the contribution of horizontal gene transfer in the expansion of protein families. We analyzed closely related prokaryote strains or species with small genomes (Helicobacter, Neisseria, Streptococcus, Sulfolobus, average-sized genomes (Bacillus, Enterobacteriaceae, and large genomes (Pseudomonas, Bradyrhizobiaceae to untangle the effects of duplication and horizontal transfer. After removing the effects of transposable elements and phages, we show that the vast majority of expansions of protein families are due to transfer, even among large genomes. Transferred genes--xenologs--persist longer in prokaryotic lineages possibly due to a higher/longer adaptive role. On the other hand, duplicated genes--paralogs--are expressed more, and, when persistent, they evolve slower. This suggests that gene transfer and gene duplication have very different roles in shaping the evolution of biological systems: transfer allows the acquisition of new functions and duplication leads to higher gene dosage. Accordingly, we show that paralogs share most protein-protein interactions and genetic regulators, whereas xenologs share very few of them. Prokaryotes invented most of life's biochemical diversity. Therefore, the study of the evolution of biology systems should explicitly account for the predominant role of horizontal gene transfer in the diversification of protein families.

  13. PePPER : a webserver for prediction of prokaryote promoter elements and regulons

    NARCIS (Netherlands)

    de Jong, Anne; Pietersma, Hilco; Cordes, Martijn; Kuipers, Oscar P.; Kok, Jan

    2012-01-01

    Background: Accurate prediction of DNA motifs that are targets of RNA polymerases, sigma factors and transcription factors (TFs) in prokaryotes is a difficult mission mainly due to as yet undiscovered features in DNA sequences or structures in promoter regions. Improved prediction and comparison

  14. Read length and repeat resolution: Exploring prokaryote genomes using next-generation sequencing technologies

    KAUST Repository

    Cahill, Matt J.

    2010-07-12

    Background: There are a growing number of next-generation sequencing technologies. At present, the most cost-effective options also produce the shortest reads. However, even for prokaryotes, there is uncertainty concerning the utility of these technologies for the de novo assembly of complete genomes. This reflects an expectation that short reads will be unable to resolve small, but presumably abundant, repeats. Methodology/Principal Findings: Using a simple model of repeat assembly, we develop and test a technique that, for any read length, can estimate the occurrence of unresolvable repeats in a genome, and thus predict the number of gaps that would need to be closed to produce a complete sequence. We apply this technique to 818 prokaryote genome sequences. This provides a quantitative assessment of the relative performance of various lengths. Notably, unpaired reads of only 150nt can reconstruct approximately 50% of the analysed genomes with fewer than 96 repeat-induced gaps. Nonetheless, there is considerable variation amongst prokaryotes. Some genomes can be assembled to near contiguity using very short reads while others require much longer reads. Conclusions: Given the diversity of prokaryote genomes, a sequencing strategy should be tailored to the organism under study. Our results will provide researchers with a practical resource to guide the selection of the appropriate read length. 2010 Cahill et al.

  15. Rumen prokaryotic communities of ruminants under different feeding paradigms on the Qinghai-Tibetan Plateau.

    Science.gov (United States)

    Xue, Dan; Chen, Huai; Zhao, Xinquan; Xu, Shixiao; Hu, Linyong; Xu, Tianwei; Jiang, Lin; Zhan, Wei

    2017-06-01

    Yak and Tibetan sheep are the major indigenous ruminants on the Qinghai-Tibetan Plateau in China. The aim of this work was to study the differences in ruminal fermentation parameters and rumen prokaryotic community composition between hosts and feeding paradigms. The 16S rRNA genes targeting bacteria and archaea were sequenced using the MiSeq platform. The results showed that the prokaryotic community structure between yak and Tibetan sheep was significantly different (PTibetan sheep of the two groups (P=0.026). The core prokaryotic populations that existed in the rumen mostly dominated the structure. There was an obvious correlation of the prokaryotic community composition at the phylum and genus levels with the host or the feeding pattern. In addition, Tibetan sheep showed significantly higher yields of volatile fatty acids (VFAs) than yak, as did the NG group compared with the TMR group. In conclusion, both the host and feeding pattern may influence rumen microbial ecology system, with host effects being more important than those of the feeding pattern. Copyright © 2017 Elsevier GmbH. All rights reserved.

  16. Focus on Membrane Differentiation and Membrane Domains in the Prokaryotic Cell

    NARCIS (Netherlands)

    Boekema, Egbert J.; Scheffers, Dirk-Jan; van Bezouwen, Laura S.; Bolhuis, Henk; Folea, I. Mihaela

    2013-01-01

    A summary is presented of membrane differentiation in the prokaryotic cell, with an emphasis on the organization of proteins in the plasma/cell membrane. Many species belonging to the Eubacteria and Archaea have special membrane domains and/or membrane proliferation, which are vital for different

  17. Global identification of prokaryotic glycoproteins based on an Escherichia coli proteome microarray.

    Directory of Open Access Journals (Sweden)

    Zong-Xiu Wang

    Full Text Available Glycosylation is one of the most abundant protein posttranslational modifications. Protein glycosylation plays important roles not only in eukaryotes but also in prokaryotes. To further understand the roles of protein glycosylation in prokaryotes, we developed a lectin binding assay to screen glycoproteins on an Escherichia coli proteome microarray containing 4,256 affinity-purified E.coli proteins. Twenty-three E.coli proteins that bound Wheat-Germ Agglutinin (WGA were identified. PANTHER protein classification analysis showed that these glycoprotein candidates were highly enriched in metabolic process and catalytic activity classes. One sub-network centered on deoxyribonuclease I (sbcB was identified. Bioinformatics analysis suggests that prokaryotic protein glycosylation may play roles in nucleotide and nucleic acid metabolism. Fifteen of the 23 glycoprotein candidates were validated by lectin (WGA staining, thereby increasing the number of validated E. coli glycoproteins from 3 to 18. By cataloguing glycoproteins in E.coli, our study greatly extends our understanding of protein glycosylation in prokaryotes.

  18. Genomics of bacteria and archaea: the emerging dynamic view of the prokaryotic world

    Science.gov (United States)

    Koonin, Eugene V.; Wolf, Yuri I.

    2008-01-01

    The first bacterial genome was sequenced in 1995, and the first archaeal genome in 1996. Soon after these breakthroughs, an exponential rate of genome sequencing was established, with a doubling time of approximately 20 months for bacteria and approximately 34 months for archaea. Comparative analysis of the hundreds of sequenced bacterial and dozens of archaeal genomes leads to several generalizations on the principles of genome organization and evolution. A crucial finding that enables functional characterization of the sequenced genomes and evolutionary reconstruction is that the majority of archaeal and bacterial genes have conserved orthologs in other, often, distant organisms. However, comparative genomics also shows that horizontal gene transfer (HGT) is a dominant force of prokaryotic evolution, along with the loss of genetic material resulting in genome contraction. A crucial component of the prokaryotic world is the mobilome, the enormous collection of viruses, plasmids and other selfish elements, which are in constant exchange with more stable chromosomes and serve as HGT vehicles. Thus, the prokaryotic genome space is a tightly connected, although compartmentalized, network, a novel notion that undermines the ‘Tree of Life’ model of evolution and requires a new conceptual framework and tools for the study of prokaryotic evolution. PMID:18948295

  19. Energy Coupling Factor-Type ABC Transporters for Vitamin Uptake in Prokaryotes

    NARCIS (Netherlands)

    Erkens, Guus B.; Dosz-Majsnerowska, Maria; ter Beek, Josy; Slotboom, Dirk Jan

    2012-01-01

    Energy coupling factor (ECF) transporters are a subgroup of ATP-binding cassette (ABC) transporters involved in the uptake of vitamins and micronutrients in prokaryotes. In contrast to classical ABC importers, ECF transporters do not make use of water-soluble substrate binding proteins or domains

  20. Lateral gene transfer between prokaryotes and multicellular eukaryotes: ongoing and significant?

    NARCIS (Netherlands)

    Ros, V.I.D.; Hurst, G.D.D.

    2009-01-01

    The expansion of genome sequencing projects has produced accumulating evidence for lateral transfer of genes between prokaryotic and eukaryotic genomes. However, it remains controversial whether these genes are of functional importance in their recipient host. Nikoh and Nakabachi, in a recent paper

  1. Prokaryotic diversity of the Saccharomyces cerevisiae Atx1p-mediated copper pathway.

    NARCIS (Netherlands)

    Bakel, H. van; Huynen, M.A.; Wijmenga, C.

    2004-01-01

    MOTIVATION: Several genes involved in the cellular import of copper and its subsequent incorporation into the high-affinity iron transport complex in Saccharomyces cerevisiae are known to be conserved between eukaryotes and prokaryotes. However, the degree to which these genes share their functional

  2. Use of prokaryotic transcriptional activators as metabolite biosensors in eukaryotic cells

    DEFF Research Database (Denmark)

    2018-01-01

    The present invention relates to the use of transcriptional activators from prokaryotic organisms for use in eukaryotic cells, such as yeast as sensors of intracellular and extracellular accumulation of a ligand or metabolite specifically activating this transcriptional activator in a eukaryot...

  3. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites

    DEFF Research Database (Denmark)

    Nielsen, Henrik; Engelbrecht, Jacob; Brunak, Søren

    1997-01-01

    We have developed a new method for the identification of signal peptides and their cleavage based on neural networks trained on separate sets of prokaryotic and eukaryotic sequence. The method performs significantly better than previous prediction schemes and can easily be applied on genome...

  4. Short Term INT-Formazan Production as a Proxy for Marine Prokaryote Respiration

    Science.gov (United States)

    Cajal-Medrano, R.; Villegas-Mendoza, J.; Maske, H.

    2016-02-01

    Prokaryotes are poisoned by the tetrazolium electron transport probe INT on time scales of less than one hour, invalidating the interpretation of the rate of in vivo INT reduction to formazan as a proxy for oxygen consumption rates (Villegas-Mendoza et al. 2015). We measured oxygen consumption rate (R; µM O2 hour-1) and electron transport activity with in vivo INT formazan production (IFP, mM formazan) at 0.5 mM INT during 1 hour exposure time of natural communities and cultures of the marine bacteria Vibrio harveyi growing in batch and continuous cultures. A strong exponential relationship R = 0.20 IFP2.15 (pgrowth rates under aerobic condition. We find that IFP and oxygen consumption increase with bacterial specific growth rates and temperature as expected from basic principles of physiology and biochemistry. Oxygen and nitrogen saturated batch cultures of V. harveyi showed that both, IFP and oxygen consumption increased for 0.8 hours but then stopped similar to natural bacterial communities supporting the above relationship of IFP to prokaryote respiration. Our method implies adding 0.5 mM INT to a plankton sample and incubating for less than 1 hour. After prokaryote separation by size filtration (0.8 mm), the formazan crystals are collected by filtration (0.2 mm) and dissolved in propanol. The absorbance at 485 nm per sample volume yields the formazan potential that is related to prokaryote respiration in the sample.

  5. Read length and repeat resolution: Exploring prokaryote genomes using next-generation sequencing technologies

    KAUST Repository

    Cahill, Matt J.; Kö ser, Claudio U.; Ross, Nicholas E.; Archer, John A.C.

    2010-01-01

    Background: There are a growing number of next-generation sequencing technologies. At present, the most cost-effective options also produce the shortest reads. However, even for prokaryotes, there is uncertainty concerning the utility of these technologies for the de novo assembly of complete genomes. This reflects an expectation that short reads will be unable to resolve small, but presumably abundant, repeats. Methodology/Principal Findings: Using a simple model of repeat assembly, we develop and test a technique that, for any read length, can estimate the occurrence of unresolvable repeats in a genome, and thus predict the number of gaps that would need to be closed to produce a complete sequence. We apply this technique to 818 prokaryote genome sequences. This provides a quantitative assessment of the relative performance of various lengths. Notably, unpaired reads of only 150nt can reconstruct approximately 50% of the analysed genomes with fewer than 96 repeat-induced gaps. Nonetheless, there is considerable variation amongst prokaryotes. Some genomes can be assembled to near contiguity using very short reads while others require much longer reads. Conclusions: Given the diversity of prokaryote genomes, a sequencing strategy should be tailored to the organism under study. Our results will provide researchers with a practical resource to guide the selection of the appropriate read length. 2010 Cahill et al.

  6. Results of a Prospective Study Concerning the Clinical Efficiency of Recent Russian-Made Specialized Foods without Phenylalanine

    Directory of Open Access Journals (Sweden)

    T. V. Bushueva

    2016-01-01

    Full Text Available Background: Specialized foods, based on aminoacids without phenilalanin, are the main source of protein for patients with phenilketonuria of all ages. Based on modern technologies, new Russian-made foods were created. They have an optimized aminoacid and micronutrient composition, which increases their bioavailability.Objective: Our aim was to investigate the clinical efficiency of the new Russian-made specialized foods based on aminoacids without phenylalanine, in patients with phenylketonuria.Methods: Evaluating physical, somatic and neuropsychic development as well as measuring phenylalanine blood level were carried out twice: before the prescription and after 1 month of using the new Russian-made specialized foods. The chemical composition of the ration was controlled using dietology methods.Results: 57 children at the age of 14 days to 15 years with phenylketonuria (which was detected in neonatal screening were picked for this study. A hypophenylalanine diet has been prescribed for all children no later than at the age of 3 months of life. In children of the first year (I group (among which there were children with high phenylalanine blood levels phenylalanine concentration decreased from 5,5 (4,0; 21,0 to 4,4 (3,7; 4,7 mg/dl (p = 0,014 while using Russian-made specialized foods. In this group of children the psychomotor and physical indices improved. In early childhood-, preschool- and school-aged patients phenylalanine blood level remained steady. The chemical composition of the ration with Russian-made foods, based on aminoacids without phenylalanine, corresponded to the reference intake of main nutrients and energy.Conclusion: Hypophenylalanine diet with new Russian-made specialized foods without phenylalanine (which is designed for phenylketonuria patients of various age groups showed high clinical efficiency of these foods.

  7. Impact of Lowland Rainforest Transformation on Diversity and Composition of Soil Prokaryotic Communities in Sumatra (Indonesia)

    Science.gov (United States)

    Schneider, Dominik; Engelhaupt, Martin; Allen, Kara; Kurniawan, Syahrul; Krashevska, Valentyna; Heinemann, Melanie; Nacke, Heiko; Wijayanti, Marini; Meryandini, Anja; Corre, Marife D.; Scheu, Stefan; Daniel, Rolf

    2015-01-01

    Prokaryotes are the most abundant and diverse group of microorganisms in soil and mediate virtually all biogeochemical cycles in terrestrial ecosystems. Thereby, they influence aboveground plant productivity and diversity. In this study, the impact of rainforest transformation to intensively managed cash crop systems on soil prokaryotic communities was investigated. The studied managed land use systems comprised rubber agroforests (jungle rubber), rubber plantations and oil palm plantations within two Indonesian landscapes Bukit Duabelas and Harapan. Soil prokaryotic community composition and diversity were assessed by pyrotag sequencing of bacterial and archaeal 16S rRNA genes. The curated dataset contained 16,413 bacterial and 1679 archaeal operational taxonomic units at species level (97% genetic identity). Analysis revealed changes in indigenous taxon-specific patterns of soil prokaryotic communities accompanying lowland rainforest transformation to jungle rubber, and intensively managed rubber and oil palm plantations. Distinct clustering of the rainforest soil communities indicated that these are different from the communities in the studied managed land use systems. The predominant bacterial taxa in all investigated soils were Acidobacteria, Actinobacteria, Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. Overall, the bacterial community shifted from proteobacterial groups in rainforest soils to Acidobacteria in managed soils. The archaeal soil communities were mainly represented by Thaumarchaeota and Euryarchaeota. Members of the Terrestrial Group and South African Gold Mine Group 1 (Thaumarchaeota) dominated in the rainforest and members of Thermoplasmata in the managed land use systems. The alpha and beta diversity of the soil prokaryotic communities was higher in managed land use systems than in rainforest. In the case of bacteria, this was related to soil characteristics such as pH value, exchangeable Ca and Fe content, C to N ratio

  8. PairWise Neighbours database: overlaps and spacers among prokaryote genomes

    Directory of Open Access Journals (Sweden)

    Garcia-Vallvé Santiago

    2009-06-01

    Full Text Available Abstract Background Although prokaryotes live in a variety of habitats and possess different metabolic and genomic complexity, they have several genomic architectural features in common. The overlapping genes are a common feature of the prokaryote genomes. The overlapping lengths tend to be short because as the overlaps become longer they have more risk of deleterious mutations. The spacers between genes tend to be short too because of the tendency to reduce the non coding DNA among prokaryotes. However they must be long enough to maintain essential regulatory signals such as the Shine-Dalgarno (SD sequence, which is responsible of an efficient translation. Description PairWise Neighbours is an interactive and intuitive database used for retrieving information about the spacers and overlapping genes among bacterial and archaeal genomes. It contains 1,956,294 gene pairs from 678 fully sequenced prokaryote genomes and is freely available at the URL http://genomes.urv.cat/pwneigh. This database provides information about the overlaps and their conservation across species. Furthermore, it allows the wide analysis of the intergenic regions providing useful information such as the location and strength of the SD sequence. Conclusion There are experiments and bioinformatic analysis that rely on correct annotations of the initiation site. Therefore, a database that studies the overlaps and spacers among prokaryotes appears to be desirable. PairWise Neighbours database permits the reliability analysis of the overlapping structures and the study of the SD presence and location among the adjacent genes, which may help to check the annotation of the initiation sites.

  9. An integrative and applicable phylogenetic footprinting framework for cis-regulatory motifs identification in prokaryotic genomes.

    Science.gov (United States)

    Liu, Bingqiang; Zhang, Hanyuan; Zhou, Chuan; Li, Guojun; Fennell, Anne; Wang, Guanghui; Kang, Yu; Liu, Qi; Ma, Qin

    2016-08-09

    Phylogenetic footprinting is an important computational technique for identifying cis-regulatory motifs in orthologous regulatory regions from multiple genomes, as motifs tend to evolve slower than their surrounding non-functional sequences. Its application, however, has several difficulties for optimizing the selection of orthologous data and reducing the false positives in motif prediction. Here we present an integrative phylogenetic footprinting framework for accurate motif predictions in prokaryotic genomes (MP(3)). The framework includes a new orthologous data preparation procedure, an additional promoter scoring and pruning method and an integration of six existing motif finding algorithms as basic motif search engines. Specifically, we collected orthologous genes from available prokaryotic genomes and built the orthologous regulatory regions based on sequence similarity of promoter regions. This procedure made full use of the large-scale genomic data and taxonomy information and filtered out the promoters with limited contribution to produce a high quality orthologous promoter set. The promoter scoring and pruning is implemented through motif voting by a set of complementary predicting tools that mine as many motif candidates as possible and simultaneously eliminate the effect of random noise. We have applied the framework to Escherichia coli k12 genome and evaluated the prediction performance through comparison with seven existing programs. This evaluation was systematically carried out at the nucleotide and binding site level, and the results showed that MP(3) consistently outperformed other popular motif finding tools. We have integrated MP(3) into our motif identification and analysis server DMINDA, allowing users to efficiently identify and analyze motifs in 2,072 completely sequenced prokaryotic genomes. The performance evaluation indicated that MP(3) is effective for predicting regulatory motifs in prokaryotic genomes. Its application may enhance

  10. Impact of lowland rainforest transformation on diversity and composition of soil prokaryotic communities in Sumatra (Indonesia

    Directory of Open Access Journals (Sweden)

    Dominik eSchneider

    2015-12-01

    Full Text Available Prokaryotes are the most abundant and diverse group of microorganisms in soil and mediate virtually all biogeochemical cycles in terrestrial ecosystems. Thereby, they influence aboveground plant productivity and diversity. In this study, the impact of rainforest transformation to intensively managed cash crop systems on soil prokaryotic communities was investigated. The studied managed land use system comprised rubber agroforests (jungle rubber, rubber plantation and oil plantations within two Indonesian landscapes Bukit Duabelas and Harapan. Soil prokaryotic community composition and diversity were assessed by pyrotag sequencing of bacterial and archaeal 16S rRNA genes. The curated dataset contained 20,494 bacterial and 1,762 archaeal Operational Taxonomic Units at species level (97% genetic identity. Analysis revealed changes in indigenous taxon-specific patterns of soil prokaryotic communities accompanying lowland rainforest transformation to jungle rubber, and intensively managed rubber and oil palm plantations. Distinct clustering of the rainforest soil communities indicated that these are different from the communities in the studied managed land use systems. The predominant bacterial taxa in all investigated soils were Acidobacteria, Actinobacteria, Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. Overall, the bacterial community shifted from proteobacterial groups in rainforest soils to Acidobacteria in managed soils. The archaeal soil communities were mainly represented by Thaumarchaeota and Euryarchaeota. Members of the Terrestrial Group and South African Gold Mine Group 1 (Thaumarchaeota dominated in the rainforest and members of Thermoplasmata in the managed land use systems. The alpha and beta diversity of the soil prokaryotic communities was higher in managed land use systems than in rainforest. In the case of bacteria, this was related to soil characteristics such as pH value, exchangeable Ca and Fe content, C to

  11. Man made disease: clinical manifestations of low phenylalanine levels in an inadequately treated phenylketonuria patient and mouse study.

    Science.gov (United States)

    Pode-Shakked, Ben; Shemer-Meiri, Lilach; Harmelin, Alon; Stettner, Noa; Brenner, Ori; Abraham, Smadar; Schwartz, Gerard; Anikster, Yair

    2013-01-01

    Phenylalanine (Phe) deficiency and its clinical manifestations have been previously described mostly as sporadic case reports dating back to the 1960's and 1970's. In these reports, low plasma Phe levels were associated with listlessness, eczematous eruptions and failure to gain weight, most often in infants in their first year of life. Herein we describe a 9 month old female patient with known phenylketonuria, who presented with an unusual constellation of symptoms, including severe erythema and desquamation, alopecia, keratomalacia, corneal perforation, failure to thrive and prolonged diarrhea. The diagnostic possibilities of acrodermatitis enteropathica and vitamin deficiencies were ruled out, and further investigation into her medical history led to the conclusion that during the weeks preceding the hospitalization, the patient's diet consisted of the phenylalanine-free medical formula alone, without the addition of a standard infant formula or food as recommended. Subsequently, dietary control of the blood phenylalanine levels brought swift and marked resolution of the dermatological lesions, with renewal of hair growth. Following this experience, and due to the relative paucity of data regarding the clinical manifestations of low serum phenylalanine levels in humans and their putative pathogenetic mechanisms, we sought to further investigate the effects of a phenylalanine-free diet in a mouse study. For this purpose, twenty mice were randomly allocated to receive either a phenylalanine-deficient diet (n=10) or a normal diet (n=10). Weight was measured weekly, and laboratory tests were obtained including complete blood count, electrolyte studies, and phenylalanine and tyrosine levels. Finally, necropsies and histopathological examinations of different tissues were performed in selected mice, either early after diet initiation, late after diet initiation or following re-introduction of normal diets. The study was then repeated in additional two groups of mice

  12. Advances in algal-prokaryotic wastewater treatment: A review of nitrogen transformations, reactor configurations and molecular tools.

    Science.gov (United States)

    Wang, Meng; Keeley, Ryan; Zalivina, Nadezhda; Halfhide, Trina; Scott, Kathleen; Zhang, Qiong; van der Steen, Peter; Ergas, Sarina J

    2018-07-01

    The synergistic activity of algae and prokaryotic microorganisms can be used to improve the efficiency of biological wastewater treatment, particularly with regards to nitrogen removal. For example, algae can provide oxygen through photosynthesis needed for aerobic degradation of organic carbon and nitrification and harvested algal-prokaryotic biomass can be used to produce high value chemicals or biogas. Algal-prokaryotic consortia have been used to treat wastewater in different types of reactors, including waste stabilization ponds, high rate algal ponds and closed photobioreactors. This review addresses the current literature and identifies research gaps related to the following topics: 1) the complex interactions between algae and prokaryotes in wastewater treatment; 2) advances in bioreactor technologies that can achieve high nitrogen removal efficiencies in small reactor volumes, such as algal-prokaryotic biofilm reactors and enhanced algal-prokaryotic treatment systems (EAPS); 3) molecular tools that have expanded our understanding of the activities of algal and prokaryotic communities in wastewater treatment processes. Copyright © 2018 Elsevier Ltd. All rights reserved.

  13. Plasma phenylalanine and tyrosine responses to different nutritional conditions (fasting/postprandial) in patients with phenylketonuria: effect of sample timing.

    Science.gov (United States)

    van Spronsen, F J; van Rijn, M; van Dijk, T; Smit, G P; Reijngoud, D J; Berger, R; Heymans, H S

    1993-10-01

    To evaluate the adequacy of dietary treatment in patients with phenylketonuria, the monitoring of plasma phenylalanine and tyrosine concentrations is of great importance. The preferable time of blood sampling in relation to the nutritional condition during the day, however, is not known. It was the aim of this study to define guidelines for the timing of blood sampling with a minimal burden for the patient. Plasma concentrations of phenylalanine and tyrosine were measured in nine patients with phenylketonuria who had no clinical evidence of tyrosine deficiency. These values were measured during the day both after a prolonged overnight fast, and before and after breakfast. Phenylalanine showed a small rise during prolonged fasting, while tyrosine decreased slightly. After an individually tailored breakfast, phenylalanine remained stable, while tyrosine showed large fluctuations. It is concluded that the patient's nutritional condition (fasting/postprandial) is not important in the evaluation of the phenylalanine intake. To detect a possible tyrosine deficiency, however, a single blood sample is not sufficient and a combination of a preprandial and postprandial blood sample on the same day is advocated.

  14. Serum phenylalanine in preterm newborns fed different diets of human milk.

    Science.gov (United States)

    Thomaz, Débora M; Serafin, Paula O; Palhares, Durval B; Tavares, Luciana V M; Grance, Thayana R S

    2014-01-01

    To evaluate phenylalanine plasma profile in preterm newborns fed different human milk diets. Twenty-four very-low weight preterm newborns were distributed randomly in three groups with different feeding types: Group I: banked human milk plus 5% commercial fortifier with bovine protein, Group II: banked human milk plus evaporated fortifier derived from modified human milk, Group III: banked human milk plus lyophilized fortifier derived from modified human milk. The newborns received the group diet when full diet was attained at 15 ± 2 days. Plasma amino acid analysis was performedon the first and last day of feeding. Comparison among groups was performed by statistical tests: one way ANOVA with Tukey's post-test using SPSS software, version 20.0 (IBM Corp, NY, USA), considering a significance level of 5%. Phenylalanine levels in the first and second analysis were, respectively, in Group I: 11.9 ± 1.22 and 29.72 ± 0.73; in Group II: 11.72 ± 1.04 and 13.44 ± 0.61; and in Group III: 11.3 ± 1.18 and 15.42 ± 0.83 μmol/L. The observed results demonstrated that human milk with fortifiers derived from human milk acted as a good substratum for preterm infant feeding both in the evaporated or the lyophilized form, without significant increases in plasma phenylalanine levels in comparison to human milk with commercial fortifier. Copyright © 2014 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

  15. Macro and Microelements Drive Diversity and Composition of Prokaryotic and Fungal Communities in Hypersaline Sediments and Saline-Alkaline Soils.

    Science.gov (United States)

    Liu, Kaihui; Ding, Xiaowei; Tang, Xiaofei; Wang, Jianjun; Li, Wenjun; Yan, Qingyun; Liu, Zhenghua

    2018-01-01

    Understanding the effects of environmental factors on microbial communities is critical for microbial ecology, but it remains challenging. In this study, we examined the diversity (alpha diversity) and community compositions (beta diversity) of prokaryotes and fungi in hypersaline sediments and salinized soils from northern China. Environmental variables were highly correlated, but they differed significantly between the sediments and saline soils. The compositions of prokaryotic and fungal communities in the hypersaline sediments were different from those in adjacent saline-alkaline soils, indicating a habitat-specific microbial distribution pattern. The macroelements (S, P, K, Mg, and Fe) and Ca were, respectively, correlated closely with the alpha diversity of prokaryotes and fungi, while the macronutrients (e.g., Na, S, P, and Ca) were correlated with the prokaryotic and fungal beta-diversity ( P ≤ 0.05). And, the nine microelements (e.g., Al, Ba, Co, Hg, and Mn) and micronutrients (Ba, Cd, and Sr) individually shaped the alpha diversity of prokaryotes and fungi, while the six microelements (e.g., As, Ba, Cr, and Ge) and only the trace elements (Cr and Cu), respectively, influenced the beta diversity of prokaryotes and fungi ( P analysis (VPA) showed that environmental variables jointly explained 55.49% and 32.27% of the total variation for the prokaryotic and fungal communities, respectively. Together, our findings demonstrate that the diversity and community composition of the prokaryotes and fungi were driven by different macro and microelements in saline habitats, and that geochemical elements could more widely regulate the diversity and community composition of prokaryotes than these of fungi.

  16. Macro and Microelements Drive Diversity and Composition of Prokaryotic and Fungal Communities in Hypersaline Sediments and Saline–Alkaline Soils

    Science.gov (United States)

    Liu, Kaihui; Ding, Xiaowei; Tang, Xiaofei; Wang, Jianjun; Li, Wenjun; Yan, Qingyun; Liu, Zhenghua

    2018-01-01

    Understanding the effects of environmental factors on microbial communities is critical for microbial ecology, but it remains challenging. In this study, we examined the diversity (alpha diversity) and community compositions (beta diversity) of prokaryotes and fungi in hypersaline sediments and salinized soils from northern China. Environmental variables were highly correlated, but they differed significantly between the sediments and saline soils. The compositions of prokaryotic and fungal communities in the hypersaline sediments were different from those in adjacent saline–alkaline soils, indicating a habitat-specific microbial distribution pattern. The macroelements (S, P, K, Mg, and Fe) and Ca were, respectively, correlated closely with the alpha diversity of prokaryotes and fungi, while the macronutrients (e.g., Na, S, P, and Ca) were correlated with the prokaryotic and fungal beta-diversity (P ≤ 0.05). And, the nine microelements (e.g., Al, Ba, Co, Hg, and Mn) and micronutrients (Ba, Cd, and Sr) individually shaped the alpha diversity of prokaryotes and fungi, while the six microelements (e.g., As, Ba, Cr, and Ge) and only the trace elements (Cr and Cu), respectively, influenced the beta diversity of prokaryotes and fungi (P analysis (VPA) showed that environmental variables jointly explained 55.49% and 32.27% of the total variation for the prokaryotic and fungal communities, respectively. Together, our findings demonstrate that the diversity and community composition of the prokaryotes and fungi were driven by different macro and microelements in saline habitats, and that geochemical elements could more widely regulate the diversity and community composition of prokaryotes than these of fungi. PMID:29535703

  17. Folate Biofortification in Hydroponically Cultivated Spinach by the Addition of Phenylalanine.

    Science.gov (United States)

    Watanabe, Sho; Ohtani, Yuta; Tatsukami, Yohei; Aoki, Wataru; Amemiya, Takashi; Sukekiyo, Yasunori; Kubokawa, Seiichi; Ueda, Mitsuyoshi

    2017-06-14

    Folate is an important vitamin mainly ingested from vegetables, and folate deficiency causes various health problems. Recently, several studies demonstrated folate biofortification in plants or food crops by metabolic engineering through genetic modifications. However, the production and sales of genetically modified foods are under strict regulation. Here, we developed a new approach to achieve folate biofortification in spinach (Spinacia oleracea) without genetic modification. We hydroponically cultivated spinach with the addition of three candidate compounds expected to fortify folate. As a result of liquid chromatography tandem mass spectrometry analysis, we found that the addition of phenylalanine increased the folate content up to 2.0-fold (306 μg in 100 g of fresh spinach), representing 76.5% of the recommended daily allowance for adults. By measuring the intermediates of folate biosynthesis, we revealed that phenylalanine activated folate biosynthesis in spinach by increasing the levels of pteridine and p-aminobenzoic acid. Our approach is a promising and practical approach to cultivate nutrient-enriched vegetables.

  18. Bioactive Phenylalanine Derivatives and Cytochalasins from the Soft Coral-Derived Fungus, Aspergillus elegans

    Directory of Open Access Journals (Sweden)

    Xue-Ping Sun

    2013-06-01

    Full Text Available One new phenylalanine derivative 4′-OMe-asperphenamate (1, along with one known phenylalanine derivative (2 and two new cytochalasins, aspochalasin A1 (3 and cytochalasin Z24 (4, as well as eight known cytochalasin analogues (5–12 were isolated from the fermentation broth of Aspergillus elegans ZJ-2008010, a fungus obtained from a soft coral Sarcophyton sp. collected from the South China Sea. Their structures and the relative configurations were elucidated using comprehensive spectroscopic methods. The absolute configuration of 1 was determined by chemical synthesis and Marfey’s method. All isolated metabolites (1–12 were evaluated for their antifouling and antibacterial activities. Cytochalasins 5, 6, 8 and 9 showed strong antifouling activity against the larval settlement of the barnacle Balanus amphitrite, with the EC50 values ranging from 6.2 to 37 μM. This is the first report of antifouling activity for this class of metabolites. Additionally, 8 exhibited a broad spectrum of antibacterial activity, especially against four pathogenic bacteria Staphylococcus albus, S. aureus, Escherichia coli and Bacillus cereus.

  19. Synthesizing aspartyl-phenylalanine methyl precursor by enzymatic method. Kosoho ni yoru asuparutemu zenkutai no gosei

    Energy Technology Data Exchange (ETDEWEB)

    Hirata, M. (Waseda University, Tokyo (Japan)); Hirata, A. (Waseda University, Tokyo (Japan). School of Science and Engineering)

    1991-10-01

    Demand for aspartyl-phenylalanine methyl, an amino acid based artificial sweetener, is increasing for use by people on diet. The material is manufactured by making a precursor through dehydrating and condensing N-protective{sub L}-asparagine acid and {sub L}-phenylalanine methyl ester, and then removing the protective group. While the manufacturing methods include chemical process and enzymatic process, this paper introduces various researches done mainly by the latter process. The enzymatic process is simpler and safer than the chemical process, allowing experiments to be carried out easily. However, since the reaction equilibrium is biased more on the decomposition side than the synthesis side, it should be shifted to the synthesis side. Such operations may be carried out as separating the products simultaneously with the reaction, adding organic solvents, and operating the reaction in an organic solvent. The last operation, for example, reduces the concentration of liquid produced by the reaction by use of either water soluble or slightly soluble organic solvent, and improves the yield. It also has an advantage that relatively stable fixing enzymes can be adjusted by use of hydrophilic carriers. 68 refs., 8 figs.

  20. Functions of phenylalanine residues within the beta-barrel stem of the anthrax toxin pore.

    Directory of Open Access Journals (Sweden)

    Jie Wang

    2009-07-01

    Full Text Available A key step of anthrax toxin action involves the formation of a protein-translocating pore within the endosomal membrane by the Protective Antigen (PA moiety. Formation of this transmembrane pore by PA involves interaction of the seven 2beta2-2beta3 loops of the heptameric precursor to generate a 14-strand transmembrane beta barrel.We examined the effects on pore formation, protein translocation, and cytotoxicity, of mutating two phenylalanines, F313 and F314, that lie at the tip the beta barrel, and a third one, F324, that lies part way up the barrel.Our results show that the function of these phenylalanine residues is to mediate membrane insertion and formation of stable transmembrane channels. Unlike F427, a key luminal residue in the cap of the pore, F313, F314, and F324 do not directly affect protein translocation through the pore. Our findings add to our knowledge of structure-function relationships of a key virulence factor of the anthrax bacillus.

  1. Influence of anoxia on the induction of mutations by phenylalanine radicals during gamma-irradiation of plasmid DNA in aqueous solution.

    Science.gov (United States)

    Kuipers, Gitta K; Slotman, Ben J; Reitsma-Wijker, Carola A; van Andel, Rob J; Poldervaart, Hester A; Lafleur, M Vincent M

    2004-12-21

    When DNA is irradiated in aqueous solution, most of the damage is inflicted by water-derived radicals. This is called the indirect effect of ionizing radiation. However in whole cells not only the primary formed water radicals play a role, because some cellular compounds form secondary radicals which can also damage DNA. It is known that the amino acid phenylalanine is able to react with water radicals, resulting in the production of secondary phenylalanine radicals which can damage and inactivate DNA. In a previous study the influence of the presence of phenylalanine during gamma-irradiation of DNA in aqueous solution under oxic conditions was studied. Under anoxic irradiation conditions different amounts and types of reactive water-derived radicals are formed compared to oxic conditions and also different phenylalanine radicals are formed. Therefore, this study examines the influence of the presence of phenylalanine under anoxic conditions on the gamma-radiation-induced mutation spectrum. The results indicate that phenylalanine radicals are damaging to DNA, but less effective compared to primary water radicals. On the mutational level, in the presence of phenylalanine radicals under anoxic conditions, the amount of mutations on G:C base pairs was significantly decreased as compared to oxic conditions. Furthermore, the results of this study indicate that nucleotide excision repair is involved in repair of both inactivating and mutagenic damage induced by phenylalanine radicals under anoxic conditions.

  2. Quantitation of deuterated and non-deuterated phenylalanine and tyrosine in human plasma using the selective ion monitoring method with combined gas chromatography-mass spectrometry

    International Nuclear Information System (INIS)

    Zagalak, M.-J.; Curtius, H.-Ch.; Leimbacher, W.; Redweik, U.

    1977-01-01

    A specific method is described for the quantitative analysis of deutarated and non-deuterated phenylalanine and tyrosine in human plasma by gas chromatography-mass spectrometry using selective ion monitoring. From the several derivatives investigated, the N- or N,O-trifluoroacetyl methyl esters were found to be the most suitable for our purposes. DL-Phenylalanine-4-d 1 and L-tyrosine-d 7 were used as internal standards. The sensitivity of this method permits the measurement of amounts as small as ca. 2.5 ng/ml in plasma for both phenylalanine and tyrosine. The coefficients of variation were found to be ca. 1.6% (n=12) for phenylalanine and 3.0% (n=12) for tyrosine. Using this method, an in vivo determination of phenylalanine-4-monooxygenase activity in humans is possible by loading the subjects with deuterated L-phenylalanine-d 5 (accepted as substrate by phenylalanine-4-monooxygenase E.C. 1.14.16.1) and the subsequent measuring of deuterated L-tyrosine-d 4 formed and residual L-phenylalanine-d 5

  3. Clustered regularly interspaced short palindromic repeats (CRISPRs): the hallmark of an ingenious antiviral defense mechanism in prokaryotes

    NARCIS (Netherlands)

    Al-Attar, S.; Westra, E.R.; Oost, van der J.; Brouns, S.J.J.

    2011-01-01

    Many prokaryotes contain the recently discovered defense system against mobile genetic elements. This defense system contains a unique type of repetitive DNA stretches, termed Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs). CRISPRs consist of identical repeated DNA sequences

  4. Footprints of Optimal Protein Assembly Strategies in the Operonic Structure of Prokaryotes

    Directory of Open Access Journals (Sweden)

    Jan Ewald

    2015-04-01

    Full Text Available In this work, we investigate optimality principles behind synthesis strategies for protein complexes using a dynamic optimization approach. We show that the cellular capacity of protein synthesis has a strong influence on optimal synthesis strategies reaching from a simultaneous to a sequential synthesis of the subunits of a protein complex. Sequential synthesis is preferred if protein synthesis is strongly limited, whereas a simultaneous synthesis is optimal in situations with a high protein synthesis capacity. We confirm the predictions of our optimization approach through the analysis of the operonic organization of protein complexes in several hundred prokaryotes. Thereby, we are able to show that cellular protein synthesis capacity is a driving force in the dissolution of operons comprising the subunits of a protein complex. Thus, we also provide a tested hypothesis explaining why the subunits of many prokaryotic protein complexes are distributed across several operons despite the presumably less precise co-regulation.

  5. SURVIVAL AND EVOLUTION OF CRISPR-CAS SYSTEM IN PROKARYOTES AND ITS APPLICATIONS

    Directory of Open Access Journals (Sweden)

    Muhammad Abu Bakr Shabbir

    2016-09-01

    Full Text Available Prokaryotes have developed numerous innate immune mechanisms in order to fend off bacteriophage or plasmid attack. One of these immune systems is Clustered regularly interspaced short palindromic repeats (CRISPR. CRISPR associated proteins play a key role in survival of prokaryotes against invaders, as these systems cleave DNA of foreign genetic elements. Beyond providing immunity, these systems have significant impact in altering the bacterial physiology in term of its virulence and pathogenicity, as well as evolution. Also, due to their diverse nature of functionality, cas9 endoribonuclease can be easily reprogrammed with the help of guide RNAs, showing unprecedented potential and significance for gene editing in treating genetic diseases. Here, we also discuss the use of NgAgo-gDNA system in genome editing of human cells.

  6. Survival and Evolution of CRISPR–Cas System in Prokaryotes and Its Applications

    Science.gov (United States)

    Shabbir, Muhammad Abu Bakr; Hao, Haihong; Shabbir, Muhammad Zubair; Hussain, Hafiz Iftikhar; Iqbal, Zahid; Ahmed, Saeed; Sattar, Adeel; Iqbal, Mujahid; Li, Jun; Yuan, Zonghui

    2016-01-01

    Prokaryotes have developed numerous innate immune mechanisms in order to fend off bacteriophage or plasmid attack. One of these immune systems is clustered regularly interspaced short palindromic repeats (CRISPR). CRISPR-associated proteins play a key role in survival of prokaryotes against invaders, as these systems cleave DNA of foreign genetic elements. Beyond providing immunity, these systems have significant impact in altering the bacterial physiology in term of its virulence and pathogenicity, as well as evolution. Also, due to their diverse nature of functionality, cas9 endoribonuclease can be easily reprogrammed with the help of guide RNAs, showing unprecedented potential and significance for gene editing in treating genetic diseases. Here, we also discuss the use of NgAgo–gDNA system in genome editing of human cells. PMID:27725818

  7. [Description of the phylogenetic structure of hydrolytic prokaryotic complex in the soils].

    Science.gov (United States)

    Lukacheva, E G; Chernov, T I; Bykova, E M; Vlasenko, A N; Manucharova, N A

    2013-01-01

    With the help of the molecular-biological method of cell hybridization in situ (FISH), the abundance of a physiologically active hydrolytic prokaryotic complex in chernozem and gley-podzolic soils is determined. The total proportion of metabolically active cells, which were detected by hybridization with universal probes as representatives of the domains Bacteria and Archaea, in samples of the studied soil, was from 38% for chernozem up to 78% for gley-podzolic soil of the total number of cells. The differences in the structure of chitinolytic and pectinolytic prokaryotic soil complexes are detected. Along with the high abundance of Actinobacteria and Firmicutes in the soils with chitin, an increase in phylogenetic groups such as Alphaproteobacteria and Bacteroidetes is observed.

  8. ConSpeciFix: Classifying prokaryotic species based on gene flow.

    Science.gov (United States)

    Bobay, Louis-Marie; Ellis, Brian Shin-Hua; Ochman, Howard

    2018-05-16

    Classification of prokaryotic species is usually based on sequence similarity thresholds, which are easy to apply but lack a biologically-relevant foundation. Here, we present ConSpeciFix, a program that classifies prokaryotes into species using criteria set forth by the Biological Species Concept, thereby unifying species definition in all domains of life. ConSpeciFix's webserver is freely available at www.conspecifix.com. The local version of the program can be freely downloaded from https://github.com/Bobay-Ochman/ConSpeciFix. ConSpeciFix is written in Python 2.7 and requires the following dependencies: Usearch, MCL, MAFFT and RAxML. ljbobay@uncg.edu.

  9. Abundance and distribution of the highly iterated palindrome 1 (HIP1) among prokaryotes

    OpenAIRE

    Delaye, Luis; Moya, Andrés

    2011-01-01

    We have studied the abundance and phylogenetic distribution of the Highly Iterated Palindrome 1 (HIP1) among sequenced prokaryotic genomes. We show that an overrepresentation of HIP1 is exclusive of some lineages of cyanobacteria, and that this abundance was gained only once during evolution and was subsequently lost in the lineage leading to marine pico-cyanobacteria. We show that among cyanobacterial protein sequences with annotated Pfam domains, only OpcA (glucose 6-phosphate dehydrogenase...

  10. Biodiversity of prokaryotic communities associated with the ectoderm of Ectopleura crocea (Cnidaria, Hydrozoa.

    Directory of Open Access Journals (Sweden)

    Cristina Gioia Di Camillo

    Full Text Available The surface of many marine organisms is colonized by complex communities of microbes, yet our understanding of the diversity and role of host-associated microbes is still limited. We investigated the association between Ectopleura crocea (a colonial hydroid distributed worldwide in temperate waters and prokaryotic assemblages colonizing the hydranth surface. We used, for the first time on a marine hydroid, a combination of electron and epifluorescence microscopy and 16S rDNA tag pyrosequencing to investigate the associated prokaryotic diversity. Dense assemblages of prokaryotes were associated with the hydrant surface. Two microbial morphotypes were observed: one horseshoe-shaped and one fusiform, worm-like. These prokaryotes were observed on the hydrozoan epidermis, but not in the portions covered by the perisarcal exoskeleton, and their abundance was higher in March while decreased in late spring. Molecular analyses showed that assemblages were dominated by Bacteria rather than Archaea. Bacterial assemblages were highly diversified, with up to 113 genera and 570 Operational Taxonomic Units (OTUs, many of which were rare and contributed to <0.4%. The two most abundant OTUs, likely corresponding to the two morphotypes present on the epidermis, were distantly related to Comamonadaceae (genus Delftia and to Flavobacteriaceae (genus Polaribacter. Epibiontic bacteria were found on E. crocea from different geographic areas but not in other hydroid species in the same areas, suggesting that the host-microbe association is species-specific. This is the first detailed report of bacteria living on the hydrozoan epidermis, and indeed the first study reporting bacteria associated with the epithelium of E. crocea. Our results provide a starting point for future studies aiming at clarifying the role of this peculiar hydrozoan-bacterial association.

  11. Particle-association lifestyle is a phylogenetically conserved trait in bathypelagic prokaryotes

    KAUST Repository

    Salazar, Guillem; Cornejo-Castillo, Francisco M.; Borrull, Encarna; Dí ez-Vives, Cristina; Lara, Elena; Vaqué , Dolors; Arrieta, J M; Duarte, Carlos M.; Gasol, Josep M.; Acinas, Silvia G.

    2015-01-01

    The free-living (FL) and particle-attached (PA) marine microbial communities have repeatedly been proved to differ in their diversity and composition in the photic ocean and also recently in the bathypelagic ocean at a global scale. However, although high taxonomic ranks exhibit preferences for a PA or FL mode of life, it remains poorly understood whether two clear lifestyles do exist and how these are distributed across the prokaryotic phylogeny. We studied the FL (<0.8 μm) and PA (0.8 – 20 μm) prokaryotes at 30 stations distributed worldwide within the bathypelagic oceanic realm (2,150 – 4,000 m depth) using high throughput sequencing of the small subunit ribosomal RNA gene (16S rRNA). A high proportion of the bathypelagic prokaryotes were mostly found either attached to particles or freely in the surrounding water but rarely in both types of environments. In particular, this trait was deeply conserved through their phylogeny suggesting that the deep-ocean particles and the surrounding water constitute two highly distinct niches and that transitions from one to the other have been rare at an evolutionary time-scale. As a consequence, PA and FL communities had clear alpha- and beta-diversity differences that exceeded the global-scale geographical variation. Our study organizes the bathypelagic prokaryotic diversity into a reasonable number of ecologically coherent taxa regarding their association to particles, a first step for understanding which are the microbes responsible for the processing of the dissolved and particulate pools of organic matter that have a very different biogeochemical role in the deep ocean.

  12. SIS: a program to generate draft genome sequence scaffolds for prokaryotes

    Directory of Open Access Journals (Sweden)

    Dias Zanoni

    2012-05-01

    Full Text Available Abstract Background Decreasing costs of DNA sequencing have made prokaryotic draft genome sequences increasingly common. A contig scaffold is an ordering of contigs in the correct orientation. A scaffold can help genome comparisons and guide gap closure efforts. One popular technique for obtaining contig scaffolds is to map contigs onto a reference genome. However, rearrangements that may exist between the query and reference genomes may result in incorrect scaffolds, if these rearrangements are not taken into account. Large-scale inversions are common rearrangement events in prokaryotic genomes. Even in draft genomes it is possible to detect the presence of inversions given sufficient sequencing coverage and a sufficiently close reference genome. Results We present a linear-time algorithm that can generate a set of contig scaffolds for a draft genome sequence represented in contigs given a reference genome. The algorithm is aimed at prokaryotic genomes and relies on the presence of matching sequence patterns between the query and reference genomes that can be interpreted as the result of large-scale inversions; we call these patterns inversion signatures. Our algorithm is capable of correctly generating a scaffold if at least one member of every inversion signature pair is present in contigs and no inversion signatures have been overwritten in evolution. The algorithm is also capable of generating scaffolds in the presence of any kind of inversion, even though in this general case there is no guarantee that all scaffolds in the scaffold set will be correct. We compare the performance of sis, the program that implements the algorithm, to seven other scaffold-generating programs. The results of our tests show that sis has overall better performance. Conclusions sis is a new easy-to-use tool to generate contig scaffolds, available both as stand-alone and as a web server. The good performance of sis in our tests adds evidence that large

  13. Particle-association lifestyle is a phylogenetically conserved trait in bathypelagic prokaryotes

    KAUST Repository

    Salazar, Guillem

    2015-10-13

    The free-living (FL) and particle-attached (PA) marine microbial communities have repeatedly been proved to differ in their diversity and composition in the photic ocean and also recently in the bathypelagic ocean at a global scale. However, although high taxonomic ranks exhibit preferences for a PA or FL mode of life, it remains poorly understood whether two clear lifestyles do exist and how these are distributed across the prokaryotic phylogeny. We studied the FL (<0.8 μm) and PA (0.8 – 20 μm) prokaryotes at 30 stations distributed worldwide within the bathypelagic oceanic realm (2,150 – 4,000 m depth) using high throughput sequencing of the small subunit ribosomal RNA gene (16S rRNA). A high proportion of the bathypelagic prokaryotes were mostly found either attached to particles or freely in the surrounding water but rarely in both types of environments. In particular, this trait was deeply conserved through their phylogeny suggesting that the deep-ocean particles and the surrounding water constitute two highly distinct niches and that transitions from one to the other have been rare at an evolutionary time-scale. As a consequence, PA and FL communities had clear alpha- and beta-diversity differences that exceeded the global-scale geographical variation. Our study organizes the bathypelagic prokaryotic diversity into a reasonable number of ecologically coherent taxa regarding their association to particles, a first step for understanding which are the microbes responsible for the processing of the dissolved and particulate pools of organic matter that have a very different biogeochemical role in the deep ocean.

  14. New insights into the structural organization of eukaryotic and prokaryotic cytoskeletons using cryo-electron tomography

    International Nuclear Information System (INIS)

    Kuerner, Julia; Medalia, Ohad; Linaroudis, Alexandros A.; Baumeister, Wolfgang

    2004-01-01

    Cryo-electron tomography (cryo-ET) is an emerging imaging technology that combines the potential of three-dimensional (3-D) imaging at molecular resolution (<5 nm) with a close-to-life preservation of the specimen. In conjunction with pattern recognition techniques, it enables us to map the molecular landscape inside cells. The application of cryo-ET to intact cells provides novel insights into the structure and the spatial organization of the cytoskeleton in prokaryotic and eukaryotic cells

  15. Colorimetric sensor for triphosphates and their application as a viable staining agent for prokaryotes and eukaryotes.

    Science.gov (United States)

    Ghosh, Amrita; Shrivastav, Anupama; Jose, D Amilan; Mishra, Sanjiv K; Chandrakanth, C K; Mishra, Sandhya; Das, Amitava

    2008-07-15

    The chromogenic complex 1 x Zn (where 1 is (E)-4-(4-dimethylamino-phenylazo)-N,N-bispyridin-2-ylmethyl-benzenesulfonamide) showed high affinity toward the phosphate ion in tetrabutylammonium phosphate in acetonitrile solution and could preferentially bind to adenosine triphosphate (ATP) in aqueous solution at physiological pH. This binding caused a visual change in color, whereas no such change was noticed with other related anions (adenosine monophosphate, adenosine diphosphate, pyrophosphate, and phosphate) of biological significance. Thus, 1 x Zn could be used as a staining agent for different biological cells through binding to the ATP, generated in situ by the mitochondria (in eukaryotes). For prokaryotes (bacteria) the cell membrane takes care of the cells' energy conversion, since they lack mitochondria. ATP is produced in their unique cell structure on the cell membrane, which is not found in any eukaryotes. These stained cells could be viewed with normal light microscopy. This reagent could even be used for distinguishing the gram-positive and the gram-negative bacteria (prokaryotes). This dye was found to be nonlipophilic in nature and nontoxic to living microbes (eukaryotes and prokaryotes). Further, stained cells were found to grow in their respective media, and this confirmed the maintenance of viability of the microbes even after staining, unlike with many other dyes available commercially.

  16. The current status of cyanobacterial nomenclature under the "prokaryotic" and the "botanical" code.

    Science.gov (United States)

    Oren, Aharon; Ventura, Stefano

    2017-10-01

    Cyanobacterial taxonomy developed in the botanical world because Cyanobacteria/Cyanophyta have traditionally been identified as algae. However, they possess a prokaryotic cell structure, and phylogenetically they belong to the Bacteria. This caused nomenclature problems as the provisions of the International Code of Nomenclature for algae, fungi, and plants (ICN; the "Botanical Code") differ from those of the International Code of Nomenclature of Prokaryotes (ICNP; the "Prokaryotic Code"). While the ICN recognises names validly published under the ICNP, Article 45(1) of the ICN has not yet been reciprocated in the ICNP. Different solutions have been proposed to solve the current problems. In 2012 a Special Committee on the harmonisation of the nomenclature of Cyanobacteria was appointed, but its activity has been minimal. Two opposing proposals to regulate cyanobacterial nomenclature were recently submitted, one calling for deletion of the cyanobacteria from the groups of organisms whose nomenclature is regulated by the ICNP, the second to consistently apply the rules of the ICNP to all cyanobacteria. Following a general overview of the current status of cyanobacterial nomenclature under the two codes we present five case studies of genera for which nomenclatural aspects have been discussed in recent years: Microcystis, Planktothrix, Halothece, Gloeobacter and Nostoc.

  17. Non-Random Inversion Landscapes in Prokaryotic Genomes Are Shaped by Heterogeneous Selection Pressures.

    Science.gov (United States)

    Repar, Jelena; Warnecke, Tobias

    2017-08-01

    Inversions are a major contributor to structural genome evolution in prokaryotes. Here, using a novel alignment-based method, we systematically compare 1,651 bacterial and 98 archaeal genomes to show that inversion landscapes are frequently biased toward (symmetric) inversions around the origin-terminus axis. However, symmetric inversion bias is not a universal feature of prokaryotic genome evolution but varies considerably across clades. At the extremes, inversion landscapes in Bacillus-Clostridium and Actinobacteria are dominated by symmetric inversions, while there is little or no systematic bias favoring symmetric rearrangements in archaea with a single origin of replication. Within clades, we find strong but clade-specific relationships between symmetric inversion bias and different features of adaptive genome architecture, including the distance of essential genes to the origin of replication and the preferential localization of genes on the leading strand. We suggest that heterogeneous selection pressures have converged to produce similar patterns of structural genome evolution across prokaryotes. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  18. Comparative evaluation of prokaryotic 16S rDNA clone libraries and SSCP in groundwater samples.

    Science.gov (United States)

    Larentis, Michael; Alfreider, Albin

    2011-06-01

    A comparison of ribosomal RNA sequence analysis methods based on clone libraries and single-strand conformational polymorphism technique (SSCP) was performed with groundwater samples obtained between 523-555 meters below surface. The coverage of analyzed clones by phylotype-richness estimates was between 88-100%, confirming that the clone libraries were adequately examined. Analysis of individual bands retrieved from SSCP gels identified 1-6 different taxonomic units per band, suggesting that a single SSCP band does often represent more than one single prokaryotic species. The prokaryotic diversity obtained by both methods showed an overall difference of 42-80%. In comparison to SSCP, clone libraries underestimated the phylogenetic diversity and only 36-66% of the phylotypes observed with SSCP were also detected with the clone libraries. An exception was a sample where the SSCP analysis of Archaea identified only half of the phylotypes retrieved by the clone library. Overall, this study suggests that the clone library and the SSCP approach do not provide an identical picture of the prokaryotic diversity in groundwater samples. The results clearly show that the SSCP method, although this approach is prone to generate methodological artifacts, was able to detect significantly more phylotypes than microbial community analysis based on clone libraries. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Cellular Viscosity in Prokaryotes and Thermal Stability of Low Molecular Weight Biomolecules.

    Science.gov (United States)

    Cuecas, Alba; Cruces, Jorge; Galisteo-López, Juan F; Peng, Xiaojun; Gonzalez, Juan M

    2016-08-23

    Some low molecular weight biomolecules, i.e., NAD(P)H, are unstable at high temperatures. The use of these biomolecules by thermophilic microorganisms has been scarcely analyzed. Herein, NADH stability has been studied at different temperatures and viscosities. NADH decay increased at increasing temperatures. At increasing viscosities, NADH decay rates decreased. Thus, maintaining relatively high cellular viscosity in cells could result in increased stability of low molecular weight biomolecules (i.e., NADH) at high temperatures, unlike what was previously deduced from studies in diluted water solutions. Cellular viscosity was determined using a fluorescent molecular rotor in various prokaryotes covering the range from 10 to 100°C. Some mesophiles showed the capability of changing cellular viscosity depending on growth temperature. Thermophiles and extreme thermophiles presented a relatively high cellular viscosity, suggesting this strategy as a reasonable mechanism to thrive under these high temperatures. Results substantiate the capability of thermophiles and extreme thermophiles (growth range 50-80°C) to stabilize and use generally considered unstable, universal low molecular weight biomolecules. In addition, this study represents a first report, to our knowledge, on cellular viscosity measurements in prokaryotes and it shows the dependency of prokaryotic cellular viscosity on species and growth temperature. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  20. A Preliminary List of Horizontally Transferred Genes in Prokaryotes Determined by Tree Reconstruction and Reconciliation

    Directory of Open Access Journals (Sweden)

    Hyeonsoo Jeong

    2017-08-01

    Full Text Available Genome-wide global detection of genes involved in horizontal gene transfer (HGT remains an active area of research in medical microbiology and evolutionary genomics. Utilizing the explicit evolutionary method of comparing topologies of a total of 154,805 orthologous gene trees against corresponding 16S rRNA “reference” trees, we previously detected a total of 660,894 candidate HGT events in 2,472 completely-sequenced prokaryotic genomes. Here, we report an HGT-index for each individual gene-reference tree pair reconciliation, representing the total number of detected HGT events on the gene tree divided by the total number of genomes (taxa member of that tree. HGT-index is thus a simple measure indicating the sensitivity of prokaryotic genes to participate (or not participate in HGT. Our preliminary list provides HGT-indices for a total of 69,365 genes (detected in >10 and <50% available prokaryotic genomes that are involved in a wide range of biological processes such as metabolism, information, and bacterial response to environment. Identification of horizontally-derived genes is important to combat antibiotic resistance and is a step forward toward reconstructions of improved phylogenies describing the history of life. Our effort is thus expected to benefit ongoing research in the fields of clinical microbiology and evolutionary biology.

  1. Prokaryotic diversity and community composition in the Salar de Uyuni, a large scale, chaotropic salt flat.

    Science.gov (United States)

    dC Rubin, Sergio S; Marín, Irma; Gómez, Manuel J; Morales, Eduardo A; Zekker, Ivar; San Martín-Uriz, Patxi; Rodríguez, Nuria; Amils, Ricardo

    2017-09-01

    Salar de Uyuni (SdU), with a geological history that reflects 50 000 years of climate change, is the largest hypersaline salt flat on Earth and is estimated to be the biggest lithium reservoir in the world. Its salinity reaches saturation levels for NaCl, a kosmotropic salt, and high concentrations of MgCL 2 and LiCl, both salts considered important chaotrophic stressors. In addition, extreme temperatures, anoxic conditions, high UV irradiance, high albedo and extremely low concentrations of phosphorous, make SdU a unique natural extreme environment in which to contrast hypotheses about limiting factors of life diversification. Geophysical studies of brines from different sampling stations show that water activity is rather constant along SdU. Geochemical measurements show significant differences in magnesium concentration, ranging from 0.2 to 2M. This work analyses the prokaryotic diversity and community structure at four SdU sampling stations, selected according to their location and ionic composition. Prokaryotic communities were composed of both Archaea (with members of the classes Halobacteria, Thermoplasmata and Nanohaloarchaea, from the Euryarchaeota and Nanohaloarcheota phyla respectively) and Bacteria (mainly belonging to Bacteroidetes and Proteobacteria phyla). The important differences in composition of microbial communities inversely correlate with Mg 2+ concentration, suggesting that prokaryotic diversity at SdU is chaotropic dependent. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  2. Differences in Prokaryotic Species Between Primary and Logged-Over Deep Peat Forest in Sarawak, Malaysia

    International Nuclear Information System (INIS)

    Mohd Shawal Thakib Maidin; Sakinah Safari; Nur Aziemah Ghani; Sharifah Azura Syed Ibrahim; Shamsilawani Ahamed Bakeri; Mohamed Mazmira Mohd Masri; Siti Ramlah Ahmad Ali

    2016-01-01

    Peat land has an important role in environmental sustainability which can be used for agricultural purposes. However, deforestation in the logged-over forest may disrupt the diversity of microbial population in peat soil. Therefore, this study focuses on the differences of microbial populations in Maludam primary forest and Cermat Ceria logged-over forest in Sarawak, Malaysia. The prokaryotic 16S rDNA region was amplified followed by denaturing gradient gel electrophoresis (16S PCR-DGGE) analysis. Berger-Parker and Shannon-Weaver Biodiversity Index showed that Maludam (0.11, 7.75) was more diverse compared to Cermat Ceria (0.19, 7.63). Sequence analysis showed that the bacterial community in Maludam and Cermat Ceria were dominated by unclassified bacteria, followed by Acidobacteria, Actinobacteria, Firmicutes and a-Proteobacteria. Based on the findings, the distinct species that can be found in Maludam were Acidobacterium capsulatum, Solibacter sp., Mycobacterium intracellulare, Rhodoplanes sp., Clostridia bacterium, Exiguobacterium sp. and Lysinibacillus fusiformis. While, the distinct species that can be found in Cermat Ceria were Telmatobacter, Mycobacterium tuberculosis and Bacillus tequilensis. Overall, the findings showed that microbial population in the logged-over forest are less diverse compared to primary forest. Higher prokaryotic diversity identified in the primary forest compared to logged-over forest showed that deforestation might cause prokaryotic population changes to both ecosystems. (author)

  3. Spatiotemporal and species variations in prokaryotic communities associated with sediments from surface-flow constructed wetlands for treating swine wastewater.

    Science.gov (United States)

    Jia, Fen; Lai, Cui; Chen, Liang; Zeng, Guangming; Huang, Danlian; Liu, Feng; Li, Xi; Luo, Pei; Wu, Jinshui; Qin, Lei; Zhang, Chen; Cheng, Min; Xu, Piao

    2017-10-01

    Microorganisms are the main mechanisms of pollutants removals in constructed wetlands (CWs) used for wastewater treatment. However, the different biological processes and variations of prokaryotic community in CWs remain poorly understood. In this study, we applied a high-throughput sequencing technique to investigate the prokaryotic communities associated with sediments from pilot-scale surface-flow constructed wetlands (SFCWs) treating swine wastewater (SW) of varying strengths. Our results revealed that highly diverse prokaryotic communities were present in the SFCWs, with Proteobacteria (16.44-44.44%), Acidobacteria (3.25-24.40%), and Chloroflexi (5.77-14.43%) being the major phyla, and Nitrospira (4.14-12.02%), the most dominant genus. The prokaryotic communities in the sediments varied greatly with location and season, which markedly altered the microenvironmental conditions. Principal co-ordinates analysis indicated that SW strength significantly influenced the community structure in sediments of the SFCWs, and canonical correspondence analysis illustrated that the shifts in prokaryotic communities were strongly related to NO 3 - -N and TN in winter; and in summer with NH 4 + N, NO 3 - -N, NO 2 - -N, TN, TP, SOM, and pH. In conclusion, the use of high-throughput sequencing greatly enhanced our understanding of prokaryotic communities with different functional groups in SFCWs. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Molecular fossils of prokaryotes in ancient authigenic minerals: archives of microbial activity in reefs and mounds?

    Science.gov (United States)

    Heindel, Katrin; Birgel, Daniel; Richoz, Sylvain; Westphal, Hildegard; Peckmann, Jörn

    2016-04-01

    Molecular fossils (lipid biomarkers) are commonly used as proxies in organic-rich sediments of various sources, including eukaryotes and prokaryotes. Usually, molecular fossils of organisms transferred from the water column to the sediment are studied to monitor environmental changes (e.g., temperature, pH). Apart from these 'allochthonous' molecular fossils, prokaryotes are active in sediments and mats on the seafloor and leave behind 'autochthonous' molecular fossils in situ. In contrast to many phototrophic organisms, most benthic sedimentary prokaryotes are obtaining their energy from oxidation or reduction of organic or inorganic substrates. A peculiarity of some of the sediment-thriving prokaryotes is their ability to trigger in situ mineral precipitation, often but not only due to metabolic activity, resulting in authigenic rocks (microbialites). During that process, prokaryotes are rapidly entombed in the mineral matrix, where the molecular fossils are protected from early (bio)degradation. In contrast to other organic compounds (DNA, proteins etc.), molecular fossils can be preserved over very long time periods (millions of years). Thus, molecular fossils in authigenic mineral phases are perfectly suitable to trace microbial activity back in time. Among the best examples of molecular fossils, which are preserved in authigenic rocks are various microbialites, forming e.g. in phototrophic microbial mats and at cold seeps. Microbialite formation is reported throughout earth history. We here will focus on reefal microbialites form the Early Triassic and the Holocene. After the End-Permian mass extinction, microbialites covered wide areas on the ocean margins. In microbialites from the Griesbachian in Iran and Turkey (both Neotethys), molecular fossils of cyanobacteria, archaea, anoxygenic phototrophs, and sulphate-reducing bacteria indicate the presence of layered microbial mats on the seafloor, in which carbonate precipitation was induced. In association with

  5. Ca2+ response in neutrophils after exposure to bacterial N-formyl-methionyl-leucyl-phenylalanine: delayed response in ulcerative colitis

    DEFF Research Database (Denmark)

    Vainer, Ben; Lamberth, Kasper; Brimnes, Jens

    2003-01-01

    In acute stages of ulcerative colitis (UC), neutrophils migrate from the circulation into inflamed colonic tissue, initiated by yet unknown stimuli. The bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) is a component of the surface membrane of colonic bacteria such as Escherichia ...

  6. Abnormal tyrosine and phenylalanine metabolism in patients with tyrosyluria and phenylketonuria; gas-liquid chromatographic analysis of urinary metabolites

    NARCIS (Netherlands)

    Wadman, S.K.; Heiden, C. van der; Ketting, D.; Sprang, F.J. van

    Gas-liquid chromatographic methods have been developed for the analysis of: urinary phenylalanine metabolites (I) in patients with phenylketonuria, tyrosine metabolites (II) in patients with a disturbed tyrosine metabolism at the level of p-hydroxyphenylpyruvate hydroxylase, and homogentisic acid in

  7. Hydrolyses of alpha-naphthyl acetate, beta-naphthyl acetate, and acetyl-DL-phenylalanine beta-naphthyl ester

    DEFF Research Database (Denmark)

    Kirkeby, S; Moe, D

    1983-01-01

    Using simultaneous coupling azo dye techniques kidney enzymes active against alpha-naphthyl acetate, beta-naphthyl acetate, and acetyl-DL-phenylalanine beta-naphthyl ester are characterized. The enzymes show identical distribution in the section. The banding patterns in zymograms are the same after...

  8. Design, synthesis and structure-activity relationships of novel phenylalanine-based amino acids as kainate receptors ligands

    DEFF Research Database (Denmark)

    Szymańska, Ewa; Chałupnik, Paulina; Szczepańska, Katarzyna

    2016-01-01

    A new series of carboxyaryl-substituted phenylalanines was designed, synthesized and pharmacologically characterized in vitro at native rat ionotropic glutamate receptors as well as at cloned homomeric kainate receptors GluK1-GluK3. Among them, six compounds bound to GluK1 receptor subtypes with ...

  9. Experimental and theoretical NMR studies of interaction between phenylalanine derivative and egg yolk lecithin.

    Science.gov (United States)

    Wałęsa, Roksana; Ptak, Tomasz; Siodłak, Dawid; Kupka, Teobald; Broda, Małgorzata A

    2014-06-01

    The interaction of phenylalanine diamide (Ac-Phe-NHMe) with egg yolk lecithin (EYL) in chloroform was studied by (1)H and (13)C NMR. Six complexes EYL-Ac-Phe-NHMe, stabilized by N-H···O or/and C-H···O hydrogen bonds, were optimized at M06-2X/6-31G(d,p) level. The assignment of EYL and Ac-Phe-NHMe NMR signals was supported using GIAO (gauge including atomic orbital) NMR calculations at VSXC and B3LYP level of theory combined with STO-3Gmag basis set. Results of our study indicate that the interaction of peptides with lecithin occurs mainly in the polar 'head' of the lecithin. Additionally, the most probable lecithin site of H-bond interaction with Ac-Phe-NHMe is the negatively charged oxygen in phosphate group that acts as proton acceptor. Copyright © 2014 John Wiley & Sons, Ltd.

  10. Sequence variation at the phenylalanine hydroxylase gene in the British Isles

    Energy Technology Data Exchange (ETDEWEB)

    Tyfield, L.A. [Southmead Hospital, Bristol (United Kingdom)]|[Univ. of Bristol (United Kingdom); Stephenson, A. [Southmead Hospital, Bristol (United Kingdom); Cockburn, F. [Royal Hospital for Sick Children, Glasgow (United Kingdom)] [and others

    1997-02-01

    Using mutation and haplotype analysis, we have examined the phenylalanine hydroxylase gene in the phenylketonuria populations of four geographical areas of the British Isles: the west of Scotland, southern Wales, and southwestern and southeastern England. The enormous genetic diversity of this locus within the British Isles is demonstrated in the large number of different mutations characterized and in the variety of genetic backgrounds on which individual mutations are found. Allele frequencies of the more common mutations exhibited significant nonrandom distribution in a north/south differentiation. Differences between the west of Scotland and southwestern England may be related to different events in the recent and past histories of their respective populations. Similarities between southern Wales and southeastern England are likely to reflect the heterogeneity that is seen in and around two large capital cities. Finally, comparison with more recently colonized areas of the world corroborates the genealogical origin by range expansion of several mutations. 38 refs., 2 tabs.

  11. Metal-Ion-Mediated Supramolecular Chirality of l-Phenylalanine Based Hydrogels.

    Science.gov (United States)

    Wang, Fang; Feng, Chuan-Liang

    2018-05-14

    For chiral hydrogels and related applications, one of the critical issues is how to control the chirality of supramolecular systems in an efficient way, including easy operation, efficient transfer of chirality, and so on. Herein, supramolecular chirality of l-phenylalanine based hydrogels can be effectively controlled by using a broad range of metal ions. The degree of twisting (twist pitch) and the diameter of the chiral nanostructures can also be efficiently regulated. These are ascribed to the synergic effect of hydrogen bonding and metal ion coordination. This study may develop a method to design a new class of electronically, optically, and biologically active materials. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Preparation of carbon-11 labelled phenylalanine and phenylglycine by a new amino acid synthesis

    International Nuclear Information System (INIS)

    Vaalburg, W.; Beerling-van der Molen, H.D.; Reiffers, S; Rijskamp, A.; Woldring, M.G.; Wynberg, H.

    1976-01-01

    Of the cyclotron-produced short-lived isotopes carbon-11 (tsub(1/2) = 20.4 min;β + ) is one of the most promising as label for radiopharmaceuticals. To prepare 11 C-labelled amino acids for evaluation as pancreas scanning agents a new rapid amino acid synthesis was developed. The method is based on the carboxylation of α-lithioisocyanides with 11 CO 2 , followed by hydrolysis of the intermediate reaction product to the desired amino acid. By this method DL-α-phenylalanine-1- 11 C and DL-α-phenylglycine-1- 11 C were prepared. The precursor 11 CO 2 was produced via the 14 N(p,α) 11 C reaction by bombardment of a flow of nitrogen gas mixed with 0.1% 0 2 with 20 MeV protons. The target system is described. (author)

  13. Synthesis of racemic [3-11C]phenylalanine and [3-11C] DOPA

    International Nuclear Information System (INIS)

    Halldin, C.; Laangstroem, B.

    1984-01-01

    The synthesis of racemic [3- 11 C]phenylalanine and [3- 11 C]DOPA is reported. The [ 11 C]benzaldehyde and [ 11 C]veratraldehyde prepared in a two-step reaction from the corresponding [ 11 C]acid salt and [ 11 C]alcohol, by means of selective oxidation with tetrabutylammonium hydrogen chromate, were reacted with 2-phenyl-5-oxazolone or 2-(4-chloro)phenyl-5-oxazolone in the presence of a tertiary amine to give the corresponding [α- 11 C]-4-arylene-2-aryl-5-oxazolones. Ring opening of these olefins, hydrogenation, and removal of protecting groups was carried out in one step using hydroiodic cid/phosphorus, with the production of the racemic [3- 11 C]amino acids in 8-30% radiochemical yield (starting with 11 CO 2 ) within 52-60 min (including LC separation). (author)

  14. Exploring the biological consequences of conformational changes in aspartame models containing constrained analogues of phenylalanine.

    Science.gov (United States)

    Mollica, Adriano; Mirzaie, Sako; Costante, Roberto; Carradori, Simone; Macedonio, Giorgia; Stefanucci, Azzurra; Dvoracsko, Szabolcs; Novellino, Ettore

    2016-12-01

    The dipeptide aspartame (Asp-Phe-OMe) is a sweetener widely used in replacement of sucrose by food industry. 2',6'-Dimethyltyrosine (DMT) and 2',6'-dimethylphenylalanine (DMP) are two synthetic phenylalanine-constrained analogues, with a limited freedom in χ-space due to the presence of methyl groups in position 2',6' of the aromatic ring. These residues have shown to increase the activity of opioid peptides, such as endomorphins improving the binding to the opioid receptors. In this work, DMT and DMP have been synthesized following a diketopiperazine-mediated route and the corresponding aspartame derivatives (Asp-DMT-OMe and Asp-DMP-OMe) have been evaluated in vivo and in silico for their activity as synthetic sweeteners.

  15. The adsorption of L-phenylalanine on oxidized single-walled carbon nanotubes.

    Science.gov (United States)

    Piao, Lingyu; Liu, Quanrun; Li, Yongdan; Wang, Chen

    2009-02-01

    A simple and green approach was proceeded to obtain a stable single-walled carbon nanotubes (SWNTs)/L-phenylalanine (Phe) solution. The oxidized SWNTs (OSWNT) were used in this work. The scanning electron microscopy (SEM), High-resolution transmission electron microscopy (HRTEM), Raman spectrometer, Fourier transform-infrared resonance (FT-IR), Ultraviolet-visible (UV-vis) spectroscopy, Thermogravimetric analysis (TGA) and High performance liquid chromatography (HPLC) were joined together to investigate the interaction between OSWNT and Phe. The OSWNT became soluble in the water and formed a stable solution since the Phe was adsorbed. The absorbed amount of Phe on the OSWNT is around 33 wt%. Adsorption of the Phe was mainly carried out on the OSWNT with smaller diameters. The Phe molecules were absorbed on the OSWNT by conjunct interaction of the pi-pi stacking, hydrogen bond and part of covalent bond.

  16. Three novel variants (p.Glu178Lys, p.Val245Met, p.Ser250Phe) of the phenylalanine hydroxylase (PAH) gene impair protein expression and function in vitro.

    Science.gov (United States)

    Zong, Yanan; Liu, Ning; Ma, Shanshan; Bai, Ying; Guan, Fangxia; Kong, Xiangdong

    2018-08-20

    Phenylketonuria (PKU) is the most common inherited metabolic disease, an autosomal recessive disorder affecting >10,000 newborns each year globally. It can be caused by over 1000 different naturally occurring mutations in the phenylalanine hydroxylase (PAH) gene. We analyzed three novel naturally occurring PAH gene variants: p.Glu178Lys (c.532G>A), p.Val245Met (c.733G>A) and p.Ser250Phe (c.749C>T). The mutant effect on the PAH enzyme structure and function was predicted by bioinformatics software. Vectors expressing the corresponding PAH variants were generated for expression in E. coli and in HEK293T cells. The RNA expression of the three PAH variants was measured by quantitative reverse transcription polymerase chain reaction (RT-qPCR). The mutant PAH protein levels were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot and enzyme-linked immunosorbent assay (ELISA). All three variants were predicted to be pathogenic by bioinformatics analysis. The transcription of the three PAH variants was similar to the wild type PAH gene in HEK293T cells. In contrast, the levels of mutant PAH proteins decreased significantly compared to the wild type control, in both E. coli and HEK293T cells. Our results indicate that the three novel PAH gene variants (p.Glu178Lys, p.Val245Met, p.Ser250Phe) impair PAH protein expression and function in prokaryotic and eukaryotic cells. Copyright © 2018. Published by Elsevier B.V.

  17. Acute phenylalanine/tyrosine depletion of phasic dopamine in the rat brain.

    Science.gov (United States)

    Shnitko, Tatiana A; Taylor, Sarah C; Stringfield, Sierra J; Zandy, Shannon L; Cofresí, Roberto U; Doherty, James M; Lynch, William B; Boettiger, Charlotte A; Gonzales, Rueben A; Robinson, Donita L

    2016-06-01

    Dopamine plays a critical role in striatal and cortical function, and depletion of the dopamine precursors phenylalanine and tyrosine is used in humans to temporarily reduce dopamine and probe the role of dopamine in behavior. This method has been shown to alter addiction-related behaviors and cognitive functioning presumably by reducing dopamine transmission, but it is unclear what specific aspects of dopamine transmission are altered. We performed this study to confirm that administration of an amino acid mixture omitting phenylalanine and tyrosine (Phe/Tyr[-]) reduces tyrosine tissue content in the prefrontal cortex (PFC) and nucleus accumbens (NAc), and to test the hypothesis that Phe/Tyr[-] administration reduces phasic dopamine release in the NAc. Rats were injected with a Phe/Tyr[-] amino acid mixture, a control amino acid mixture, or saline. High-performance liquid chromatography was used to determine the concentration of tyrosine, dopamine, or norepinephrine in tissue punches from the PFC and ventral striatum. In a separate group of rats, phasic dopamine release was measured with fast-scan cyclic voltammetry in the NAc core after injection with either the Phe/Tyr[-] mixture or the control amino acid solution. Phe/Tyr[-] reduced tyrosine content in the PFC and NAc, but dopamine and norepinephrine tissue content were not reduced. Moreover, Phe/Tyr[-] decreased the frequency of dopamine transients, but not their amplitude, in freely moving rats. These results indicate that depletion of tyrosine via Phe/Tyr[-] decreases phasic dopamine transmission, providing insight into the mechanism by which this method modifies dopamine-dependent behaviors in human imaging studies.

  18. Protein Based Molecular Markers Provide Reliable Means to Understand Prokaryotic Phylogeny and Support Darwinian Mode of Evolution

    Directory of Open Access Journals (Sweden)

    Vaibhav eBhandari

    2012-07-01

    Full Text Available The analyses of genome sequences have led to the proposal that lateral gene transfers (LGTs among prokaryotes are so widespread that they disguise the interrelationships among these organisms. This has led to questioning whether the Darwinian model of evolution is applicable to the prokaryotic organisms. In this review, we discuss the usefulness of taxon-specific molecular markers such as conserved signature indels (CSIs and conserved signature proteins (CSPs for understanding the evolutionary relationships among prokaryotes and to assess the influence of LGTs on prokaryotic evolution. The analyses of genomic sequences have identified large numbers of CSIs and CSPs that are unique properties of different groups of prokaryotes ranging from phylum to genus levels. The species distribution patterns of these molecular signatures strongly support a tree-like vertical inheritance of the genes containing these molecular signatures that is consistent with phylogenetic trees. Recent detailed studies in this regard on Thermotogae and Archaea, which are reviewed here, have identified large numbers of CSIs and CSPs that are specific for the species from these two taxa and a number of their major clades. The genetic changes responsible for these CSIs (and CSPs initially likely occurred in the common ancestors of these taxa and then vertically transferred to various descendants. Although some CSIs and CSPs in unrelated groups of prokaryotes were identified, their small numbers and random occurrence has no apparent influence on the consistent tree-like branching pattern emerging from other markers. These results provide evidence that although LGT is an important evolutionary force, it does not mask the tree-like branching pattern of prokaryotes or understanding of their evolutionary relationships. The identified CSIs and CSPs also provide novel and highly specific means for identification of different groups of microbes and for taxonomical and biochemical

  19. Protein based molecular markers provide reliable means to understand prokaryotic phylogeny and support Darwinian mode of evolution.

    Science.gov (United States)

    Bhandari, Vaibhav; Naushad, Hafiz S; Gupta, Radhey S

    2012-01-01

    The analyses of genome sequences have led to the proposal that lateral gene transfers (LGTs) among prokaryotes are so widespread that they disguise the interrelationships among these organisms. This has led to questioning of whether the Darwinian model of evolution is applicable to prokaryotic organisms. In this review, we discuss the usefulness of taxon-specific molecular markers such as conserved signature indels (CSIs) and conserved signature proteins (CSPs) for understanding the evolutionary relationships among prokaryotes and to assess the influence of LGTs on prokaryotic evolution. The analyses of genomic sequences have identified large numbers of CSIs and CSPs that are unique properties of different groups of prokaryotes ranging from phylum to genus levels. The species distribution patterns of these molecular signatures strongly support a tree-like vertical inheritance of the genes containing these molecular signatures that is consistent with phylogenetic trees. Recent detailed studies in this regard on the Thermotogae and Archaea, which are reviewed here, have identified large numbers of CSIs and CSPs that are specific for the species from these two taxa and a number of their major clades. The genetic changes responsible for these CSIs (and CSPs) initially likely occurred in the common ancestors of these taxa and then vertically transferred to various descendants. Although some CSIs and CSPs in unrelated groups of prokaryotes were identified, their small numbers and random occurrence has no apparent influence on the consistent tree-like branching pattern emerging from other markers. These results provide evidence that although LGT is an important evolutionary force, it does not mask the tree-like branching pattern of prokaryotes or understanding of their evolutionary relationships. The identified CSIs and CSPs also provide novel and highly specific means for identification of different groups of microbes and for taxonomical and biochemical studies.

  20. Prokaryotic communities in pit mud from different-aged cellars used for the production of Chinese strong-flavored liquor.

    Science.gov (United States)

    Tao, Yong; Li, Jiabao; Rui, Junpeng; Xu, Zhancheng; Zhou, Yan; Hu, Xiaohong; Wang, Xiang; Liu, Menghua; Li, Daping; Li, Xiangzhen

    2014-04-01

    Chinese strong-flavored liquor (CSFL) accounts for more than 70% of all Chinese liquor production. Microbes in pit mud play key roles in the fermentation cellar for the CSFL production. However, microbial diversity, community structure, and cellar-age-related changes in pit mud are poorly understood. Here, we investigated the prokaryotic community structure and diversity in pit-mud samples with different cellar ages (1, 10, 25, and 50 years) using the pyrosequencing technique. Results indicated that prokaryotic diversity increased with cellar age until the age reached 25 years and that prokaryotic community structure changed significantly between three cellar ages (1, 10, and 25 years). Significant correlations between prokaryotic communities and environmental variables (pH, NH4(+), lactic acid, butyric acid, and caproic acid) were observed. Overall, our study results suggested that the long-term brewing operation shapes unique prokaryotic community structure and diversity as well as pit-mud chemistry. We have proposed a three-phase model to characterize the changes of pit-mud prokaryotic communities. (i) Phase I is an initial domestication period. Pit mud is characterized by abundant Lactobacillus and high lactic acid and low pH levels. (ii) Phase II is a transition period. While Lactobacillus abundance decreases dramatically, that of Bacteroidetes and methanogens increases. (iii) Phase III is a relative mature period. The prokaryotic community shows the highest diversity and capability to produce more caproic acid as a precursor for synthesis of ethyl caproate, the main flavor component in CSFL. This research provides scientific evidence to support the practical experience that old fermentation cellars produce high-quality liquor.

  1. Microbial biomass and viral infections of heterotrophic prokaryotes in the sub-surface layer of the central Arctic Ocean

    Science.gov (United States)

    Steward, Grieg F.; Fandino, Laura B.; Hollibaugh, James T.; Whitledge, Terry E.; Azam, Farooq

    2007-10-01

    Seawater samples were collected for microbial analyses between 55 and 235 m depth across the Arctic Ocean during the SCICEX 97 expedition (03 September-02 October 1997) using a nuclear submarine as a research platform. Abundances of prokaryotes (range 0.043-0.47×10 9 dm -3) and viruses (range 0.68-11×10 9 dm -3) were correlated ( r=0.66, n=150) with an average virus:prokaryote ratio of 26 (range 5-70). Biomass of prokaryotes integrated from 55 to 235 m ranged from 0.27 to 0.85 g C m -2 exceeding that of phytoplankton (0.005-0.2 g C m -2) or viruses (0.02-0.05 g C m -2) over the same depth range by an order of magnitude on average. Using transmission electron microscopy (TEM), we estimated that 0.5% of the prokaryote community on average (range 0-1.4%) was visibly infected with viruses, which suggests that very little of prokaryotic secondary production was lost due to viral lysis. Intracellular viruses ranged from 5 to >200/cell, with an average apparent burst size of 45±38 (mean±s.d.; n=45). TEM also revealed the presence of putative metal-precipitating bacteria in 8 of 13 samples, which averaged 0.3% of the total prokaryote community (range 0-1%). If these prokaryotes are accessible to protistan grazers, the Fe and Mn associated with their capsules might be an important source of trace metals to the planktonic food web. After combining our abundance and mortality data with data from the literature, we conclude that the biomass of prokaryoplankton exceeds that of phytoplankton when averaged over the upper 250 m of the central Arctic Ocean and that the fate of this biomass is poorly understood.

  2. A survey of aspartate-phenylalanine and glutamate-phenylalanine interactions in the protein data bank: searching for anion-π pairs.

    Science.gov (United States)

    Philip, Vivek; Harris, Jason; Adams, Rachel; Nguyen, Don; Spiers, Jeremy; Baudry, Jerome; Howell, Elizabeth E; Hinde, Robert J

    2011-04-12

    Protein structures are stabilized using noncovalent interactions. In addition to the traditional noncovalent interactions, newer types of interactions are thought to be present in proteins. One such interaction, an anion-π pair, in which the positively charged edge of an aromatic ring interacts with an anion, forming a favorable anion-quadrupole interaction, has been previously proposed [Jackson, M. R., et al. (2007) J. Phys. Chem. B111, 8242-8249]. To study the role of anion-π interactions in stabilizing protein structure, we analyzed pairwise interactions between phenylalanine (Phe) and the anionic amino acids, aspartate (Asp) and glutamate (Glu). Particular emphasis was focused on identification of Phe-Asp or -Glu pairs separated by less than 7 Å in the high-resolution, nonredundant Protein Data Bank. Simplifying Phe to benzene and Asp or Glu to formate molecules facilitated in silico analysis of the pairs. Kitaura-Morokuma energy calculations were performed on roughly 19000 benzene-formate pairs and the resulting energies analyzed as a function of distance and angle. Edgewise interactions typically produced strongly stabilizing interaction energies (-2 to -7.3 kcal/mol), while interactions involving the ring face resulted in weakly stabilizing to repulsive interaction energies. The strongest, most stabilizing interactions were identified as preferentially occurring in buried residues. Anion-π pairs are found throughout protein structures, in helices as well as β strands. Numerous pairs also had nearby cation-π interactions as well as potential π-π stacking. While more than 1000 structures did not contain an anion-π pair, the 3134 remaining structures contained approximately 2.6 anion-π pairs per protein, suggesting it is a reasonably common motif that could contribute to the overall structural stability of a protein.

  3. A Survey of Aspartate-Phenylalanine and Glutamate-Phenylalanine Interactions in the Protein Data Bank: Searching for Anion-pi Pairs

    Energy Technology Data Exchange (ETDEWEB)

    Philip, Vivek M [ORNL; Harris, Jason B [ORNL; Adams, Rachel M [ORNL; Nguyen, Don [University of Tennessee; Spiers, Jeremy D [ORNL; Baudry, Jerome Y [ORNL; Howell, Elizabeth E [ORNL; Hinde, Robert J [ORNL

    2011-01-01

    Protein structures are stabilized using noncovalent interactions. In addition to the traditional noncovalent interactions, newer types of interactions are thought to be present in proteins. One such interaction, an anion-{pi} pair, in which the positively charged edge of an aromatic ring interacts with an anion, forming a favorable anion-quadrupole interaction, has been previously proposed [Jackson, M. R., et al. (2007) J. Phys. Chem. B111, 8242-8249]. To study the role of anion-{pi} interactions in stabilizing protein structure, we analyzed pairwise interactions between phenylalanine (Phe) and the anionic amino acids, aspartate (Asp) and glutamate (Glu). Particular emphasis was focused on identification of Phe-Asp or -Glu pairs separated by less than 7 {angstrom} in the high-resolution, nonredundant Protein Data Bank. Simplifying Phe to benzene and Asp or Glu to formate molecules facilitated in silico analysis of the pairs. Kitaura-Morokuma energy calculations were performed on roughly 19000 benzene-formate pairs and the resulting energies analyzed as a function of distance and angle. Edgewise interactions typically produced strongly stabilizing interaction energies (-2 to -7.3 kcal/mol), while interactions involving the ring face resulted in weakly stabilizing to repulsive interaction energies. The strongest, most stabilizing interactions were identified as preferentially occurring in buried residues. Anion-{pi} pairs are found throughout protein structures, in helices as well as {beta} strands. Numerous pairs also had nearby cation-{pi} interactions as well as potential {pi}-{pi} stacking. While more than 1000 structures did not contain an anion-{pi} pair, the 3134 remaining structures contained approximately 2.6 anion-{pi} pairs per protein, suggesting it is a reasonably common motif that could contribute to the overall structural stability of a protein.

  4. The measurement of muscle protein synthesis in broilers with a flooding dose technique: use of 15N-labelled phenylalanine, GC-MS and GC-C-IRMS.

    Science.gov (United States)

    Dänicke, S; Böttcher, W; Simon, O; Jeroch, H

    2001-01-01

    An experiment was carried out to measure fractional muscle protein synthesis rates (k(s)) in broilers with injection of a flooding dose of phenylalanine (1 ml/100 g body weight of 150 mM phenylalanine; 38 atom percent excess (APE) [15N]phenylalanine). K(s) was calculated from the [15N] enrichment in phenylalanine of tissue-free and protein-bound phenylalanine using both gas chromatography mass spectrometry (GC-MS) and gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) for measurements after a 10 min isotope incorporation period. The tertiary-butyldimethylsilyl (t-BDMS) derivatives of phenylalanine were used for gas chromatographic separation in both systems. GC-MS and GC-C-IRMS were calibrated for a range of 7 to 37 [15N]APE and 0 to 0.62 [15N]APE, respectively, and for sample sizes of 0.45 to 4.5 nmol phenylalanine and 7 to 40 nmol phenylalanine, respectively. Reproducibility of standards as a measure of precision varied from 0.06 to 0.29 [15N]APE and from 0.0004 to 0.0018 [15N]APE in GC-MS and GC-C-IRMS, respectively. K(s) was measured in the m. pectoralis major of broilers fed rye based diets (56%) which were provided either unsupplemented (-) or supplemented (+) with an enzyme preparation containing xylanase. K(s) in breast muscles was significantly increased from 21.8%/d to 23.9%/d due to enzyme supplementation. It can be concluded from the study that the measurement of protein synthesis in broilers with the flooding dose technique can be carried out by using [15N]phenylalanine, GC-MS and GC-C-IRMS.

  5. Effect of metabolic regulators on aluminium uptake and toxicity in Matricaria chamomilla plants

    Czech Academy of Sciences Publication Activity Database

    Kováčik, J.; Štork, F.; Klejdus, B.; Grúz, Jiří; Hedbavny, J.

    2012-01-01

    Roč. 54, May 2012 (2012), s. 140-148 ISSN 0981-9428 R&D Projects: GA AV ČR KAN200380801 Grant - others:GA ČR(CZ) GA525/07/0338; GA MŠk(CZ) ED1.1.00/02.0068 Program:GA; ED Institutional research plan: CEZ:AV0Z50380511 Keywords : Cinnamic acid * Mineral nutrients * Phenylalanine ammonia-lyase Subject RIV: ED - Physiology Impact factor: 2.775, year: 2012

  6. Dicty_cDB: Contig-U09447-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 1( AB300202 |pid:none) Ephedra sinica Espal4 mRNA for phe... 100 1e-19 AB283036_1( AB283036 |pid:none) Lotus..... 99 1e-19 ( P45734 ) RecName: Full=Phenylalanine ammonia-lyase; EC=... 99 1e-19 AB300201_1( AB300201 |pid:none) Ephedra sinica Espa...9 complet... 99 2e-19 AB300199_1( AB300199 |pid:none) Ephedra sinica Espal1 mRNA for phe... 99 2e-19 AY32108

  7. Induction of anthocyanin formation and of enzymes related to its biosynthesis by UV light in cell cultures of Haplopappus gracilis

    International Nuclear Information System (INIS)

    Wellmann, E.; Hrazdina, G.; Grisebach, H.

    1976-01-01

    Only UV light below 345 nm stimulates anthocyanin formation in dark grown cell suspension cultures of Haplopappus gracilis. A linear relationship between UV dose and flavonoid accumulation, as found previously with parsley cell cultures was not observed with the H.gracilis cells. Only continuous irradiation with high doses of UV was effective. Drastic increases in the activities of the enzymes phenylalanine ammonia-lyase, chalcone isomerase and flavanone synthase were observed under continuous UV light. The increase in enzyme activities paralleled anthocyanin formation. (author)

  8. Ultrasound treatment on phenolic metabolism and antioxidant capacity of fresh-cut pineapple during cold storage.

    Science.gov (United States)

    Yeoh, Wei Keat; Ali, Asgar

    2017-02-01

    Ultrasound treatment at different power output (0, 25 and 29W) and exposure time (10 and 15min) was used to investigate its effect on the phenolic metabolism enzymes, total phenolic content and antioxidant capacity of fresh-cut pineapple. Following ultrasound treatment at 25 and 29W, the activity of phenylalanine ammonia lyase (PAL) was increased significantly (Ppineapple was significantly (Ppineapple. Results suggest that hormetic dosage of ultrasound treatment can enhance the activity of PAL and total phenolic content and hence the total antioxidant capacity to encounter with oxidative stress. Copyright © 2016. Published by Elsevier Ltd.

  9. Metabolically engineered cells for the production of resveratrol or an oligomeric or glycosidically-bound derivative thereof

    DEFF Research Database (Denmark)

    2006-01-01

    A from said 4-coumaric acid, and resveratrol synthase (VST) produces said resveratrol from said 4- coumaroyl CoA, or in which L-phenylalanine- or tyrosine- ammonia lyase (PAL/TAL) produces 4-coumaric acid, 4- coumarate-CoA ligase (4CL) produces 4-coumaroyl CoA from said 4-coumaric acid, and resveratrol...... synthase (VST) produces said resveratrol from said 4-coumaroyl CoA. The micro-organism may be a yeast, fungus or bacterium including Saccharomyces cerevisiae, E. coli, Lactococcus lactis, Aspergillus niger, or Aspergillus oryzae....

  10. Viruses and Protists Induced-mortality of Prokaryotes around the Antarctic Peninsula during the Austral Summer

    KAUST Repository

    Vaque, Dolors; Boras, Julia A.; Torrent-Llagostera, Francesc; Agusti, Susana; Arrieta, J M; Lara, Elena; Castillo, Yaiza M.; Duarte, Carlos M.; Sala, Maria M.

    2017-01-01

    During the Austral summer 2009 we studied three areas surrounding the Antarctic Peninsula: the Bellingshausen Sea, the Bransfield Strait and the Weddell Sea. We aimed to investigate, whether viruses or protists were the main agents inducing prokaryotic mortality rates, and the sensitivity to temperature of prokaryotic heterotrophic production and mortality based on the activation energy (Ea) for each process. Seawater samples were taken at seven depths (0.1-100 m) to quantify viruses, prokaryotes and protists abundances, and heterotrophic prokaryotic production (PHP). Viral lytic production, lysogeny, and mortality rates of prokaryotes due to viruses and protists were estimated at surface (0.1-1 m) and at the Deep Fluorescence Maximum (DFM, 12-55 m) at eight representative stations of the three areas. The average viral lytic production ranged from 1.0 +/- 0.3 x 10(7) viruses ml(-1) d(-1) in the Bellingshausen Sea to1.3 +/- 0.7 x 10(7) viruses ml(-1) d(-1) in the Bransfield Strait, while lysogeny, when detectable, recorded the lowest value in the Bellingshausen Sea (0.05 +/- 0.05 x 10(7) viruses ml(-1) d(-1)) and the highest in the Weddell Sea (4.3 +/- 3.5 x 10(7) viruses ml(-1) d(-1)). Average mortality rates due to viruses ranged from 9.7 +/- 6.1 x 10(4) cells ml(-1) d(-1) in the Weddell Sea to 14.3 +/- 4.0 x 10(4) cells ml(-1) d(-1) in the Bellingshausen Sea, and were higher than averaged grazing rates in the Weddell Sea (5.9 +/- 1.1 x 10(4) cells ml(-1) d(-1)) and in the Bellingshausen Sea (6.8 +/- 0.9 x 10(4) cells ml-1 d(-1)). The highest impact on prokaryotes by viruses and main differences between viral and protists activities were observed in surface samples: 17.8 +/- 6.8 x 10(4) cells ml(-1) d(-1) and 6.5 +/- 3.9 x 10(4) cells ml(-1) d(-1) in the Weddell Sea; 22.1 +/- 9.6 x 10(4) cells ml(-1) d(-1) and 11.6 +/- 1.4 x 10(4) cells ml(-1) d(-1) in the Bransfield Strait; and 16.1 +/- 5.7 x 10(4) cells ml(-1) d(-1) and 7.9 +/- 2.6 x 10(4) cells ml(-1) d(-1) in

  11. Viruses and Protists Induced-mortality of Prokaryotes around the Antarctic Peninsula during the Austral Summer

    KAUST Repository

    Vaque, Dolors

    2017-03-27

    During the Austral summer 2009 we studied three areas surrounding the Antarctic Peninsula: the Bellingshausen Sea, the Bransfield Strait and the Weddell Sea. We aimed to investigate, whether viruses or protists were the main agents inducing prokaryotic mortality rates, and the sensitivity to temperature of prokaryotic heterotrophic production and mortality based on the activation energy (Ea) for each process. Seawater samples were taken at seven depths (0.1-100 m) to quantify viruses, prokaryotes and protists abundances, and heterotrophic prokaryotic production (PHP). Viral lytic production, lysogeny, and mortality rates of prokaryotes due to viruses and protists were estimated at surface (0.1-1 m) and at the Deep Fluorescence Maximum (DFM, 12-55 m) at eight representative stations of the three areas. The average viral lytic production ranged from 1.0 +/- 0.3 x 10(7) viruses ml(-1) d(-1) in the Bellingshausen Sea to1.3 +/- 0.7 x 10(7) viruses ml(-1) d(-1) in the Bransfield Strait, while lysogeny, when detectable, recorded the lowest value in the Bellingshausen Sea (0.05 +/- 0.05 x 10(7) viruses ml(-1) d(-1)) and the highest in the Weddell Sea (4.3 +/- 3.5 x 10(7) viruses ml(-1) d(-1)). Average mortality rates due to viruses ranged from 9.7 +/- 6.1 x 10(4) cells ml(-1) d(-1) in the Weddell Sea to 14.3 +/- 4.0 x 10(4) cells ml(-1) d(-1) in the Bellingshausen Sea, and were higher than averaged grazing rates in the Weddell Sea (5.9 +/- 1.1 x 10(4) cells ml(-1) d(-1)) and in the Bellingshausen Sea (6.8 +/- 0.9 x 10(4) cells ml-1 d(-1)). The highest impact on prokaryotes by viruses and main differences between viral and protists activities were observed in surface samples: 17.8 +/- 6.8 x 10(4) cells ml(-1) d(-1) and 6.5 +/- 3.9 x 10(4) cells ml(-1) d(-1) in the Weddell Sea; 22.1 +/- 9.6 x 10(4) cells ml(-1) d(-1) and 11.6 +/- 1.4 x 10(4) cells ml(-1) d(-1) in the Bransfield Strait; and 16.1 +/- 5.7 x 10(4) cells ml(-1) d(-1) and 7.9 +/- 2.6 x 10(4) cells ml(-1) d(-1) in

  12. Viruses and Protists Induced-mortality of Prokaryotes around the Antarctic Peninsula during the Austral Summer.

    Science.gov (United States)

    Vaqué, Dolors; Boras, Julia A; Torrent-Llagostera, Francesc; Agustí, Susana; Arrieta, Jesús M; Lara, Elena; Castillo, Yaiza M; Duarte, Carlos M; Sala, Maria M

    2017-01-01

    During the Austral summer 2009 we studied three areas surrounding the Antarctic Peninsula: the Bellingshausen Sea, the Bransfield Strait and the Weddell Sea. We aimed to investigate, whether viruses or protists were the main agents inducing prokaryotic mortality rates, and the sensitivity to temperature of prokaryotic heterotrophic production and mortality based on the activation energy (Ea) for each process. Seawater samples were taken at seven depths (0.1-100 m) to quantify viruses, prokaryotes and protists abundances, and heterotrophic prokaryotic production (PHP). Viral lytic production, lysogeny, and mortality rates of prokaryotes due to viruses and protists were estimated at surface (0.1-1 m) and at the Deep Fluorescence Maximum (DFM, 12-55 m) at eight representative stations of the three areas. The average viral lytic production ranged from 1.0 ± 0.3 × 10 7 viruses ml -1 d -1 in the Bellingshausen Sea to1.3 ± 0.7 × 10 7 viruses ml -1 d -1 in the Bransfield Strait, while lysogeny, when detectable, recorded the lowest value in the Bellingshausen Sea (0.05 ± 0.05 × 10 7 viruses ml -1 d -1 ) and the highest in the Weddell Sea (4.3 ± 3.5 × 10 7 viruses ml -1 d -1 ). Average mortality rates due to viruses ranged from 9.7 ± 6.1 × 10 4 cells ml -1 d -1 in the Weddell Sea to 14.3 ± 4.0 × 10 4 cells ml -1 d -1 in the Bellingshausen Sea, and were higher than averaged grazing rates in the Weddell Sea (5.9 ± 1.1 × 10 4 cells ml -1 d -1 ) and in the Bellingshausen Sea (6.8 ± 0.9 × 10 4 cells ml -1 d -1 ). The highest impact on prokaryotes by viruses and main differences between viral and protists activities were observed in surface samples: 17.8 ± 6.8 × 10 4 cells ml -1 d -1 and 6.5 ± 3.9 × 10 4 cells ml -1 d -1 in the Weddell Sea; 22.1 ± 9.6 × 10 4 cells ml -1 d -1 and 11.6 ± 1.4 × 10 4 cells ml -1 d -1 in the Bransfield Strait; and 16.1 ± 5.7 × 10 4 cells ml -1 d -1 and 7.9 ± 2.6 × 10 4 cells ml -1 d -1 in the Bellingshausen Sea, respectively

  13. Relationships between meiofaunal biodiversity and prokaryotic heterotrophic production in different tropical habitats and oceanic regions.

    Science.gov (United States)

    Pusceddu, Antonio; Gambi, Cristina; Corinaldesi, Cinzia; Scopa, Mariaspina; Danovaro, Roberto

    2014-01-01

    Tropical marine ecosystems are among the most diverse of the world oceans, so that assessing the linkages between biodiversity and ecosystem functions (BEF) is a crucial step to predict consequences of biodiversity loss. Most BEF studies in marine ecosystems have been carried out on macrobenthic diversity, whereas the influence of the meiofauna on ecosystem functioning has received much less attention. We compared meiofaunal and nematode biodiversity and prokaryotic heterotrophic production across seagrass, mangrove and reef sediments in the Caribbean, Celebes and Red Seas. For all variables we report the presence of differences among habitats within the same region, and among regions within the same habitat. In all regions, the richness of meiofaunal taxa in reef and seagrass sediments is higher than in mangrove sediments. The sediments of the Celebes Sea show the highest meiofaunal biodiversity. The composition of meiofaunal assemblages varies significantly among habitats in the same region. The nematode beta diversity among habitats within the same region is higher than the beta diversity among regions. Although one site per habitat was considered in each region, these results suggest that the composition of meiofaunal assemblages varies primarily among biogeographic regions, whereas the composition of nematode assemblages varies more considerably among habitats. Meiofauna and nematode biodiversity and prokaryotic heterotrophic production, even after the removal of covariate effects linked with longitude and the quantity and nutritional quality of organic matter, are positively and linearly linked both across regions and within each habitat type. Our results confirm that meiofauna and nematode biodiversity may influence benthic prokaryotic activity, which, in turn, implies that diversity loss could have negative impacts on ecosystem functioning in these systems.

  14. Relationships between meiofaunal biodiversity and prokaryotic heterotrophic production in different tropical habitats and oceanic regions.

    Directory of Open Access Journals (Sweden)

    Antonio Pusceddu

    Full Text Available Tropical marine ecosystems are among the most diverse of the world oceans, so that assessing the linkages between biodiversity and ecosystem functions (BEF is a crucial step to predict consequences of biodiversity loss. Most BEF studies in marine ecosystems have been carried out on macrobenthic diversity, whereas the influence of the meiofauna on ecosystem functioning has received much less attention. We compared meiofaunal and nematode biodiversity and prokaryotic heterotrophic production across seagrass, mangrove and reef sediments in the Caribbean, Celebes and Red Seas. For all variables we report the presence of differences among habitats within the same region, and among regions within the same habitat. In all regions, the richness of meiofaunal taxa in reef and seagrass sediments is higher than in mangrove sediments. The sediments of the Celebes Sea show the highest meiofaunal biodiversity. The composition of meiofaunal assemblages varies significantly among habitats in the same region. The nematode beta diversity among habitats within the same region is higher than the beta diversity among regions. Although one site per habitat was considered in each region, these results suggest that the composition of meiofaunal assemblages varies primarily among biogeographic regions, whereas the composition of nematode assemblages varies more considerably among habitats. Meiofauna and nematode biodiversity and prokaryotic heterotrophic production, even after the removal of covariate effects linked with longitude and the quantity and nutritional quality of organic matter, are positively and linearly linked both across regions and within each habitat type. Our results confirm that meiofauna and nematode biodiversity may influence benthic prokaryotic activity, which, in turn, implies that diversity loss could have negative impacts on ecosystem functioning in these systems.

  15. Prokaryotic Diversity in the Rhizosphere of Organic, Intensive, and Transitional Coffee Farms in Brazil.

    Science.gov (United States)

    Caldwell, Adam Collins; Silva, Lívia Carneiro Fidéles; da Silva, Cynthia Canêdo; Ouverney, Cleber Costa

    2015-01-01

    Despite a continuous rise in consumption of coffee over the past 60 years and recent studies showing positive benefits linked to human health, intensive coffee farming practices have been associated with environmental damage, risks to human health, and reductions in biodiversity. In contrast, organic farming has become an increasingly popular alternative, with both environmental and health benefits. This study aimed to characterize and determine the differences in the prokaryotic soil microbiology of three Brazilian coffee farms: one practicing intensive farming, one practicing organic farming, and one undergoing a transition from intensive to organic practices. Soil samples were collected from 20 coffee plant rhizospheres (soil directly influenced by the plant root exudates) and 10 control sites (soil 5 m away from the coffee plantation) at each of the three farms for a total of 90 samples. Profiling of 16S rRNA gene V4 regions revealed high levels of prokaryotic diversity in all three farms, with thousands of species level operational taxonomic units identified in each farm. Additionally, a statistically significant difference was found between each farm's coffee rhizosphere microbiome, as well as between coffee rhizosphere soils and control soils. Two groups of prokaryotes associated with the nitrogen cycle, the archaeal genus Candidatus Nitrososphaera and the bacterial order Rhizobiales were found to be abundant and statistically different in composition between the three farms and in inverse relationship to each other. Many of the nitrogen-fixing genera known to enhance plant growth were found in low numbers (e.g. Rhizobium, Agrobacter, Acetobacter, Rhodospirillum, Azospirillum), but the families in which they belong had some of the highest relative abundance in the dataset, suggesting many new groups may exist in these samples that can be further studied as potential plant growth-promoting bacteria to improve coffee production while diminishing negative

  16. [Prokaryotic expression of Leptospira interrogans groEL gene and immunoprotection of its products in hamsters].

    Science.gov (United States)

    Li, Xiaoyu; Wang, Yinhuan; Yan, Jie; Cheng, Dongqing

    2013-03-01

    To construct a prokaryotic expression system of groEL gene of Leptospira interrogans serogroup Icterohaemorrhagia serovar Lai strain Lai, and to determine the immunoprotective effect of recombinant GroEL protein (rGroEL) in LVG hamsters. The groEL gene was amplified by high fidelity PCR and the amplification products were then sequenced. A prokaryotic expression system of groEL gene was constructed using routine genetic engineering technique. SDS-PAGE plus Bio-Rad Gel Image Analyzer was applied to examine the expression and dissolubility of rGroEL protein while Ni-NTA affinity chromatography was used to extract the expressed rGroEL. The immunoprotective rate in rGroEL-immunized LVG hamsters was determined after challenge with L.interrogans strain Lai. The cross agglutination titers of sera from immunized hamsters with different L.interrogans serogroups were detected using MAT. The nucleotide and amino acid sequences of the cloned groEL gene were the same as those reported in GenBank. The constructed prokaryotic expression system of groEL gene expressed soluble rGroEL. The immunoprotective rates of 100 and 200 μg rGroEL in LVG hamsters were 50.0 % and 75.0%, respectively. The sera from the rGroEL-immunized LVG hamsters agglutinated all the L.interrogans serogroups tested with different levels. The GroEL protein is a genus-specific immunoprotective antigen of L.interrogans and can be used to develop an universal genetically engineering vaccine of Leptospira.

  17. Analyzing the Differences and Preferences of Pathogenic and Nonpathogenic Prokaryote Species

    Science.gov (United States)

    Nolen, L.; Duong, K.; Heim, N. A.; Payne, J.

    2015-12-01

    A limited amount of knowledge exists on the large-scale characteristics and differences of pathogenic species in comparison to all prokaryotes. Pathogenic species, like other prokaryotes, have attributes specific to their environment and lifestyles. However, because they have evolved to coexist inside their hosts, the conditions they occupy may be more limited than those of non-pathogenic species. In this study we investigate the possibility of divergent evolution between pathogenic and non-pathogenic species by examining differences that may have evolved as a result of the need to adapt to their host. For this research we analyzed data collected from over 1900 prokaryotic species and performed t-tests using R to quantify potential differences in preferences. To examine the possible divergences from nonpathogenic bacteria, we focused on three variables: cell biovolume, preferred environmental pH, and preferred environmental temperature. We also looked at differences between pathogenic and nonpathogenic species belonging to the same phylum. Our results suggest a strong divergence in abiotic preferences between the two groups, with pathogens occupying a much smaller range of temperatures and pHs than their non-pathogenic counterparts. However, while the median biovolume is different when comparing pathogens and nonpathogens, we cannot conclude that the mean values are significantly different from each other. In addition, we found evidence of convergent evolution, as the temperature and pH preferences of pathogenic bacteria species from different phlya all approach the same values. Pathogenic species do not, however, all approach the same biovolume values, suggesting that specific pH and temperature preferences are more characteristic of pathogens than certain biovolumes.

  18. Comparative genomic analysis of translation initiation mechanisms for genes lacking the Shine–Dalgarno sequence in prokaryotes

    KAUST Repository

    Nakagawa, So

    2017-02-15

    In prokaryotes, translation initiation is believed to occur through an interaction between the 3\\' tail of a 16S rRNA and a corresponding Shine-Dalgarno (SD) sequence in the 5\\' untranslated region (UTR) of an mRNA. However, some genes lack SD sequences (non-SD genes), and the fraction of non-SD genes in a genome varies depending on the prokaryotic species. To elucidate non-SD translation initiation mechanisms in prokaryotes from an evolutionary perspective, we statistically examined the nucleotide frequencies around the initiation codons in non-SD genes from 260 prokaryotes (235 bacteria and 25 archaea). We identified distinct nucleotide frequency biases upstream of the initiation codon in bacteria and archaea, likely because of the presence of leaderless mRNAs lacking a 5\\' UTR. Moreover, we observed overall similarities in the nucleotide patterns between upstream and downstream regions of the initiation codon in all examined phyla. Symmetric nucleotide frequency biases might facilitate translation initiation by preventing the formation of secondary structures around the initiation codon. These features are more prominent in species\\' genomes that harbor large fractions of non-SD sequences, suggesting that a reduced stability around the initiation codon is important for efficient translation initiation in prokaryotes.

  19. Comparative genomic analysis of translation initiation mechanisms for genes lacking the Shine–Dalgarno sequence in prokaryotes

    KAUST Repository

    Nakagawa, So; Niimura, Yoshihito; Gojobori, Takashi

    2017-01-01

    In prokaryotes, translation initiation is believed to occur through an interaction between the 3' tail of a 16S rRNA and a corresponding Shine-Dalgarno (SD) sequence in the 5' untranslated region (UTR) of an mRNA. However, some genes lack SD sequences (non-SD genes), and the fraction of non-SD genes in a genome varies depending on the prokaryotic species. To elucidate non-SD translation initiation mechanisms in prokaryotes from an evolutionary perspective, we statistically examined the nucleotide frequencies around the initiation codons in non-SD genes from 260 prokaryotes (235 bacteria and 25 archaea). We identified distinct nucleotide frequency biases upstream of the initiation codon in bacteria and archaea, likely because of the presence of leaderless mRNAs lacking a 5' UTR. Moreover, we observed overall similarities in the nucleotide patterns between upstream and downstream regions of the initiation codon in all examined phyla. Symmetric nucleotide frequency biases might facilitate translation initiation by preventing the formation of secondary structures around the initiation codon. These features are more prominent in species' genomes that harbor large fractions of non-SD sequences, suggesting that a reduced stability around the initiation codon is important for efficient translation initiation in prokaryotes.

  20. Metabolic engineering with systems biology tools to optimize production of prokaryotic secondary metabolites

    DEFF Research Database (Denmark)

    Kim, Hyun Uk; Charusanti, Pep; Lee, Sang Yup

    2016-01-01

    Metabolic engineering using systems biology tools is increasingly applied to overproduce secondary metabolites for their potential industrial production. In this Highlight, recent relevant metabolic engineering studies are analyzed with emphasis on host selection and engineering approaches...... for the optimal production of various prokaryotic secondary metabolites: native versus heterologous hosts (e.g., Escherichia coli) and rational versus random approaches. This comparative analysis is followed by discussions on systems biology tools deployed in optimizing the production of secondary metabolites....... The potential contributions of additional systems biology tools are also discussed in the context of current challenges encountered during optimization of secondary metabolite production....

  1. Keeping the wolves at bay: antitoxins of prokaryotic type II toxin-antitoxin systems

    Directory of Open Access Journals (Sweden)

    Wai Ting eChan

    2016-03-01

    Full Text Available In their initial stages of discovery, prokaryotic toxin-antitoxin (TA systems were confined to bacterial plasmids where they function to mediate the maintenance and stability of usually low- to medium-copy number plasmids through the post-segregational killing of any plasmid-free daughter cells that developed. Their eventual discovery as nearly ubiquitous and repetitive elements in bacterial chromosomes led to a wealth of knowledge and scientific debate as to their diversity and functionality in the prokaryotic lifestyle. Currently categorized into six different types designated types I – VI, type II TA systems are the best characterized. These generally comprised of two genes encoding a proteic toxin and its corresponding proteic antitoxin, respectively. Under normal growth conditions, the stable toxin is prevented from exerting its lethal effect through tight binding with the less stable antitoxin partner, forming a non-lethal TA protein complex. Besides binding with its cognate toxin, the antitoxin also plays a role in regulating the expression of the type II TA operon by binding to the operator site, thereby repressing transcription from the TA promoter. In most cases, full repression is observed in the presence of the TA complex as binding of the toxin enhances the DNA binding capability of the antitoxin. TA systems have been implicated in a gamut of prokaryotic cellular functions such as being mediators of programmed cell death as well as persistence or dormancy, biofilm formation, as defensive weapons against bacteriophage infections and as virulence factors in pathogenic bacteria. It is thus apparent that these antitoxins, as DNA-binding proteins, play an essential role in modulating the prokaryotic lifestyle whilst at the same time preventing the lethal action of the toxins under normal growth conditions, i.e., keeping the proverbial wolves at bay. In this review, we will cover the diversity and characteristics of various type II TA

  2. Mesopelagic Prokaryotes Alter Surface Phytoplankton Production during Simulated Deep Mixing Experiments in Eastern Mediterranean Sea Waters

    Directory of Open Access Journals (Sweden)

    Or Hazan

    2018-01-01

    Full Text Available Mesopelagic prokaryotes (archaea and bacteria, which are transported together with nutrient-rich intermediate-water to the surface layer by deep convection in the oceans (e.g., winter mixing, upwelling systems, can interact with surface microbial populations. This interaction can potentially affect production rates and biomass of surface microbial populations, and thus play an important role in the marine carbon cycle and oceanic carbon sequestration. The Eastern Mediterranean Sea (EMS is one of the most oligotrophic and warm systems in the world's oceans, with usually very shallow winter mixing (<200 m and lack of large-size spring algal blooms. In this study, we collected seawater (0–1,500 m in 9 different cruises at the open EMS during both the stratified and the mixed seasons. We show that the EMS is a highly oligotrophic regime, resulting in low autotrophic biomass and primary productivity and relatively high heterotrophic prokaryotic biomass and production. Further, we simulated deep water mixing in on-board microcosms using Levantine surface (LSW, ~0.5 m and intermediate (LIW, ~400 m waters at a 9:1 ratio, respectively and examined the responses of the microbial populations to such a scenario. We hypothesized that the LIW, being nutrient-rich (e.g., N, P and a “hot-spot” for microbial activity (due to the warm conditions that prevail in these depths, may supply the LSW with not only key-limiting nutrients but also with viable and active heterotrophic prokaryotes that can interact with the ambient surface microbial population. Indeed, we show that LIW heterotrophic prokaryotes negatively affected the surface phytoplankton populations, resulting in lower chlorophyll-a levels and primary production rates. This may be due to out-competition of phytoplankton by LIW populations for resources and/or by a phytoplankton cell lysis via viral infection. Our results suggest that phytoplankton in the EMS may not likely form blooms, even after

  3. [Prokaryotic expression of trigeminy artificial fusion gene of Leptospira interrogans and the immunogenicity of its products].

    Science.gov (United States)

    Luo, Dong-jiao; Qiu, Xiao-feng; Wang, Jiang; Yan, Jin; Wang, Hai-bin; Zhou, Jin-cheng; Yan, Jie

    2008-11-01

    To construct lipL32/1-lipL21-OmpL1/2 fusion gene of Leptospira interrogans and its prokaryotic expression system, and to identify the immunogenicity of its products. PCR using linking primers was applied to construct lipL32/1-lipL21-OmpL1/2 fusion gene and a prokaryotic expression system of the fusion gene was then established using routine genetic engineering technique. SDS-PAGE was used to examine output of the target recombinant protein rLipL32/1-LipL21-OmpL1/2. Double immunodiffusion and Western Blot assay were applied to identify immunogenicity of rLipL32/1-LipL21-OmpL1/2. lipL32/1-lipL21-OmpL1/2 fusion gene with correct sequence and its prokaryotic expression system E.coli BL21DE3pET42a-lipL32/1-lipL21-ompL1/2 was obtained in this study. The output of rLipL32/1-LipL21- OmpL1/2 after optimisation was 37.78 mg/L. The immunodiffusion titer of rabbit antiserum against rLipL32/1-LipL21-OmpL1/2 was 1:4. The rLipL32/1-LipL21-OmpL1/2 antiserum was able to recognize rLipL32/1-LipL21-OmpL1/2, rLipL32/1, rLipL21 and rOmpL1/2. Positive Western hybridization signals were found among rLipL32/1-LipL21-OmpL1/2 and rabbit antiserum against whole cell of strain 56601 and serum from patients infected with L.interrogans serogroups Icterohaemorrhagiae, Grippotyphosa, Autumnalis and Pomona. The fusion gene lipL32/1-lipL21-OmpL1/2 and its prokaryotic expression system were successfully constructed in this study. The expressed fusion protein can be used as the antigen for developing universal genetic engineering vaccine and universal serological tests of leptospirosis.

  4. Adaptation of the short intergenic spacers between co-directional genes to the Shine-Dalgarno motif among prokaryote genomes

    DEFF Research Database (Denmark)

    Caro, Albert Pallejà; García-Vallvé, Santiago; Romeu, Antoni

    2009-01-01

    ABSTRACT: BACKGROUND: In prokaryote genomes most of the co-directional genes are in close proximity. Even the coding sequence or the stop codon of a gene can overlap with the Shine-Dalgarno (SD) sequence of the downstream co-directional gene. In this paper we analyze how the presence of SD may...... influence the stop codon usage or the spacing lengths between co-directional genes. RESULTS: The SD sequences for 530 prokaryote genomes have been predicted using computer calculations of the base-pairing free energy between translation initiation regions and the 16S rRNA 3' tail. Genomes with a large...... to the discussion of which factors affect the intergenic lengths, which cannot be totally explained by the pressure to compact the prokaryote genomes....

  5. Diversity, evolution, and therapeutic applications of small RNAs in prokaryotic and eukaryotic immune systems

    Science.gov (United States)

    Cooper, Edwin L.; Overstreet, Nicola

    2014-03-01

    Recent evidence supports that prokaryotes exhibit adaptive immunity in the form of CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) and Cas (CRISPR associated proteins). The CRISPR-Cas system confers resistance to exogenous genetic elements such as phages and plasmids by allowing for the recognition and silencing of these genetic elements. Moreover, CRISPR-Cas serves as a memory of past exposures. This suggests that the evolution of the immune system has counterparts among the prokaryotes, not exclusively among eukaryotes. Mathematical models have been proposed which simulate the evolutionary patterns of CRISPR, however large gaps in our understanding of CRISPR-Cas function and evolution still exist. The CRISPR-Cas system is analogous to small RNAs involved in resistance mechanisms throughout the tree of life, and a deeper understanding of the evolution of small RNA pathways is necessary before the relationship between these convergent systems is to be determined. Presented in this review are novel RNAi therapies based on CRISPR-Cas analogs and the potential for future therapies based on CRISPR-Cas system components.

  6. Establishing homology between mitochondrial calcium uniporters, prokaryotic magnesium channels and chlamydial IncA proteins.

    Science.gov (United States)

    Lee, Andre; Vastermark, Ake; Saier, Milton H

    2014-08-01

    Mitochondrial calcium uniporters (MCUs) (TC no. 1.A.77) are oligomeric channel proteins found in the mitochondrial inner membrane. MCUs have two well-conserved transmembrane segments (TMSs), connected by a linker, similar to bacterial MCU homologues. These proteins and chlamydial IncA proteins (of unknown function; TC no. 9.B.159) are homologous to prokaryotic Mg(2+) transporters, AtpI and AtpZ, based on comparison scores of up to 14.5 sds. A phylogenetic tree containing all of these proteins showed that the AtpZ proteins cluster coherently as a subset within the large and diverse AtpI cluster, which branches separately from the MCUs and IncAs, both of which cluster coherently. The MCUs and AtpZs share the same two TMS topology, but the AtpIs have four TMSs, and IncAs can have either two (most frequent) or four (less frequent) TMSs. Binary alignments, comparison scores and motif analyses showed that TMSs 1 and 2 align with TMSs 3 and 4 of the AtpIs, suggesting that the four TMS AtpI proteins arose via an intragenic duplication event. These findings establish an evolutionary link interconnecting eukaryotic and prokaryotic Ca(2+) and Mg(2+) transporters with chlamydial IncAs, and lead us to suggest that all members of the MCU superfamily, including IncAs, function as divalent cation channels. © 2014 The Authors.

  7. [Characterization of the Structure of the Prokaryotic Complex of Antarctic Permafrost by Molecular Genetic Techniques].

    Science.gov (United States)

    Manucharova, N A; Trosheva, E V; Kol'tsova, E M; Demkina, E V; Karaevskaya, E V; Rivkina, E M; Mardanov, A V; El'-Registan, G I

    2016-01-01

    A prokaryotic mesophilic organotrophic community responsible for 10% of the total microbial number determined by epifluorescence microscopy was reactivated in the samples ofAntarctic permafrost retrieved from the environment favoring long-term preservation of microbial communities (7500 years). No culturable forms were obtained without resuscitation procedures (CFU = 0). Proteobacteria, Actinobacteria, and Firmicutes were the dominant microbial groups in the complex. Initiation of the reactivated microbial complex by addition of chitin (0.1% wt/vol) resulted in an increased share of metabolically active biomass (up to 50%) due to the functional domination of chitinolytics caused by the target resource. Thus, sequential application of resuscitation procedures and initiation of a specific physiological group (in this case, chitinolytics) to a permafrost-preserved microbial community made it possible to reveal a prokaryotic complex capable of reversion of metabolic activity (FISH data), to determine its phylogenetic structure by metagenomic anal-ysis, and to isolate a pure culture of the dominant microorganism with high chitinolytic activity.

  8. Prokaryotic Phylogenies Inferred from Whole-Genome Sequence and Annotation Data

    Directory of Open Access Journals (Sweden)

    Wei Du

    2013-01-01

    Full Text Available Phylogenetic trees are used to represent the evolutionary relationship among various groups of species. In this paper, a novel method for inferring prokaryotic phylogenies using multiple genomic information is proposed. The method is called CGCPhy and based on the distance matrix of orthologous gene clusters between whole-genome pairs. CGCPhy comprises four main steps. First, orthologous genes are determined by sequence similarity, genomic function, and genomic structure information. Second, genes involving potential HGT events are eliminated, since such genes are considered to be the highly conserved genes across different species and the genes located on fragments with abnormal genome barcode. Third, we calculate the distance of the orthologous gene clusters between each genome pair in terms of the number of orthologous genes in conserved clusters. Finally, the neighbor-joining method is employed to construct phylogenetic trees across different species. CGCPhy has been examined on different datasets from 617 complete single-chromosome prokaryotic genomes and achieved applicative accuracies on different species sets in agreement with Bergey's taxonomy in quartet topologies. Simulation results show that CGCPhy achieves high average accuracy and has a low standard deviation on different datasets, so it has an applicative potential for phylogenetic analysis.

  9. Prediction of Metabolic Pathway Involvement in Prokaryotic UniProtKB Data by Association Rule Mining

    KAUST Repository

    Boudellioua, Imene; Saidi, Rabie; Hoehndorf, Robert; Martin, Maria J.; Solovyev, Victor

    2016-01-01

    The widening gap between known proteins and their functions has encouraged the development of methods to automatically infer annotations. Automatic functional annotation of proteins is expected to meet the conflicting requirements of maximizing annotation coverage, while minimizing erroneous functional assignments. This trade-off imposes a great challenge in designing intelligent systems to tackle the problem of automatic protein annotation. In this work, we present a system that utilizes rule mining techniques to predict metabolic pathways in prokaryotes. The resulting knowledge represents predictive models that assign pathway involvement to UniProtKB entries. We carried out an evaluation study of our system performance using cross-validation technique. We found that it achieved very promising results in pathway identification with an F1-measure of 0.982 and an AUC of 0.987. Our prediction models were then successfully applied to 6.2 million UniProtKB/TrEMBL reference proteome entries of prokaryotes. As a result, 663,724 entries were covered, where 436,510 of them lacked any previous pathway annotations.

  10. A Comprehensive Curation Shows the Dynamic Evolutionary Patterns of Prokaryotic CRISPRs

    Directory of Open Access Journals (Sweden)

    Guoqin Mai

    2016-01-01

    Full Text Available Motivation. Clustered regularly interspaced short palindromic repeat (CRISPR is a genetic element with active regulation roles for foreign invasive genes in the prokaryotic genomes and has been engineered to work with the CRISPR-associated sequence (Cas gene Cas9 as one of the modern genome editing technologies. Due to inconsistent definitions, the existing CRISPR detection programs seem to have missed some weak CRISPR signals. Results. This study manually curates all the currently annotated CRISPR elements in the prokaryotic genomes and proposes 95 updates to the annotations. A new definition is proposed to cover all the CRISPRs. The comprehensive comparison of CRISPR numbers on the taxonomic levels of both domains and genus shows high variations for closely related species even in the same genus. The detailed investigation of how CRISPRs are evolutionarily manipulated in the 8 completely sequenced species in the genus Thermoanaerobacter demonstrates that transposons act as a frequent tool for splitting long CRISPRs into shorter ones along a long evolutionary history.

  11. Prokaryotic community composition in alkaline-fermented skate (Raja pulchra).

    Science.gov (United States)

    Jang, Gwang Il; Kim, Gahee; Hwang, Chung Yeon; Cho, Byung Cheol

    2017-02-01

    Prokaryotes were extracted from skates and fermented skates purchased from fish markets and a local manufacturer in South Korea. The prokaryotic community composition of skates and fermented skates was investigated using 16S rRNA pyrosequencing. The ranges for pH and salinity of the grinded tissue extract from fermented skates were 8.4-8.9 and 1.6-6.6%, respectively. Urea and ammonia concentrations were markedly low and high, respectively, in fermented skates compared to skates. Species richness was increased in fermented skates compared to skates. Dominant and predominant bacterial groups present in the fermented skates belonged to the phylum Firmicutes, whereas those in skates belonged to Gammaproteobacteria. The major taxa found in Firmicutes were Atopostipes (Carnobacteriaceae, Lactobacillales) and/or Tissierella (Tissierellaceae, Tissierellales). A combination of RT-PCR and pyrosequencing for active bacterial composition showed that the dominant taxa i.e., Atopostipes and Tissierella, were active in fermented skate. Those dominant taxa are possibly marine lactic acid bacteria. Marine bacteria of the taxa Lactobacillales and/or Clostridia seem to be important in alkaline fermentation of skates. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Resilience of the prokaryotic microbial community of Acropora digitifera to elevated temperature.

    Science.gov (United States)

    Gajigan, Andrian P; Diaz, Leomir A; Conaco, Cecilia

    2017-08-01

    The coral is a holobiont formed by the close interaction between the coral animal and a diverse community of microorganisms, including dinoflagellates, bacteria, archaea, fungi, and viruses. The prokaryotic symbionts of corals are important for host fitness but are also highly sensitive to changes in the environment. In this study, we used 16S ribosomal RNA (rRNA) sequencing to examine the response of the microbial community associated with the coral, Acropora digitifera, to elevated temperature. The A. digitifera microbial community is dominated by operational taxonomic unit (OTUs) affiliated with classes Alphaproteobacteria and Gammaproteobacteria. The prokaryotic community in the coral tissue is distinct from that of the mucus and the surrounding seawater. Remarkably, the overall microbial community structure of A. digitifera remained stable for 10 days of continuous exptosure at 32°C compared to corals maintained at 27°C. However, the elevated temperature regime resulted in a decrease in the abundance of OTUs affiliated with certain groups of bacteria, such as order Rhodobacterales. On the other hand, some OTUs affiliated with the orders Alteromonadales, Vibrionales, and Flavobacteriales, which are often associated with diseased and stressed corals, increased in abundance. Thus, while the A. digitifera bacterial community structure appears resilient to higher temperature, prolonged exposure and intensified stress results in changes in the abundance of specific microbial community members that may affect the overall metabolic state and health of the coral holobiont. © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  13. Amino acid composition in endothermic vertebrates is biased in the same direction as in thermophilic prokaryotes

    Directory of Open Access Journals (Sweden)

    Wang Guang-Zhong

    2010-08-01

    Full Text Available Abstract Background Among bacteria and archaea, amino acid usage is correlated with habitat temperatures. In particular, protein surfaces in species thriving at higher temperatures appear to be enriched in amino acids that stabilize protein structure and depleted in amino acids that decrease thermostability. Does this observation reflect a causal relationship, or could the apparent trend be caused by phylogenetic relatedness among sampled organisms living at different temperatures? And do proteins from endothermic and exothermic vertebrates show similar differences? Results We find that the observed correlations between the frequencies of individual amino acids and prokaryotic habitat temperature are strongly influenced by evolutionary relatedness between the species analysed; however, a proteome-wide bias towards increased thermostability remains after controlling for phylogeny. Do eukaryotes show similar effects of thermal adaptation? A small shift of amino acid usage in the expected direction is observed in endothermic ('warm-blooded' mammals and chicken compared to ectothermic ('cold-blooded' vertebrates with lower body temperatures; this shift is not simply explained by nucleotide usage biases. Conclusion Protein homologs operating at different temperatures have different amino acid composition, both in prokaryotes and in vertebrates. Thus, during the transition from ectothermic to endothermic life styles, the ancestors of mammals and of birds may have experienced weak genome-wide positive selection to increase the thermostability of their proteins.

  14. New Insight Into the Diversity of SemiSWEET Sugar Transporters and the Homologs in Prokaryotes

    Directory of Open Access Journals (Sweden)

    Baolei Jia

    2018-05-01

    Full Text Available Sugars will eventually be exported transporters (SWEETs and SemiSWEETs represent a family of sugar transporters in eukaryotes and prokaryotes, respectively. SWEETs contain seven transmembrane helices (TMHs, while SemiSWEETs contain three. The functions of SemiSWEETs are less studied. In this perspective article, we analyzed the diversity and conservation of SemiSWEETs and further proposed the possible functions. 1,922 SemiSWEET homologs were retrieved from the UniProt database, which is not proportional to the sequenced prokaryotic genomes. However, these proteins are very diverse in sequences and can be classified into 19 clusters when >50% sequence identity is required. Moreover, a gene context analysis indicated that several SemiSWEETs are located in the operons that are related to diverse carbohydrate metabolism. Several proteins with seven TMHs can be found in bacteria, and sequence alignment suggested that these proteins in bacteria may be formed by the duplication and fusion. Multiple sequence alignments showed that the amino acids for sugar translocation are still conserved and coevolved, although the sequences show diversity. Among them, the functions of a few amino acids are still not clear. These findings highlight the challenges that exist in SemiSWEETs and provide future researchers the foundation to explore these uncharted areas.

  15. Deep-biosphere consortium of fungi and prokaryotes in Eocene subseafloor basalts.

    Science.gov (United States)

    Bengtson, S; Ivarsson, M; Astolfo, A; Belivanova, V; Broman, C; Marone, F; Stampanoni, M

    2014-11-01

    The deep biosphere of the subseafloor crust is believed to contain a significant part of Earth's biomass, but because of the difficulties of directly observing the living organisms, its composition and ecology are poorly known. We report here a consortium of fossilized prokaryotic and eukaryotic micro-organisms, occupying cavities in deep-drilled vesicular basalt from the Emperor Seamounts, Pacific Ocean, 67.5 m below seafloor (mbsf). Fungal hyphae provide the framework on which prokaryote-like organisms are suspended like cobwebs and iron-oxidizing bacteria form microstromatolites (Frutexites). The spatial inter-relationships show that the organisms were living at the same time in an integrated fashion, suggesting symbiotic interdependence. The community is contemporaneous with secondary mineralizations of calcite partly filling the cavities. The fungal hyphae frequently extend into the calcite, indicating that they were able to bore into the substrate through mineral dissolution. A symbiotic relationship with chemoautotrophs, as inferred for the observed consortium, may be a pre-requisite for the eukaryotic colonization of crustal rocks. Fossils thus open a window to the extant as well as the ancient deep biosphere. © 2014 The Authors. Geobiology Published by John Wiley & Sons Ltd.

  16. Prokaryotic diversity, composition structure, and phylogenetic analysis of microbial communities in leachate sediment ecosystems.

    Science.gov (United States)

    Liu, Jingjing; Wu, Weixiang; Chen, Chongjun; Sun, Faqian; Chen, Yingxu

    2011-09-01

    In order to obtain insight into the prokaryotic diversity and community in leachate sediment, a culture-independent DNA-based molecular phylogenetic approach was performed with archaeal and bacterial 16S rRNA gene clone libraries derived from leachate sediment of an aged landfill. A total of 59 archaeal and 283 bacterial rDNA phylotypes were identified in 425 archaeal and 375 bacterial analyzed clones. All archaeal clones distributed within two archaeal phyla of the Euryarchaeota and Crenarchaeota, and well-defined methanogen lineages, especially Methanosaeta spp., are the most numerically dominant species of the archaeal community. Phylogenetic analysis of the bacterial library revealed a variety of pollutant-degrading and biotransforming microorganisms, including 18 distinct phyla. A substantial fraction of bacterial clones showed low levels of similarity with any previously documented sequences and thus might be taxonomically new. Chemical characteristics and phylogenetic inferences indicated that (1) ammonium-utilizing bacteria might form consortia to alleviate or avoid the negative influence of high ammonium concentration on other microorganisms, and (2) members of the Crenarchaeota found in the sediment might be involved in ammonium oxidation. This study is the first to report the composition of the microbial assemblages and phylogenetic characteristics of prokaryotic populations extant in leachate sediment. Additional work on microbial activity and contaminant biodegradation remains to be explored.

  17. Prediction of Metabolic Pathway Involvement in Prokaryotic UniProtKB Data by Association Rule Mining

    KAUST Repository

    Boudellioua, Imene

    2016-07-08

    The widening gap between known proteins and their functions has encouraged the development of methods to automatically infer annotations. Automatic functional annotation of proteins is expected to meet the conflicting requirements of maximizing annotation coverage, while minimizing erroneous functional assignments. This trade-off imposes a great challenge in designing intelligent systems to tackle the problem of automatic protein annotation. In this work, we present a system that utilizes rule mining techniques to predict metabolic pathways in prokaryotes. The resulting knowledge represents predictive models that assign pathway involvement to UniProtKB entries. We carried out an evaluation study of our system performance using cross-validation technique. We found that it achieved very promising results in pathway identification with an F1-measure of 0.982 and an AUC of 0.987. Our prediction models were then successfully applied to 6.2 million UniProtKB/TrEMBL reference proteome entries of prokaryotes. As a result, 663,724 entries were covered, where 436,510 of them lacked any previous pathway annotations.

  18. Relationship between viral and prokaryotic abundance on the Bajo O’Higgins 1 Seamount (Humboldt Current System off Chile

    Directory of Open Access Journals (Sweden)

    Oscar E. Chiang

    2007-03-01

    Full Text Available There is little known about the ecology of microbial communities living in the water column over seamounts. Here, for the first time, the spatial distribution and abundance of virus-like particles (VLP are described over a seamount. The association between VLP distribution, prokaryotic abundance, and environmental variables is also analyzed. Sampling was conducted in December 2004 on the Bajo O’Higgins 1 seamount (32°54’S, 73°53’W located in the Humboldt Current System off Chile. A oxygen minimum layer (OMZ was clearly present between 130 and 280 m in the water column over the seamount. Water samples were taken with Niskin bottles at 10 oceanographic stations over the seamount at depths of 5, 20, 50, 75, 100, and 150 m and at the benthic boundary layer (BBL; 5-12 m over the sediments. Temperature, salinity, oxygen, chlorophyll , and phaeopigments were measured at each station. Viral and prokaryotic abundances were determined with fluorochrome SYBR Green I. Viral abundance ranged from 1.53 x 109 VLP L-1 - 16.48 x 109 VLP L-1, whereas prokaryotic abundance ranged from 1.78 x 10 8 cell L-1 - 14.91 x 108 cell L-1. The virus-like particle/prokaryote ratio varied widely among the analyzed layers (i.e. surface, OMZ, and BBL, probably due to the presence of different prokaryotic and viral assemblages in each layer. Our results indicate that the environmental conditions, mainly the concentration of dissolved oxygen in the water column over Bajo O’Higgins 1 seamount, shape the association between viral and prokaryotic abundance.

  19. Splicing of phenylalanine hydroxylase (PAH) exon 11 is vulnerable - Molecular pathology of mutations in PAH exon 11

    DEFF Research Database (Denmark)

    Heintz, Caroline; Dobrowolski, Steven F.; Andersen, Henriette Skovgaard

    2012-01-01

    as a vulnerable exon and used patient derived lymphoblast cell lines and PAH minigenes to study the molecular defect that impacted pre-mRNA processing. We showed that the c.1144T>C and c.1066-3C>T mutations cause exon 11 skipping, while the c.1139C>T mutation is neutral or slightly beneficial. The c.1144T......In about 20-30% of phenylketonuria (PKU) patients, phenylalanine (Phe) levels can be controlled by cofactor 6R-tetrahydrobiopterin (BH(4)) administration. The phenylalanine hydroxylase (PAH) genotype has a predictive value concerning BH(4)-response and therefore a correct assessment of the mutation...... molecular pathology is important. Mutations that disturb the splicing of exons (e.g. interplay between splice site strength and regulatory sequences like exon splicing enhancers (ESEs)/exon splicing silencers (ESSs)) may cause different severity of PKU. In this study, we identified PAH exon 11...

  20. The metabolism of L-phenylalanine and L-tyrosine by liver cells isolated from adrenalectomized rats and from streptozotocin-diabetic rats.

    OpenAIRE

    Stanley, J C; Fisher, M J; Pogson, C I

    1985-01-01

    Flux through, and maximal activities of, key enzymes of phenylalanine and tyrosine degradation were measured in liver cells prepared from adrenalectomized rats and from streptozotocin-diabetic rats. Adrenalectomy decreased the phenylalanine hydroxylase flux/activity ratio; this was restored by steroid treatment in vivo. Changes in the phosphorylation state of the hydroxylase may mediate these effects; there was no significant change in the maximal activity of the hydroxylase. Tyrosine metabol...

  1. Tracer experiment administering L-phenylalanine-U-14C and L-tyrosine-U-14C to the tissue slices of bamboo shoots

    International Nuclear Information System (INIS)

    Kozukue, E.; Mizuno, S.

    1987-01-01

    Uniformly 14 C-labeled L-phenylalanine and L-tyrosine were administered to tissue slices of both top and base sections of bamboo shoots. Alcohol soluble substances were extracted and then separated into organic acid, sugar and amino acid fractions by ion exchange chromatography. The homogentisic acid fraction among the organic acids was collected by high-performance liquid chromatography (HPLC) and its radioactivity was measured, while the alcohol insoluble residue was used for the analysis of lignin aldehyde by the method of alkaline nitrobenzene oxidation. 1. The two labeled amino acids were steadily incorporated into the tissues during incubation and rapidly converted to organic acid, sugar and alcohol insoluble residue, especially the latter. 2. On determining the amount of phenylalanine converted to tyrosine, it was found that this was extremely small. 3. The incorporation of phenylalanine-U- 14 C into alcohol insoluble residue was higher than that of tyrosine in both sections. 4. Although the conversion into lignin aldehyde from phenylalanine-U- 14 C was higher than that from tyrosine-U- 14 C, it was found that tyrosine incorporated into the shoots was converted to a remarkable extent for formation of lignin aldehyde. 5. The incorporation of phenylalanine and tyrosine into homogentisic acid was very low. From these results, we assume that the conversion of phenylalanine to tyrosine or of tyrosine to homogentisic acid is very small, and that a part of the high amount of tyrosine in the shoots may be used for formation of lignin

  2. Enhancement of L-phenylalanine production by engineered Escherichia coli using phased exponential L-tyrosine feeding combined with nitrogen source optimization.

    Science.gov (United States)

    Yuan, Peipei; Cao, Weijia; Wang, Zhen; Chen, Kequan; Li, Yan; Ouyang, Pingkai

    2015-07-01

    Nitrogen source optimization combined with phased exponential L-tyrosine feeding was employed to enhance L-phenylalanine production by a tyrosine-auxotroph strain, Escherichia coli YP1617. The absence of (NH4)2SO4, the use of corn steep powder and yeast extract as composite organic nitrogen source were more suitable for cell growth and L-phenylalanine production. Moreover, the optimal initial L-tyrosine level was 0.3 g L(-1) and exponential L-tyrosine feeding slightly improved L-phenylalanine production. Nerveless, L-phenylalanine production was greatly enhanced by a strategy of phased exponential L-tyrosine feeding, where exponential feeding was started at the set specific growth rate of 0.08, 0.05, and 0.02 h(-1) after 12, 32, and 52 h, respectively. Compared with exponential L-tyrosine feeding at the set specific growth rate of 0.08 h(-1), the developed strategy obtained a 15.33% increase in L-phenylalanine production (L-phenylalanine of 56.20 g L(-1)) and a 45.28% decrease in L-tyrosine supplementation. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  3. Evaluation of an Adsorbent Based on Agricultural Waste (Corn Cobs for Removal of Tyrosine and Phenylalanine from Aqueous Solutions

    Directory of Open Access Journals (Sweden)

    Cibele C. O. Alves

    2013-01-01

    Full Text Available Adsorption of phenolic amino acids, such as phenylalanine and tyrosine, is quite relevant for the production of protein hydrolysates used as dietary formulations for patients suffering from congenital disorders of amino acid metabolism, such as phenylketonuria. In this study, an adsorbent prepared from corn cobs was evaluated for the removal of tyrosine (Tyr from both a single component solution and a binary aqueous solution with phenylalanine (Phe. The adsorption behavior of tyrosine was similar to that of phenylalanine in single component solutions, however, with a much lower adsorption capacity (14 mg g−1 for Tyr compared to 109 mg g−1 for Phe. Tyr adsorption kinetics was satisfactorily described by a pseudosecond-order model as it was for Phe. In adsorption equilibrium studies for binary mixtures, the presence of Tyr in Phe solutions favored Phe faster adsorption whereas the opposite behavior was observed for the presence of Phe in Tyr solutions. Such results indicate that, in binary systems, Phe will be adsorbed preferably to Tyr, and this is a welcome feature when employing the prepared adsorbent for the removal of Phe from protein hydrolysates to be used in dietary formulations for phenylketonuria treatment.

  4. A phenylalanine in DGAT is a key determinant of oil content and composition in maize.

    Science.gov (United States)

    Zheng, Peizhong; Allen, William B; Roesler, Keith; Williams, Mark E; Zhang, Shirong; Li, Jiming; Glassman, Kimberly; Ranch, Jerry; Nubel, Douglas; Solawetz, William; Bhattramakki, Dinakar; Llaca, Victor; Deschamps, Stéphane; Zhong, Gan-Yuan; Tarczynski, Mitchell C; Shen, Bo

    2008-03-01

    Plant oil is an important renewable resource for biodiesel production and for dietary consumption by humans and livestock. Through genetic mapping of the oil trait in plants, studies have reported multiple quantitative trait loci (QTLs) with small effects, but the molecular basis of oil QTLs remains largely unknown. Here we show that a high-oil QTL (qHO6) affecting maize seed oil and oleic-acid contents encodes an acyl-CoA:diacylglycerol acyltransferase (DGAT1-2), which catalyzes the final step of oil synthesis. We further show that a phenylalanine insertion in DGAT1-2 at position 469 (F469) is responsible for the increased oil and oleic-acid contents. The DGAT1-2 allele with F469 is ancestral, whereas the allele without F469 is a more recent mutant selected by domestication or breeding. Ectopic expression of the high-oil DGAT1-2 allele increases oil and oleic-acid contents by up to 41% and 107%, respectively. This work provides insights into the molecular basis of natural variation of oil and oleic-acid contents in plants and highlights DGAT as a promising target for increasing oil and oleic-acid contents in other crops.

  5. Thermodynamic and spectroscopic studies of alanine and phenylalanine in aqueous β-cyclodextrin solutions

    Directory of Open Access Journals (Sweden)

    Anwar Ali

    2017-01-01

    Full Text Available Ultrasonic speed, u and density, ρ have been measured for dl-alanine (Ala and l-phenylalanine (Phe in aqueous β-cyclodextrin (β-CD at 298.15, 303.15, 308.15, and 313.15 K. The complexation of Ala and Phe with β-CD has been studied by means of UV–vis and thermodynamic (ultrasonic speed and density studies. Using the measured ultrasonic speed and density data the apparent molar compressibility (κS,φ, apparent molar volume (Vφ, limiting apparent molar compressibility (κS,φ0, limiting apparent molar volume (Vφ0, their constants (SK and Sv, and hydration number (nH have been obtained. The positive values of transfer properties at infinite dilution for Ala and Phe in β-CD is the outcome of the balance between released water molecules from β-CD cavity and hydrophobic groups of Ala and Phe that enter into the macrocycle β-CD cavity. The experimental results have also been discussed on the basis of UV–vis absorbance. The results indicate the formation of a more stable host–guest complex between Phe-β-CD than between Ala-β-CD.

  6. Regional mapping of the phenylalanine hydroxylase gene and the phenylketonuria locus in the human genome

    Energy Technology Data Exchange (ETDEWEB)

    Lidsky, A.S.; Law, M.L.; Morse, H.G.; Kao, F.T.; Rabin, M.; Ruddle, F.H.; Woo, S.L.C.

    1985-09-01

    Phenylketonuria (PKU) is an autosomal recessive disorder of amino acid metabolism caused by a deficiency of the hepatic enzyme phenylalanine hydroxylase. To define the regional map position of the disease locus and the PAH gene on human chromosome 12, DNA was isolated from human-hamster somatic cell hybrids with various deletions of human chromosome 12 and was analyzed by Southern blot analysis using the human cDNA PAH clone as a hybridization probe. From these results, together with detailed biochemical and cytogenetic characterization of the hybrid cells, the region on chromosome 12 containing the human PAH gene has been defined as 12q14.3..-->..qter. The PAH map position on chromosome 12 was further localized by in situ hybridization of /sup 125/I-labeled human PAH cDNA to chromosomes prepared from a human lymphoblastoid cell line. Results of these experiments demonstrated that the region on chromosome 12 containing the PAH gene and the PKU locus in man is 12q22..-->..12q24.1. These results not only provide a regionalized map position for a major human disease locus but also can serve as a reference point for linkage analysis with other DNA markers on human chromosome 12.

  7. Regional mapping of the phenylalanine hydroxylase gene and the phenylketonuria locus in the human genome

    International Nuclear Information System (INIS)

    Lidsky, A.S.; Law, M.L.; Morse, H.G.; Kao, F.T.; Rabin, M.; Ruddle, F.H.; Woo, S.L.C.

    1985-01-01

    Phenylketonuria (PKU) is an autosomal recessive disorder of amino acid metabolism caused by a deficiency of the hepatic enzyme phenylalanine hydroxylase. To define the regional map position of the disease locus and the PAH gene on human chromosome 12, DNA was isolated from human-hamster somatic cell hybrids with various deletions of human chromosome 12 and was analyzed by Southern blot analysis using the human cDNA PAH clone as a hybridization probe. From these results, together with detailed biochemical and cytogenetic characterization of the hybrid cells, the region on chromosome 12 containing the human PAH gene has been defined as 12q14.3→qter. The PAH map position on chromosome 12 was further localized by in situ hybridization of 125 I-labeled human PAH cDNA to chromosomes prepared from a human lymphoblastoid cell line. Results of these experiments demonstrated that the region on chromosome 12 containing the PAH gene and the PKU locus in man is 12q22→12q24.1. These results not only provide a regionalized map position for a major human disease locus but also can serve as a reference point for linkage analysis with other DNA markers on human chromosome 12

  8. Gypsy Phenylketonuria: A point mutation of the phenylalanine hydroxylase gene in Gypsy families from Slovakia

    Energy Technology Data Exchange (ETDEWEB)

    Kalanin, J. [Institute for Clinical and Experical Medicine, Praha (Czechoslovakia); Takarada, Y. [Toyobo Research Center, Shiga (Japan); Kagawa, S.; Yamashita, K.; Ohtsuka, N.; Matsuoka, A. [Hyogo College of Medicine, Nishinomiya (Japan)

    1994-01-15

    A direct mutational analysis of the phenylalanine hydroxylase gene (PAH) in Gypsy families with phenylketonuria (PKU) has not yet been presented. However, they obviously represent a group at high risk for this inherited disease. The authors analyzed the PAH loci of 65 Gypsies originating from Eastern Slovakia by a combination of PCR amplification, direct sequencing and ASO hybridization. These studies uncovered 10 {open_quotes}classical PKU{close_quotes} patients to be homozygous for a R252W (CGG-TGG) transition, and 29 heterozygous carriers of this mutation. Fifteen control Caucasoid PKU patients from the Czech and Slovak Republics were selected. In this group they detected R252W mutation in two subjects (6.67% of all mutant alleles). Both were compound heterozygous for two different mutations. Previous haplotype studies of Welsh Gypsies with PKU were uninformative in the determination of heterozygosity. ASO hybridization served effectively for the consequent analyses in Gypsy PKU-related families and to identify the carriers among the unrelated subjects. 19 refs., 2 figs.

  9. Specificity of the Acute Tryptophan and Tyrosine plus Phenylalanine Depletion and Loading Tests Part II:

    Directory of Open Access Journals (Sweden)

    Abdulla A.-B. Badawy

    2010-01-01

    Full Text Available Current formulations for acute tryptophan (Trp or tyrosine (Tyr plus phenylalanine (Phe depletion and loading cause undesirable decreases in ratios of Trp or Tyr + Phe to competing amino acids (CAA, thus undermining the specificities of these tests. Branched-chain amino acids (BCAA cause these unintended decreases, and lowering their content in a new balanced control formulation in the present study led to normalization of all ratios. Four groups (n = 12 each of adults each received one of four 50 g control formulations, with 0% (traditional, 20%, 30%, or 40% less of the BCAA. The free and total [Trp]/[CAA] and [Phe + Tyr]/[BCAA+ Trp] ratios all decreased significantly during the first 5 h following the traditional formulation, but were fully normalized by the formulation containing 40% less of the BCAA. We recommend the latter as a balanced control formulation and propose adjustments in the depletion and loading formulations to enhance their specificities for 5-HT and the catecholamines.

  10. Molecularly imprinted polymer based on chemiluminescence imaging for the chiral recognition of dansyl-phenylalanine.

    Science.gov (United States)

    Wang, Li; Zhang, Zhujun; Huang, Lianggao

    2008-03-01

    A new molecularly imprinted polymer (MIP)-chemiluminescence (CL) imaging detection approach towards chiral recognition of dansyl-phenylalanine (Phe) is presented. The polymer microspheres were synthesized using precipitation polymerization with dansyl-L-Phe as template. Polymer microspheres were immobilized in microtiter plates (96 wells) using poly(vinyl alcohol) (PVA) as glue. The analyte was selectively adsorbed on the MIP microspheres. After washing, the bound fraction was quantified based on peroxyoxalate chemiluminescence (PO-CL) analysis. In the presence of dansyl-Phe, bis(2,4,6-trichlorophenyl)oxalate (TCPO) reacted with hydrogen peroxide (H2O2) to emit chemiluminescence. The signal was detected and quantified with a highly sensitive cooled charge-coupled device (CCD). Influencing factors were investigated and optimized in detail. Control experiments using capillary electrophoresis showed that there was no significant difference between the proposed method and the control method at a confidence level of 95%. The method can perform 96 independent measurements simultaneously in 30 min and the limits of detection (LODs) for dansyl-L-Phe and dansyl-D-Phe were 0.025 micromol L(-1) and 0.075 micromol L(-1) (3sigma), respectively. The relative standard deviation (RSD) for 11 parallel measurements of dansyl-L-Phe (0.78 micromol L(-1)) was 8%. The results show that MIP-based CL imaging can become a useful analytical technology for quick chiral recognition.

  11. Exploration and pharmacokinetic profiling of phenylalanine based carbamates as novel substance p 1-7 analogues.

    Science.gov (United States)

    Fransson, Rebecca; Nordvall, Gunnar; Bylund, Johan; Carlsson-Jonsson, Anna; Kratz, Jadel M; Svensson, Richard; Artursson, Per; Hallberg, Mathias; Sandström, Anja

    2014-12-11

    The bioactive metabolite of Substance P, the heptapeptide SP1-7 (H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH), has been shown to attenuate signs of hyperalgesia in diabetic mice, which indicate a possible use of compounds targeting the SP1-7 binding site as analgesics for neuropathic pain. Aiming at the development of drug-like SP1-7 peptidomimetics we have previously reported on the discovery of H-Phe-Phe-NH2 as a high affinity lead compound. Unfortunately, the pharmacophore of this compound was accompanied by a poor pharmacokinetic (PK) profile. Herein, further lead optimization of H-Phe-Phe-NH2 by substituting the N-terminal phenylalanine for a benzylcarbamate group giving a new type of SP1-7 analogues with good binding affinities is reported. Extensive in vitro as well as in vivo PK characterization is presented for this compound. Evaluation of different C-terminal functional groups, i.e., hydroxamic acid, acyl sulfonamide, acyl cyanamide, acyl hydrazine, and oxadiazole, suggested hydroxamic acid as a bioisosteric replacement for the original primary amide.

  12. Measurement and prediction of physical properties of aqueous sodium salt of L-phenylalanine

    Directory of Open Access Journals (Sweden)

    Garg Sahil

    2017-01-01

    Full Text Available Physical properties, such as density, refractive index and viscosity, of an aqueous sodium salt of (S-2-amino-3-phenylpropionic acid (L-phenylalanine, Na-Phe were investigated in this work. These properties were measured over a temperature range of 298.15–343.15 K at atmospheric pressure. The mass fractions (w of Na-Phe were 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35 and 0.40. The analysis of the experimental data showed that the values of density, refractive index and viscosity decreased with increasing temperature at any constant concentration of Na-Phe. However, these values increased with increasing concentration at any constant temperature. The density values were used to estimate the thermal expansion coefficient. The thermal expansion coefficient increased slightly with increasing temperature and concentration. The density and refractive index data were correlated using a modified Graber equation, while, the viscosity data were correlated using a modified Vogel–Tamman–Fulcher (VTF equation. In all the cases, quantitative analyses of the influence of temperature and concentration were performed.

  13. Asymmetric synthesis of L-[3-11C]phenylalanine using chiral hydrogenation catalysts

    International Nuclear Information System (INIS)

    Halldin, C.; Langstroem, B.

    1984-01-01

    The seven-step synthesis of L-[3- 11 C]phenylalanine using chiral diphosphines as ligands in rhodium catalysts is reported. [ 11 C]Benzaldehyde, prepared in a three-step reaction from [ 11 C]carbon dioxide, as reported elsewhere, was reacted with 2-phenyl-5-oxazolone or 2-(4-chloro)phenyl-5-oxazolone in the presence of the tertiary amine diazabicyclooctane (DABCO). The resultant [α- 11 C]-4-arylene-2-atyl-5-oxazolones were hydrogenated after ring opening, using the chiral rhodium complex of (R)-1,2-bis(diphenylphosphino)propane [(R)-PROPHOS] or (+)-2,3-isopropylidene-2,3-dihydroxy-1,4-bis(diphenylphosphino)butane [(+)-DIOP]. After removal of the amino protecting group, the labelled amino acid was obtained on purification by preparative LC in 10-15% radiochemical yield, and radiochemical purity higher than 95% from [ 11 C]carbon dioxide within 60 min. The optical purity of the products determined by the tRNA method and capillary GC, was 80 and 60% e.e., respectively (i.e. L/D=90/10 and 80/20). (author)

  14. Phenotypic stability of B16-BL6 melanoma exposed to low levels of tyrosine and phenylalanine.

    Science.gov (United States)

    Elstad, C A; Meadows, G G

    1990-01-01

    We previously demonstrated that tyrosine (Tyr) and phenylalanine (Phe) restriction suppresses metastatic heterogeneity of B16-BL6 (BL6) melanoma and selects for tumor variants with decreased metastatic potential. In this study, we investigate stability of this Tyr- and Phe-modulated tumor phenotype by sequentially transplanting BL6 in vivo into mice fed Low Tyr and Phe Diet. Metastatic potential of BL6 is suppressed after one subcutaneous passage. Suppression is unlikely to result from inhibition of tumor growth, since growth in vitro is significantly increased. The metastatic potential of the Tyr- and Phe-modulated tumor is unstable after in vivo passage, and lung colonizing ability is regenerated after ten in vivo passages. Conversely, the antimetastatic effect of Tyr and Phe restriction is stable after prolonged in vitro passage. The metastatic potential of tumors from mice fed Normal Diet is unstable after long-term in vitro culture. Sensitivity to adriamycin of BL6 from mice fed Low Tyr and Phe Diet is increased and is not altered by change in metastatic potential.

  15. Development of an electrochemical biosensor for vitamin B12 using D-phenylalanine nanotubes

    Science.gov (United States)

    Moazeni, Maryam; Karimzadeh, Fathallah; Kermanpur, Ahmad; Allafchian, Alireza

    2018-01-01

    In the past decades, biosensors are one of the most interesting topics among researchers and scientist. The biosensors are used in several applications such as determining food quality, control and diagnose clinical problems and metabolic control. Therefore, many efforts have been carried out to design and develop a new generation of these systems. On the other hand nanotechnology by improving the performance of sensors has created an excellent outlook. Using nanomaterials such as nanoparticles, nanotubes, nanowires, and nanorods in diagnostic tools has been significantly increased accuracy, sensitivity and improved detection limits in sensors. In this study, the one-dimensional morphology of the D-phenylalanine was assembled on the surface of the gold electrode. In the next step electrochemical performance of the modified electrode was investigated by Cyclic Voltammetry (CV), Electrochemical Impedance Spectroscopy (EIS) and Differential Pals Voltammograms (DPV). Finally, by measuring the different concentrations of vitamin B12, the detection limit of the biosensor was obtained 1.6 µM.

  16. A paradigm study for assessment of phenylalanine's damage under arc-discharge irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Ke Zhigang [Laboratory of Ion Beam Bio-engineering, Institute of Plasma Physics of Chinese Academy of Sciences, P.O. Box 1126, Shushanhu Road 350, Hefei 230031 (China); Huang Qing, E-mail: huangq@ipp.ac.c [Laboratory of Ion Beam Bio-engineering, Institute of Plasma Physics of Chinese Academy of Sciences, P.O. Box 1126, Shushanhu Road 350, Hefei 230031 (China); Su Xi; Jiang Jiang; Wang Xiangqin; Yu Zengliang [Laboratory of Ion Beam Bio-engineering, Institute of Plasma Physics of Chinese Academy of Sciences, P.O. Box 1126, Shushanhu Road 350, Hefei 230031 (China)

    2010-05-15

    Energetic ions induce important biological effects and the research into radiolysis of amino acids can help to clarify radiolysis of proteins. For this purpose, arc-discharge induced radiolysis of the benzyl-containing aromatic amino acid phenylalanine in aqueous solution was studied and the damage was assessed quantitatively. The energetic ions were produced by arc-discharge in nitrogen and argon atmosphere. The arc-discharge induced chemical reactions of the biomolecule in aqueous solution were detected and analyzed by means of UV-Vis absorption, fluorescence, Fourier transformation infrared (FTIR) spectroscopy and high performance liquid chromatography (HPLC). Based on the multiple spectroscopic approach, the damage can be evaluated in a more reliable and convenient way. The fluorescence analysis in this case appears to be a more direct indicator for the assessment of damage, where it reveals that the damage increases with the irradiation time exponentially. On the contrary, the 'plateau region' or 'saddle-shape' apparently shown both in our rough absorption analysis and ninhydrin reaction test, similar to the previously reported feature of the dose effect for low-energy-ion induced damage, may be just a consequence with varied reactions and processes involved at different stages. This work thus demonstrated that application of appropriate combination of spectroscopic tools can effectively dissect the diversity of the radiolysis reaction system and assess the biomolecular damage properly.

  17. Preparation of carbon-11 labelled phenylalanine and phenylglycine by a new amino acid synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Vaalburg, W; Beerling-van der Molen, H D; Reiffers, S; Rijskamp, A; Woldring, M G; Wynberg, H [Academic Hospital, Groningen (Netherlands). Central Isotope Lab.

    1976-03-01

    Of the cyclotron-produced short-lived isotopes carbon-11 (tsub(1/2) = 20.4 min;..beta../sup +/) is one of the most promising as label for radiopharmaceuticals. To prepare /sup 11/C-labelled amino acids for evaluation as pancreas scanning agents a new rapid amino acid synthesis was developed. The method is based on the carboxylation of ..cap alpha..-lithioisocyanides with /sup 11/CO/sub 2/, followed by hydrolysis of the intermediate reaction product to the desired amino acid. By this method DL-..cap alpha..-phenylalanine-1-/sup 11/C and DL-..cap alpha..-phenylglycine-1-/sup 11/C were prepared. The precursor /sup 11/CO/sub 2/ was produced via the /sup 14/N(p,..cap alpha..)/sup 11/C reaction by bombardment of a flow of nitrogen gas mixed with 0.1% 0/sub 2/ with 20 MeV protons. The target system is described.

  18. Assessing the evolutionary rate of positional orthologous genes in prokaryotes using synteny data

    Directory of Open Access Journals (Sweden)

    Lespinet Olivier

    2007-11-01

    Full Text Available Abstract Background Comparison of completely sequenced microbial genomes has revealed how fluid these genomes are. Detecting synteny blocks requires reliable methods to determining the orthologs among the whole set of homologs detected by exhaustive comparisons between each pair of completely sequenced genomes. This is a complex and difficult problem in the field of comparative genomics but will help to better understand the way prokaryotic genomes are evolving. Results We have developed a suite of programs that automate three essential steps to study conservation of gene order, and validated them with a set of 107 bacteria and archaea that cover the majority of the prokaryotic taxonomic space. We identified the whole set of shared homologs between two or more species and computed the evolutionary distance separating each pair of homologs. We applied two strategies to extract from the set of homologs a collection of valid orthologs shared by at least two genomes. The first computes the Reciprocal Smallest Distance (RSD using the PAM distances separating pairs of homologs. The second method groups homologs in families and reconstructs each family's evolutionary tree, distinguishing bona fide orthologs as well as paralogs created after the last speciation event. Although the phylogenetic tree method often succeeds where RSD fails, the reverse could occasionally be true. Accordingly, we used the data obtained with either methods or their intersection to number the orthologs that are adjacent in for each pair of genomes, the Positional Orthologous Genes (POGs, and to further study their properties. Once all these synteny blocks have been detected, we showed that POGs are subject to more evolutionary constraints than orthologs outside synteny groups, whichever the taxonomic distance separating the compared organisms. Conclusion The suite of programs described in this paper allows a reliable detection of orthologs and is useful for evaluating gene

  19. PSAT: A web tool to compare genomic neighborhoods of multiple prokaryotic genomes

    Directory of Open Access Journals (Sweden)

    Wasnick Michael

    2008-03-01

    Full Text Available Abstract Background The conservation of gene order among prokaryotic genomes can provide valuable insight into gene function, protein interactions, or events by which genomes have evolved. Although some tools are available for visualizing and comparing the order of genes between genomes of study, few support an efficient and organized analysis between large numbers of genomes. The Prokaryotic Sequence homology Analysis Tool (PSAT is a web tool for comparing gene neighborhoods among multiple prokaryotic genomes. Results PSAT utilizes a database that is preloaded with gene annotation, BLAST hit results, and gene-clustering scores designed to help identify regions of conserved gene order. Researchers use the PSAT web interface to find a gene of interest in a reference genome and efficiently retrieve the sequence homologs found in other bacterial genomes. The tool generates a graphic of the genomic neighborhood surrounding the selected gene and the corresponding regions for its homologs in each comparison genome. Homologs in each region are color coded to assist users with analyzing gene order among various genomes. In contrast to common comparative analysis methods that filter sequence homolog data based on alignment score cutoffs, PSAT leverages gene context information for homologs, including those with weak alignment scores, enabling a more sensitive analysis. Features for constraining or ordering results are designed to help researchers browse results from large numbers of comparison genomes in an organized manner. PSAT has been demonstrated to be useful for helping to identify gene orthologs and potential functional gene clusters, and detecting genome modifications that may result in loss of function. Conclusion PSAT allows researchers to investigate the order of genes within local genomic neighborhoods of multiple genomes. A PSAT web server for public use is available for performing analyses on a growing set of reference genomes through any

  20. Evolutionary consequences of polyploidy in prokaryotes and the origin of mitosis and meiosis.

    Science.gov (United States)

    Markov, Alexander V; Kaznacheev, Ilya S

    2016-06-08

    The origin of eukaryote-specific traits such as mitosis and sexual reproduction remains disputable. There is growing evidence that both mitosis and eukaryotic sex (i.e., the alternation of syngamy and meiosis) may have already existed in the basal eukaryotes. The mating system of the halophilic archaeon Haloferax volcanii probably represents an intermediate stage between typical prokaryotic and eukaryotic sex. H. volcanii is highly polyploid, as well as many other Archaea. Here, we use computer simulation to explore genetic and evolutionary outcomes of polyploidy in amitotic prokaryotes and its possible role in the origin of mitosis, meiosis and eukaryotic sex. Modeling suggests that polyploidy can confer strong short-term evolutionary advantage to amitotic prokaryotes. However, it also promotes the accumulation of recessive deleterious mutations and the risk of extinction in the long term, especially in highly mutagenic environment. There are several possible strategies that amitotic polyploids can use in order to reduce the genetic costs of polyploidy while retaining its benefits. Interestingly, most of these strategies resemble different components or aspects of eukaryotic sex. They include asexual ploidy cycles, equalization of genome copies by gene conversion, high-frequency lateral gene transfer between relatives, chromosome exchange coupled with homologous recombination, and the evolution of more accurate chromosome distribution during cell division (mitosis). Acquisition of mitosis by an amitotic polyploid results in chromosome diversification and specialization. Ultimately, it transforms a polyploid cell into a functionally monoploid one with multiple unique, highly redundant chromosomes. Specialization of chromosomes makes the previously evolved modes of promiscuous chromosome shuffling deleterious. This can result in selective pressure to develop accurate mechanisms of homolog pairing, and, ultimately, meiosis. Emergence of mitosis and the first

  1. Clustered regularly interspaced short palindromic repeats (CRISPRs): the hallmark of an ingenious antiviral defense mechanism in prokaryotes.

    Science.gov (United States)

    Al-Attar, Sinan; Westra, Edze R; van der Oost, John; Brouns, Stan J J

    2011-04-01

    Many prokaryotes contain the recently discovered defense system against mobile genetic elements. This defense system contains a unique type of repetitive DNA stretches, termed Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs). CRISPRs consist of identical repeated DNA sequences (repeats), interspaced by highly variable sequences referred to as spacers. The spacers originate from either phages or plasmids and comprise the prokaryotes' 'immunological memory'. CRISPR-associated (cas) genes encode conserved proteins that together with CRISPRs make-up the CRISPR/Cas system, responsible for defending the prokaryotic cell against invaders. CRISPR-mediated resistance has been proposed to involve three stages: (i) CRISPR-Adaptation, the invader DNA is encountered by the CRISPR/Cas machinery and an invader-derived short DNA fragment is incorporated in the CRISPR array. (ii) CRISPR-Expression, the CRISPR array is transcribed and the transcript is processed by Cas proteins. (iii) CRISPR-Interference, the invaders' nucleic acid is recognized by complementarity to the crRNA and neutralized. An application of the CRISPR/Cas system is the immunization of industry-relevant prokaryotes (or eukaryotes) against mobile-genetic invasion. In addition, the high variability of the CRISPR spacer content can be exploited for phylogenetic and evolutionary studies. Despite impressive progress during the last couple of years, the elucidation of several fundamental details will be a major challenge in future research.

  2. Rational development of an attenuated recombinant cyprinid herpesvirus 3 vaccine using prokaryotic mutagenesis and in vivo bioluminescent imaging

    Science.gov (United States)

    Cyprinid herpesvirus 3 (CyHV-3) is causing severe economic losses worldwide in the carp industry, and a safe and efficacious attenuated vaccine compatible with mass vaccination is needed. We produced single deleted recombinants using prokaryotic mutagenesis. When producing a recombinant lacking open...

  3. Metagenomic Sequence of Prokaryotic Microbiota from an Intermediate-Salinity Pond of a Saltern in Isla Cristina, Spain

    OpenAIRE

    Fernández, Ana B.; León, María José; Vera, Blanca; Sánchez-Porro, Cristina; Ventosa, Antonio

    2014-01-01

    Marine salterns are artificial multipond systems designed for the commercial production of salt by evaporation of seawater. We report here the metagenomic sequence of the prokaryotic microbiota of a pond with intermediate salinity (21% total salts) of a saltern located in Isla Cristina, Huelva, southwest Spain.

  4. Metagenomic sequence of prokaryotic microbiota from an intermediate-salinity pond of a saltern in isla cristina, Spain.

    Science.gov (United States)

    Fernández, Ana B; León, María José; Vera, Blanca; Sánchez-Porro, Cristina; Ventosa, Antonio

    2014-02-13

    Marine salterns are artificial multipond systems designed for the commercial production of salt by evaporation of seawater. We report here the metagenomic sequence of the prokaryotic microbiota of a pond with intermediate salinity (21% total salts) of a saltern located in Isla Cristina, Huelva, southwest Spain.

  5. A neural network method for identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites

    DEFF Research Database (Denmark)

    Nielsen, Henrik; Engelbrecht, Jacob; Brunak, Søren

    1997-01-01

    We have developed a new method for the identication of signal peptides and their cleavage sites based on neural networks trained on separate sets of prokaryotic and eukaryotic sequences. The method performs signicantly better than previous prediction schemes, and can easily be applied to genome...

  6. Cloning, expression, and purification of recombinant protein MPT-64 from a virulent strain of Mycobacterium bovis in a prokaryotic system

    Directory of Open Access Journals (Sweden)

    Maryam Mohammadi Tashakkori

    2016-01-01

    Conclusion: These results indicated successful expression and purification of recombinant MPT-64 protein in a prokaryotic system. This protein can be used for serological diagnosis, improved detection of pathogenicity and non-pathogenicity between infected cattle, and for verification of suspected cases of bovine TB.

  7. Emerging experimental and computational technologies for purpose designed engineering of photosynthetic prokaryotes

    KAUST Repository

    Lindblad, Peter

    2016-01-25

    With recent advances in synthetic molecular tools to be used in photosynthetic prokaryotes, like cyanobacteria, it is possible to custom design and construct microbial cells for specific metabolic functions. This cross-disciplinary area of research has emerged within the interfaces of advanced genetic engineering, computational science, and molecular biotechnology. We have initiated the development of a genetic toolbox, using a synthetic biology approach, to custom design, engineer and construct cyanobacteria for selected function and metabolism. One major bottleneck is a controlled transcription and translation of introduced genetic constructs. An additional major issue is genetic stability. I will present and discuss recent progress in our development of genetic tools for advanced cyanobacterial biotechnology. Progress on understanding the electron pathways in native and engineered cyanobacterial enzymes and heterologous expression of non-native enymzes in cyanobacterial cells will be highlighted. Finally, I will discuss our attempts to merge synthetic biology with synthetic chemistry to explore fundamantal questions of protein design and function.

  8. Production of isotopically labeled heterologous proteins in non-E. coli prokaryotic and eukaryotic cells

    International Nuclear Information System (INIS)

    Takahashi, Hideo; Shimada, Ichio

    2010-01-01

    The preparation of stable isotope-labeled proteins is necessary for the application of a wide variety of NMR methods, to study the structures and dynamics of proteins and protein complexes. The E. coli expression system is generally used for the production of isotope-labeled proteins, because of the advantages of ease of handling, rapid growth, high-level protein production, and low cost for isotope-labeling. However, many eukaryotic proteins are not functionally expressed in E. coli, due to problems related to disulfide bond formation, post-translational modifications, and folding. In such cases, other expression systems are required for producing proteins for biomolecular NMR analyses. In this paper, we review the recent advances in expression systems for isotopically labeled heterologous proteins, utilizing non-E. coli prokaryotic and eukaryotic cells.

  9. Powered by light: Phototrophy and photosynthesis in prokaryotes and its evolution.

    Science.gov (United States)

    Nowicka, Beatrycze; Kruk, Jerzy

    2016-01-01

    Photosynthesis is a complex metabolic process enabling photosynthetic organisms to use solar energy for the reduction of carbon dioxide into biomass. This ancient pathway has revolutionized life on Earth. The most important event was the development of oxygenic photosynthesis. It had a tremendous impact on the Earth's geochemistry and the evolution of living beings, as the rise of atmospheric molecular oxygen enabled the development of a highly efficient aerobic metabolism, which later led to the evolution of complex multicellular organisms. The mechanism of photosynthesis has been the subject of intensive research and a great body of data has been accumulated. However, the evolution of this process is not fully understood, and the development of photosynthesis in prokaryota in particular remains an unresolved question. This review is devoted to the occurrence and main features of phototrophy and photosynthesis in prokaryotes. Hypotheses concerning the origin and spread of photosynthetic traits in bacteria are also discussed. Copyright © 2016 Elsevier GmbH. All rights reserved.

  10. Binding site of ribosomal proteins on prokaryotic 5S ribonucleic acids: a study with ribonucleases

    DEFF Research Database (Denmark)

    Douthwaite, S; Christensen, A; Garrett, R A

    1982-01-01

    The binding sites of ribosomal proteins L18 and L25 on 5S RNA from Escherichia coli were probed with ribonucleases A, T1, and T2 and a double helix specific cobra venom endonuclease. The results for the protein-RNA complexes, which were compared with those for the free RNA [Douthwaite, S...... stearothermophilus 5S RNA. Several protein-induced changes in the RNA structures were identified; some are possibly allosteric in nature. The two prokaryotic 5S RNAs were also incubated with total 50S subunit proteins from E. coli and B. stearothermophilus ribosomes. Homologous and heterologous reconstitution....... stearothermophilus 5S RNA, which may have been due to a third ribosomal protein L5....

  11. [Prokaryote diversity in water environment of land-ocean ecotone of Zhuhai City].

    Science.gov (United States)

    Huang, Xiao-Lan; Chen, Jian-Yao; Zhou, Shi-Ning; Xie, Li-Chun; Fu, Cong-Sheng

    2010-02-01

    By constructing 16S rDNA clone library with PCR-RFLP, the prokaryote diversity in the seawater and groundwater of land-ocean ecotone of Zhuhai City was investigated, and the similarity and cluster analyses were implemented with the database of the sequences in Genbank. In the seawater, Proteobacteria was dominant, followed by Archaeon, Gemmatimonadetes, Candidate division OP3 and OP8, and Planctomycetes, etc.; while in the groundwater, Archaeon was dominant, followed by Proteobacteria, Sphingobacteria, Candidate division OP3, Actinobacterium, and Pseudomonas. The dominant taxa in the groundwater had high similarity to the unculturable groups of marine microorganisms. Large amount of bacteria capable of degrading organic matter and purifying water body existed in the groundwater, suggesting that after long-term evolution, the land-ocean ecotone of Zhuhai City had the characteristics of both land and ocean.

  12. [A study of recombinant human sestrin 1 and sestrin 2 proteins produced in a prokaryotic system].

    Science.gov (United States)

    Rai, N; Kumar, R; Haque, Md A; Hassan, Md I; Dey, S

    2017-01-01

    Sestrins are highly conserved stress-inducible proteins capable of suppressing the production of ROS and signalling through mTORC1. Here we report a study of human sestrin1 (sesn1) and sestrin2 (sesn2) proteins produced in a pET28^(+) vector based prokaryotic system. Mass spectrometry analysis, western blot and surface plasmon resonance (SPR) of affinity purified sesn1 and sesn2 proteins confirmed their identity; biophysical characteristics were observed using circular dichroism (CD) showing that sesn1 and sesn2 have a predominant α-helical structure. Here we describe a simple, one step purification process to purify a large amount of sestrin proteins with significant yield. Further study of recombinant human sestrins may further facilitate the understanding of their roles in eukaryotic cells.

  13. [Ultrastructural basis of interactions between prokaryotes and eukaryotes in different symbiotic models].

    Science.gov (United States)

    Sacchi, L

    2004-06-01

    This paper reviews the Author's contribution to the knowledge of the ultrastructural basis of the prokaryote-eukaryote interactions in different models assessed by an ultrastructural approach. In agreement with the hypothesis of the origin of eukaryotic cells, which are chimeras of several prokaryotes with different morpho-functional specializations, symbiosis had major consequence for evolution of life. In Arthropods, one of the most successful lifestyles, the presence of endosymbiotic prokaryotes, plays an important role in their metabolism. In some cases, genome integration has occurred in the endosymbiotic relationships with the host, proving that intracellular symbiosis is not merely a nutritional supplement. Intracellular symbiotic bacteria are also described in nematodes. In particular, the presence of intracellular Wolbachia in filariae, even if its function is not yet completely known, influences positively the reproductive biology and the survival of the host, as proved by antibiotic treatment against this bacterium. The ultrastructural images reported in this review were obtained using different species of cockroaches, termites, ticks and filarial nematodes. The traditional methods of transmission (TEM), scansion (SEM) and immuno electron microscopy were used. In addition, also freeze-fracture and deep-etching techniques were employed. The cockroaches and the primitive termite Mastotermes darwiniensis host symbiotic bacteria in the ovary and in specialized cells (bacteriocytes) of the fat body. These bacteria have the typical cell boundary profile of gram-negative bacteria and are enveloped in a vacuolar membrane produced by the host cell. Molecular sequence data of 16S rDNA of endosymbionts of five species of cockroaches and M. darwiniensis indicate that they are members of the Flavobacteria-bacteroides group and that the infection occurred in an ancestor common to cockroaches and termites probably after the end of the Paleozoic (250 Ma BP). The

  14. Pangenomic Definition of Prokaryotic Species and the Phylogenetic Structure of Prochlorococcus spp.

    Directory of Open Access Journals (Sweden)

    Mikhail A. Moldovan

    2018-03-01

    Full Text Available The pangenome is the collection of all groups of orthologous genes (OGGs from a set of genomes. We apply the pangenome analysis to propose a definition of prokaryotic species based on identification of lineage-specific gene sets. While being similar to the classical biological definition based on allele flow, it does not rely on DNA similarity levels and does not require analysis of homologous recombination. Hence this definition is relatively objective and independent of arbitrary thresholds. A systematic analysis of 110 accepted species with the largest numbers of sequenced strains yields results largely consistent with the existing nomenclature. However, it has revealed that abundant marine cyanobacteria Prochlorococcus marinus should be divided into two species. As a control we have confirmed the paraphyletic origin of Yersinia pseudotuberculosis (with embedded, monophyletic Y. pestis and Burkholderia pseudomallei (with B. mallei. We also demonstrate that by our definition and in accordance with recent studies Escherichia coli and Shigella spp. are one species.

  15. Nitrate addition has minimal short-term impacts on greenland ice sheet supraglacial prokaryotes

    DEFF Research Database (Denmark)

    Cameron, Karen A.; Stibal, Marek; Chrismas, Nathan

    2017-01-01

    Tropospheric nitrate levels are predicted to increase throughout the 21st century, with potential effects on terrestrial ecosystems, including the Greenland ice sheet (GrIS). This study considers the impacts of elevated nitrate concentrations on the abundance and composition of dominant bulk...... and active prokaryotic communities sampled from in situ nitrate fertilization plots on the GrIS surface. Nitrate concentrations were successfully elevated within sediment-filled meltwater pools, known as cryoconite holes; however, nitrate additions applied to surface ice did not persist. Estimated bulk...... cryoconite communities were not nitrate limited at the time of sampling. Instead, temporal changes in biomass and community composition were more pronounced. As these in situ incubations were short (6 weeks), and the community composition across GrIS surface ice is highly variable, we suggest that further...

  16. GenomePeek—an online tool for prokaryotic genome and metagenome analysis

    Directory of Open Access Journals (Sweden)

    Katelyn McNair

    2015-06-01

    Full Text Available As more and more prokaryotic sequencing takes place, a method to quickly and accurately analyze this data is needed. Previous tools are mainly designed for metagenomic analysis and have limitations; such as long runtimes and significant false positive error rates. The online tool GenomePeek (edwards.sdsu.edu/GenomePeek was developed to analyze both single genome and metagenome sequencing files, quickly and with low error rates. GenomePeek uses a sequence assembly approach where reads to a set of conserved genes are extracted, assembled and then aligned against the highly specific reference database. GenomePeek was found to be faster than traditional approaches while still keeping error rates low, as well as offering unique data visualization options.

  17. Genome-based analysis of heme biosynthesis and uptake in prokaryotic systems.

    Science.gov (United States)

    Cavallaro, Gabriele; Decaria, Leonardo; Rosato, Antonio

    2008-11-01

    Heme is the prosthetic group of many proteins that carry out a variety of key biological functions. In addition, for many pathogenic organisms, heme (acquired from the host) may constitute a very important source of iron. Organisms can meet their heme demands by taking it up from external sources, by producing the cofactor through a dedicated biosynthetic pathway, or both. Here we analyzed the distribution of proteins specifically involved in the processes of heme biosynthesis and heme uptake in 474 prokaryotic organisms. These data allowed us to identify which organisms are capable of performing none, one, or both processes, based on the similarity to known systems. Some specific instances where one or more proteins along the pathways had unusual modifications were singled out. For two key protein domains involved in heme uptake, we could build a series of structural models, which suggested possible alternative modes of heme binding. Future directions for experimental work are given.

  18. Prokaryotic Abundance and Activity in Permafrost of the Northern Victoria Land and Upper Victoria Valley (Antarctica).

    Science.gov (United States)

    La Ferla, Rosabruna; Azzaro, Maurizio; Michaud, Luigi; Caruso, Gabriella; Lo Giudice, Angelina; Paranhos, Rodolfo; Cabral, Anderson S; Conte, Antonella; Cosenza, Alessandro; Maimone, Giovanna; Papale, Maria; Rappazzo, Alessandro Ciro; Guglielmin, Mauro

    2017-08-01

    Victoria Land permafrost harbours a potentially large pool of cold-affected microorganisms whose metabolic potential still remains underestimated. Three cores (BC-1, BC-2 and BC-3) drilled at different depths in Boulder Clay (Northern Victoria Land) and one sample (DY) collected from a core in the Dry Valleys (Upper Victoria Valley) were analysed to assess the prokaryotic abundance, viability, physiological profiles and potential metabolic rates. The cores drilled at Boulder Clay were a template of different ecological conditions (different temperature regime, ice content, exchanges with atmosphere and with liquid water) in the same small basin while the Dry Valleys site was very similar to BC-2 conditions but with a complete different geological history and ground ice type. Image analysis was adopted to determine cell abundance, size and shape as well as to quantify the potential viable and respiring cells by live/dead and 5-cyano-2,3-ditolyl-tetrazolium chloride staining, respectively. Subpopulation recognition by apparent nucleic acid contents was obtained by flow cytometry. Moreover, the physiological profiles at community level by Biolog-Ecoplate™ as well as the ectoenzymatic potential rates on proteinaceous (leucine-aminopeptidase) and glucidic (ß-glucosidase) organic matter and on organic phosphates (alkaline-phosphatase) by fluorogenic substrates were tested. The adopted methodological approach gave useful information regarding viability and metabolic performances of microbial community in permafrost. The occurrence of a multifaceted prokaryotic community in the Victoria Land permafrost and a large number of potentially viable and respiring cells (in the order of 10 4 -10 5 ) were recognised. Subpopulations with a different apparent DNA content within the different samples were observed. The physiological profiles stressed various potential metabolic pathways among the samples and intense utilisation rates of polymeric carbon compounds and carbohydrates

  19. Pyrosequencing assessment of prokaryotic and eukaryotic diversity in biofilm communities from a French river.

    Science.gov (United States)

    Bricheux, Geneviève; Morin, Loïc; Le Moal, Gwenaël; Coffe, Gérard; Balestrino, Damien; Charbonnel, Nicolas; Bohatier, Jacques; Forestier, Christiane

    2013-06-01

    Despite the recent and significant increase in the study of aquatic microbial communities, little is known about the microbial diversity of complex ecosystems such as running waters. This study investigated the biodiversity of biofilm communities formed in a river with 454 Sequencing™. This river has the particularity of integrating both organic and microbiological pollution, as receiver of agricultural pollution in its upstream catchment area and urban pollution through discharges of the wastewater treatment plant of the town of Billom. Different regions of the small subunit (SSU) ribosomal RNA gene were targeted using nine pairs of primers, either universal or specific for bacteria, eukarya, or archaea. Our aim was to characterize the widest range of rDNA sequences using different sets of polymerase chain reaction (PCR) primers. A first look at reads abundance revealed that a large majority (47-48%) were rare sequences (<5 copies). Prokaryotic phyla represented the species richness, and eukaryotic phyla accounted for a small part. Among the prokaryotic phyla, Proteobacteria (beta and alpha) predominated, followed by Bacteroidetes together with a large number of nonaffiliated bacterial sequences. Bacillariophyta plastids were abundant. The remaining bacterial phyla, Verrucomicrobia and Cyanobacteria, made up the rest of the bulk biodiversity. The most abundant eukaryotic phyla were annelid worms, followed by Diatoms, and Chlorophytes. These latter phyla attest to the abundance of plastids and the importance of photosynthetic activity for the biofilm. These findings highlight the existence and plasticity of multiple trophic levels within these complex biological systems. © 2013 The Authors. Microbiology Open published by John Wiley & Sons Ltd.

  20. Diversity of 23S rRNA genes within individual prokaryotic genomes.

    Directory of Open Access Journals (Sweden)

    Anna Pei

    Full Text Available BACKGROUND: The concept of ribosomal constraints on rRNA genes is deduced primarily based on the comparison of consensus rRNA sequences between closely related species, but recent advances in whole-genome sequencing allow evaluation of this concept within organisms with multiple rRNA operons. METHODOLOGY/PRINCIPAL FINDINGS: Using the 23S rRNA gene as an example, we analyzed the diversity among individual rRNA genes within a genome. Of 184 prokaryotic species containing multiple 23S rRNA genes, diversity was observed in 113 (61.4% genomes (mean 0.40%, range 0.01%-4.04%. Significant (1.17%-4.04% intragenomic variation was found in 8 species. In 5 of the 8 species, the diversity in the primary structure had only minimal effect on the secondary structure (stem versus loop transition. In the remaining 3 species, the diversity significantly altered local secondary structure, but the alteration appears minimized through complex rearrangement. Intervening sequences (IVS, ranging between 9 and 1471 nt in size, were found in 7 species. IVS in Deinococcus radiodurans and Nostoc sp. encode transposases. T. tengcongensis was the only species in which intragenomic diversity >3% was observed among 4 paralogous 23S rRNA genes. CONCLUSIONS/SIGNIFICANCE: These findings indicate tight ribosomal constraints on individual 23S rRNA genes within a genome. Although classification using primary 23S rRNA sequences could be erroneous, significant diversity among paralogous 23S rRNA genes was observed only once in the 184 species analyzed, indicating little overall impact on the mainstream of 23S rRNA gene-based prokaryotic taxonomy.

  1. Prokaryotic diversity in one of the largest hypersaline coastal lagoons in the world.

    Science.gov (United States)

    Clementino, M M; Vieira, R P; Cardoso, A M; Nascimento, A P A; Silveira, C B; Riva, T C; Gonzalez, A S M; Paranhos, R; Albano, R M; Ventosa, A; Martins, O B

    2008-07-01

    Araruama Lagoon is an environment characterized by high salt concentrations. The low raining and high evaporation rates in this region favored the development of many salty ponds around the lagoon. In order to reveal the microbial composition of this system, we performed a 16S rRNA gene survey. Among archaea, most clones were related to uncultured environmental Euryarchaeota. In lagoon water, we found some clones related to Methanomicrobia and Methanothermococcus groups, while in the saline pond water members related to the genus Haloarcula were detected. Bacterial community was dominated by clones related to Gamma-proteobacteria, Actinobacteria, and Synechococcus in lagoon water, while Salinibacter ruber relatives dominated in saline pond. We also detected the presence of Alpha-proteobacteria, Pseudomonas-like bacteria and Verrucomicrobia. Only representatives of the genus Ralstonia were cosmopolitan, being observed in both systems. The detection of a substantial number of clones related to uncultured archaea and bacteria suggest that the hypersaline waters of Araruama harbor a pool of novel prokaryotic phylotypes, distinct from those observed in other similar systems. We also observed clones related to halophilic genera of cyanobacteria that are specific for each habitat studied. Additionally, two bacterioplankton molecular markers with ecological relevance were analyzed, one is linked to nitrogen fixation (nifH) and the other is linked to carbon fixation by bacterial photosynthesis, the protochlorophyllide genes, revealing a specific genetic distribution in this ecosystem. This is the first study of the biogeography and community structure of microbial assemblages in Brazilian tropical hypersaline environments. This work is directed towards a better understanding of the free-living prokaryotic diversity adapted to life in hypersaline waters.

  2. OxyGene: an innovative platform for investigating oxidative-response genes in whole prokaryotic genomes

    Directory of Open Access Journals (Sweden)

    Barloy-Hubler Frédérique

    2008-12-01

    Full Text Available Abstract Background Oxidative stress is a common stress encountered by living organisms and is due to an imbalance between intracellular reactive oxygen and nitrogen species (ROS, RNS and cellular antioxidant defence. To defend themselves against ROS/RNS, bacteria possess a subsystem of detoxification enzymes, which are classified with regard to their substrates. To identify such enzymes in prokaryotic genomes, different approaches based on similarity, enzyme profiles or patterns exist. Unfortunately, several problems persist in the annotation, classification and naming of these enzymes due mainly to some erroneous entries in databases, mistake propagation, absence of updating and disparity in function description. Description In order to improve the current annotation of oxidative stress subsystems, an innovative platform named OxyGene has been developed. It integrates an original database called OxyDB, holding thoroughly tested anchor-based signatures associated to subfamilies of oxidative stress enzymes, and a new anchor-driven annotator, for ab initio detection of ROS/RNS response genes. All complete Bacterial and Archaeal genomes have been re-annotated, and the results stored in the OxyGene repository can be interrogated via a Graphical User Interface. Conclusion OxyGene enables the exploration and comparative analysis of enzymes belonging to 37 detoxification subclasses in 664 microbial genomes. It proposes a new classification that improves both the ontology and the annotation of the detoxification subsystems in prokaryotic whole genomes, while discovering new ORFs and attributing precise function to hypothetical annotated proteins. OxyGene is freely available at: http://www.umr6026.univ-rennes1.fr/english/home/research/basic/software

  3. Phylogenetic and Comparative Sequence Analysis of Thermostable Alpha Amylases of kingdom Archea, Prokaryotes and Eukaryotes.

    Science.gov (United States)

    Huma, Tayyaba; Maryam, Arooma; Rehman, Shahid Ur; Qamar, Muhammad Tahir Ul; Shaheen, Tayyaba; Haque, Asma; Shaheen, Bushra

    2014-01-01

    Alpha amylase family is generally defined as a group of enzymes that can hydrolyse and transglycosylase α-(1, 4) or α-(1, 6) glycosidic bonds along with the preservation of anomeric configuration. For the comparative analysis of alpha amylase family, nucleotide sequences of seven thermo stable organisms of Kingdom Archea i.e. Pyrococcus furiosus (100-105°C), Kingdom Prokaryotes i.e. Bacillus licheniformis (90-95°C), Geobacillus stearothermophilus (75°C), Bacillus amyloliquefaciens (72°C), Bacillus subtilis (70°C) and Bacillus KSM K38 (55°C) and Eukaryotes i.e. Aspergillus oryzae (60°C) were selected from NCBI. Primary structure composition analysis and Conserved sequence analysis were conducted through Bio Edit tools. Results from BioEdit shown only three conserved regions of base pairs and least similarity in MSA of the above mentioned alpha amylases. In Mega 5.1 Phylogeny of thermo stable alpha amylases of Kingdom Archea, Prokaryotes and Eukaryote was handled by Neighbor-Joining (NJ) algorithm. Mega 5.1 phylogenetic results suggested that alpha amylases of thermo stable organisms i.e. Pyrococcus furiosus (100-105°C), Bacillus licheniformis (90-95°C), Geobacillus stearothermophilus (75°C) and Bacillus amyloliquefaciens (72°C) are more distantly related as compared to less thermo stable organisms. By keeping in mind the characteristics of most thermo stable alpha amylases novel and improved features can be introduced in less thermo stable alpha amylases so that they become more thermo tolerant and productive for industry.

  4. SERS study of transformation of phenylalanine to tyrosine under particle irradiation

    Science.gov (United States)

    Zhang, Jingjing; Huang, Qing; Yao, Guohua; Ke, Zhigang; Zhang, Hong; Lu, Yilin

    2014-08-01

    Surface enhanced Raman scattering or spectroscopy (SERS) is a very powerful analytical tool which has been widely applied in many scientific research and application fields. It is therefore also very intriguing for us to introduce SERS technique in the radiobiological research, where in many cases only a very few of biomolecules are subjected to changes which however can lead to significant biological effects. The radiation induced biochemical reactions are normally very sophisticated with different substances produced in the system, so currently it is still a big challenge for SERS to analyze such a mixture system which contains multiple analytes. In this context, this work aimed to establish and consolidate the feasibility of SERS as an effective tool in radiation chemistry, and this purpose, we employed SERS as a sensitive probe to a known process, namely, the oxidation of phenylalanine (Phe) under particle irradiation, where the energetic particles were obtained from either plasma discharge or electron-beam. During the irradiation, three types of tyrosine (Tyr), namely, p-Tyr, m-Tyr and o-Tyr were produced, and all these tyrosine isomers together with Phe could be identified and measured based on the SERS spectral analysis of the corresponding enhanced characteristic signals, namely, 1002 cm-1 for Phe, 1161 cm-1 for p-Tyr, 990 cm-1 for m-Tyr, and 970 cm-1 for o-Tyr, respectively. The estimation of the quantities of different tyrosine isomers were also given and verified by conventional method such as high performance liquid chromatography (HPLC). As for comparison of different ways of particle irradiation, our results also indicated that electron-beam irradiation was more efficient for converting Phe into Tyr than plasma discharge treatment, confirming the role of hydroxyl radicals in the Phe-Tyr conformation. Therefore, our work has not only demonstrated that SERS can be successfully applied in the radiobiological study, but also given insights into the

  5. Mutations of the phenylalanine hydroxylase gene in patients with phenylketonuria in Shanxi, China

    Directory of Open Access Journals (Sweden)

    Yong-An Zhou

    2012-01-01

    Full Text Available The variation in mutations in exons 3, 6, 7, 11 and 12 of the phenylalanine hydroxylase (PAH gene was investigated in 59 children with phenylketonuria (PKU and 100 normal children. Three single nucleotide polymorphisms were detected by sequence analysis. The mutational frequencies of cDNA 696, cDNA 735 and cDNA 1155 in patients were 96.2%, 76.1% and 7.6%, respectively, whereas in healthy children the corresponding frequencies were 97.0%, 77.3% and 8.3%. In addition, 81 mutations accounted for 61.0% of the mutant alleles. R111X, H64 > TfsX9 and S70 del accounted for 5.1%, 0.8% and 0.8% mutation of alleles in exon 3, whereas EX6-96A > G accounted for 10.2% mutation of alleles in exon 6. R243Q had the highest incidence in exon 7 (12.7%, followed by Ivs7 +2T>A (5.1% and T278I (2.5%. G247V, R252Q, L255S, R261Q and E280K accounted for 0.8% while Y356X and V399V accounted for 5.9% and 5.1%, respectively, in exon 11. R413P and A434D accounted for 5.9% and 2.5%, respectively, in exon 12. Seventy-two variant alleles accounted for the 16 mutations observed here. The mutation characteristics and distributions demonstrated that EX6-96A > G and R243Q were the hot regions for mutations in the PAH gene in Shanxi patients with PKU.

  6. Phenylalanine hydroxylase gene mutations in the United States: Report from the maternal PKU collaborative study

    Energy Technology Data Exchange (ETDEWEB)

    Guldberg, P.; Henriksen, K.F.; Guettler, F. [John F. Kennedy Inst., Glostrup (Denmark)] [and others

    1996-07-01

    The major cause of hyperphenylalaninemia is mutations in the gene encoding phenylalanine hydroxylase (PAH). The known mutations have been identified primarily in European patients. The purpose of this study was to determine the spectrum of mutations responsible for PAH deficiency in the United States. One hundred forty-nine patients enrolled in the Maternal PKU Collaborative Study were subjects for clinical and molecular investigations. PAH gene mutations associated with phenylketonuria (PKU) or mild hyperphenylalaninemia (MHP) were identified on 279 of 294 independent mutant chromosomes, a diagnostic efficiency of 95%. The spectrum is composed of 71 different mutations, including 47 missense mutations, 11 splice mutations, 5 nonsense mutations, and 8 microdeletions. Sixteen previously unreported mutations were identified. Among the novel mutations, five were found in patients with MHP, and the remainder were found in patients with PKU. The most common mutations were R408W, IVS12nt1g{r_arrow}a, and Y414C, accounting for 18.7%, 7.8% and 5.4% of the mutant chromosomes, respectively. Thirteen mutations had relative frequencies of 1%-5%, and 55 mutations each had frequencies {le}1%. The mutational spectrum corresponded to that observed for the European ancestry of the U.S. population. To evaluate the extent of allelic variation at the PAH locus within the United States in comparison with other populations, we used allele frequencies to calculate the homozygosity for 11 populations where >90% ascertainment has been obtained. The United States was shown to contain one of the most heterogeneous populations, with homozygosity values similar to Sicily and ethnically mixed sample populations in Europe. The extent of allelic heterogeneity must be a major determining factor in the choice of mutation-detection methodology for molecular diagnosis in PAH deficiency. 47 refs., 1 fig., 5 tabs.

  7. 1H MR chemical shift imaging detection of phenylalanine in patients suffering from phenylketonuria (PKU)

    International Nuclear Information System (INIS)

    Sijens, Paul E.; Oudkerk, Matthijs; Reijngoud, Dirk-Jan; Spronsen, Francjan J. van; Leenders, Klaas L.; Valk, Harold W. de

    2004-01-01

    Short echo time single voxel methods were used in previous MR spectroscopy studies of phenylalanine (Phe) levels in phenylketonuria (PKU) patients. In this study, apparent T 2 relaxation time of the 7.3-ppm Phe multiplet signal in the brain of PKU patients was assessed in order to establish which echo time would be optimal. 1 H chemical shift imaging (CSI) examinations of a transverse plain above the ventricles of the brain were performed in 10 PKU patients and 11 persons not suffering from PKU at 1.5 T, using four echo times (TE 20, 40, 135 and 270 ms). Phe was detectable only when the signals from all CSI voxels were summarized. In patients suffering from PKU the T 2 relaxation times of choline, creatine and N-acetyl aspartate (NAA) were similar to those previously reported for healthy volunteers (between 200 and 325 ms). The T 2 of Phe in brain tissue was 215±120 ms (standard deviation). In the PKU patients the brain tissue Phe concentrations were 141±69 μM as opposed to 58±23 μM in the persons not suffering from PKU. In the detection of Phe, MR spectroscopy performed at TE 135 or 270 ms is not inferior to that performed at TE 20 or 40 ms (all previous studies). Best results were obtained at TE=135 ms, relating to the fact that at that particular TE, the visibility of a compound with a T 2 of 215 ms still is good, while interfering signals from short-TE compounds are negligible. (orig.)

  8. {sup 1}H MR chemical shift imaging detection of phenylalanine in patients suffering from phenylketonuria (PKU)

    Energy Technology Data Exchange (ETDEWEB)

    Sijens, Paul E.; Oudkerk, Matthijs [University Hospital Groningen, Department of Radiology, Hanzeplein 1, P.O. Box 30001, Groningen (Netherlands); Reijngoud, Dirk-Jan; Spronsen, Francjan J. van [University Hospital Groningen, Department of Pediatrics, Groningen (Netherlands); Leenders, Klaas L. [University Hospital Groningen, Department of Neurology, Groningen (Netherlands); Valk, Harold W. de [University Medical Centre of Utrecht, Department of Internal Medicine, Utrecht (Netherlands)

    2004-10-01

    Short echo time single voxel methods were used in previous MR spectroscopy studies of phenylalanine (Phe) levels in phenylketonuria (PKU) patients. In this study, apparent T{sub 2} relaxation time of the 7.3-ppm Phe multiplet signal in the brain of PKU patients was assessed in order to establish which echo time would be optimal. {sup 1}H chemical shift imaging (CSI) examinations of a transverse plain above the ventricles of the brain were performed in 10 PKU patients and 11 persons not suffering from PKU at 1.5 T, using four echo times (TE 20, 40, 135 and 270 ms). Phe was detectable only when the signals from all CSI voxels were summarized. In patients suffering from PKU the T{sub 2} relaxation times of choline, creatine and N-acetyl aspartate (NAA) were similar to those previously reported for healthy volunteers (between 200 and 325 ms). The T{sub 2} of Phe in brain tissue was 215{+-}120 ms (standard deviation). In the PKU patients the brain tissue Phe concentrations were 141{+-}69 {mu}M as opposed to 58{+-}23 {mu}M in the persons not suffering from PKU. In the detection of Phe, MR spectroscopy performed at TE 135 or 270 ms is not inferior to that performed at TE 20 or 40 ms (all previous studies). Best results were obtained at TE=135 ms, relating to the fact that at that particular TE, the visibility of a compound with a T{sub 2} of 215 ms still is good, while interfering signals from short-TE compounds are negligible. (orig.)

  9. Associations between mutations and a VNTR in the human phenylalanine hydroxylase gene

    Energy Technology Data Exchange (ETDEWEB)

    Goltsov, A.A.; Eisensmith, R.C.; Woo, S.L.C. (Baylor College of Medicine, Houston, TX (United States)); Konecki, D.S.; Lichter-Konecki, U.

    1992-09-01

    The HindIII RFLP in the human phenylalanine hydroxylase (PAH) gene is caused by the presence of an AT-rich (70%) minisatellite region. This region contains various multiples of 30-bp tandem repeats and is located 3 kb downstream of the final exon of the gene. PCR-mediated amplification of this region from haplotyped PAH chromosomes indicates that the previously reported 4.0-kb HindIII allele contains three of these repeats, while the 4.4-kb HindIII allele contains 12 of these repeats. The 4.2-kb HindIII fragment can contain six, seven, eight, or nine copies of this repeat. These variations permit more detailed analysis of mutant haplotypes 1, 5, 6, and, possibly, others. Kindred analysis in phenylketonuria families demonstrates Mendelian segregation of these VNTR alleles, as well as associations between theses alleles and certain PAH mutations. The R261Q mutation, associated with haplotype 1, is associated almost exclusively with an allele containing eight repeats; the R408W mutation, when occurring on a haplotype 1 background, may also be associated with the eight-repeat VNTR allele. Other PAH mutations associated with haplotype 1, R252W and P281L, do not appear to segregate with specific VNTR alleles. The IVS-10 mutation, when associated with haplotype 6, is associated exclusively with an allele containing seven repeats. The combined use of this VNTR system and the existing RFLP haplotype system will increase the performance of prenatal diagnostic tests based on haplotype analysis. In addition, this VNTR may prove useful in studies concerning the origins and distributions of PAH mutations in different human populations. 32 refs., 3 figs., 3 tabs.

  10. Application of SAIL phenylalanine and tyrosine with alternative isotope-labeling patterns for protein structure determination

    Energy Technology Data Exchange (ETDEWEB)

    Takeda, Mitsuhiro [Nagoya University, Structural Biology Research Center, Graduate School of Science (Japan); Ono, Akira M.; Terauchi, Tsutomu [SAIL Technologies Co., Inc. (Japan); Kainosho, Masatsune, E-mail: kainosho@nmr.chem.metro-u.ac.j [Nagoya University, Structural Biology Research Center, Graduate School of Science (Japan)

    2010-01-15

    The extensive collection of NOE constraint data involving the aromatic ring signals is essential for accurate protein structure determination, although it is often hampered in practice by the pervasive signal overlapping and tight spin couplings for aromatic rings. We have prepared various types of stereo-array isotope labeled phenylalanines ({epsilon}- and {zeta}-SAIL Phe) and tyrosine ({epsilon}-SAIL Tyr) to overcome these problems (Torizawa et al. 2005), and proven that these SAIL amino acids provide dramatic spectral simplification and sensitivity enhancement for the aromatic ring NMR signals. In addition to these SAIL aromatic amino acids, we recently synthesized {delta}-SAIL Phe and {delta}-SAIL Tyr, which allow us to observe and assign {delta}-{sup 13}C/{sup 1}H signals very efficiently. Each of the various types of SAIL Phe and SAIL Tyr yields well-resolved resonances for the {delta}-, {epsilon}- or {zeta}-{sup 13}C/{sup 1}H signals, respectively, which can readily be assigned by simple and robust pulse sequences. Since the {delta}-, {epsilon}-, and {zeta}-proton signals of Phe/Tyr residues give rise to complementary NOE constraints, the concomitant use of various types of SAIL-Phe and SAIL-Tyr would generate more accurate protein structures, as compared to those obtained by using conventional uniformly {sup 13}C, {sup 15}N-double labeled proteins. We illustrated this with the case of an 18.2 kDa protein, Escherichia coli peptidyl-prolyl cis-trans isomerase b (EPPIb), and concluded that the combined use of {zeta}-SAIL Phe and {epsilon}-SAIL Tyr would be practically the best choice for protein structural determinations.

  11. Elevated phenylalanine on newborn screening: follow-up testing may reveal undiagnosed galactosaemia.

    Science.gov (United States)

    Shakespeare, Lynette; Downing, Melanie; Allen, Joyce; Casbolt, Ann-Marie; Ellin, Sheila; Maloney, Martin; Race, Gillian; Bonham, Jim

    2010-11-01

    Introduction Newborn screening for phenylketonuria (PKU) can reveal other conditions which lead to an increased blood spot phenylalanine (Phe) concentration. We have investigated the proportion of blood spot samples that gave a positive screen due to clinically significant conditions other than PKU, compared the positive predictive value (PPV) of our referral Phe cut-off with that recommended by the UK Newborn Screening Programme Centre (UKNSPC) (>210 and >240 μmol/L, respectively) and evaluated the effectiveness of reflex testing for galactosaemia using a lower blood spot Phe cut-off concentration of 130 μmol/L. All blood spot samples that screened positive, for an increased Phe concentration, between April 2001 and March 2008, were identified from the records of the Sheffield Newborn Screening Laboratory and the diagnoses noted. In addition, all cases of galactosaemia detected in or notified to our screening laboratory within this time were also examined and the screened Phe concentrations compared. Out of 438,674 babies who were screened, 67 had Phe concentration >210 μmol/L (15 per 100,000). Of these, 40 had PKU or persistent hyperphenylalaninaemia with a Phe concentration identified by screening between 270 and 2350 μmol/L. A further 11 were diagnosed with another clinically significant disorder: galactosaemia (n = 8), biopterin defects (n = 2), tyrosinaemia Type 1 (n = 1). In addition, 16 had transient elevations in Phe. In total, nine cases of galactosaemia were identified, of whom, three had Phe concentrations 240 μmol/L) will not affect the detection rate of classical PKU, but will improve the PPV from 76% to 80%. The use of a lower cut-off (130 μmol/L) for reflex galactosaemia testing enables the timely identification of asymptomatic cases that benefit particularly from early treatment, without prompting any unnecessary clinical referrals or delaying any referrals. This intervention may reduce mortality in this vulnerable group.

  12. Economic impact of feeding a phenylalanine-restricted diet to adults with previously untreated phenylketonuria.

    Science.gov (United States)

    Brown, M C; Guest, J F

    1999-02-01

    The aim of the present study was to estimate the direct healthcare cost of managing adults with previously untreated phenylketonuria (PKU) for one year before any dietary restrictions and for the first year after a phenylalanine- (PHE-) restricted diet was introduced. The resource use and corresponding costs were estimated from medical records and interviews with health care professionals experienced in caring for adults with previously untreated PKU. The mean annual cost of caring for a client being fed an unrestricted diet was estimated to be 83 996 pound silver. In the first year after introducing a PHE-restricted diet, the mean annual cost was reduced by 20 647 pound silver to 63 348 pound silver as a result of a reduction in nursing time, hospitalizations, outpatient clinic visits and medications. However, the economic benefit of the diet depended on whether the clients were previously high or low users of nursing care. Nursing time was the key cost-driver, accounting for 79% of the cost of managing high users and 31% of the management cost for low users. In contrast, the acquisition cost of a PHE-restricted diet accounted for up to 6% of the cost for managing high users and 15% of the management cost for low users. Sensitivity analyses showed that introducing a PHE-restricted diet reduces the annual cost of care, provided that annual nursing time was reduced by more than 8% or more than 5% of clients respond to the diet. The clients showed fewer negative behaviours when being fed a PHE-restricted diet, which may account for the observed reduction in nursing time needed to care for these clients. In conclusion, feeding a PHE-restricted diet to adults with previously untreated PKU leads to economic benefits to the UK's National Health Service and society in general.

  13. Effect of inhaled formyl-methionyl-leucyl-phenylalanine on airway function.

    Science.gov (United States)

    Berend, N; Peters, M J; Armour, C L; Black, J L; Ward, H E

    1988-01-01

    Formyl-methionyl-leucyl-phenylalanine (FMLP), a synthetic, acylated tripeptide analogous to bacterial chemotactic factors, has been shown to cause bronchoconstriction in guinea pig, rabbit, and human airways in vitro. To determine whether FMLP causes bronchoconstriction in man in vivo, a preliminary study was undertaken in which five non-smokers (mean age 35 years, FEV1 94% (SEM 5%) predicted) and five smokers (mean age 34 years, FEV1 93% (6%) predicted) inhaled aerosols of FMLP. None of the subjects showed airway hyperresponsiveness to histamine (the provocative concentrations of histamine causing a fall of greater than or equal to 20% in FEV1 (PC20) were over 8 mg/ml). FMLP dissolved in 50% dimethylsulphoxide and 50% saline in concentrations of 0, 0.06, 0.12, 0.25, 0.5, 1.0, 2.0, and 4.0 mg/ml was administered to the subjects by means of a French-Rosenthal dosimeter, FEV1 being recorded after inhalation of each concentration. Dose dependent falls in FEV1 occurred in five non-smokers (geometric mean 1.76, 95% confidence limits 0.87-3.53 mg/ml) and three smokers (0.23, 0.07-0.78 mg/ml), with two smokers not responding by 20% to the highest concentration of FMLP. On a separate day the FMLP dose-response curves were repeated after nebulisation of 500 micrograms of ipratropium bromide. The PC20 FMLP in the responders more than doubled. In six additional normal subjects a histamine inhalation test was performed before and four and 24 hours after inhalation of FMLP. All subjects remained unresponsive to histamine. These results show that FMLP is a potent bronchoconstrictor in some non-asthmatic individuals in vivo and this may be important in bronchoconstriction related to infection in patients with chronic obstructive lung disease.

  14. No Tryptophan, Tyrosine and Phenylalanine Abnormalities in Children with Attention-Deficit/Hyperactivity Disorder.

    Directory of Open Access Journals (Sweden)

    Catharina Elisabeth Bergwerff

    Full Text Available The aim of the current study was to explore the role of aromatic amino acids (AAAs in blood in relation to attention-deficit/hyperactivity disorder (ADHD. Given their impact on the synthesis of serotonin and dopamine, decreased concentrations of the AAAs tryptophan, tyrosine and phenylalanine in blood may contribute to the expression of ADHD symptoms. Decreased AAA blood concentrations, in turn, may be related to lowered dietary protein intake or to abnormal AAA catabolism, as evidenced by increased urinary AAA concentrations.Eighty-three children with ADHD (75% males and 72 typically developing (TD children (51% males, aged 6 to 13 years, participated in the study. AAA concentrations were assessed in blood spots and an 18-hour urinary sample. A nutritional diary was filled out by parents to calculate dietary protein intake. Parent and teacher questionnaires assessed symptoms of ADHD, oppositional defiant disorder, conduct disorder, and autism spectrum disorder.Children with ADHD showed normal AAA concentrations in blood spots and urine, as well as normal protein intake compared to controls. No associations between AAA concentrations and symptoms of ADHD or comorbid psychiatric disorders were found.This study is the first to explore AAA metabolism in children with ADHD using a well-defined and relatively large sample. We found that AAA deficiencies are not related to ADHD. The results do not support treatment with AAA supplements in children with ADHD. Future studies regarding the cause of serotonin and dopamine alterations in ADHD should focus on other explanations, such as effects of altered transport of AAAs.

  15. Application of SAIL phenylalanine and tyrosine with alternative isotope-labeling patterns for protein structure determination.

    Science.gov (United States)

    Takeda, Mitsuhiro; Ono, Akira M; Terauchi, Tsutomu; Kainosho, Masatsune

    2010-01-01

    The extensive collection of NOE constraint data involving the aromatic ring signals is essential for accurate protein structure determination, although it is often hampered in practice by the pervasive signal overlapping and tight spin couplings for aromatic rings. We have prepared various types of stereo-array isotope labeled phenylalanines (epsilon- and zeta-SAIL Phe) and tyrosine (epsilon-SAIL Tyr) to overcome these problems (Torizawa et al. 2005), and proven that these SAIL amino acids provide dramatic spectral simplification and sensitivity enhancement for the aromatic ring NMR signals. In addition to these SAIL aromatic amino acids, we recently synthesized delta-SAIL Phe and delta-SAIL Tyr, which allow us to observe and assign delta-(13)C/(1)H signals very efficiently. Each of the various types of SAIL Phe and SAIL Tyr yields well-resolved resonances for the delta-, epsilon- or zeta-(13)C/(1)H signals, respectively, which can readily be assigned by simple and robust pulse sequences. Since the delta-, epsilon-, and zeta-proton signals of Phe/Tyr residues give rise to complementary NOE constraints, the concomitant use of various types of SAIL-Phe and SAIL-Tyr would generate more accurate protein structures, as compared to those obtained by using conventional uniformly (13)C, (15)N-double labeled proteins. We illustrated this with the case of an 18.2 kDa protein, Escherichia coli peptidyl-prolyl cis-trans isomerase b (EPPIb), and concluded that the combined use of zeta-SAIL Phe and epsilon-SAIL Tyr would be practically the best choice for protein structural determinations.

  16. Application of SAIL phenylalanine and tyrosine with alternative isotope-labeling patterns for protein structure determination

    International Nuclear Information System (INIS)

    Takeda, Mitsuhiro; Ono, Akira M.; Terauchi, Tsutomu; Kainosho, Masatsune

    2010-01-01

    The extensive collection of NOE constraint data involving the aromatic ring signals is essential for accurate protein structure determination, although it is often hampered in practice by the pervasive signal overlapping and tight spin couplings for aromatic rings. We have prepared various types of stereo-array isotope labeled phenylalanines (ε- and ζ-SAIL Phe) and tyrosine (ε-SAIL Tyr) to overcome these problems (Torizawa et al. 2005), and proven that these SAIL amino acids provide dramatic spectral simplification and sensitivity enhancement for the aromatic ring NMR signals. In addition to these SAIL aromatic amino acids, we recently synthesized δ-SAIL Phe and δ-SAIL Tyr, which allow us to observe and assign δ- 13 C/ 1 H signals very efficiently. Each of the various types of SAIL Phe and SAIL Tyr yields well-resolved resonances for the δ-, ε- or ζ- 13 C/ 1 H signals, respectively, which can readily be assigned by simple and robust pulse sequences. Since the δ-, ε-, and ζ-proton signals of Phe/Tyr residues give rise to complementary NOE constraints, the concomitant use of various types of SAIL-Phe and SAIL-Tyr would generate more accurate protein structures, as compared to those obtained by using conventional uniformly 13 C, 15 N-double labeled proteins. We illustrated this with the case of an 18.2 kDa protein, Escherichia coli peptidyl-prolyl cis-trans isomerase b (EPPIb), and concluded that the combined use of ζ-SAIL Phe and ε-SAIL Tyr would be practically the best choice for protein structural determinations.

  17. The in vivo anti-fibrotic function of calcium sensitive receptor (CaSR) modulating poly(p-dioxanone-co-l-phenylalanine) prodrug.

    Science.gov (United States)

    Wang, Bing; Wen, Aiping; Feng, Chengmin; Niu, Lijing; Xiao, Xin; Luo, Le; Shen, Chengyi; Zhu, Jiang; Lei, Jun; Zhang, Xiaoming

    2018-04-13

    In present study, the apoptosis induction and proliferation suppression effects of l-phenylalanine (l-Phe) on fibroblasts were confirmed. The action sites of l-Phe on fibroblasts suppression were deduced to be calcium sensitive receptor (CaSR) which could cause the release of endoplasmic reticulum (ER) Ca 2+ stores; disruption of intracellular Ca 2+ homeostasis triggers cell apoptosis via the ER or mitochondrial pathways. The down-regulation of CaSR were observed after the application of l-Phe, and the results those l-Phe triggered the increasing of intracellular Ca 2+ concentration and calcineurin expression, and then the apoptosis and increasing G1 fraction of fibroblasts have verified our deduction. Hence, l-Phe could be seen as a kind of anti-fibrotic drugs for the crucial participation of fibroblast in the occurrence of fibrosis. And then, poly(p-dioxanone-co-l-phenylalanine) (PDPA) which could prolong the in-vivo anti-fibrotic effect of l-Phe for the sustained release of l-Phe during its degradation could be treated as anti-fibrotic polymer prodrugs. Based on the above, the in vivo anti-fibrotic function of PDPA was evaluated in rabbit ear scarring, rat peritoneum lipopolysaccharide, and rat sidewall defect/cecum abrasion models. PDPA reduced skin scarring and suppressed peritoneal fibrosis and post operation adhesion as well as secretion of transforming growth factor-β1 in injured tissue. These results indicate that PDPA is an effective agent for preventing fibrosis following tissue injury. We have previously demonstrated that poly(p-dioxanone-co-l-phenylalanine) (PDPA) could induce apoptosis to fibroblast and deduced that the inhibitory effect comes from l-phenylalanine. In present study, the inhibition mechanism of l-phenylalanine on fibroblast proliferation was demonstrated. The calcium sensitive receptor (CaSR) was found to be the action site. The CaSR was downregulated after the application of l-phenylalanine, and then the ER Ca 2+ stores were released

  18. Influence of the composition of aqueous-alcohol solvents on the thermodynamic characteristics of L-phenylalanine dissolution at 298.15 K

    International Nuclear Information System (INIS)

    Badelin, Valentin G.; Smirnov, Valeriy I.

    2011-01-01

    Highlights: ► Enthalpies of L-phenylalanine dissolution have been measured in aqueous methanol, ethanol, 1-propanol and 2-propanol. ► The measured data were reported as functions of composition of water + alcohol mixtures. ► Enthalpy coefficients of pair-wise interactions have been analyzed in terms of McMillan-Mayer theory. ► A comparative analysis of the characteristics of dissolution of L-phenylalanine and some other L-amino acids in the similar systems has been made. - Abstract: The enthalpies of L-phenylalanine dissolution in aqueous methanol, ethanol, 1-propanol and 2-propanol have been determined by calorimetry at 298.15 K and alcohol mole fractions up to x 2 ∼0.4. The standard enthalpies of solution Δ sol H° and transfer Δ tr H° from water to the mixed solvent as well as the enthalpy coefficients of L-phenylalanine–alcohol pair-wise interactions were calculated. The interrelation of the enthalpies of dissolution and transfer for L-phenylalanine with structural features of alcohols has been determined. A comparative analysis of the thermodynamic characteristics of dissolution of L-phenylalanine and some other amino acids (glycine, L-alanine, L-threonine and L-valine) in the mixtures studied has been made.

  19. Synthesis, uptake mechanism characterization and biological evaluation of {sup 18}F labeled fluoroalkyl phenylalanine analogs as potential PET imaging agents

    Energy Technology Data Exchange (ETDEWEB)

    Wang Limin [Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104 (United States); Qu Wenchao; Lieberman, Brian P.; Ploessl, Karl [Department of Radiology, University of Pennsylvania, Philadelphia, PA 19104 (United States); Kung, Hank F., E-mail: kunghf@gmail.co [Department of Radiology, University of Pennsylvania, Philadelphia, PA 19104 (United States); Department of Pharmacology, University of Pennsylvania, Philadelphia, PA 19104 (United States)

    2011-01-15

    Introduction: Amino acids based tracers represent a promising class of tumor metabolic imaging agents with successful clinical applications. Two new phenylalanine derivatives, p-(2-[{sup 18}F]fluoroethyl)-L-phenylalanine (FEP, [{sup 18}F]2) and p-(3-[{sup 18}F]fluoropropyl)-L-phenylalanine (FPP, [{sup 18}F]3) were synthesized and evaluated in comparison to clinically utilized O-(2-[{sup 18}F]fluoroethyl)-L-tyrosine (FET, [{sup 18}F]1). Methods: FEP ([{sup 18}F]2) and FPP ([{sup 18}F]3) were successfully synthesized by a rapid and efficient two-step nucleophilic fluorination of tosylate precursors and deprotection reaction. In vitro cell uptake studies were carried out in 9L glioma cells. In vivo studies, 9L tumor xenografts were implanted in Fisher 344 rats. Results: FEP ([{sup 18}F]2) and FPP ([{sup 18}F]3) could be efficiently labeled within 90 min with good enantiomeric purity (>95%), good yield (11-37%) and high specific activity (21-69 GBq/{mu}mol). Cell uptake studies showed FEP had higher uptake than FPP as well as reference ligand FET ([{sup 18}F]1). Uptake mechanism studies suggested that FEP is a selective substrate for system L and prefers its subtype LAT1. In vivo biodistribution studies demonstrated FEP had specific accumulation in tumor cells and tumor to background ratio reached 1.45 at 60 min. Small animal positron emission tomography (PET) imaging studies showed FEP was comparable to FET for imaging rats bearing 9L tumor model. FEP had high uptake in 9L tumor compared to surrounding tissue and was quickly excreted through urinary tract. Conclusion: Biological evaluations indicate that FEP ([{sup 18}F]2) is a potential useful tracer for tumor imaging with PET.

  20. Fasting Increases Human Skeletal Muscle Net Phenylalanine Release and This Is Associated with Decreased mTOR Signaling

    Science.gov (United States)

    Vendelbo, Mikkel Holm; Møller, Andreas Buch; Christensen, Britt; Nellemann, Birgitte; Clasen, Berthil Frederik Forrest; Nair, K. Sreekumaran; Jørgensen, Jens Otto Lunde; Jessen, Niels; Møller, Niels

    2014-01-01

    Aim Fasting is characterised by profound changes in energy metabolism including progressive loss of body proteins. The underlying mechanisms are however unknown and we therefore determined the effects of a 72-hour-fast on human skeletal muscle protein metabolism and activation of mammalian target of rapamycin (mTOR), a key regulator of cell growth. Methods Eight healthy male volunteers were studied twice: in the postabsorptive state and following 72 hours of fasting. Regional muscle amino acid kinetics was measured in the forearm using amino acid tracers. Signaling to protein synthesis and breakdown were assessed in skeletal muscle biopsies obtained during non-insulin and insulin stimulated conditions on both examination days. Results Fasting significantly increased forearm net phenylalanine release and tended to decrease phenylalanine rate of disappearance. mTOR phosphorylation was decreased by ∼50% following fasting, together with reduced downstream phosphorylation of 4EBP1, ULK1 and rpS6. In addition, the insulin stimulated increase in mTOR and rpS6 phosphorylation was significantly reduced after fasting indicating insulin resistance in this part of the signaling pathway. Autophagy initiation is in part regulated by mTOR through ULK1 and fasting increased expression of the autophagic marker LC3B-II by ∼30%. p62 is degraded during autophagy but was increased by ∼10% during fasting making interpretation of autophagic flux problematic. MAFbx and MURF1 ubiquitin ligases remained unaltered after fasting indicating no change in protesomal protein degradation. Conclusions Our results show that during fasting increased net phenylalanine release in skeletal muscle is associated to reduced mTOR activation and concomitant decreased downstream signaling to cell growth. PMID:25020061

  1. Fasting increases human skeletal muscle net phenylalanine release and this is associated with decreased mTOR signaling.

    Directory of Open Access Journals (Sweden)

    Mikkel Holm Vendelbo

    Full Text Available Fasting is characterised by profound changes in energy metabolism including progressive loss of body proteins. The underlying mechanisms are however unknown and we therefore determined the effects of a 72-hour-fast on human skeletal muscle protein metabolism and activation of mammalian target of rapamycin (mTOR, a key regulator of cell growth.Eight healthy male volunteers were studied twice: in the postabsorptive state and following 72 hours of fasting. Regional muscle amino acid kinetics was measured in the forearm using amino acid tracers. Signaling to protein synthesis and breakdown were assessed in skeletal muscle biopsies obtained during non-insulin and insulin stimulated conditions on both examination days.Fasting significantly increased forearm net phenylalanine release and tended to decrease phenylalanine rate of disappearance. mTOR phosphorylation was decreased by ∼50% following fasting, together with reduced downstream phosphorylation of 4EBP1, ULK1 and rpS6. In addition, the insulin stimulated increase in mTOR and rpS6 phosphorylation was significantly reduced after fasting indicating insulin resistance in this part of the signaling pathway. Autophagy initiation is in part regulated by mTOR through ULK1 and fasting increased expression of the autophagic marker LC3B-II by ∼30%. p62 is degraded during autophagy but was increased by ∼10% during fasting making interpretation of autophagic flux problematic. MAFbx and MURF1 ubiquitin ligases remained unaltered after fasting indicating no change in protesomal protein degradation.Our results show that during fasting increased net phenylalanine release in skeletal muscle is associated to reduced mTOR activation and concomitant decreased downstream signaling to cell growth.

  2. De Novo Synthesis of Benzenoid Compounds by the Yeast Hanseniaspora vineae Increases the Flavor Diversity of Wines.

    Science.gov (United States)

    Martin, Valentina; Giorello, Facundo; Fariña, Laura; Minteguiaga, Manuel; Salzman, Valentina; Boido, Eduardo; Aguilar, Pablo S; Gaggero, Carina; Dellacassa, Eduardo; Mas, Albert; Carrau, Francisco

    2016-06-08

    Benzyl alcohol and other benzenoid-derived metabolites of particular importance in plants confer floral and fruity flavors to wines. Among the volatile aroma components in Vitis vinifera grape varieties, benzyl alcohol is present in its free and glycosylated forms. These compounds are considered to originate from grapes only and not from fermentative processes. We have found increased levels of benzyl alcohol in red Tannat wine compared to that in grape juice, suggesting de novo formation of this metabolite during vinification. In this work, we show that benzyl alcohol, benzaldehyde, p-hydroxybenzaldehyde, and p-hydroxybenzyl alcohol are synthesized de novo in the absence of grape-derived precursors by Hanseniaspora vineae. Levels of benzyl alcohol produced by 11 different H. vineae strains were 20-200 times higher than those measured in fermentations with Saccharomyces cerevisiae strains. These results show that H. vineae contributes to flavor diversity by increasing grape variety aroma concentration in a chemically defined medium. Feeding experiments with phenylalanine, tryptophan, tyrosine, p-aminobenzoic acid, and ammonium in an artificial medium were tested to evaluate the effect of these compounds either as precursors or as potential pathway regulators for the formation of benzenoid-derived aromas. Genomic analysis shows that the phenylalanine ammonia-lyase (PAL) and tyrosine ammonia lyase (TAL) pathways, used by plants to generate benzyl alcohols from aromatic amino acids, are absent in the H. vineae genome. Consequently, alternative pathways derived from chorismate with mandelate as an intermediate are discussed.

  3. Biochemical changes involved in browning of gamma-irradiated cut witloof chicory

    International Nuclear Information System (INIS)

    Hanotel, L.; Fleuriet, A.; Boisseau, P.

    1995-01-01

    The increasing browning phenomenon of cut witloof chicory (Cichorium intybus) irradiated with a dose of 3 kGy and stored five days at 10 °C was investigated in relation to total phenolic concentration, phenylalanine ammonia-lyase activity, total and reduced glutathione content, polyphenol oxidase and peroxidase activities. Phenylalanine ammonia-lyase activity underwent a strong activation with a maximum on the second day of storage, and total phenolic compounds tended to accumulate from the third day. Glutathione content was monitored as indicator of antioxidant capacities. Total glutathione decreased by 25% after irradiation and was restored on the third day of storage, while the 50% decrease of reduced glutathione was maintained along the five days. Conversely, a 30% inhibition of polyphenol oxidase and peroxidase activities occured after irradiation. We concluded first that irradiation-induced browning of cut chicory may be due to both an increase of phenolic metabolism and a reduction in antioxidant capacities, and second that increased membrane permeability may allow substrate-enzyme contact

  4. Thiamine primed defense provides reliable alternative to systemic fungicide carbendazim against sheath blight disease in rice (Oryza sativa L.).

    Science.gov (United States)

    Bahuguna, Rajeev Nayan; Joshi, Rohit; Shukla, Alok; Pandey, Mayank; Kumar, J

    2012-08-01

    A novel pathogen defense strategy by thiamine priming was evaluated for its efficacy against sheath blight pathogen, Rhizoctonia solani AG-1A, of rice and compared with that of systemic fungicide, carbendazim (BCM). Seeds of semidwarf, high yielding, basmati rice variety Vasumati were treated with thiamine (50 mM) and BCM (4 mM). The pot cultured plants were challenge inoculated with R. solani after 40 days of sowing and effect of thiamine and BCM on rice growth and yield traits was examined. Higher hydrogen peroxide content, total phenolics accumulation, phenylalanine ammonia lyase (PAL) activity and superoxide dismutase (SOD) activity under thiamine treatment displayed elevated level of systemic resistance, which was further augmented under challenging pathogen infection. High transcript level of phenylalanine ammonia lyase (PAL) and manganese superoxide dismutase (MnSOD) validated mode of thiamine primed defense. Though minimum disease severity was observed under BCM treatment, thiamine produced comparable results, with 18.12 per cent lower efficacy. Along with fortifying defense components and minor influence on photosynthetic pigments and nitrate reductase (NR) activity, thiamine treatment significantly reduced pathogen-induced loss in photosynthesis, stomatal conductance, chlorophyll fluorescence, NR activity and NR transcript level. Physiological traits affected under pathogen infection were found signatory for characterizing plant's response under disease and were detectable at early stage of infection. These findings provide a novel paradigm for developing alternative, environmentally safe strategies to control plant diseases. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  5. Probe the Binding Mode of Aristololactam-β-D-glucoside to Phenylalanine Transfer RNA in Silico

    DEFF Research Database (Denmark)

    Xiao, Xingqing; Zhao, Binwu; Yang, Li

    2016-01-01

    Understanding the interactions of drug molecules with biomacromolecules at a micro-scale level is essential to design potent drugs for the treatments of human genome diseases. To unravel the mechanism of binding of aristololactam-β-D-glucoside (ADG) and phenylalanine transfer RNA (t...... on the tRNAPhe, and atomistic MD simulations were conducted to examine the thermal stability of five predicted binding poses for the complex of ADG and the tRNAPhe. The binding free energies of the five complexes were then calculated using the molecular mechanics/generalized born surface area approach...

  6. The GPR139 reference agonists 1a and 7c, and tryptophan and phenylalanine share a common binding site

    DEFF Research Database (Denmark)

    Shehata, Mohamed A.; Jensen, Anne Cathrine Nøhr; Jespers, Willem

    2017-01-01

    for GPR139. Two series published by Shi et al. and Dvorak et al. included agonists 1a and 7c respectively, with potencies in the ten-nanomolar range. Furthermore, Isberg et al. and Liu et al. have previously shown that tryptophan (Trp) and phenylalanine (Phe) can activate GPR139 in the hundred...... important for the activation of GPR139 as predicted by the receptor model. The initial ligand-receptor complex was optimized through free energy perturbation simulations, generating a refined GPR139 model in agreement with experimental data. In summary, the GPR139 reference surrogate agonists 1a and 7c...

  7. Glacier inputs influence organic matter composition and prokaryotic distribution in a high Arctic fjord (Kongsfjorden, Svalbard)

    KAUST Repository

    Bourgeois, Solveig

    2016-08-23

    With climate change, the strong seasonality and tight pelagic-benthic coupling in the Arctic is expected to change in the next few decades. It is currently unclear how the benthos will be affected by changes of environmental conditions such as supplies of organic matter (OM) from the water column. In the last decade, Kongsfjorden (79°N), a high Arctic fjord in Svalbard influenced by several glaciers and Atlantic water inflow, has been a site of great interest owing to its high sensitivity to climate change, evidenced by a reduction in ice cover and an increase in melting freshwater. To investigate how spatial and seasonal changes in vertical fluxes can impact the benthic compartment of Kongsfjorden, we studied the organic matter characteristics (in terms of quantity and quality) and prokaryotic distribution in sediments from 3 stations along a transect extending from the glacier into the outer fjord in 4 different seasons (spring, summer, autumn and winter) in 2012–2013. The biochemical parameters used to describe the sedimentary organic matter were organic carbon (OC), total nitrogen, bulk stable isotope ratios, pigments (chorophyll-a and phaeopigments) and biopolymeric carbon (BPC), which is the sum of the main macromolecules, i.e. lipids, proteins and carbohydrates. Prokaryotic abundance and distribution were estimated by 4′,6-diamidino-2-phenylindole (DAPI) staining. This study identifies a well-marked quantitative gradient of biogenic compounds throughout all seasons and also highlights a discrepancy between the quantity and quality of sedimentary organic matter within the fjord. The sediments near the glacier were organic-poor (< 0.3%OC), however the high primary productivity in the water column displayed during spring was reflected in summer sediments, and exhibited higher freshness of material at the inner station compared to the outer basin (means C-chlorophyll-a/OC ~ 5 and 1.5%, respectively). However, sediments at the glacier front were depleted

  8. Spatial changes in the prokaryotic community structure across a soil catena

    Science.gov (United States)

    Semenov, Mikhail; Zhuravleva, Anna; Tkhakakhova, Azida

    2017-04-01

    ; Verrucomicrobia, Proteobacteria and Acidobacteria - in transitional site (both soils with the total dominance of Chthoniobacter flavus). In Fluvisol of accumulative landscape position, it was revealed a completely different prokaryotic community with the dominance of Bacillus, Clostridium, Desulfovibrio, Saccharopolyspora, and Gallionella. B. longiquaesitum and B. nealsonii were the two most abundant species. In general, prokaryotic community of Fluvisol was characterized by a wide range of microorganisms involved in the biogeochemical cycles of iron (Gallionella ferruginea, Rhodoferax ferrireducens, Carboxydocella ferrireducens, Gallionella capsiferriformans, etc.) and sulfur (Desulfomonile iedjei, Sulfurospirillum sp., Desulfonatronum thiosulfatophilum, Thermodesulfovibrio thiophilus, Thermodesulfovibrio aggregans, Ammonifex thiophilus, etc.). Metabolically active archaea of soils across the catena included Thaumarchaeota and Euryarchaeota phyla. In general, 23 species of methanogens were detected in AC position characterized by excessive moisture which explains prevailing of methane emission over consumption. It was also revealed that Methanolobus taylori, Methanococcoides methylutens, and Methanosaeta concilii were the dominant methanogens, while Methylosinus pucelana and Methylosinus acidophilus were the main methanotrophs in prokaryotic communities of studied soils. This research was supported by the Russian Science Foundation, Projects No 14-26-00625 and No 14-26-00079.

  9. Validation of brain tumour imaging with p-[123I]iodo-l-phenylalanine and SPECT

    International Nuclear Information System (INIS)

    Hellwig, Dirk; Sell, Nadja; Schaefer, Andrea; Kirsch, Carl-Martin; Samnick, Samuel; Ketter, Ralf; Moringlane, Jean R.; Romeike, Bernd F.M.

    2005-01-01

    The aims of this prospective study were to validate single-photon emission computed tomography (SPECT) with p-[ 123 I]iodo-l-phenylalanine (IPA) in brain tumours and to evaluate its potential for the characterisation of indeterminate brain lesions. In 45 patients with indeterminate brain lesions or suspected progression of glioma, amino acid uptake was studied using IPA-SPECT and compared with the final diagnosis established by biopsy or serial imaging. After image fusion of IPA-SPECT and magnetic resonance imaging, the presence of tumour was visually determined by two independent observers. IPA uptake was quantified as the ratio between maximum uptake in the suspicious lesion and mean uptake in unaffected brain. Primary brain tumours were present in 35 cases (12 low-grade and 23 high-grade gliomas). Non-neoplastic brain lesions were confirmed in seven cases (three dysplasias, three inflammatory lesions, one lesion after effective therapy). Visual analysis showed a high concordance between the two observers (kappa=0.90, p<0.001), with sensitivity and specificity of 86% and 100% for the discrimination of primary brain tumours and non-neoplastic lesions. At 30 min p.i., IPA uptake in primary brain tumours was higher than that in non-neoplastic lesions (1.70±0.36 vs 1.14±0.18, p<0.05). Brain metastases showed no increased uptake (1.13±0.22, n=3). The persistent retention of IPA in low-grade gliomas without disruption of the blood-brain barrier was visualised up to 24 h p.i. Low-grade and high-grade gliomas showed equivalent IPA uptake (1.72±0.37 vs 1.67±0.36 at 30 min, p=0.745). IPA shows long and specific retention in gliomas. IPA is a promising and safe radiopharmaceutical for the visualisation of gliomas and the characterisation of indeterminate brain lesions. (orig.)

  10. Metabolic transcription analysis of engineered Escherichia coli strains that overproduce L-phenylalanine

    Directory of Open Access Journals (Sweden)

    Gosset Guillermo

    2007-09-01

    Full Text Available Abstract Background The rational design of L-phenylalanine (L-Phe overproducing microorganisms has been successfully achieved by combining different genetic strategies such as inactivation of the phosphoenolpyruvate: phosphotransferase transport system (PTS and overexpression of key genes (DAHP synthase, transketolase and chorismate mutase-prephenate dehydratase, reaching yields of 0.33 (g-Phe/g-Glc, which correspond to 60% of theoretical maximum. Although genetic modifications introduced into the cell for the generation of overproducing organisms are specifically targeted to a particular pathway, these can trigger unexpected transcriptional responses of several genes. In the current work, metabolic transcription analysis (MTA of both L-Phe overproducing and non-engineered strains using Real-Time PCR was performed, allowing the detection of transcriptional responses to PTS deletion and plasmid presence of genes related to central carbon metabolism. This MTA included 86 genes encoding enzymes of glycolysis, gluconeogenesis, pentoses phosphate, tricarboxylic acid cycle, fermentative and aromatic amino acid pathways. In addition, 30 genes encoding regulatory proteins and transporters for aromatic compounds and carbohydrates were also analyzed. Results MTA revealed that a set of genes encoding carbohydrate transporters (galP, mglB, gluconeogenic (ppsA, pckA and fermentative enzymes (ldhA were significantly induced, while some others were down-regulated such as ppc, pflB, pta and ackA, as a consequence of PTS inactivation. One of the most relevant findings was the coordinated up-regulation of several genes that are exclusively gluconeogenic (fbp, ppsA, pckA, maeB, sfcA, and glyoxylate shunt in the best PTS- L-Phe overproducing strain (PB12-ev2. Furthermore, it was noticeable that most of the TCA genes showed a strong up-regulation in the presence of multicopy plasmids by an unknown mechanism. A group of genes exhibited transcriptional responses to

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

    Science.gov (United States)

    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. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Influence of additive L-phenylalanine on stabilization of metastable α-form of L-glutamic acid in cooling crystallization

    Science.gov (United States)

    Quang, Khuu Chau; Nhan, Le Thi Hong; Huyen, Trinh Thi Thanh; Tuan, Nguyen Anh

    2017-09-01

    The influence of additive amino acid L-phenylalanine on stabilization of metastable α-form of L-glutamic acid was investigated in cooling crystallization. The present study found that the additive L-phenylalanine could be used to stabilize the pure metastable α-form in L-glutamic acid crystallization, where the additive concentration of 0.05-0.1 (g/L) was sufficient to stabilize the 100% wt metastable α-form in solid product at L-glutamic acid concentration of 30-45 (g/L). Additionally, the present results indicated that the adsorption of additive L-phenylalanine on the (001) surface of α-form was more favorable than that of the β-form molecular, so the nucleation sites of stable β-form was occupied by additive molecular, which resulted in inhibition of nucleation and growth of β-form, allowing stabilization of metastable α-form.

  13. Succession within the prokaryotic communities during the VAHINE mesocosms experiment in the New Caledonia lagoon

    Science.gov (United States)

    Pfreundt, Ulrike; Van Wambeke, France; Caffin, Mathieu; Bonnet, Sophie; Hess, Wolfgang R.

    2016-04-01

    N2 fixation fuels ˜ 50 % of new primary production in the oligotrophic South Pacific Ocean. The VAHINE experiment has been designed to track the fate of diazotroph-derived nitrogen (DDN) and carbon within a coastal lagoon ecosystem in a comprehensive way. For this, large-volume ( ˜ 50 m3) mesocosms were deployed in the New Caledonian lagoon and were intentionally fertilized with dissolved inorganic phosphorus (DIP) to stimulate N2 fixation. This study examined the temporal dynamics of the prokaryotic community together with the evolution of biogeochemical parameters for 23 consecutive days in one of these mesocosms (M1) and in the Nouméa lagoon using MiSeq 16S rRNA gene sequencing and flow cytometry. Combining these methods allowed for inference of absolute cell numbers from 16S data. We observed clear successions within M1, some of which were not mirrored in the lagoon. The dominating classes in M1 were Alpha- and Gammaproteobacteria, Cyanobacteria, eukaryotic microalgae, Marine Group II Euryarchaeota, Flavobacteriia, and Acidimicrobia. Enclosure led to significant changes in the M1 microbial community, probably initiated by the early decay of Synechococcus and diatoms. However, we did not detect a pronounced bottle effect with a copiotroph-dominated community. The fertilization with ˜ 0.8 µM DIP on day 4 did not have directly observable effects on the overall community within M1, as the data samples obtained from before and 4 days after fertilization clustered together, but likely influenced the development of individual populations later on, like Defluviicoccus-related bacteria and UCYN-C-type diazotrophic cyanobacteria (Cyanothece). Growth of UCYN-C led to among the highest N2-fixation rates ever measured in this region and enhanced growth of nearly all abundant heterotrophic groups in M1. We further show that different Rhodobacteraceae were the most efficient heterotrophs in the investigated system and we observed niche partitioning within the SAR86 clade

  14. The impact of road salt runoff on methanogens and other lacustrine prokaryotes

    Science.gov (United States)

    Sprague, E.; Dupuis, D.; Koretsky, C.; Docherty, K. M.

    2017-12-01

    Road salt deicers are widely used in regions that experience icy winters. The resulting saline runoff can negatively impact freshwater lake ecosystems. Saline runoff can cause density stratification, resulting in persistently anoxic hypolimnia. This may result in a shift in the structure of the hypolimnetic prokaryotic community, with potential increases in anaerobic and halotolerant taxa. Specifically, anoxia creates a habitat suitable for the proliferation of obligately anaerobic Archaeal methanogens. As a result, more persistent and expanded anoxic zones due to road salt runoff have the potential to increase hypolimnetic methane concentrations. If a portion of this methane is released to the atmosphere, it could be a currently uncharacterized contributor to atmospheric greenhouse gas emissions. This study examines two urban, eutrophic lakes with significant road salt influx and one rural, eutrophic lake with little road salt influx. All three lakes are located in southwest Michigan. Samples were taken from the water column at every meter at the deepest part of each lake, with a sample from the sediment-water interface, in May, August, and November 2016 and February 2017. The V4 and V5 hypervariable regions of the 16S rRNA gene in Bacteria and Archaea were amplified and sequenced using an Illumina MiSeq approach. Abundance of the mcrA gene, a marker for Archaeal methyl coenzyme A reductase, was quantified using qPCR. Water column methane levels, sediment methane production, water surface methane flux and a suite of supporting geochemical parameters were measured to determine changes in redox stratification in each lake and across seasons. Results indicate significant changes in the 16S rRNA-based community associated with depth, season, salinity and lake. Cyanobacteria, Actinobacteria, and Proteobacteria were among the phyla with the highest overall relative abundance. Sediment samples had more copies of the mcrA gene than the water column samples. In most

  15. Tracer experiment administering L-phenylalanine-U-{sup 14}C and L-tyrosine-U-{sup 14}C to the tissue slices of bamboo shoots

    Energy Technology Data Exchange (ETDEWEB)

    Kozukue, E. [Kenmei Women' s Junior Coll., Himeji, Hyogo (Japan); Mizuno, S.

    1987-09-15

    Uniformly {sup 14}C-labeled L-phenylalanine and L-tyrosine were administered to tissue slices of both top and base sections of bamboo shoots. Alcohol soluble substances were extracted and then separated into organic acid, sugar and amino acid fractions by ion exchange chromatography. The homogentisic acid fraction among the organic acids was collected by high-performance liquid chromatography (HPLC) and its radioactivity was measured, while the alcohol insoluble residue was used for the analysis of lignin aldehyde by the method of alkaline nitrobenzene oxidation. 1. The two labeled amino acids were steadily incorporated into the tissues during incubation and rapidly converted to organic acid, sugar and alcohol insoluble residue, especially the latter. 2. On determining the amount of phenylalanine converted to tyrosine, it was found that this was extremely small. 3. The incorporation of phenylalanine-U-{sup 14}C into alcohol insoluble residue was higher than that of tyrosine in both sections. 4. Although the conversion into lignin aldehyde from phenylalanine-U-{sup 14}C was higher than that from tyrosine-U-{sup 14}C, it was found that tyrosine incorporated into the shoots was converted to a remarkable extent for formation of lignin aldehyde. 5. The incorporation of phenylalanine and tyrosine into homogentisic acid was very low. From these results, we assume that the conversion of phenylalanine to tyrosine or of tyrosine to homogentisic acid is very small, and that a part of the high amount of tyrosine in the shoots may be used for formation of lignin.

  16. Prokaryote community dynamics in anaerobic co-digestion of swine manure, rice straw and industrial clay residuals.

    Science.gov (United States)

    Jiménez, Janet; Theuerl, Susanne; Bergmann, Ingo; Klocke, Michael; Guerra, Gilda; Romero-Romero, Osvaldo

    The aim of this study was to analyze the effect of the addition of rice straw and clay residuals on the prokaryote methane-producing community structure in a semi-continuously stirred tank reactor fed with swine manure. Molecular techniques, including terminal restriction fragment length polymorphism and a comparative nucleotide sequence analyses of the prokaryotic 16S rRNA genes, were performed. The results showed a positive effect of clay addition on methane yield during the co-digestion of swine manure and rice straw. At the digestion of swine manure, the bacterial phylum Firmicutes and the archaeal family Methanosarcinaceae, particularly Methanosarcina species, were predominant. During the co-digestion of swine manure and rice straw the microbial community changed, and with the addition of clay residual, the phylum Bacteroidetes predominated. The new nutritional conditions resulted in a shift in the archaeal family Methanosarcinaceae community as acetoclastic Methanosaeta species became dominant.

  17. Muscle-directed gene therapy for phenylketonuria (PKU): Development of transgenic mice with muscle-specific phenylalanine hydroxylase expression

    Energy Technology Data Exchange (ETDEWEB)

    Harding, C.O.; Messing, A.; Wolff, J.A. [Univ. of Wisconsin, Madison, WI (United States)

    1994-09-01

    Phenylketonuria (PKU) is an attractive target for gene therapy because of shortcomings in current therapy including lifelong commitment to a difficult and expensive diet, persistent mild cognitive deficits in some children despite adequate dietary therapy, and maternal PKU syndrome. Phenylalanine hydroxylase (PAH) is normally expressed only in liver, but we propose to treat PKU by introducing the gene for PAH into muscle. In order to evaluate both the safety and efficacy of this approach, we have a developed a trangenic mouse which expresses PAH in both cardiac and skeletal muscle. The transgene includes promoter and enhancer sequences from the mouse muscle creatine kinase (MCK) gene fused to the mouse liver PAH cDNA. Mice which have inherited the transgene are healthy, active, and do not exhibit any signs of muscle weakness or wasting. Ectopic PAH expression in muscle is not detrimental to the health, neurologic function, or reproduction of the mice. Pah{sup enu2} hyperphenylalaninemic mice, a model of human PAH deficiency, bred to carry the transgene have substantial PAH expression in cardiac and skeletal muscle but none in liver. Muscle PAH expression alone does not complement the hyperphenylalaninemic phenotype of Pah{sup enu2} mice. However, administration of reduced tetrahydrobiopterin to transgenic Pah{sup enu2} mice is associated with a 25% mean decrease in serum phenylalanine levels. We predict that ectopic expression of PAH in muscle along with adequate muscle supplies of reduced biopterin cofactor will decrease hyperphenylalaninemia in PKU.

  18. The Fas/Fas ligand death receptor pathway contributes to phenylalanine-induced apoptosis in cortical neurons.

    Directory of Open Access Journals (Sweden)

    Xiaodong Huang

    Full Text Available Phenylketonuria (PKU, an autosomal recessive disorder of amino acid metabolism caused by mutations in the phenylalanine hydroxylase (PAH gene, leads to childhood mental retardation by exposing neurons to cytotoxic levels of phenylalanine (Phe. A recent study showed that the mitochondria-mediated (intrinsic apoptotic pathway is involved in Phe-induced apoptosis in cultured cortical neurons, but it is not known if the death receptor (extrinsic apoptotic pathway and endoplasmic reticulum (ER stress-associated apoptosis also contribute to neurodegeneration in PKU. To answer this question, we used specific inhibitors to block each apoptotic pathway in cortical neurons under neurotoxic levels of Phe. The caspase-8 inhibitor Z-IETD-FMK strongly attenuated apoptosis in Phe-treated neurons (0.9 mM, 18 h, suggesting involvement of the Fas receptor (FasR-mediated cell death receptor pathway in Phe toxicity. In addition, Phe significantly increased cell surface Fas expression and formation of the Fas/FasL complex. Blocking Fas/FasL signaling using an anti-Fas antibody markedly inhibited apoptosis caused by Phe. In contrast, blocking the ER stress-induced cell death pathway with salubrinal had no effect on apoptosis in Phe-treated cortical neurons. These experiments demonstrate that the Fas death receptor pathway contributes to Phe-induced apoptosis and suggest that inhibition of the death receptor pathway may be a novel target for neuroprotection in PKU patients.

  19. Molecular complexes of L-phenylalanine with substituted 1,4-benzoquinones in aqueous medium: Spectral and theoretical investigations

    Science.gov (United States)

    Ganesh, K.; El-Mossalamy, E. H.; Satheshkumar, A.; Balraj, C.; Elango, K. P.

    2013-12-01

    Various spectral techniques such as UV-Vis, FT-IR, and fluorescence have been employed to investigate the charge transfer interaction of L-phenylalanine (LPA) with substituted 1,4-benzoquinones (MQ1-4). Kinetic and thermodynamic properties of the complexes were determined in aqueous medium at physiological condition (pH = 7). The interaction of MQ1-4 with L-phenylalanine (LPA) was found to proceed through the formation of donor-acceptor complex, yielding a radical anion. The stoichiometry of the complexes was determined by Jobs continuous variation method and was found to be 1:1 in all the cases. Fluorescence quenching studies showed that the interaction between the donor and the acceptors is spontaneous. The results indicated that the progressive replacement of chlorine atom (-I effect) by methoxy group (+M effect) in the quinone decreased the electron acceptor property of the quinone. The order of the experimentally measured association constant of these complexes was well supported by DFT/B3LYP calculations.

  20. Culturable prokaryotic diversity of deep, gas hydrate sediments: first use of a continuous high-pressure, anaerobic, enrichment and isolation system for subseafloor sediments (DeepIsoBUG)

    OpenAIRE

    Parkes, R John; Sellek, Gerard; Webster, Gordon; Martin, Derek; Anders, Erik; Weightman, Andrew J; Sass, Henrik

    2009-01-01

    Deep subseafloor sediments may contain depressurization-sensitive, anaerobic, piezophilic prokaryotes. To test this we developed the DeepIsoBUG system, which when coupled with the HYACINTH pressure-retaining drilling and core storage system and the PRESS core cutting and processing system, enables deep sediments to be handled without depressurization (up to 25 MPa) and anaerobic prokaryotic enrichments and isolation to be conducted up to 100 MPa. Here, we describe the system and its first use...

  1. Diversity of sulfur-cycle prokaryotes in freshwater lake sediments investigated using aprA as the functional marker gene.

    Science.gov (United States)

    Watanabe, Tomohiro; Kojima, Hisaya; Takano, Yoshinori; Fukui, Manabu

    2013-09-01

    The diversity of sulfate-reducing prokaryotes (SRPs) and sulfur-oxidizing prokaryotes (SOPs) in freshwater lake ecosystems was investigated by cloning and sequencing of the aprA gene, which encodes for a key enzyme in dissimilatory sulfate reduction and sulfur oxidation. To understand their diversity better, the spatial distribution of aprA genes was investigated in sediments collected from six geographically distant lakes in Antarctica and Japan, including a hypersaline lake for comparison. The microbial community compositions of freshwater sediments and a hypersaline sediment showed notable differences. The clones affiliated with Desulfobacteraceae and Desulfobulbaceae were frequently detected in all freshwater lake sediments. The SOP community was mainly composed of four major phylogenetic groups. One of them formed a monophyletic cluster with a sulfur-oxidizing betaproteobacterium, Sulfuricella denitrificans, but the others were not assigned to specific genera. In addition, the AprA sequences, which were not clearly affiliated to either SRP or SOP lineages, dominated the libraries from four freshwater lake sediments. The results showed the wide distribution of some sulfur-cycle prokaryotes across geographical distances and supported the idea that metabolic flexibility is an important feature for SRP survival in low-sulfate environments. Copyright © 2013 Elsevier GmbH. All rights reserved.

  2. Chronic polyaromatic hydrocarbon (PAH contamination is a marginal driver for community diversity and prokaryotic predicted functioning in coastal sediments

    Directory of Open Access Journals (Sweden)

    Mathilde Jeanbille

    2016-08-01

    Full Text Available Benthic microorganisms are key players in the recycling of organic matter and recalcitrant compounds such as polyaromatic hydrocarbons (PAHs in coastal sediments. Despite their ecological importance, the response of microbial communities to chronic PAH pollution, one of the major threats to coastal ecosystems, has received very little attention. In one of the largest surveys performed so far on coastal sediments, the diversity and composition of microbial communities inhabiting both chronically contaminated and non-contaminated coastal sediments were investigated using high-throughput sequencing on the 18S and 16S rRNA genes. Prokaryotic alpha-diversity showed significant association with salinity, temperature, and organic carbon content. The effect of particle size distribution was strong on eukaryotic diversity. Similarly to alpha-diversity, beta-diversity patterns were strongly influenced by the environmental filter, while PAHs had no influence on the prokaryotic community structure and a weak impact on the eukaryotic community structure at the continental scale. However, at the regional scale, PAHs became the main driver shaping the structure of bacterial and eukaryotic communities. These patterns were not found for PICRUSt predicted prokaryotic functions, thus indicating some degree of functional redundancy. Eukaryotes presented a greater potential for their use as PAH contamination biomarkers, owing to their stronger response at both regional and continental scales.

  3. CVTree3 Web Server for Whole-genome-based and Alignment-free Prokaryotic Phylogeny and Taxonomy

    Directory of Open Access Journals (Sweden)

    Guanghong Zuo

    2015-10-01

    Full Text Available A faithful phylogeny and an objective taxonomy for prokaryotes should agree with each other and ultimately follow the genome data. With the number of sequenced genomes reaching tens of thousands, both tree inference and detailed comparison with taxonomy are great challenges. We now provide one solution in the latest Release 3.0 of the alignment-free and whole-genome-based web server CVTree3. The server resides in a cluster of 64 cores and is equipped with an interactive, collapsible, and expandable tree display. It is capable of comparing the tree branching order with prokaryotic classification at all taxonomic ranks from domains down to species and strains. CVTree3 allows for inquiry by taxon names and trial on lineage modifications. In addition, it reports a summary of monophyletic and non-monophyletic taxa at all ranks as well as produces print-quality subtree figures. After giving an overview of retrospective verification of the CVTree approach, the power of the new server is described for the mega-classification of prokaryotes and determination of taxonomic placement of some newly-sequenced genomes. A few discrepancies between CVTree and 16S rRNA analyses are also summarized with regard to possible taxonomic revisions. CVTree3 is freely accessible to all users at http://tlife.fudan.edu.cn/cvtree3/ without login requirements.

  4. Prokaryotic and eukaryotic features observed on the secondary structures of Giardia SSU rRNAs and its phylogenetic implications.

    Science.gov (United States)

    Hwang, Ui Wook

    2007-04-01

    Phylogenetic position of a diplomonad protist Giardia, a principle cause of diarrhea, among eukaryotes has been vigorously debated so far. Through the comparisons of primary and secondary structures of SSU rRNAs of G. intestinalis, G. microti, G. ardeae, and G. muris, I found two major indel regions (a 6-nt indel and a 22-26-nt indel), which correspond to the helix 10 of the V2 region and helices E23-8 to E23-9 of the V4 region, respectively. As generally shown in eukaryotes, G. intestinalis and G. microti have commonly a relatively longer helix 10 (a 7-bp stem and a 4-nt loop), and also the eukaryote-specific helices E23-6 to E23-9. On the other hand, G. muris and G. ardeae have a shorter helix 10: a 2-bp stem and a 6-nt loop in G. ardeae and a 3-bp stem and a 6-nt loop in G. muris. In the V4, they have a single long helix (like the P23-1 helix in prokaryotes) instead of the helices E23-6 to E23-9. Among the four Giardia species, co-appearance of prokaryote- and eukaryote-typical features might be significant evidence to suggest that Giardia (Archezoa) is a living fossil showing an "intermediate stage" during the evolution from prokaryotes to eukaryotes.

  5. CVTree3 Web Server for Whole-genome-based and Alignment-free Prokaryotic Phylogeny and Taxonomy.

    Science.gov (United States)

    Zuo, Guanghong; Hao, Bailin

    2015-10-01

    A faithful phylogeny and an objective taxonomy for prokaryotes should agree with each other and ultimately follow the genome data. With the number of sequenced genomes reaching tens of thousands, both tree inference and detailed comparison with taxonomy are great challenges. We now provide one solution in the latest Release 3.0 of the alignment-free and whole-genome-based web server CVTree3. The server resides in a cluster of 64 cores and is equipped with an interactive, collapsible, and expandable tree display. It is capable of comparing the tree branching order with prokaryotic classification at all taxonomic ranks from domains down to species and strains. CVTree3 allows for inquiry by taxon names and trial on lineage modifications. In addition, it reports a summary of monophyletic and non-monophyletic taxa at all ranks as well as produces print-quality subtree figures. After giving an overview of retrospective verification of the CVTree approach, the power of the new server is described for the mega-classification of prokaryotes and determination of taxonomic placement of some newly-sequenced genomes. A few discrepancies between CVTree and 16S rRNA analyses are also summarized with regard to possible taxonomic revisions. CVTree3 is freely accessible to all users at http://tlife.fudan.edu.cn/cvtree3/ without login requirements. Copyright © 2015 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.

  6. Soil Nematodes and Their Prokaryotic Prey Along an Elevation Gradient in The Mojave Desert (Death Valley National Park, California, USA

    Directory of Open Access Journals (Sweden)

    Alyxandra Pikus

    2012-10-01

    Full Text Available We characterized soil communities in the Mojave Desert across an elevation gradient. Our goal was to test the hypothesis that as soil quality improved with increasing elevation (due to increased productivity, the diversity of soil prokaryotes and nematodes would also increase. Soil organic matter and soil moisture content increased with elevation as predicted. Soil salinity did not correlate to elevation, but was highest at a mid-gradient, alluvial site. Soil nematode density, community trophic structure, and diversity did not show patterns related to elevation. Similar results were obtained for diversity of bacteria and archaea. Relationships between soil properties, nematode communities, and prokaryotic diversity were site-specific. For example, at the lowest elevation site, nematode communities contained a high proportion of fungal-feeding species and diversity of bacteria was lowest. At a high-salinity site, nematode density was highest, and overall, nematode density showed an unexpected, positive correlation to salinity. At the highest elevation site, nematode density and species richness were attenuated, despite relatively high moisture and organic matter content for the soils. Our results support emerging evidence for the lack of a relationship between productivity and the diversity of soil nematodes and prokaryotes.

  7. DFAST: a flexible prokaryotic genome annotation pipeline for faster genome publication.

    Science.gov (United States)

    Tanizawa, Yasuhiro; Fujisawa, Takatomo; Nakamura, Yasukazu

    2018-03-15

    We developed a prokaryotic genome annotation pipeline, DFAST, that also supports genome submission to public sequence databases. DFAST was originally started as an on-line annotation server, and to date, over 7000 jobs have been processed since its first launch in 2016. Here, we present a newly implemented background annotation engine for DFAST, which is also available as a standalone command-line program. The new engine can annotate a typical-sized bacterial genome within 10 min, with rich information such as pseudogenes, translation exceptions and orthologous gene assignment between given reference genomes. In addition, the modular framework of DFAST allows users to customize the annotation workflow easily and will also facilitate extensions for new functions and incorporation of new tools in the future. The software is implemented in Python 3 and runs in both Python 2.7 and 3.4-on Macintosh and Linux systems. It is freely available at https://github.com/nigyta/dfast_core/under the GPLv3 license with external binaries bundled in the software distribution. An on-line version is also available at https://dfast.nig.ac.jp/. yn@nig.ac.jp. Supplementary data are available at Bioinformatics online.

  8. Copahue Geothermal System: A Volcanic Environment with Rich Extreme Prokaryotic Biodiversity.

    Science.gov (United States)

    Urbieta, María Sofía; Porati, Graciana Willis; Segretín, Ana Belén; González-Toril, Elena; Giaveno, María Alejandra; Donati, Edgardo Rubén

    2015-07-08

    The Copahue geothermal system is a natural extreme environment located at the northern end of the Cordillera de los Andes in Neuquén province in Argentina. The geochemistry and consequently the biodiversity of the area are dominated by the activity of the Copahue volcano. The main characteristic of Copahue is the extreme acidity of its aquatic environments; ponds and hot springs of moderate and high temperature as well as Río Agrio. In spite of being an apparently hostile location, the prokaryotic biodiversity detected by molecular ecology techniques as well as cultivation shows a rich and diverse environment dominated by acidophilic, sulphur oxidising bacteria or archaea, depending on the conditions of the particular niche studied. In microbial biofilms, found in the borders of the ponds where thermal activity is less intense, the species found are completely different, with a high presence of cyanobacteria and other photosynthetic species. Our results, collected during more than 10 years of work in Copahue, have enabled us to outline geomicrobiological models for the different environments found in the ponds and Río Agrio. Besides, Copahue seems to be the habitat of novel, not yet characterised autochthonous species, especially in the domain Archaea.

  9. Copahue Geothermal System: A Volcanic Environment with Rich Extreme Prokaryotic Biodiversity

    Directory of Open Access Journals (Sweden)

    María Sofía Urbieta

    2015-07-01

    Full Text Available The Copahue geothermal system is a natural extreme environment located at the northern end of the Cordillera de los Andes in Neuquén province in Argentina. The geochemistry and consequently the biodiversity of the area are dominated by the activity of the Copahue volcano. The main characteristic of Copahue is the extreme acidity of its aquatic environments; ponds and hot springs of moderate and high temperature as well as Río Agrio. In spite of being an apparently hostile location, the prokaryotic biodiversity detected by molecular ecology techniques as well as cultivation shows a rich and diverse environment dominated by acidophilic, sulphur oxidising bacteria or archaea, depending on the conditions of the particular niche studied. In microbial biofilms, found in the borders of the ponds where thermal activity is less intense, the species found are completely different, with a high presence of cyanobacteria and other photosynthetic species. Our results, collected during more than 10 years of work in Copahue, have enabled us to outline geomicrobiological models for the different environments found in the ponds and Río Agrio. Besides, Copahue seems to be the habitat of novel, not yet characterised autochthonous species, especially in the domain Archaea.

  10. [Cloning, prokaryotic expression and antibacterial assay of Tenecin gene encoding an antibacterial peptide from Tenebrio molitor].

    Science.gov (United States)

    Liu, Ying; Jiang, Yu-xin; Li, Chao-pin

    2011-12-01

    To clone tenecin gene, an antibacterial peptide gene, from Tenebrio molitor for its prokaryotic expression and explore the molecular mechanism for regulating the expression of antibacterial peptide in Tenebrio molitor larvae. The antibacterial peptide was induced from the larvae of Tenebrio molitor by intraperitoneal injection of Escherichia coli DH-5α (1×10(8)/ml). RT-PCR was performed 72 h after the injection to clone Tenecin gene followed by sequencing and bioinformatic analysis. The recombinant expression vector pET-28a(+)-Tenecin was constructed and transformed into E. coli BL21(DE3) cells and the expression of tenecin protein was observed after IPTG induction. Tenecin expression was detected in transformed E.coli using SDS-PAGE after 1 mmol/L IPTG induction. Tenecin gene, which was about 255 bp in length, encoded Tenecin protein with a relative molecular mass of 9 kD. Incubation of E.coli with 80, 60, 40, and 20 µg/ml tenecin for 18 h resulted in a diameter of the inhibition zone of 25.1∓0.03, 20.7∓0.06, 17.2∓0.11 and 9.3∓0.04 mm, respectively. Tenecin protein possesses strong antibacterial activity against E. coli DH-5α, which warrants further study of this protein for its potential as an antibacterial agent in clinical application.

  11. Structure of a preternary complex involving a prokaryotic NHEJ DNA polymerase.

    Science.gov (United States)

    Brissett, Nigel C; Martin, Maria J; Pitcher, Robert S; Bianchi, Julie; Juarez, Raquel; Green, Andrew J; Fox, Gavin C; Blanco, Luis; Doherty, Aidan J

    2011-01-21

    In many prokaryotes, a specific DNA primase/polymerase (PolDom) is required for nonhomologous end joining (NHEJ) repair of DNA double-strand breaks (DSBs). Here, we report the crystal structure of a catalytically active conformation of Mycobacterium tuberculosis PolDom, consisting of a polymerase bound to a DNA end with a 3' overhang, two metal ions, and an incoming nucleotide but, significantly, lacking a primer strand. This structure represents a polymerase:DNA complex in a preternary intermediate state. This polymerase complex occurs in solution, stabilizing the enzyme on DNA ends and promoting nucleotide extension of short incoming termini. We also demonstrate that the invariant Arg(220), contained in a conserved loop (loop 2), plays an essential role in catalysis by regulating binding of a second metal ion in the active site. We propose that this NHEJ intermediate facilitates extension reactions involving critically short or noncomplementary DNA ends, thus promoting break repair and minimizing sequence loss during DSB repair. Copyright © 2011 Elsevier Inc. All rights reserved.

  12. Selection on start codons in prokaryotes and potential compensatory nucleotide substitutions.

    Science.gov (United States)

    Belinky, Frida; Rogozin, Igor B; Koonin, Eugene V

    2017-09-29

    Reconstruction of the evolution of start codons in 36 groups of closely related bacterial and archaeal genomes reveals purifying selection affecting AUG codons. The AUG starts are replaced by GUG and especially UUG significantly less frequently than expected under the neutral expectation derived from the frequencies of the respective nucleotide triplet substitutions in non-coding regions and in 4-fold degenerate sites. Thus, AUG is the optimal start codon that is actively maintained by purifying selection. However, purifying selection on start codons is significantly weaker than the selection on the same codons in coding sequences, although the switches between the codons result in conservative amino acid substitutions. The only exception is the AUG to UUG switch that is strongly selected against among start codons. Selection on start codons is most pronounced in evolutionarily conserved, highly expressed genes. Mutation of the start codon to a sub-optimal form (GUG or UUG) tends to be compensated by mutations in the Shine-Dalgarno sequence towards a stronger translation initiation signal. Together, all these findings indicate that in prokaryotes, translation start signals are subject to weak but significant selection for maximization of initiation rate and, consequently, protein production.

  13. Next-generation systematics: An innovative approach to resolve the structure of complex prokaryotic taxa

    Science.gov (United States)

    Sangal, Vartul; Goodfellow, Michael; Jones, Amanda L.; Schwalbe, Edward C.; Blom, Jochen; Hoskisson, Paul A.; Sutcliffe, Iain C.

    2016-12-01

    Prokaryotic systematics provides the fundamental framework for microbiological research but remains a discipline that relies on a labour- and time-intensive polyphasic taxonomic approach, including DNA-DNA hybridization, variation in 16S rRNA gene sequence and phenotypic characteristics. These techniques suffer from poor resolution in distinguishing between closely related species and often result in misclassification and misidentification of strains. Moreover, guidelines are unclear for the delineation of bacterial genera. Here, we have applied an innovative phylogenetic and taxogenomic approach to a heterogeneous actinobacterial taxon, Rhodococcus, to identify boundaries for intrageneric and supraspecific classification. Seven species-groups were identified within the genus Rhodococcus that are as distantly related to one another as they are to representatives of other mycolic acid containing actinobacteria and can thus be equated with the rank of genus. It was also evident that strains assigned to rhodococcal species-groups are underspeciated with many misclassified using conventional taxonomic criteria. The phylogenetic and taxogenomic methods used in this study provide data of theoretical value for the circumscription of generic and species boundaries and are also of practical significance as they provide a robust basis for the classification and identification of rhodococci of agricultural, industrial and medical/veterinary significance.

  14. Structure elucidation and chemical synthesis of stigmolone, a novel type of prokaryotic pheromone.

    Science.gov (United States)

    Hull, W E; Berkessel, A; Plaga, W

    1998-09-15

    Approximately 2 micromol of a novel prokaryotic pheromone, involved in starvation-induced aggregation and formation of fruiting bodies by the myxobacterium Stigmatella aurantiaca, were isolated by a large-scale elution procedure. The pheromone was purified by HPLC, and high-resolution MS, IR, 1H-NMR, and 13C-NMR were used to identify the active substance as the hydroxy ketone 2,5, 8-trimethyl-8-hydroxy-nonan-4-one, which has been named stigmolone. The analysis was complicated by a solvent-dependent equilibrium between stigmolone and the cyclic enol-ether 3,4-dihydro-2,2, 5-trimethyl-6-(2-methylpropyl)-2H-pyran formed by intramolecular nucleophilic attack of the 8-OH group at the ketone C4 followed by loss of H2O. Both compounds were synthesized chemically, and their structures were confirmed by NMR analysis. Natural and synthetic stigmolone have the same biological activity at ca. 1 nM concentration.

  15. Co(II) Coordination in Prokaryotic Zinc Finger Domains as Revealed by UV-Vis Spectroscopy

    Science.gov (United States)

    Sivo, Valeria; D'Abrosca, Gianluca; Russo, Luigi; Iacovino, Rosa; Pedone, Paolo Vincenzo; Fattorusso, Roberto

    2017-01-01

    Co(II) electronic configuration allows its use as a spectroscopic probe in UV-Vis experiments to characterize the metal coordination sphere that is an essential component of the functional structure of zinc-binding proteins and to evaluate the metal ion affinities of these proteins. Here, exploiting the capability of the prokaryotic zinc finger to use different combinations of residues to properly coordinate the structural metal ion, we provide the UV-Vis characterization of Co(II) addition to Ros87 and its mutant Ros87_C27D which bears an unusual CysAspHis2 coordination sphere. Zinc finger sites containing only one cysteine have been infrequently characterized. We show for the CysAspHis2 coordination an intense d-d transition band, blue-shifted with respect to the Cys2His2 sphere. These data complemented by NMR and CD data demonstrate that the tetrahedral geometry of the metal site is retained also in the case of a single-cysteine coordination sphere. PMID:29386985

  16. [Prokaryotic expression and histological localization of the Taenia solium CDC37 gene].

    Science.gov (United States)

    Huang, Jiang; Li, Bo; Dai, Jia-Lin; Zhang, Ai-Hua

    2013-02-01

    To express Taenia solium gene encoding cell division cycle 37 protein (TsCDC37) and investigate its antigenicity and localization in adults of Taenia solium. The complete coding sequence of TsCDC37 was amplified by PCR based on the recombinant plasmid clone from the cDNA library of adult Taenia solium. The PCR product was cloned into a prokaryotic expression vector pET-28a (+). The recombinant expression plasmid was identified by PCR, double endonuclease digestion and sequencing. The recombinant plasmid was transformed into E. coli BL21/DE3 and followed by expression of the protein induced by IPTG. The mice were immunized subcutaneously with purified recombinant TsCDC37 formulated in Freund's adjuvant. The antigenicity of the recombinant protein was examined by Western blotting. The localization of TsCDC37 in adult worms was demonstrated by immunofluorescent technique. The recombinant expression vector was constructed successfully. The recombinant protein was about M(r) 52 000, it was then purified and specifically recognized by immuno sera of SD rats and sera from patients infected with Taenia solium, Taenia saginata or Taenia asiatica. The immunofluorescence assay revealed that TsCDC37 located at the tegument of T. solium adult and the eggs. TsCDC37 gene has been expressed with immunoreactivity. The recombinant protein is mainly expressed in tegument and egg, and is a common antigen of the three human taenia cestodes.

  17. Similarities and Differences in the Glycosylation Mechanisms in Prokaryotes and Eukaryotes

    Directory of Open Access Journals (Sweden)

    Anne Dell

    2010-01-01

    Full Text Available Recent years have witnessed a rapid growth in the number and diversity of prokaryotic proteins shown to carry N- and/or O-glycans, with protein glycosylation now considered as fundamental to the biology of these organisms as it is in eukaryotic systems. This article overviews the major glycosylation pathways that are known to exist in eukarya, bacteria and archaea. These are (i oligosaccharyltransferase (OST-mediated N-glycosylation which is abundant in eukarya and archaea, but is restricted to a limited range of bacteria; (ii stepwise cytoplasmic N-glycosylation that has so far only been confirmed in the bacterial domain; (iii OST-mediated O-glycosylation which appears to be characteristic of bacteria; and (iv stepwise O-glycosylation which is common in eukarya and bacteria. A key aim of the review is to integrate information from the three domains of life in order to highlight commonalities in glycosylation processes. We show how the OST-mediated N- and O-glycosylation pathways share cytoplasmic assembly of lipid-linked oligosaccharides, flipping across the ER/periplasmic/cytoplasmic membranes, and transferring “en bloc” to the protein acceptor. Moreover these hallmarks are mirrored in lipopolysaccharide biosynthesis. Like in eukaryotes, stepwise O-glycosylation occurs on diverse bacterial proteins including flagellins, adhesins, autotransporters and lipoproteins, with O-glycosylation chain extension often coupled with secretory mechanisms.

  18. Comparison of prokaryotic community structure from Mediterranean and Atlantic saltern concentrator ponds by a metagenomic approach

    Directory of Open Access Journals (Sweden)

    Ana Beatriz eFernández

    2014-05-01

    Full Text Available We analyzed the prokaryotic community structure of a saltern pond with 21 % total salts located in Isla Cristina, Huelva, Southwest Spain, close to the Atlantic ocean coast. For this purpose, we constructed a metagenome (designated as IC21 obtained by pyrosequencing consisting of 486 Mb with an average read length of 397 bp and compared it with other metagenomic datasets obtained from ponds with 19, 33 and 37 % total salts acquired from Santa Pola marine saltern, located in Alicante, East Spain, on the Mediterranean coast. Although the salinity in IC21 is closer to the pond with 19 % total salts from Santa Pola saltern (designated as SS19, IC21 is more similar at higher taxonomic levels to the pond with 33 % total salts from Santa Pola saltern (designated as SS33, since both are predominated by the phylum Euryarchaeota. However, there are significant differences at lower taxonomic levels where most sequences were related to the genus Halorubrum in IC21 and to Haloquadratum in SS33. Within the Bacteroidetes, the genus Psychroflexus is the most abundant in IC21 while Salinibacter dominates in SS33. Sequences related to bacteriorhodopsins and halorhodopsins correlate with the abundance of Haloquadratum in Santa Pola SS19 to SS33 and of Halorubrum in Isla Cristina IC21 dataset, respectively. Differences in composition might be attributed to local ecological conditions since IC21 showed a decrease in the number of sequences related to the synthesis of compatible solutes and in the utilization of phosphonate.

  19. GAAP: Genome-organization-framework-Assisted Assembly Pipeline for prokaryotic genomes.

    Science.gov (United States)

    Yuan, Lina; Yu, Yang; Zhu, Yanmin; Li, Yulai; Li, Changqing; Li, Rujiao; Ma, Qin; Siu, Gilman Kit-Hang; Yu, Jun; Jiang, Taijiao; Xiao, Jingfa; Kang, Yu

    2017-01-25

    Next-generation sequencing (NGS) technologies have greatly promoted the genomic study of prokaryotes. However, highly fragmented assemblies due to short reads from NGS are still a limiting factor in gaining insights into the genome biology. Reference-assisted tools are promising in genome assembly, but tend to result in false assembly when the assigned reference has extensive rearrangements. Herein, we present GAAP, a genome assembly pipeline for scaffolding based on core-gene-defined Genome Organizational Framework (cGOF) described in our previous study. Instead of assigning references, we use the multiple-reference-derived cGOFs as indexes to assist in order and orientation of the scaffolds and build a skeleton structure, and then use read pairs to extend scaffolds, called local scaffolding, and distinguish between true and chimeric adjacencies in the scaffolds. In our performance tests using both empirical and simulated data of 15 genomes in six species with diverse genome size, complexity, and all three categories of cGOFs, GAAP outcompetes or achieves comparable results when compared to three other reference-assisted programs, AlignGraph, Ragout and MeDuSa. GAAP uses both cGOF and pair-end reads to create assemblies in genomic scale, and performs better than the currently available reference-assisted assembly tools as it recovers more assemblies and makes fewer false locations, especially for species with extensive rearranged genomes. Our method is a promising solution for reconstruction of genome sequence from short reads of NGS.

  20. Genome-wide comparative analysis of phylogenetic trees: the prokaryotic forest of life.

    Science.gov (United States)

    Puigbò, Pere; Wolf, Yuri I; Koonin, Eugene V

    2012-01-01

    Genome-wide comparison of phylogenetic trees is becoming an increasingly common approach in evolutionary genomics, and a variety of approaches for such comparison have been developed. In this article, we present several methods for comparative analysis of large numbers of phylogenetic trees. To compare phylogenetic trees taking into account the bootstrap support for each internal branch, the Boot-Split Distance (BSD) method is introduced as an extension of the previously developed Split Distance method for tree comparison. The BSD method implements the straightforward idea that comparison of phylogenetic trees can be made more robust by treating tree splits differentially depending on the bootstrap support. Approaches are also introduced for detecting tree-like and net-like evolutionary trends in the phylogenetic Forest of Life (FOL), i.e., the entirety of the phylogenetic trees for conserved genes of prokaryotes. The principal method employed for this purpose includes mapping quartets of species onto trees to calculate the support of each quartet topology and so to quantify the tree and net contributions to the distances between species. We describe the application of these methods to analyze the FOL and the results obtained with these methods. These results support the concept of the Tree of Life (TOL) as a central evolutionary trend in the FOL as opposed to the traditional view of the TOL as a "species tree."

  1. Thermophilic Adaptation in Prokaryotes Is Constrained by Metabolic Costs of Proteostasis

    Science.gov (United States)

    Venev, Sergey V; Zeldovich, Konstantin B

    2018-01-01

    Abstract Prokaryotes evolved to thrive in an extremely diverse set of habitats, and their proteomes bear signatures of environmental conditions. Although correlations between amino acid usage and environmental temperature are well-documented, understanding of the mechanisms of thermal adaptation remains incomplete. Here, we couple the energetic costs of protein folding and protein homeostasis to build a microscopic model explaining both the overall amino acid composition and its temperature trends. Low biosynthesis costs lead to low diversity of physical interactions between amino acid residues, which in turn makes proteins less stable and drives up chaperone activity to maintain appropriate levels of folded, functional proteins. Assuming that the cost of chaperone activity is proportional to the fraction of unfolded client proteins, we simulated thermal adaptation of model proteins subject to minimization of the total cost of amino acid synthesis and chaperone activity. For the first time, we predicted both the proteome-average amino acid abundances and their temperature trends simultaneously, and found strong correlations between model predictions and 402 genomes of bacteria and archaea. The energetic constraint on protein evolution is more apparent in highly expressed proteins, selected by codon adaptation index. We found that in bacteria, highly expressed proteins are similar in composition to thermophilic ones, whereas in archaea no correlation between predicted expression level and thermostability was observed. At the same time, thermal adaptations of highly expressed proteins in bacteria and archaea are nearly identical, suggesting that universal energetic constraints prevail over the phylogenetic differences between these domains of life. PMID:29106597

  2. Biodiversity of thermophilic prokaryotes with hydrolytic activities in hot springs of Uzon Caldera, Kamchatka (Russia).

    Science.gov (United States)

    Kublanov, Ilya V; Perevalova, Anna A; Slobodkina, Galina B; Lebedinsky, Aleksander V; Bidzhieva, Salima K; Kolganova, Tatyana V; Kaliberda, Elena N; Rumsh, Lev D; Haertlé, Thomas; Bonch-Osmolovskaya, Elizaveta A

    2009-01-01

    Samples of water from the hot springs of Uzon Caldera with temperatures from 68 to 87 degrees C and pHs of 4.1 to 7.0, supplemented with proteinaceous (albumin, casein, or alpha- or beta-keratin) or carbohydrate (cellulose, carboxymethyl cellulose, chitin, or agarose) biological polymers, were filled with thermal water and incubated at the same sites, with the contents of the tubes freely accessible to the hydrothermal fluid. As a result, several enrichment cultures growing in situ on different polymeric substrates were obtained. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene fragments obtained after PCR with Bacteria-specific primers showed that the bacterial communities developing on carbohydrates included the genera Caldicellulosiruptor and Dictyoglomus and that those developing on proteins contained members of the Thermotogales order. DGGE analysis performed after PCR with Archaea- and Crenarchaeota-specific primers showed that archaea related to uncultured environmental clones, particularly those of the Crenarchaeota phylum, were present in both carbohydrate- and protein-degrading communities. Five isolates obtained from in situ enrichments or corresponding natural samples of water and sediments represented the bacterial genera Dictyoglomus and Caldanaerobacter as well as new archaea of the Crenarchaeota phylum. Thus, in situ enrichment and consequent isolation showed the diversity of thermophilic prokaryotes competing for biopolymers in microbial communities of terrestrial hot springs.

  3. RegPredict: an integrated system for regulon inference in prokaryotes by comparative genomics approach

    Energy Technology Data Exchange (ETDEWEB)

    Novichkov, Pavel S.; Rodionov, Dmitry A.; Stavrovskaya, Elena D.; Novichkova, Elena S.; Kazakov, Alexey E.; Gelfand, Mikhail S.; Arkin, Adam P.; Mironov, Andrey A.; Dubchak, Inna

    2010-05-26

    RegPredict web server is designed to provide comparative genomics tools for reconstruction and analysis of microbial regulons using comparative genomics approach. The server allows the user to rapidly generate reference sets of regulons and regulatory motif profiles in a group of prokaryotic genomes. The new concept of a cluster of co-regulated orthologous operons allows the user to distribute the analysis of large regulons and to perform the comparative analysis of multiple clusters independently. Two major workflows currently implemented in RegPredict are: (i) regulon reconstruction for a known regulatory motif and (ii) ab initio inference of a novel regulon using several scenarios for the generation of starting gene sets. RegPredict provides a comprehensive collection of manually curated positional weight matrices of regulatory motifs. It is based on genomic sequences, ortholog and operon predictions from the MicrobesOnline. An interactive web interface of RegPredict integrates and presents diverse genomic and functional information about the candidate regulon members from several web resources. RegPredict is freely accessible at http://regpredict.lbl.gov.

  4. Prokaryotic Expression of Rice Ospgip1 Gene and Bioinformatic Analysis of Encoded Product

    Directory of Open Access Journals (Sweden)

    Xi-jun CHEN

    2011-12-01

    Full Text Available Using the reference sequences of pgip genes in GenBank, a fragment of 930 bp covering the open reading frame (ORF of rice Ospgip1 (Oryza sativa polygalacturonase-inhibiting protein 1 was amplified. The prokaryotic expression product of the gene inhibited the growth of Rhizoctonia solani, the causal agent of rice sheath blight, and reduced its polygalacturonase activity. Bioinformatic analysis showed that OsPGIP1 is a hydrophobic protein with a molecular weight of 32.8 kDa and an isoelectric point (pI of 7.26. The protein is mainly located in the cell wall of rice, and its signal peptide cleavage site is located between the 17th and 18th amino acids. There are four cysteines in both the N- and C-termini of the deduced protein, which can form three disulfide bonds (between the 56th and 63rd, the 278th and 298th, and the 300th and 308th amino acids. The protein has a typical leucine-rich repeat (LRR domain, and its secondary structure comprises α-helices, β-sheets and irregular coils. Compared with polygalacturonase-inhibiting proteins (PGIPs from other plants, the 7th LRR is absent in OsPGIP1. The nine LRRs could form a cleft that might associate with proteins from pathogenic fungi, such as polygalacturonase.

  5. Characteristic Variations and Similarities in Biochemical, Molecular, and Functional Properties of Glyoxalases across Prokaryotes and Eukaryotes.

    Science.gov (United States)

    Kaur, Charanpreet; Sharma, Shweta; Hasan, Mohammad Rokebul; Pareek, Ashwani; Singla-Pareek, Sneh L; Sopory, Sudhir K

    2017-03-30

    The glyoxalase system is the ubiquitous pathway for the detoxification of methylglyoxal (MG) in the biological systems. It comprises two enzymes, glyoxalase I (GLYI) and glyoxalase II (GLYII), which act sequentially to convert MG into d-lactate, thereby helping living systems get rid of this otherwise cytotoxic byproduct of metabolism. In addition, a glutathione-independent GLYIII enzyme activity also exists in the biological systems that can directly convert MG to d-lactate. Humans and Escherichia coli possess a single copy of GLYI (encoding either the Ni- or Zn-dependent form) and GLYII genes, which through MG detoxification provide protection against various pathological and disease conditions. By contrast, the plant genome possesses multiple GLYI and GLYII genes with a role in abiotic stress tolerance. Plants possess both Ni 2+ - and Zn 2+ -dependent forms of GLYI, and studies on plant glyoxalases reveal the various unique features of these enzymes distinguishing them from prokaryotic and other eukaryotic glyoxalases. Through this review, we provide an overview of the plant glyoxalase family along with a comparative analysis of glyoxalases across various species, highlighting similarities as well as differences in the biochemical, molecular, and physiological properties of these enzymes. We believe that the evolution of multiple glyoxalases isoforms in plants is an important component of their robust defense strategies.

  6. Melatonin immunoreactivity in the photosynthetic prokaryote Rhodospirillum rubrum: implications for an ancient antioxidant system.

    Science.gov (United States)

    Manchester, L C; Poeggeler, B; Alvares, F L; Ogden, G B; Reiter, R J

    1995-01-01

    Rhodospirillum rubrum is a spiral anoxygenic photosynthetic bacterium that can exist under either aerobic or anaerobic conditions. The organism thrives in the presence of light or complete darkness and represents one of the oldest species of living organisms, possibly 2-3.5 billion years old. The success of this prokaryotic species may be attributed to the evolution of certain indole compounds that offer protection against life-threatening oxygen radicals produced by an evolutionary harsh environment. Melatonin, N-acetyl-5-methoxytryptamine, is an indolic highly conserved molecule that exists in protists, plants, and animals. This study was undertaken to determine the presence of an immunoreactive melatonin in the kingdom Monera and particularly in the photosynthetic bacterium, R. rubrum, under conditions of prolonged darkness or prolonged light. Immunoreactive melatonin was measured during both the extended day and extended night. Significantly more melatonin was observed during the scotophase than the photophase. This study marks the first demonstration of melatonin in a bacterium. The high level of melatonin observed in bacteria may provide on-site protection of bacterial DNA against free radical attack.

  7. Tetrapyrroles as Endogenous TSPO Ligands in Eukaryotes and Prokaryotes: Comparisons with Synthetic Ligands

    Directory of Open Access Journals (Sweden)

    Leo Veenman

    2016-06-01

    Full Text Available The 18 kDa translocator protein (TSPO is highly 0conserved in eukaryotes and prokaryotes. Since its discovery in 1977, numerous studies established the TSPO’s importance for life essential functions. For these studies, synthetic TSPO ligands typically are applied. Tetrapyrroles present endogenous ligands for the TSPO. Tetrapyrroles are also evolutionarily conserved and regulate multiple functions. TSPO and tetrapyrroles regulate each other. In animals TSPO-tetrapyrrole interactions range from effects on embryonic development to metabolism, programmed cell death, response to stress, injury and disease, and even to life span extension. In animals TSPOs are primarily located in mitochondria. In plants TSPOs are also present in plastids, the nuclear fraction, the endoplasmic reticulum, and Golgi stacks. This may contribute to translocation of tetrapyrrole intermediates across organelles’ membranes. As in animals, plant TSPO binds heme and protoporphyrin IX. TSPO-tetrapyrrole interactions in plants appear to relate to development as well as stress conditions, including salt tolerance, abscisic acid-induced stress, reactive oxygen species homeostasis, and finally cell death regulation. In bacteria, TSPO is important for switching from aerobic to anaerobic metabolism, including the regulation of photosynthesis. As in mitochondria, in bacteria TSPO is located in the outer membrane. TSPO-tetrapyrrole interactions may be part of the establishment of the bacterial-eukaryote relationships, i.e., mitochondrial-eukaryote and plastid-plant endosymbiotic relationships.

  8. Construction of a phylogenetic tree of photosynthetic prokaryotes based on average similarities of whole genome sequences.

    Directory of Open Access Journals (Sweden)

    Soichirou Satoh

    Full Text Available Phylogenetic trees have been constructed for a wide range of organisms using gene sequence information, especially through the identification of orthologous genes that have been vertically inherited. The number of available complete genome sequences is rapidly increasing, and many tools for construction of genome trees based on whole genome sequences have been proposed. However, development of a reasonable method of using complete genome sequences for construction of phylogenetic trees has not been established. We have developed a method for construction of phylogenetic trees based on the average sequence similarities of whole genome sequences. We used this method to examine the phylogeny of 115 photosynthetic prokaryotes, i.e., cyanobacteria, Chlorobi, proteobacteria, Chloroflexi, Firmicutes and nonphotosynthetic organisms including Archaea. Although the bootstrap values for the branching order of phyla were low, probably due to lateral gene transfer and saturated mutation, the obtained tree was largely consistent with the previously reported phylogenetic trees, indicating that this method is a robust alternative to traditional phylogenetic methods.

  9. Human phenylalanine hydroxylase is activated by H2O2: a novel mechanism for increasing the L-tyrosine supply for melanogenesis in melanocytes

    International Nuclear Information System (INIS)

    Schallreuter, Karin U.; Wazir, Umar; Kothari, Sonal; Gibbons, Nicholas C.J.; Moore, Jeremy; Wood, John M.

    2004-01-01

    Epidermal phenylalanine hydroxylase (PAH) produces L-tyrosine from the essential amino acid L-phenylalanine supporting melanogenesis in human melanocytes. Those PAH activities increase linearly in the different skin phototypes I-VI (Fitzpatrick classification) and also increase up to 24 h after UVB light with only one minimal erythemal dose. Since UVB generates also H 2 O 2 , we here asked the question whether this reactive oxygen species could influence the activity of pure recombinant human PAH. Under saturating conditions with the substrate L-phenylalanine (1 x 10 -3 M), the V max for enzyme activity increased 4-fold by H 2 O 2 (>2.0 x 10 -3 M). Lineweaver-Burk analysis identified a mixed activation mechanism involving both the regulatory and catalytic domains of PAH. Hyperchem molecular modelling and Deep View analysis support oxidation of the single Trp 120 residue to 5-OH-Trp 120 by H 2 O 2 causing a conformational change in the regulatory domain. PAH was still activated by H 2 O 2 in the presence of the electron donor/cofactor 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin despite slow oxidation of this cofactor. In vivo FT-Raman spectroscopy confirmed decreased epidermal phenylalanine in association with increased tyrosine after UVB exposure. Hence, generation of H 2 O 2 by UVB can activate epidermal PAH leading to an increased L-tyrosine pool for melanogenesis

  10. Manipulation of [C-11]-5-hydroxytryptophan and 6-[F-18]fluoro-3,4-dihydroxy-L-phenylalanine accumulation in neuroendocrine tumor cells

    NARCIS (Netherlands)

    Neels, Oliver C.; Koopmans, Klaas P.; Jager, Pieter L.; Vercauteren, Laya; van Waarde, Aren; Doorduin, Janine; Timmer-Bosscha, Hetty; Brouwers, Adrienne H.; de Vries, Elisabeth G. E.; Dierckx, Rudi A. J. O.; Kema, Ido P.; Elsinga, Philip H.

    2008-01-01

    [C-11]-5-Hydroxytryptophan ([C-11]HTP) and 6-[F-18]fluoro-3,4-dihydroxy-L-phenylalanine [F-18]FDOPA) are used to image neuroendocrine tumors with positron emission tomography. The precise mechanism by which these tracers accumulate in tumor cells is unknown. We aimed to study tracer uptake via large

  11. Event-related potential correlates of selective processing in early- and continuously-treated children with phenylketonuria : Effects of concurrent phenylalanine level and dietary control

    NARCIS (Netherlands)

    de Sonneville, Leo M. J.; Huijbregts, Stephan C. J.; van Spronsen, Francjan J.; Verkerk, Paul H.; Sergeant, Joseph A.; Licht, Robert

    2010-01-01

    This Study focused on important characteristics of attentional (selective) processing in children with early-treated phenylketonuria (PKU). Seven to 14-year-old children with PKU were allocated to high phenylalanine (Phe) and low Phe groups and compared with control children on amplitudes and

  12. Event-related potential correlates of selective processing in early- and continuously-treated children with phenylketonuria: Effects of concurrent phenylalanine level and dietary control

    NARCIS (Netherlands)

    Sonneville, L.M.J. de; Huijbregts, S.C.J.; Spronsen, F.J. van; Verkerk, P.H.; Sergeant, J.A.; Licht, R.

    2009-01-01

    This study focused on important characteristics of attentional (selective) processing in children with early-treated phenylketonuria (PKU). Seven to 14-year-old children with PKU were allocated to high phenylalanine (Phe) and low Phe groups and compared with control children on amplitudes and

  13. Photoinduced Intramolecular Tryptophan Oxidation and Excited-State Behavior of [Re(L-AA)(CO)3(r-diimine)] þ (L = Pyridine or Imidazole, AA = Tryptophan, Tyrosine, Phenylalanine)

    Czech Academy of Sciences Publication Activity Database

    Blanco-Rodríguez, A. M.; Towrie, M.; Sýkora, Jan; Záliš, Stanislav; Vlček, Antonín

    2011-01-01

    Roč. 50, č. 13 (2011), s. 6122-6134 ISSN 0020-1669 R&D Projects: GA MŠk(CZ) LD11082 Institutional research plan: CEZ:AV0Z40400503 Keywords : tryptophan * tyrosine * phenylalanine Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 4.601, year: 2011

  14. Costs and Outcomes over 36 Years of Patients with Phenylketonuria Who Do and Do Not Remain on a Phenylalanine-Restricted Diet

    Science.gov (United States)

    Guest, J. F.; Bai, J. J.; Taylor, R. R.; Sladkevicius, E.; Lee, P. J.; Lachmann, R. H.

    2013-01-01

    Background: To quantify the costs and consequences of managing phenylketonuria (PKU) in the UK and to estimate the potential implications to the UK's National Health Service (NHS) of keeping patients on a phenylalanine-restricted diet for life. Methods: A computer-based model was constructed depicting the management of PKU patients over the first…

  15. A highly conserved phenylalanine in the alpha, beta-T cell receptor (TCR) constant region determines the integrity of TCR/CD3 complexes

    DEFF Research Database (Denmark)

    Caspar-Bauguil, S; Arnaud, J; Huchenq, A

    1994-01-01

    In the present study, we have investigated the importance of a phenylalanine (phe195) in the Tcr-C alpha region on Tcr-alpha,beta/CD3 membrane expression. An exchange of phe195 with a tyrosine residue does not affect Tcr/CD3 membrane expression; however, exchange with aspartic acid, histidine or ...

  16. 21 CFR 201.21 - Declaration of presence of phenylalanine as a component of aspartame in over-the-counter and...

    Science.gov (United States)

    2010-04-01

    ... component of aspartame in over-the-counter and prescription drugs for human use. 201.21 Section 201.21 Food... component of aspartame in over-the-counter and prescription drugs for human use. (a) Aspartame is the... acids are so combined to form aspartame (1-methyl N-L-α-aspartyl-L-phenylalanine), they produce an...

  17. Simultaneous determination of tyrosine, phenylalanine and deoxyguanosine oxidation products by liquid chromatography-tandem mass spectrometry as non-invasive biomarkers for oxidative damage

    NARCIS (Netherlands)

    Orhan, Hilmi; Vermeulen, Nico P E; Tump, Cornelis; Zappey, Herman; Meerman, John H N

    2004-01-01

    We developed an isotope dilution HPLC-atmospheric pressure chemical ionization-tandem mass spectrometry (HPLC-APCI-MS/MS) method for the simultaneous determination of p-tyrosine, phenylalanine, o,o'-dityrosine, m-tyrosine, o-tyrosine, 3-chlorotyrosine and 3-nitrotyrosine and

  18. Sulfate-Reducing Prokaryotes from North Sea Oil reservoirs; organisms, distribution and origin

    Energy Technology Data Exchange (ETDEWEB)

    Beeder, Janiche

    1996-12-31

    During oil production in the North Sea, anaerobic seawater is pumped in which stimulates the growth of sulphate-reducing prokaryotes that produce hydrogen sulphide. This sulphide causes major health hazards, economical and operational problems. As told in this thesis, several strains of sulphate reducers have been isolated from North Sea oil field waters. Antibodies have been produced against these strains and used to investigate the distribution of sulphate reducers in a North Sea oil reservoir. The result showed a high diversity among sulphate reducers, with different strains belonging to different parts of the reservoir. Some of these strains have been further characterized. The physiological and phylogenetic characterization showed that strain 7324 was an archaean. Strain A8444 was a bacterium, representing a new species of a new genus. A benzoate degrading sulphate reducing bacterium was isolated from injection water, and later the same strain was detected in produced water. This is the first field observations indicating that sulphate reducers are able to penetrate an oil reservoir. It was found that the oil reservoir contains a diverse population of thermophilic sulphate reducers able to grow on carbon sources in the oil reservoir, and to live and grow in this extreme environment of high temperature and pressure. The mesophilic sulphate reducers are established in the injection water system and in the reservoir near the injection well during oil production. The thermophilic sulphate reducers are able to grow in the reservoir prior to, as well as during production. It appears that the oil reservoir is a natural habitat for thermophilic sulphate reducers and that they have been present in the reservoir long before production started. 322 refs., 9 figs., 11 tabs.

  19. Molecular interactions within the halophilic, thermophilic, and mesophilic prokaryotic ribosomal complexes: clues to environmental adaptation.

    Science.gov (United States)

    Mallik, Saurav; Kundu, Sudip

    2015-01-01

    Using the available crystal structures of 50S ribosomal subunits from three prokaryotic species: Escherichia coli (mesophilic), Thermus thermophilus (thermophilic), and Haloarcula marismortui (halophilic), we have analyzed different structural features of ribosomal RNAs (rRNAs), proteins, and of their interfaces. We have correlated these structural features with the environmental adaptation strategies of the corresponding species. While dense intra-rRNA packing is observed in thermophilic, loose intra-rRNA packing is observed in halophilic (both compared to mesophilic). Interestingly, protein-rRNA interfaces of both the extremophiles are densely packed compared to that of the mesophilic. The intersubunit bridge regions are almost devoid of cavities, probably ensuring the proper formation of each bridge (by not allowing any loosely packed region nearby). During rRNA binding, the ribosomal proteins experience some structural transitions. Here, we have analyzed the intrinsically disordered and ordered regions of the ribosomal proteins, which are subjected to such transitions. The intrinsically disordered and disorder-to-order transition sites of the thermophilic and mesophilic ribosomal proteins are simultaneously (i) highly conserved and (ii) slowly evolving compared to rest of the protein structure. Although high conservation is observed at such sites of halophilic ribosomal proteins, but slow rate of evolution is absent. Such differences between thermophilic, mesophilic, and halophilic can be explained from their environmental adaptation strategy. Interestingly, a universal biophysical principle evident by a linear relationship between the free energy of interface formation, interface area, and structural changes of r-proteins during assembly is always maintained, irrespective of the environmental conditions.

  20. CRISPR-Cas: evolution of an RNA-based adaptive immunity system in prokaryotes.

    Science.gov (United States)

    Koonin, Eugene V; Makarova, Kira S

    2013-05-01

    The CRISPR-Cas (clustered regularly interspaced short palindromic repeats, CRISPR-associated genes) is an adaptive immunity system in bacteria and archaea that functions via a distinct self-non-self recognition mechanism that is partially analogous to the mechanism of eukaryotic RNA interference (RNAi). The CRISPR-Cas system incorporates fragments of virus or plasmid DNA into the CRISPR repeat cassettes and employs the processed transcripts of these spacers as guide RNAs to cleave the cognate foreign DNA or RNA. The Cas proteins, however, are not homologous to the proteins involved in RNAi and comprise numerous, highly diverged families. The majority of the Cas proteins contain diverse variants of the RNA recognition motif (RRM), a widespread RNA-binding domain. Despite the fast evolution that is typical of the cas genes, the presence of diverse versions of the RRM in most Cas proteins provides for a simple scenario for the evolution of the three distinct types of CRISPR-cas systems. In addition to several proteins that are directly implicated in the immune response, the cas genes encode a variety of proteins that are homologous to prokaryotic toxins that typically possess nuclease activity. The predicted toxins associated with CRISPR-Cas systems include the essential Cas2 protein, proteins of COG1517 that, in addition to a ligand-binding domain and a helix-turn-helix domain, typically contain different nuclease domains and several other predicted nucleases. The tight association of the CRISPR-Cas immunity systems with predicted toxins that, upon activation, would induce dormancy or cell death suggests that adaptive immunity and dormancy/suicide response are functionally coupled. Such coupling could manifest in the persistence state being induced and potentially providing conditions for more effective action of the immune system or in cell death being triggered when immunity fails.