The Antiproliferative Effect of Cyclodipeptides from Pseudomonas aeruginosa PAO1 on HeLa Cells Involves Inhibition of Phosphorylation of Akt and S6k Kinases.

PubMed

Hernández-Padilla, Laura; Vázquez-Rivera, Dolores; Sánchez-Briones, Luis A; Díaz-Pérez, Alma L; Moreno-Rodríguez, José; Moreno-Eutimio, Mario A; Meza-Carmen, Victor; Cruz, Homero Reyes-De la; Campos-García, Jesús

2017-06-20

Pseudomonas aeruginosa PAO1, a potential pathogen of plants and animals, produces the cyclodipeptides cyclo(l-Pro-l-Tyr), cyclo(l-Pro-l-Phe), and cyclo(l-Pro-l-Val) (PAO1-CDPs), whose effects have been implicated in inhibition of human tumor cell line proliferation. Our purpose was to investigate in depth in the mechanisms of HeLa cell proliferation inhibition by the PAO1-CDPs. The results indicate that PAO1-CDPs, both purified individually and in mixtures, inhibited HeLa cell proliferation by arresting the cell cycle at the G0-G1 transition. The crude PAO1-CDPs mixture promoted cell death in HeLa cells in a dose-dependent manner, showing efficacy similar to that of isolated PAO1-CDPs (LD 50 of 60-250 µM) and inducing apoptosis with EC 50 between 0.6 and 3.0 µM. Moreover, PAO1-CDPs showed a higher proapoptotic activity (~10³-10⁵ fold) than their synthetic analogs did. Subsequently, the PAO1-CDPs affected mitochondrial membrane potential and induced apoptosis by caspase-9-dependent pathway. The mechanism of inhibition of cells proliferation in HeLa cells involves inhibition of phosphorylation of both Akt-S473 and S6k-T389 protein kinases, showing a cyclic behavior of their expression and phosphorylation in a time and concentration-dependent fashion. Taken together our findings indicate that PI3K-Akt-mTOR-S6k signaling pathway blockage is involved in the antiproliferative effect of the PAO1-CDPs.

Antitumor and Antimicrobial Activity of Some Cyclic Tetrapeptides and Tripeptides Derived from Marine Bacteria

PubMed Central

Chakraborty, Subrata; Tai, Dar-Fu; Lin, Yi-Chun; Chiou, Tzyy-Wen

2015-01-01

Marine derived cyclo(Gly-l-Ser-l-Pro-l-Glu) was selected as a lead to evaluate antitumor-antibiotic activity. Histidine was chosen to replace the serine residue to form cyclo(Gly-l-His-l-Pro-l-Glu). Cyclic tetrapeptides (CtetPs) were then synthesized using a solution phase method, and subjected to antitumor and antibiotic assays. The benzyl group protected CtetPs derivatives, showed better activity against antibiotic-resistant Staphylococcus aureus in the range of 60–120 μM. Benzyl group protected CtetPs 3 and 4, exhibited antitumor activity against several cell lines at a concentration of 80–108 μM. However, shortening the size of the ring to the cyclic tripeptide (CtriP) scaffold, cyclo(Gly-l-Ser-l-Pro), cyclo(Ser-l-Pro-l-Glu) and their analogues showed no antibiotic or antitumor activity. This phenomenon can be explained from their backbone structures. PMID:25988520

Antitumor and antimicrobial activity of some cyclic tetrapeptides and tripeptides derived from marine bacteria.

PubMed

Chakraborty, Subrata; Tai, Dar-Fu; Lin, Yi-Chun; Chiou, Tzyy-Wen

2015-05-15

Marine derived cyclo(Gly-l-Ser-l-Pro-l-Glu) was selected as a lead to evaluate antitumor-antibiotic activity. Histidine was chosen to replace the serine residue to form cyclo(Gly-l-His-l-Pro-l-Glu). Cyclic tetrapeptides (CtetPs) were then synthesized using a solution phase method, and subjected to antitumor and antibiotic assays. The benzyl group protected CtetPs derivatives, showed better activity against antibiotic-resistant Staphylococcus aureus in the range of 60-120 μM. Benzyl group protected CtetPs 3 and 4, exhibited antitumor activity against several cell lines at a concentration of 80-108 μM. However, shortening the size of the ring to the cyclic tripeptide (CtriP) scaffold, cyclo(Gly-l-Ser-l-Pro), cyclo(Ser-l-Pro-l-Glu) and their analogues showed no antibiotic or antitumor activity. This phenomenon can be explained from their backbone structures.

Immunostimulatory activities of a decapeptide derived from Alcaligenes faecalis FY-3 to crucian carp.

PubMed

Wang, G-X; Li, F-Y; Cui, J; Wang, Y; Liu, Y-T; Han, J; Lei, Y

2011-07-01

A strain was isolated from a soil sample collected from Weihe river in Shaanxi province (108°03'E 34°14'N), which was identified as Alcaligenes faecalis by 16S rRNA analysis. A compound M showing potent immune activity was isolated from secondary metabolites of the strain through bioassay-guided isolation techniques. The structure of the compound M was elucidated using FT-IR, EI-MS, 1H NMR and 13C NMR spectra and identified as cyclo-(L-Pro-Gly)5 which was first time reported as a natural product. We evaluated the immune effects of the cyclo-(L-Pro-Gly)5 on the basis of serum lysozyme activity, bacterial agglutination titre assay, superoxide anion production and phagocytic activity assay, and they were found to be significantly increased by cyclo-(L-Pro-Gly)5. The effects of cyclo-(L-Pro-Gly)5 on immune-related gene expression were further investigated. The outcomes of real-time quantitative polymerase chain reaction (RQ-PCR) proved that the transcribing level of interleukin 6β (IL-6β) and inducible nitric oxide synthase 1β (iNOS-1β) mRNA in the blood leucocytes have been augmented by cyclo-(L-Pro-Gly)5. The challenge experiment showed that crucian carp injected the cyclo-(L-Pro-Gly)5 had significantly (P < 0.05) lower cumulative mortality (13.0%) compared with the control (45.4%) after infection with live Aeromonas hydrophila. These results suggested that cyclo-(L-Pro-Gly)5 is a possible immunostimulant and may strengthen the immune response and protect the heath status of crucian carp against A. hydrophila. © 2011 The Authors. Scandinavian Journal of Immunology © 2011 Blackwell Publishing Ltd.

Activation of dormant secondary metabolite production by introducing neomycin resistance into the deep-sea fungus, Aspergillus versicolor ZBY-3.

PubMed

Dong, Yuan; Cui, Cheng-Bin; Li, Chang-Wei; Hua, Wei; Wu, Chang-Jing; Zhu, Tian-Jiao; Gu, Qian-Qun

2014-07-29

A new ultrasound-mediated approach has been developed to introduce neomycin-resistance to activate silent pathways for secondary metabolite production in a bio-inactive, deep-sea fungus, Aspergillus versicolor ZBY-3. Upon treatment of the ZBY-3 spores with a high concentration of neomycin by proper ultrasound irradiation, a total of 30 mutants were obtained by single colony isolation. The acquired resistance of the mutants to neomycin was confirmed by a resistance test. In contrast to the ZBY-3 strain, the EtOAc extracts of 22 of the 30 mutants inhibited the human cancer K562 cells, indicating that these mutants acquired a capability to produce antitumor metabolites. HPLC-photodiode array detector (PDAD)-UV and HPLC-electron spray ionization (ESI)-MS analyses of the EtOAc extracts of seven bioactive mutants and the ZBY-3 strain indicated that diverse secondary metabolites have been newly produced in the mutant extracts in contrast to the ZBY-3 extract. The followed isolation and characterization demonstrated that six metabolites, cyclo(D-Pro-D-Phe) (1), cyclo(D-Tyr-D-Pro) (2), phenethyl 5-oxo-L-prolinate (3), cyclo(L-Ile-L-Pro) (4), cyclo(L-Leu-L-Pro) (5) and 3β,5α,9α-trihydroxy-(22E,24R)-ergosta-7,22-dien-6-one (6), were newly produced by the mutant u2n2h3-3 compared to the parent ZBY-3 strain. Compound 3 was a new compound; 2 was isolated from a natural source for the first time, and all of these compounds were also not yet found in the metabolites of other A. versicolor strains. Compounds 1-6 inhibited the K562 cells, with inhibition rates of 54.6% (1), 72.9% (2), 23.5% (3), 29.6% (4), 30.9% (5) and 51.1% (6) at 100 μg/mL, and inhibited also other human cancer HL-60, BGC-823 and HeLa cells, to some extent. The present study demonstrated the effectiveness of the ultrasound-mediated approach to activate silent metabolite production in fungi by introducing acquired resistance to aminoglycosides and its potential for discovering new compounds from silent fungal metabolic pathways. This approach could be applied to elicit the metabolic potentials of other fungal isolates to discover new compounds from cryptic secondary metabolites.

The Aminolysis Reaction of Streptomyces S9 Aminopeptidase Promotes the Synthesis of Diverse Prolyl Dipeptides▿ †

PubMed Central

Arima, Jiro; Morimoto, Masazumi; Usuki, Hirokazu; Mori, Nobuhiro; Hatanaka, Tadashi

2010-01-01

Prolyl dipeptide synthesis by S9 aminopeptidase from Streptomyces thermocyaneoviolaceus (S9AP-St) has been demonstrated. In the synthesis, S9AP-St preferentially used l-Pro-OBzl as the acyl donor, yielding synthesized dipeptides having an l-Pro-Xaa structure. In addition, S9AP-St showed broad specificity toward the acyl acceptor. Furthermore, S9AP-St produced cyclo (l-Pro-l-His) with a conversion ratio of substrate to cyclo (l-Pro-l-His) higher than 40%. PMID:20418423

Non-ribosomal Peptide Synthases from Pseudomonas aeruginosa Play a Role in Cyclodipeptide Biosynthesis, Quorum-Sensing Regulation, and Root Development in a Plant Host.

PubMed

González, Omar; Ortíz-Castro, Randy; Díaz-Pérez, César; Díaz-Pérez, Alma L; Magaña-Dueñas, Viridiana; López-Bucio, José; Campos-García, Jesús

2017-04-01

Diverse molecules mediate cross-kingdom communication between bacteria and their eukaryotic partners and determine pathogenic or symbiotic relationships. N-acyl-L-homoserine lactone-dependent quorum-sensing signaling represses the biosynthesis of bacterial cyclodipeptides (CDPs) that act as auxin signal mimics in the host plant Arabidopsis thaliana. In this work, we performed bioinformatics, biochemical, and plant growth analyses to identify non-ribosomal peptide synthase (NRPS) proteins of Pseudomonas aeruginosa, which are involved in CDP synthesis. A reverse genetics strategy allowed the identification of the genes encoding putative multi-modular-NRPS (MM-NRPS). Mutations in these genes affected the synthesis of the CDPs cyclo(L-Pro-L-Val), cyclo(L-Pro-L-Leu), and cyclo(L-Pro-L-Tyr), while showing wild-type-like levels of virulence factors, such as violacein, elastase, and pyocyanin. When analyzing the bioactivity of purified, naturally produced CDPs, it was found that cyclo(L-Pro-L-Tyr) and cyclo(L-Pro-L-Val) were capable of antagonizing quorum-sensing-LasR (QS-LasR)-dependent signaling in a contrasting manner in the cell-free supernatants of the selected NRPS mutants, which showed QS induction. Using a bacteria-plant interaction system, we further show that the pvdJ, ambB, and pchE P. aeruginosa mutants failed to repress primary root growth, but improved root branching in A. thaliana seedlings. These results indicated that the CDP production in P. aeruginosa depended on the functional MM-NRPS, which influences quorum-sensing of bacteria and plays a role in root architecture remodeling.

Lactobacillus reuteri-produced cyclic dipeptides quench agr-mediated expression of toxic shock syndrome toxin-1 in staphylococci

PubMed Central

Li, Jingru; Wang, Wenliang; Xu, Stacey X.; Magarvey, Nathan A.; McCormick, John K.

2011-01-01

The production of the staphylococcal exotoxin toxic shock syndrome toxin-1 (TSST-1) by Staphylococcus aureus has been associated with essentially all cases of menstruation-associated toxic shock syndrome (TSS). In this work, we show that the human vaginal isolate Lactobacillus reuteri RC-14 produces small signaling molecules that are able to interfere with the staphylococcal quorum-sensing system agr, a key regulator of virulence genes, and repress the expression of TSST-1 in S. aureus MN8, a prototype of menstrual TSS S. aureus strains. Quantitative real-time PCR data showed that transcription from the Ptst promoter, as well as the P2 and P3 promoters of the agr system from all four agr subgroups of S. aureus, was strongly inhibited in response to growth with L. reuteri RC-14 cultural supernatant. Alterations in the transcriptional levels of two other virulence-associated regulators sarA and saeRS were also observed, indicating a potential overall influence of L. reuteri RC-14 signals on the production of virulence factors in S. aureus. S. aureus promoter-lux reporter strains were used to screen biochemically fractionated L. reuteri RC-14 supernatant, and the cyclic dipeptides cyclo(l-Phe-l-Pro) and cyclo(l-Tyr-l-Pro) were identified as the signaling molecules. The results from this work contribute to a better understanding of interspecies cell-to-cell communication between Lactobacillus and Staphylococcus, and provide a unique mechanism by which endogenous or probiotic strains may attenuate virulence factor production by bacterial pathogens. PMID:21282650

Lactobacillus reuteri-produced cyclic dipeptides quench agr-mediated expression of toxic shock syndrome toxin-1 in staphylococci.

PubMed

Li, Jingru; Wang, Wenliang; Xu, Stacey X; Magarvey, Nathan A; McCormick, John K

2011-02-22

The production of the staphylococcal exotoxin toxic shock syndrome toxin-1 (TSST-1) by Staphylococcus aureus has been associated with essentially all cases of menstruation-associated toxic shock syndrome (TSS). In this work, we show that the human vaginal isolate Lactobacillus reuteri RC-14 produces small signaling molecules that are able to interfere with the staphylococcal quorum-sensing system agr, a key regulator of virulence genes, and repress the expression of TSST-1 in S. aureus MN8, a prototype of menstrual TSS S. aureus strains. Quantitative real-time PCR data showed that transcription from the Ptst promoter, as well as the P2 and P3 promoters of the agr system from all four agr subgroups of S. aureus, was strongly inhibited in response to growth with L. reuteri RC-14 cultural supernatant. Alterations in the transcriptional levels of two other virulence-associated regulators sarA and saeRS were also observed, indicating a potential overall influence of L. reuteri RC-14 signals on the production of virulence factors in S. aureus. S. aureus promoter-lux reporter strains were used to screen biochemically fractionated L. reuteri RC-14 supernatant, and the cyclic dipeptides cyclo(L-Phe-L-Pro) and cyclo(L-Tyr-L-Pro) were identified as the signaling molecules. The results from this work contribute to a better understanding of interspecies cell-to-cell communication between Lactobacillus and Staphylococcus, and provide a unique mechanism by which endogenous or probiotic strains may attenuate virulence factor production by bacterial pathogens.

Lysobacter capsici AZ78 produces cyclo(L-Pro-L-Tyr), a 2,5-diketopiperazine with toxic activity against sporangia of Phytophthora infestans and Plasmopara viticola.

PubMed

Puopolo, G; Cimmino, A; Palmieri, M C; Giovannini, O; Evidente, A; Pertot, I

2014-10-01

To investigate low molecular weight compounds produced in vitro by Lysobacter capsici AZ78 and their toxic activity against sporangia of plant pathogenic oomycetes. Assays carried out in vitro showed that L. capsici AZ78 drastically inhibits the growth of plant pathogenic oomycetes. Accordingly, the preventive application of culture filtrates of L. capsici AZ78 on grapevine and tomato plants reduced the infections, respectively, caused by Plasmopara (Pl.) viticola and Phytophthora infestans. The subsequent chemical analysis of the culture filtrates of L. capsici AZ78 by spectroscopic (essentially 1D and 2D (1)H NMR and (13)C NMR and ESI MS spectra) and optical methods led to the identification of the 2,5-diketopiperazine cyclo(L-Pro-L-Tyr) that inhibited the development of P. infestans sporangia in vitro and on tomato leaves. Furthermore, a genomic region with high sequence identity with genes coding for a hybrid polyketide synthase and nonribosomal peptide synthetase was detected in L. capsici AZ78. Lysobacter capsici AZ78 produces cyclo(L-Pro-L-Tyr) in vitro that was effective in killing the sporangia of P. infestans and Pl. viticola in vitro. Moreover, this low molecular weight compound prevents the occurrence of late blight lesions when applied on tomato leaves. The application of L. capsici AZ78 cells or its own culture filtrates effectively controls both P. infestans and Pl. viticola. Cyclo(L-Pro-L-Tyr) produced by L. capsici AZ78 is toxic against sporangia of both these oomycetes. These data enforce the potential in the use of Lysobacter members for the control of plant pathogenic oomycetes and provide the basis for the development of new low-impact fungicides based on cyclo(L-Pro-L-Tyr). © 2014 The Society for Applied Microbiology.

A lead (II) 3D coordination polymer based on a marine cyclic peptide motif.

PubMed

Chakraborty, Subrata; Tyagi, Pooja; Tai, Dar-Fu; Lee, Gene-Hsiang; Peng, Shie-Ming

2013-04-26

The crystal structure of a naturally occurring cyclic tetrapeptide cyclo(Gly-L-Ser-L-Pro-L-Glu) [cyclo(GSPE)] was obtained. The conformation of synthesized cyclo(GSPE) fixes the coordination to lead ion in a 1:1 ratio. This cyclo(GSPE)-Pb complex was constructed as an asymmetric 3D network in the crystalline state. The polymerization of a heavy metal ion with a rigid asymmetric cyclic tetrapeptide represents the first example of a new class of macrocyclic complexes.

[Study on chemical constituents from leaves of Tripterygium wilfordii].

PubMed

Cao, Xu; Li, Chuangjun; Yang, Jingzhi; Wei, Baixing; Luo, Yongming; Zhang, Dongming

2011-04-01

In order to study the chemical constituents of the leaves of Tripterygium wilfordii and provide references for the bio-active study, we isolated nine compounds from the dried leaves of Tripterygium wilfordii. Their structures were determined by application of spectroscopic (NMR, MS) and chemical methods. These compounds were isolated and identified as (+)-lyoniresinol (1), (+)-isolariciresinol (2), burselignan (3), dibutyl phthalate (4), cyclo-(S-Pro-R-Phe) (5), cyclo-(S-Pro-R-Leu) (6), cyclo-(S-Pro-S-Ile) (7), 3-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-1-propanone (8) and daucosterol (9). Compounds 1-3, 5-8 were isolated from this plant for the first time.

New cyclic peptides with osteoblastic proliferative activity from Dianthus superbus.

PubMed

Tong, Yun; Luo, Jian-Guang; Wang, Rui; Wang, Xiao-Bing; Kong, Ling-Yi

2012-03-01

Two new cyclic peptides, dianthins G-H (1 and 2), together with the known dianthin E (3), were isolated from the traditional Chinese medicinal plant Dianthus superbus. The sequences of cyclic peptides 1 and 2 were elucidated as cyclo (-Gly(1)-Pro(2)-Leu(3)-Thr(4)-Leu(5)-Phe(6)-) and cyclo (-Gly(1)-Pro(2)-Val(3)-Thr(4)-Ile(5)-Phe(6)-), on the basis of ESI tandem mass fragmentation analysis, extensive 2D NMR methods and X-ray diffraction. The isolated three compounds all increase proliferation of MC3T3-E1 cells in vitro using MTT method. Copyright © 2012 Elsevier Ltd. All rights reserved.

Cyclic Dipeptides from Bacillus vallismortis BS07 Require Key Components of Plant Immunity to Induce Disease Resistance in Arabidopsis against Pseudomonas Infection

PubMed Central

Noh, Seong Woo; Seo, Rira; Park, Jung-Kwon; Manir, Md. Maniruzzaman; Park, Kyungseok; Sang, Mee Kyung; Moon, Surk-Sik; Jung, Ho Won

2017-01-01

Cyclic dipeptides (CDPs) are one of the simplest compounds produced by living organisms. Plant-growth promoting rhizobacteria (PGPRs) also produce CDPs that can induce disease resistance. Bacillus vallismortis strain BS07 producing various CDPs has been evaluated as a potential biocontrol agent against multiple plant pathogens in chili pepper. However, plant signal pathway triggered by CDPs has not been fully elucidated yet. Here we introduce four CDPs, cyclo(Gly-L-Pro) previously identified from Aspergillus sp., and cyclo(L-Ala-L-Ile), cyclo(L-Ala-L-Leu), and cyclo(LLeu-L-Pro) identified from B. vallismortis BS07, which induce disease resistance in Arabidopsis against Pseudomonas syringae infection. The CDPs do not directly inhibit fungal and oomycete growth in vitro. These CDPs require PHYTOALEXIN DEFICIENT4, SALICYLIC ACID INDUCTION DEFICIENT2, and NONEXPRESSOR OF PATHOGENESIS-RELATED PROTEINS1 important for salicylic acid-dependent defense to induce resistance. On the other hand, regulators involved in jasmonate-dependent event, such as ETHYLENE RECEPTOR1, JASMONATE RESPONSE1, and JASMONATE INSENSITIVE1, are necessary to the CDP-induced resistance. Furthermore, treatment of these CDPs primes Arabidopsis plants to rapidly express PATHOGENESIS-RELATED PROTEIN4 at early infection phase. Taken together, we propose that these CDPs from PGPR strains accelerate activation of jasmonate-related signaling pathway during infection. PMID:28811757

NMR analysis of cross strand aromatic interactions in an 8 residue hairpin and a 14 residue three stranded β-sheet peptide.

PubMed

Sonti, Rajesh; Rai, Rajkishor; Ragothama, Srinivasarao; Balaram, Padmanabhan

2012-12-13

Cross strand aromatic interactions between a facing pair of phenylalanine residues in antiparallel β-sheet structures have been probed using two structurally defined model peptides. The octapeptide Boc-LFV(D)P(L)PLFV-OMe (peptide 1) favors the β-hairpin conformation nucleated by the type II' β-turn formed by the (D)Pro-(L)Pro segment, placing Phe2 and Phe7 side chains in proximity. Two centrally positioned (D)Pro-(L)Pro segments facilitate the three stranded β-sheet formation in the 14 residue peptide Boc-LFV(D)P(L)PLFVA(D)P(L)PLFV-OMe (peptide 2) in which the Phe2/Phe7 orientations are similar to that in the octapeptide. The anticipated folded conformations of peptides 1 and 2 are established by the delineation of intramolecularly hydrogen bonded NH groups and by the observation of specific cross strand NOEs. The observation of ring current shifted aromatic protons is a diagnostic of close approach of the Phe2 and Phe7 side chains. Specific assignment of aromatic proton resonances using HSQC and HSQC-TOCSY methods allow an analysis of interproton NOEs between the spatially proximate aromatic rings. This approach facilitates specific assignments in systems containing multiple aromatic rings in spectra at natural abundance. Evidence is presented for a dynamic process which invokes a correlated conformational change about the C(α)-C(β)(χ(1)) bond for the pair of interacting Phe residues. NMR results suggest that aromatic ring orientations observed in crystals are maintained in solution. Anomalous temperature dependence of ring current induced proton chemical shifts suggests that solvophobic effects may facilitate aromatic ring clustering in apolar solvents.

Vibrio vulnificus quorum-sensing molecule cyclo(Phe-Pro) inhibits RIG-I-mediated antiviral innate immunity.

PubMed

Lee, Wooseong; Lee, Seung-Hoon; Kim, Minwoo; Moon, Jae-Su; Kim, Geon-Woo; Jung, Hae-Gwang; Kim, In Hwang; Oh, Ji Eun; Jung, Hi Eun; Lee, Heung Kyu; Ku, Keun Bon; Ahn, Dae-Gyun; Kim, Seong-Jun; Kim, Kun-Soo; Oh, Jong-Won

2018-04-23

The recognition of pathogen-derived ligands by pattern recognition receptors activates the innate immune response, but the potential interaction of quorum-sensing (QS) signaling molecules with host anti-viral defenses remains largely unknown. Here we show that the Vibrio vulnificus QS molecule cyclo(Phe-Pro) (cFP) inhibits interferon (IFN)-β production by interfering with retinoic-acid-inducible gene-I (RIG-I) activation. Binding of cFP to the RIG-I 2CARD domain induces a conformational change in RIG-I, preventing the TRIM25-mediated ubiquitination to abrogate IFN production. cFP enhances susceptibility to hepatitis C virus (HCV), as well as Sendai and influenza viruses, each known to be sensed by RIG-I but did not affect the melanoma-differentiation-associated gene 5 (MDA5)-recognition of norovirus. Our results reveal an inter-kingdom network between bacteria, viruses and host that dysregulates host innate responses via a microbial quorum-sensing molecule modulating the response to viral infection.

Conformational analysis of α-helical polypeptide included L-proline residue by high-resolution solid-state NMR measurement and quantum chemical calculation

NASA Astrophysics Data System (ADS)

Souma, Hiroyuki; Shoji, Akira; Kurosu, Hiromichi

2008-10-01

We challenged the problem about the stabilization mechanism of an α-helix formation for polypeptides containing L-proline (Pro) residue. We computed the optimized structure of α-helical poly( L-alanine) molecules including a Pro residue, H-(Ala) 8-Pro-(Ala) 9-OH, based on the molecular orbital calculation with density functional theory, B3LYP/6-31G(d) and the 13C and 15N chemical shift values based on the GIAO-CHF method with B3LYP/6-311G(d,p), respectively. It was found that two kinds of optimized structures, 'Bent structure' and 'Included α-helix structure', were preferred structures in H-(Ala) 8-Pro-(Ala) 9-OH. In addition, based on the precise 13C and 15N chemical shift data of the simple model, we successfully analyzed the secondary structure of well-defined synthetic polypeptide H-(Phe-Leu-Ala) 3-Phe C-Pro-Ala N-(Phe-Leu-Ala) 2-OH (FLA-11P), the secondary structure of which was proven to the 'Included α-helix structure'.

Discovery of potent HDAC inhibitors based on chlamydocin with inhibitory effects on cell migration.

PubMed

Wang, Shimiao; Li, Xiaohui; Wei, Yingdong; Xiu, Zhilong; Nishino, Norikazu

2014-03-01

The histone deacetylase (HDAC) family is a promising drug target class owing to the importance of these enzymes in a variety of cellular processes. Docking studies were conducted to identify novel HDAC inhibitors. Subtle modifications in the recognition domain were introduced into a series of chlamydocin analogues, and the resulting scaffolds were combined with various zinc binding domains. Remarkably, cyclo(L-Asu(NHOH)-L-A3mc6c-L-Phe-D-Pro, compound 1 b), with a methyl group at positions 3 or 5 on the aliphatic ring, exhibited better antiproliferative effects than trichostatin A (TSA) against MCF-7 and K562 cell lines. In addition to cell-cycle arrest and apoptosis, cell migration inhibition was observed in cells treated with compound 1 b. Subsequent western blot analysis revealed that the balance between matrix metalloproteinase 2 (MMP2) and tissue inhibitors of metalloproteinase 1 (TIMP1) determines the degree of metalloproteinase activity in MCF-7 cells, thereby regulating cell migration. The improved inhibitory activity imparted by altering the hydrophobic substitution pattern at the bulky cap group is a valuable approach in the development of novel HDAC inhibitors. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Behavioural and biochemical responses following activation of midbrain dopamine pathways by receptor selective neurokinin agonists.

PubMed

Elliott, P J; Mason, G S; Stephens-Smith, M; Hagan, R M

1991-06-01

Preferential activation of mesolimbic and nigro-striatal dopamine (DA) pathways by receptor-selective and peptidase-resistant neurokinin (NK) agonists is reported. The DA cell body region of the mesolimbic pathway appears to be activated by NK agonists selective for NK-1 and NK-3 receptors whereas the DA cell bodies in the substantia nigra are under an excitatory NK-2 receptor-mediated influence. Stimulation of the mesolimbic DA pathway by NK-1 (Ava[L-Pro9,N-Me-Leu10]SP (7-11) [GR73632]) or NK-3 (Senktide) agonists increase locomotor activity. Additional studies showed that this elevated motor response observed after intra-VTA infusion of GR73632 was accompanied by a corresponding increase in DA turnover in the terminal fields of this pathway. Similarly, unilateral activation of the nigro-striatal DA pathway by NK-2 selective agonists (Ava (D-Pro9) SP (7-11) [GR51667] or [Lys3,Gly8,R-Lac-Leu9]NKA (3-10) [GR64349]) elicit contralateral rotational activity and an increase in DA turnover in the ipsilateral striatum. The rotational response was attenuated by prior administration of an NK-2 antagonist (cyclo (Gln, Trp, Phe, Gly, Leu, Met)] L-659877]) into the nigra. Peripheral injection of haloperidol, a DA antagonist, also blocked the NK-2 agonist induced rotations.

Association of Synergistetes and Cyclodipeptides with Periodontitis.

PubMed

Marchesan, J T; Morelli, T; Moss, K; Barros, S P; Ward, M; Jenkins, W; Aspiras, M B; Offenbacher, S

2015-10-01

The purpose of this study was to evaluate the microbial community (MC) composition as it relates to salivary metabolites and periodontal clinical parameters in a 21-d biofilm-overgrowth model. Subjects (N = 168) were enrolled equally into 5 categories of periodontal status per the biofilm-gingival interface classification. Microbial species within subgingival plaque samples were identified by human microbiome identification microarray. Whole saliva was analyzed by liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry for metabolite identification. Phylum was grouped into MCs according to principal component analysis. Generalized linear and regression models were used to examine the association among MC, species, periodontal clinical parameters, and salivary metabolome. Multiple comparisons were adjusted with the false discovery rate. The study population was distributed into 8 distinct MC profiles, designated MC-1 to MC-8. MC-2 explained 14% of the variance and was dominated by Synergistetes and Spirochaetes. It was the only community structure significantly associated with high probing depth (P = 0.02) and high bleeding on probing (P = 0.008). MC-2 was correlated with traditional periodontal pathogens and several newly identified putative periodontal pathogens: Fretibacterium fastidiosum, Fretibacterium sp. OT360/OT362, Filifactor alocis, Treponema lecithinolyticum, Eubacterium saphenum, Desulfobulbus sp./OT041, and Mogibacterium timidum. Synergistetes phylum was strongly associated with 2 novel metabolites-cyclo (-leu-pro) and cyclo (-phe-pro)-at 21 d of biofilm overgrowth (P = 0.02). In subjects with severe periodontitis (P2 and P3), cyclo (-leu-pro) and cyclo (-phe-pro) were significantly associated with increased changes in probing depth at 21 d of biofilm overgrowth (P ≤ 0.05). The analysis identified a MC dominated by Synergistetes, with classic and putative newly identified pathogens/pathobionts associated with clinical disease. The metabolomic discovery of 2 novel cyclodipeptides that have been reported to serve as quorum-sensing and/or bacteriocidal/bacteriostatic molecules, in association with Synergistetes, suggests a potential role in periodontal biofilm dysbiosis and periodontal disease that warrants further investigation. © International & American Associations for Dental Research 2015.

Macrocyclic peptides decrease c-Myc protein levels and reduce prostate cancer cell growth.

PubMed

Mukhopadhyay, Archana; Hanold, Laura E; Thayele Purayil, Hamsa; Gisemba, Solomon A; Senadheera, Sanjeewa N; Aldrich, Jane V

2017-08-03

The oncoprotein c-Myc is often overexpressed in cancer cells, and the stability of this protein has major significance in deciding the fate of a cell. Thus, targeting c-Myc levels is an attractive approach for developing therapeutic agents for cancer treatment. In this study, we report the anti-cancer activity of the macrocyclic peptides [D-Trp]CJ-15,208 (cyclo[Phe-D-Pro-Phe-D-Trp]) and the natural product CJ-15,208 (cyclo[Phe-D-Pro-Phe-Trp]). [D-Trp]CJ-15,208 reduced c-Myc protein levels in prostate cancer cells and decreased cell proliferation with IC 50 values ranging from 2.0 to 16 µM in multiple PC cell lines. [D-Trp]CJ-15,208 induced early and late apoptosis in PC-3 cells following 48 hours treatment, and growth arrest in the G2 cell cycle phase following both 24 and 48 hours treatment. Down regulation of c-Myc in PC-3 cells resulted in loss of sensitivity to [D-Trp]CJ-15,208 treatment, while overexpression of c-Myc in HEK-293 cells imparted sensitivity of these cells to [D-Trp]CJ-15,208 treatment. This macrocyclic tetrapeptide also regulated PP2A by reducing the levels of its phosphorylated form which regulates the stability of cellular c-Myc protein. Thus [D-Trp]CJ-15,208 represents a new lead compound for the potential development of an effective treatment of prostate cancer.

Secondary metabolites from marine-derived Streptomyces antibioticus strain H74-21.

PubMed

Fu, Shuna; Wang, Fan; Li, Hongyu; Bao, Yixuan; Yang, Yu; Shen, Huifang; Lin, Birun; Zhou, Guangxiong

2016-11-01

A new secondary metabolite, (2S,3R)-l-threonine, N-[3-(formylamino)-2-hydroxybenzoyl]-ethyl ester (streptomyceamide C, 1), together with four known compounds 1, 4-dimethyl-3-isopropyl-2,5-piperidinedione (2), cyclo-((S)-Pro-8- hydroxy-(R)-Ile (3), cyclo-((S)-Pro-(R)-Leu (4), and seco-((S)-Pro-(R)-Val) (5), were isolated from the EtOH extract of the fermented mycelium of the marine-derived streptomycete strain H74-21, which was isolated from sea sediment in a mangrove site. The structure of the new compound was established on the basis of its spectroscopic data, including 1D and 2D NMR, HR-TOF-MS. Their antifungal activities against Candida albicans and cytotoxicities against human breast adenocarcinoma cell line MCF-7, human glioblastoma cell line SF-268 and human lung cancer cell line NCI-H460 were tested. Compounds 1 only displayed cytotoxicity against human breast adenocarcinoma cell line MCF-7 with the IC50 value of 27.0 μg/mL. However, compounds 1-5 do not show antifungal activities at the test concentration of 1 mg/mL, and 2-5 have no cytotoxicities at the test concentration of 50 μg/mL.

A Comparative biochemical study on two marine endophytes, Bacterium SRCnm and Bacillus sp. JS, Isolated from red sea algae.

PubMed

Ahmed, Eman Fadl; Hassan, Hossam Mokhtar; Rateb, Mostafa Ezzat; Abdel-Wahab, Noha; Sameer, Somayah; Aly Taie, Hanan Anwar; Abdel-Hameed, Mohammed Sayed; Hammouda, Ola

2016-01-01

Two marine endophytic bacteria were isolated from the Red Sea algae; a red alga; Acanthophora dendroides and the brown alga Sargassum sabrepandum. The isolates were identified based on their 16SrRNA sequences as Bacterium SRCnm and Bacillus sp. JS. The objective of this study was to investigate the potential anti-microbial and antioxidant activities of the extracts of the isolated bacteria grown in different nutrient conditions. Compared to amoxicillin (25μg/disk) and erythromycin (15μg/disk), the extracts of Bacterium SRCn min media II, III, IV and V were potent inhibitors of the gram-positive bacterium Sarcina maxima even at low concentrations. Also, the multidrug resistant Staphylococcus aureus(MRSA) was more sensitive to the metabolites produced in medium (II) of the same endophyte than erythromycin (15μg/disk). A moderate activity of the Bacillus sp. JS extracts of media I and II was obtained against the same pathogen. The total compounds (500ug/ml) of both isolated endophytes showed moderate antioxidant activities (48.9% and 46.1%, respectively). LC/MS analysis of the bacterial extracts was carried out to investigate the likely natural products produced. Cyclo(D-cis-Hyp-L-Leu), dihydrosphingosine and 2-Amino-1,3-hexadecanediol were identified in the fermentation medium of Bacterium SRCnm, whereas cyclo (D-Pro-L-Tyr) and cyclo (L-Leu-L-Pro) were the suggested compounds of Bacillus sp. JS.

Longicalycinin A, a new cytotoxic cyclic peptide from Dianthus superbus var. longicalycinus (MAXIM.) WILL.

PubMed

Hsieh, Pei-Wen; Chang, Fang-Rong; Wu, Ching-Chung; Li, Chien-Ming; Wu, Kuen-Yuh; Chen, Su-Li; Yen, Hsin-Fu; Wu, Yang-Chang

2005-03-01

A new cyclic peptide, longicalycinin A (1), and six known compounds, vaccaroside A, dianoside A, dianoside G, 3-(4-hydroxy-3-methoxy-phenyl)propionic acid methyl ester, p-hydroxybenzoic acid, and p-hydroxybenzaldehyde were isolated from the MeOH extract of Dianthus superbus var. longicalycinus. The amino acid sequences of 1 was elucidated as cyclo(Gly(1)-Phe(2)-Tyr(3)-Pro(4)-Phe(5)-) on the basis of ESI tandem mass fragmentation analysis, chemical evidence, and extensive 2D NMR methods. Furthermore, compound 1 showed cytotoxicity to Hep G2 cancer cell line.

Protective effects of diketopiperazines from Moslae Herba against influenza A virus-induced pulmonary inflammation via inhibition of viral replication and platelets aggregation.

PubMed

Zhang, Huan-Huan; Yu, Wen-Ying; Li, Lan; Wu, Fang; Chen, Qin; Yang, Yang; Yu, Chen-Huan

2018-04-06

Moslae Herba (MH) is broadly used as an antiviral, antipyretic and anticoagulant drug which effectively treats respiratory diseases including cough, asthma, throat, cold and flu. The excessive inflammation of the lungs is the hallmark of severe influenza A virus (IAV) infection, while platelet aggregation and its subsequent microvascular thrombosis can exacerbate IAV-induced lung injury. Thus, inhibition of platelet aggregation can be a potential target for IAV treatment. Previous studies focus on the flavonoids from MH and their anti-inflammatory activities, but the anticoagulant compounds and potential molecular mechanism of MH remains unclear. This study was to isolate and characterize diketopiperazines (DKPs) from MH and to explore the underlying anticoagulant mechanism on IAV infection models. EtOAc sub-extract separated from MH ethanolic extract was subjected to fractionation through column chromatography. The chemical structures of pure compounds were characterized by the spectral analysis. Antiviral activities of DKPs were assayed in IAV-infected Madin-Darby canine kidney (MDCK) cells and mice. Anticoagulant effects of DKPs were investigated on adenosine 5'-diphosphate (ADP)-induced acute pulmonary embolism and IAV-induced lung injury in vivo, as well as the inhibition on platelet activating factor (PAF), arachidonic acid (AA) and ADP-induced platelet aggregation in vitro. The serum levels of thromboxane B 2 (TXB 2 ) and 6-keto-PGF 1α were detected by ELISA. The expressions of key proteins in CD41-mediated PI3K/AKT pathways were determined by western blotting analysis. Six DKPs were, for the first time, isolated from MH and identified as cyclo(Tyr-Leu) (1), cyclo(Phe-Phe) (2), cyclo(Phe-Tyr) (3), cyclo(Ala-Ile) (4), cyclo(Ala-Leu) (5) and Bz-Phe-Phe-OMe (6). Among these DKPs, cyclo(Ala-Ile) and Bz-Phe-Phe-OMe possessed low cytotoxicities and significant inhibition against cytopathic effects induced by IAV (H1N1 and H3N2) replication in MDCK cells. Furthermore, cyclo(Ala-Ile) and Bz-Phe-Phe-OMe significantly alleviated IAV-induced platelet activation and lung inflammation in mice. They could reduce the expression of CD41 and the phosphorylation of PI3K and AKT in PLTs of IAV-infected mice. These results suggested that cyclo(Ala-Ile) and Bz-Phe-Phe-OMe isolated from MH have antiviral and anticoagulant effects against IAV-induced PLT aggregation and lung inflammation via regulating CD41/PI3K/AKT pathway, and could be used as the potential agents for IAV treatment. Copyright © 2018 Elsevier B.V. All rights reserved.

New dicyclopeptides from Dianthus chinensis.

PubMed

Han, Jing; Wang, Zhe; Zheng, Yu-Qing; Zeng, Guang-Zhi; He, Wen-Jun; Tan, Ning-Hua

2014-05-01

One new dicyclopeptide cyclo-(L-N-methyl Glu-L-N-methyl Glu) (1), together with one new natural dicyclopeptide cyclo-(L-methyl Glu ester-L-methyl Glu ester) (2), and two known dicyclopeptides cyclo-(L-methyl Glu ester-L-Glu) (3), and cyclo-(L-Glu-L-Glu) (4), were isolated from the aerial parts of Dianthus chinensis L. Their structures were determined by spectroscopic analyses and chemical methods.

Development of a robust, sensitive and selective liquid chromatography-tandem mass spectrometry assay for the quantification of the novel macrocyclic peptide kappa opioid receptor antagonist [D-Trp]CJ-15,208 in plasma and application to an initial pharmacokinetic study.

PubMed

Khaliq, Tanvir; Williams, Todd D; Senadheera, Sanjeewa N; Aldrich, Jane V

2016-08-15

Selective kappa opioid receptor (KOR) antagonists may have therapeutic potential as treatments for substance abuse and mood disorders. Since [D-Trp]CJ-15,208 (cyclo[Phe-d-Pro-Phe-d-Trp]) is a novel potent KOR antagonist in vivo, it is imperative to evaluate its pharmacokinetic properties to assist the development of analogs as potential therapeutic agents, necessitating the development and validation of a quantitative method for determining its plasma levels. A method for quantifying [D-Trp]CJ-15,208 was developed employing high performance liquid chromatography-tandem mass spectrometry in mouse plasma. Sample preparation was accomplished through a simple one-step protein precipitation method with acetonitrile, and [D-Trp]CJ-15,208 analyzed following HPLC separation on a Hypersil BDS C8 column. Multiple reaction monitoring (MRM), based on the transitions m/z 578.1→217.1 and 245.0, was specific for [D-Trp]CJ-15,208, and MRM based on the transition m/z 566.2→232.9 was specific for the internal standard without interference from endogenous substances in blank mouse plasma. The assay was linear over the concentration range 0.5-500ng/mL with a mean r(2)=0.9987. The mean inter-day accuracy and precision for all calibration standards were 93-118% and 8.9%, respectively. The absolute recoveries were 85±6% and 81±9% for [D-Trp]CJ-15,208 and the internal standard, respectively. The analytical method had excellent sensitivity with a lower limit of quantification of 0.5ng/mL using a sample volume of 20μL. The method was successfully applied to an initial pharmacokinetic study of [D-Trp]CJ-15,208 following intravenous administration to mice. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of a robust, sensitive and selective liquid chromatography-tandem mass spectrometry assay for the quantification of the novel macrocyclic peptide kappa opioid receptor antagonist [D-Trp]CJ-15,208 in plasma and application to an initial pharmacokinetic study

PubMed Central

Khaliq, Tanvir; Williams, Todd D.; Senadheera, Sanjeewa N.; Aldrich, Jane V.

2016-01-01

Selective kappa opioid receptor (KOR) antagonists may have therapeutic potential as treatments for substance abuse and mood disorders. Since [D-Trp]CJ-15,208 (cyclo[Phe-D-Pro-Phe-D-Trp]) is a novel potent KOR antagonist in vivo, it is imperative to evaluate its pharmacokinetic properties to assist the development of analogs as potential therapeutic agents, necessitating the development and validation of a quantitative method for determining its plasma levels. A method for quantifying [D-Trp]CJ-15,208 was developed employing high performance liquid chromatography-tandem mass spectrometry in mouse plasma. Sample preparation was accomplished through a simple one-step protein precipitation method with acetonitrile, and [D-Trp]CJ-15,208 analyzed following HPLC separation on a Hypersil BDS C8 column. Multiple reaction monitoring (MRM), based on the transitions m/z 578.1 → 217.1 and 245.0, was specific for [D-Trp]CJ-15,208, and MRM based on the transition m/z 566.2 → 232.9 was specific for the internal standard without interference from endogenous substances in blank mouse plasma. The assay was linear over the concentration range 0.5–500 ng/mL with a mean r2 = 0.9987. The mean inter-day accuracy and precision for all calibration standards was 93–118% and 8.9%, respectively. The absolute recoveries were 85±6% and 81±9% for [D-Trp]CJ-15,208 and the internal standard, respectively. The analytical method had excellent sensitivity with a lower limit of quantification of 0.5 ng/mL using a sample volume of 20 μL. The method was successfully applied to an initial pharmacokinetic study of [D-Trp]CJ-15,208 following intravenous administration to mice. PMID:27318293

Therapeutic index of gramicidin S is strongly modulated by D-phenylalanine analogues at the beta-turn.

PubMed

Solanas, Concepción; de la Torre, Beatriz G; Fernández-Reyes, María; Santiveri, Clara M; Jiménez, M Angeles; Rivas, Luis; Jiménez, Ana I; Andreu, David; Cativiela, Carlos

2009-02-12

Analogues of the cationic antimicrobial peptide gramicidin S (GS), cyclo(Val-Orn-Leu-D-Phe-Pro)2, with d-Phe residues replaced by different (restricted mobility, mostly) surrogates have been synthesized and used in SAR studies against several pathogenic bacteria. While all D-Phe substitutions are shown by NMR to preserve the overall beta-sheet conformation, they entail subtle structural alterations that lead to significant modifications in biological activity. In particular, the analogue incorporating D-Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) shows a modest but significant increase in therapeutic index, mostly due to a sharp decrease in hemolytic effect. The fact that NMR data show a shortened distance between the D-Tic aromatic ring and the Orn delta-amino group may help explain the improved antibiotic profile of this analogue.

Therapeutic index of gramicidin S is strongly modulated by d-phenylalanine analogues at the β-turn

PubMed Central

Solanas, Concepción; de la Torre, Beatriz G.; Fernández-Reyes, María; Santiveri, Clara M.; Jiménez, M. Ángeles; Rivas, Luis; Jiménez, Ana I.; Andreu, David; Cativiela, Carlos

2009-01-01

Analogues of the cationic antimicrobial peptide gramicidin S (GS), cyclo(Val-Orn-Leu-d-Phe-Pro)2, with d-Phe residues replaced by different (restricted mobility, mostly) surrogates have been synthesized and used in SAR studies against several pathogenic bacteria. While all d-Phe substitutions are shown by NMR to preserve the overall β-sheet conformation, they entail subtle structural alterations that lead to significant modifications in biological activity. In particular, the analogue incorporating d-Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) shows a modest but significant increase in therapeutic index, mostly due to a sharp decrease in hemolytic effect. The fact that NMR data show a shortened distance between the d-Tic aromatic ring and the Orn δ-amino group may help explain the improved antibiotic profile of this analogue. PMID:19132829

Photosynthetic inhibition and oxidative stress to the toxic Phaeocystis globosa caused by a diketopiperazine isolated from products of algicidal bacterium metabolism.

PubMed

Tan, Shuo; Hu, Xiaoli; Yin, Pinghe; Zhao, Ling

2016-05-01

Algicidal bacteria have been turned out to be available for inhibiting Phaeocystis globosa which frequently caused harmful algal blooms and threatened to economic development and ecological balance. A marine bacterium Bacillus sp. Ts-12 exhibited significant algicidal activity against P. globosa by indirect attack. In present study, an algicidal compound was isolated by silica gel column, Sephadex G-15 column and HPLC, further identified as hexahydropyrrolo[1,2-a]pyrazine-1,4-dione, cyclo-(Pro-Gly), by GC-MS and (1)H-NMR. Cyclo-(Pro-Gly) significantly increased the level of reactive oxygen species (ROS) within P. globosa cells, further activating the enzymatic and non-enzymatic antioxidant systems, including superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and ascorbic acid (AsA). The increase in methane dicarboxylic aldehyde (MDA) content showed that the surplus ROS induced lipid peroxidation on membrane system. Transmission electron microscope (TEM) and flow cytometry (FCM) analysis revealed that cyclo-(Pro-Gly) caused reduction of Chl-a content, destruction of cell membrane integrity, chloroplasts and nuclear structure. Real-time PCR assay showed that the transcriptions of photosynthesis related genes (psbA, psbD, rbcL) were significantly inhibited. This study indicated that cyclo-(Pro-Gly) from marine Bacillus sp. Ts-12 exerted photosynthetic inhibition and oxidative stress to P. globosa and eventually led to the algal cells lysis. This algicidal compound might be potential bio-agent for controlling P. globosa red tide.

Cyclo(l-Leucyl-l-Prolyl) Produced by Achromobacter xylosoxidans Inhibits Aflatoxin Production by Aspergillus parasiticus

PubMed Central

Yan, Pei-Sheng; Song, Yuan; Sakuno, Emi; Nakajima, Hiromitsu; Nakagawa, Hiroyuki; Yabe, Kimiko

2004-01-01

Aflatoxins are potent carcinogenic and toxic substances that are produced primarily by Aspergillus flavus and Aspergillus parasiticus. We found that a bacterium remarkably inhibited production of norsolorinic acid, a precursor of aflatoxin, by A. parasiticus. This bacterium was identified as Achromobacter xylosoxidans based on its 16S ribosomal DNA sequence and was designated A. xylosoxidans NFRI-A1. A. xylosoxidans strains commonly showed similar inhibition. The inhibitory substance(s) was excreted into the medium and was stable after heat, acid, or alkaline treatment. Although the bacterium appeared to produce several inhibitory substances, we finally succeeded in purifying a major inhibitory substance from the culture medium using Diaion HP20 column chromatography, thin-layer chromatography, and high-performance liquid chromatography. The purified inhibitory substance was identified as cyclo(l-leucyl-l-prolyl) based on physicochemical methods. The 50% inhibitory concentration for aflatoxin production by A. parasiticus SYS-4 (= NRRL2999) was 0.20 mg ml−1, as determined by the tip culture method. High concentrations (more than 6.0 mg ml−1) of cyclo(l-leucyl-l-prolyl) further inhibited fungal growth. Similar inhibitory activities were observed with cyclo(d-leucyl-d-prolyl) and cyclo(l-valyl-l-prolyl), whereas cyclo(d-prolyl-l-leucyl) and cyclo(l-prolyl-d-leucyl) showed weaker activities. Reverse transcription-PCR analyses showed that cyclo(l-leucyl-l-prolyl) repressed transcription of the aflatoxin-related genes aflR, hexB, pksL1, and dmtA. This is the first report of a cyclodipeptide that affects aflatoxin production. PMID:15574949

The macrocyclic peptide natural product CJ-15,208 is orally active and prevents reinstatement of extinguished cocaine-seeking behavior.

PubMed

Aldrich, Jane V; Senadheera, Sanjeewa N; Ross, Nicolette C; Ganno, Michelle L; Eans, Shainnel O; McLaughlin, Jay P

2013-03-22

The macrocyclic tetrapeptide natural product CJ-15,208 (cyclo[Phe-d-Pro-Phe-Trp]) exhibited both dose-dependent antinociception and kappa opioid receptor (KOR) antagonist activity after oral administration. CJ-15,208 antagonized a centrally administered KOR selective agonist, providing strong evidence it crosses the blood-brain barrier to reach KOR in the CNS. Orally administered CJ-15,208 also prevented both cocaine- and stress-induced reinstatement of extinguished cocaine-seeking behavior in the conditioned place preference assay in a time- and dose-dependent manner. Thus, CJ-15,208 is a promising lead compound with a unique activity profile for potential development, particularly as a therapeutic to prevent relapse to drug-seeking behavior in abstinent subjects.

Stereochemical studies of the monocyclic agouti-related protein (103-122) Arg-Phe-Phe residues: conversion of a melanocortin-4 receptor antagonist into an agonist and results in the discovery of a potent and selective melanocortin-1 agonist.

PubMed

Joseph, Christine G; Wang, Xiang S; Scott, Joseph W; Bauzo, Rayna M; Xiang, Zhimin; Richards, Nigel G; Haskell-Luevano, Carrie

2004-12-30

The agouti-related protein (AGRP) is an endogenous antagonist of the centrally expressed melanocortin receptors. The melanocortin-4 receptor (MC4R) is involved in energy homeostasis, food intake, sexual function, and obesity. The endogenous hAGRP protein is 132 amino acids in length, possesses five disulfide bridges at the C-terminus of the molecule, and is expressed in the hypothalamus of the brain. We have previously reported that a monocyclic hAGRP(103-122) peptide is an antagonist at the melanocortin receptors expressed in the brain. Stereochemical inversion from the endogenous l- to d-isomers of single or multiple amino acid modifications in this monocyclic truncated hAGRP sequence resulted in molecules that are converted from melanocortin receptor antagonists into melanocortin receptor agonists. The Asp-Pro-Ala-Ala-Thr-Ala-Tyr-cyclo[Cys-Arg-DPhe-DPhe-Asn-Ala-Phe-Cys]-Tyr-Ala-Arg-Lys-Leu peptide resulted in a 60 nM melanocortin-1 receptor agonist that is 100-fold selective versus the mMC4R, 1000-fold selective versus the mMC3R, and ca. 180-fold selective versus the mMC5R. In attempts to identify putative ligand-receptor interactions that may be participating in the agonist induced stimulation of the MC4R, selected ligands were docked into a homology molecular model of the mMC4R. These modeling studies have putatively identified hAGRP ligand DArg111-mMC4RAsn115 (TM3) and the hAGRP DPhe113-mMC4RPhe176 (TM4) interactions as important for agonist activity.

The use of parenteral nutrition for the management of PKU patient undergoing chemotherapy for lymphoma: a case report.

PubMed

Salvarinova-Zivkovic, R; Hartnett, C; Sinclair, G; Dix, D; Horvath, G; Lillquist, Y; Stockler-Ipsiroglu, S

2012-04-01

The metabolic control of phenylalanine levels is a challenge during illness. We present the metabolic management of a 6 year old boy with classical PKU who was diagnosed with stage III intraabdominal Burkit's lymphoma and underwent surgical resection and chemotherapy. The metabolic control during chemotherapy was achieved by the use of parenteral custom made amino acid solution and pro-active adjustment of intake. From the 94 obtained plasma phenylalanine (Phe) levels, 18.4% were above our clinic's recommended upper limit (360 μmol/L, 6 mg/dL) while 52.7% of Phe levels were below the recommended lower limit (120 μmol/L, 2 mg/dL). Phe levels above recommended range were associated with low caloric/protein intake, while levels below recommended range reflected the difficulty in achieving the full prescribed Phe intake. We recommend early institution of custom made amino acid solution with maximum amino acid content and caloric intake to provide optimal phenylalanine control. Administration of phenylalanine via regular intravenous amino acid solution may assist in avoiding low Phe levels when prescribed intake is compromised due to vomiting and other disease related illnesses. Use of custom made, phenylalanine free amino acid solution proved beneficial in the management of blood phenylalanine levels in a PKU patient during chemotherapy for Burkitt lymphoma. Copyright © 2012 Elsevier Inc. All rights reserved.

The role of limited proteolysis of thyrotropin-releasing hormone in thermoregulation. Final report

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

Prasad, C.

1982-01-01

Cyclo (His-Pro) is a biologiclly active cyclic dipeptide derived from thyrotropin-releasing hormone by its limited proteolysis. We have developed a specific radioimmunoassay for this cyclic peptide and shown its presence throughout rat and monkey brains. The normal rat brain concentration of cyclo (His-Pro) ranged from 35-61 pmols/brain. The elution profiles of rat brain cyclo (His-Pro)-like immunoreactivity and synthetic radioactive cyclo (His-Pro) following gel filtration, ion-exchange chromatography and high pressure liquid chromatography were similar. An analysis of the regional distribution of cyclo (His-Pro) and TRH in rat and monkey brains exhibited no apparent precursor-product relationship. Studies on the neuroanatomic sites formore » the thermoregulatory effects of cyclo (His-Pro) suggested that the neural loci responsible for cyclo (His-Pro)-induced hypothermia resides within POA/AHA. The endogenous levels of brain cyclo (His-Pro) were elevated when rats were made either hypothyroid by surgical thyroidectomy or forced to drink alcohol for six weeks. These studies demonstrate that cyclo (His-Pro) is present throughout the central nervous system in physiologically relevant concentrations which can be modified by appropriate physiological and pharamacological manipulations. These data in conjunction with earlier reports of multiple biological activities of exogenous cyclo (His-Pro), suggest that endogenous cyclo (His-Pro) is a biological active peptide and it may play a neurotransmitter or neuromodulator role in the central nervous system.« less

Proline-Based Cyclic Dipeptides from Korean Fermented Vegetable Kimchi and from Leuconostoc mesenteroides LBP-K06 Have Activities against Multidrug-Resistant Bacteria.

PubMed

Liu, Rui; Kim, Andrew H; Kwak, Min-Kyu; Kang, Sa-Ouk

2017-01-01

Lactobacillus plantarum and Leuconostoc mesenteroides play a prominent role as functional starters and predominant isolates in the production of various types of antimicrobial compound-containing fermented foods, especially including kimchi. In the case of the bioactive cyclic dipeptides, their racemic diastereomers inhibitory to bacteria and fungi have been suggested to come solely from Lactobacillus spp. of these strains. We previously demonstrated the antifungal and antiviral activities of proline-based cyclic dipeptides, which were fractionated from culture filtrates of Lb. plantarum LBP-K10 originated from kimchi. However, cyclic dipeptides have not been identified in the filtrates, either from cultures or fermented subject matter, driven by Ln. mesenteroides , which have been widely used as starter cultures for kimchi fermentation. Most importantly, the experimental verification of cyclic dipeptide-content changes during kimchi fermentation have also not been elucidated. Herein, the antibacterial fractions, including cyclo(Leu-Pro) and cyclo(Phe-Pro), from Ln. mesenteroides LBP-K06 culture filtrates, which exhibited a typical chromatographic retention behavior (t R ), were identified by using semi-preparative high-performance liquid chromatography and gas chromatography-mass spectrometry. Based on this finding, the proline-based cyclic dipeptides, including cyclo(Ser-Pro), cyclo(Tyr-Pro), and cyclo(Leu-Pro), were additionally identified in the filtrates only when fermenting Chinese cabbage produced with Ln. mesenteroides LBP-K06 starter cultures. The detection and isolation of cyclic dipeptides solely in controlled fermented cabbage were conducted under the control of fermentation-process parameters concomitantly with strong CDP selectivity by using a two-consecutive-purification strategy. Interestingly, cyclic dipeptides in the filtrates, when using this strain as a starter, increased with fermentation time. However, no cyclic dipeptides were observed in the filtrates of other fermented products, including other types of kimchi and fermented materials of plant and animal origin. This is the first report to conclusively demonstrate evidence for the existence of antimicrobial cyclic dipeptides produced by Ln. mesenteroides in kimchi. Through filtrates from lactic acid bacterial cultures and from fermented foods, we have also proved a method of combining chromatographic fractionation and mass spectrometry-based analysis for screening cyclic dipeptide profiling, which may allow evaluation of the fermented dairy foods from a new perspective.

Effect of 2',6'-dimethyl-L-tyrosine (Dmt) on pharmacological activity of cyclic endomorphin-2 and morphiceptin analogs.

PubMed

Fichna, Jakub; Perlikowska, Renata; Wyrębska, Anna; Gach, Katarzyna; Piekielna, Justyna; do-Rego, Jean Claude; Toth, Geza; Kluczyk, Alicja; Janecki, Tomasz; Janecka, Anna

2011-12-01

This study reports the synthesis and biological evaluation of a series of new side-chain-to-side-chain cyclized endomorphin-2 (EM-2) and morphiceptin analogs of a general structure Tyr-c(Xaa-Phe-Phe-Yaa)NH(2) or Tyr-c(Xaa-Phe-D-Pro-Yaa)NH(2), respectively, where Xaa and Yaa were L/D Asp or L/D Lys. Further modification of these analogs was achieved by introduction of 2',6'-dimethyl-L-tyrosine (Dmt) instead of Tyr in position 1. Peptides were synthesized by solid phase method and cleaved from the resin by a microwave-assisted procedure. Dmt(1)-substituted analogs displayed high affinity at the μ-opioid receptors, remained intact after incubation with the rat brain homogenate and showed remarkable, long-lasting μ-opioid receptor-mediated antinociceptive activity after central, but not peripheral administration. Our results demonstrate that cyclization is a promising strategy in the development of new opioid analgesics, but further modifications are necessary to enhance the blood-brain barrier permeability. Copyright © 2011. Published by Elsevier Ltd.

Synergistic algicidal effect and mechanism of two diketopiperazines produced by Chryseobacterium sp. strain GLY-1106 on the harmful bloom-forming Microcystis aeruginosa

NASA Astrophysics Data System (ADS)

Guo, Xingliang; Liu, Xianglong; Pan, Jianliang; Yang, Hong

2015-10-01

A potent algicidal bacterium isolated from Lake Taihu, Chryseobacterium sp. strain GLY-1106, produces two algicidal compounds: 1106-A (cyclo(4-OH-Pro-Leu)) and 1106-B (cyclo(Pro-Leu)). Both diketopiperazines showed strong algicidal activities against Microcystis aeruginosa, the dominant bloom-forming cyanobacterium in Lake Taihu. Interestingly, these two algicidal compounds functioned synergistically. Compared with individual treatment, combined treatment with cyclo(4-OH-Pro-Leu) and cyclo(Pro-Leu) significantly enhanced algicidal activity, accelerated the increase in intracellular reactive oxygen species (ROS) levels in M. aeruginosa, and further decreased the activities of antioxidases, effective quantum yield and maximal electron transport rate of M. aeruginosa. The results also showed that the algicidal characteristics of cyclo(4-OH-Pro-Leu) are distinct from those of cyclo(Pro-Leu). Cyclo(4-OH-Pro-Leu) mainly interrupted the flux of electron transport in the cyanobacterial photosynthetic system, whereas cyclo(Pro-Leu) mainly inhibited the activity of cyanobacterial intracellular antioxidases. A possible algicidal mechanism for the synergism between cyclo(4-OH-Pro-Leu) and cyclo(Pro-Leu) is proposed, which is in accordance with their distinct algicidal characteristics in individual and combined treatment. These findings suggest that synergism between algicidal compounds might be used as an effective strategy for the future control of Microcystis blooms.

Synergistic algicidal effect and mechanism of two diketopiperazines produced by Chryseobacterium sp. strain GLY-1106 on the harmful bloom-forming Microcystis aeruginosa

PubMed Central

Guo, Xingliang; Liu, Xianglong; Pan, Jianliang; Yang, Hong

2015-01-01

A potent algicidal bacterium isolated from Lake Taihu, Chryseobacterium sp. strain GLY-1106, produces two algicidal compounds: 1106-A (cyclo(4-OH-Pro-Leu)) and 1106-B (cyclo(Pro-Leu)). Both diketopiperazines showed strong algicidal activities against Microcystis aeruginosa, the dominant bloom-forming cyanobacterium in Lake Taihu. Interestingly, these two algicidal compounds functioned synergistically. Compared with individual treatment, combined treatment with cyclo(4-OH-Pro-Leu) and cyclo(Pro-Leu) significantly enhanced algicidal activity, accelerated the increase in intracellular reactive oxygen species (ROS) levels in M. aeruginosa, and further decreased the activities of antioxidases, effective quantum yield and maximal electron transport rate of M. aeruginosa. The results also showed that the algicidal characteristics of cyclo(4-OH-Pro-Leu) are distinct from those of cyclo(Pro-Leu). Cyclo(4-OH-Pro-Leu) mainly interrupted the flux of electron transport in the cyanobacterial photosynthetic system, whereas cyclo(Pro-Leu) mainly inhibited the activity of cyanobacterial intracellular antioxidases. A possible algicidal mechanism for the synergism between cyclo(4-OH-Pro-Leu) and cyclo(Pro-Leu) is proposed, which is in accordance with their distinct algicidal characteristics in individual and combined treatment. These findings suggest that synergism between algicidal compounds might be used as an effective strategy for the future control of Microcystis blooms. PMID:26423356

Synergistic algicidal effect and mechanism of two diketopiperazines produced by Chryseobacterium sp. strain GLY-1106 on the harmful bloom-forming Microcystis aeruginosa.

PubMed

Guo, Xingliang; Liu, Xianglong; Pan, Jianliang; Yang, Hong

2015-10-01

A potent algicidal bacterium isolated from Lake Taihu, Chryseobacterium sp. strain GLY-1106, produces two algicidal compounds: 1106-A (cyclo(4-OH-Pro-Leu)) and 1106-B (cyclo(Pro-Leu)). Both diketopiperazines showed strong algicidal activities against Microcystis aeruginosa, the dominant bloom-forming cyanobacterium in Lake Taihu. Interestingly, these two algicidal compounds functioned synergistically. Compared with individual treatment, combined treatment with cyclo(4-OH-Pro-Leu) and cyclo(Pro-Leu) significantly enhanced algicidal activity, accelerated the increase in intracellular reactive oxygen species (ROS) levels in M. aeruginosa, and further decreased the activities of antioxidases, effective quantum yield and maximal electron transport rate of M. aeruginosa. The results also showed that the algicidal characteristics of cyclo(4-OH-Pro-Leu) are distinct from those of cyclo(Pro-Leu). Cyclo(4-OH-Pro-Leu) mainly interrupted the flux of electron transport in the cyanobacterial photosynthetic system, whereas cyclo(Pro-Leu) mainly inhibited the activity of cyanobacterial intracellular antioxidases. A possible algicidal mechanism for the synergism between cyclo(4-OH-Pro-Leu) and cyclo(Pro-Leu) is proposed, which is in accordance with their distinct algicidal characteristics in individual and combined treatment. These findings suggest that synergism between algicidal compounds might be used as an effective strategy for the future control of Microcystis blooms.

The Macrocyclic Peptide Natural Product CJ-15,208 is Orally Active and Prevents Reinstatement of Extinguished Cocaine Seeking Behavior1

PubMed Central

Aldrich, Jane V.; Senadheera, Sanjeewa N.; Ross, Nicolette C.; Ganno, Michelle L.; Eans, Shainnel O.; McLaughlin, Jay P.

2013-01-01

The macrocyclic tetrapeptide natural product CJ-15,208 (cyclo[Phe-D-Pro-Phe-Trp]) exhibited both dose-dependent antinociception and kappa opioid receptor (KOR) antagonist activity after oral administration. CJ-15,208 antagonized a centrally administered KOR selective agonist, providing strong evidence it crosses the blood-brain barrier to reach KOR in the CNS. Orally administered CJ-15,208 also prevented both cocaine- and stress-induced reinstatement of extinguished cocaine seeking behavior in the conditioned place preference assay in a time- and dose-dependent manner. Thus, CJ-15,208 is a promising lead compound with a unique activity profile for potential development, particularly as a therapeutic to prevent relapse to drug seeking behavior in abstinent subjects. PMID:23327691

Metabolism of aspartame by human and pig intestinal microvillar peptidases.

PubMed Central

Hooper, N M; Hesp, R J; Tieku, S

1994-01-01

The artificial sweetener aspartame (N-L-alpha-aspartyl-L-phenyl-alanine-1-methyl ester; Nutrasweet), its decomposition product alpha Asp-Phe and the related peptide alpha Asp-PheNH2 were rapidly hydrolysed by microvillar membranes prepared from human duodenum, jejunum and ileum, and from pig duodenum and kidney. The metabolism of aspartame by the human and pig intestinal microvillar membrane preparations was inhibited significantly (> 78%) by amastatin or 1,10-phenanthroline, and partially (> 38%) by actinonin or bestatin, and was activated 2.9-4.5-fold by CaCl2. The inhibition by amastatin and 1,10-phenanthroline, and the activation by CaCl2 are characteristic of the cell-surface ectoenzyme aminopeptidase A (EC 3.4.11.7) and a purified preparation of this enzyme hydrolysed aspartame with a Km of 0.25 mM and a Vmax of 126 mumol/min per mg. A purified preparation of aminopeptidase W (EC 3.4.11.16) also hydrolysed aspartame but with a Km of 4.96 mM and a Vmax of 110 mumol/min per mg. However, rentiapril, an inhibitor of aminopeptidase W, caused only slight inhibition (maximally 19%) of the hydrolysis of aspartame by the microvillar membrane preparations. Similar patterns of inhibition and kinetic parameters were observed for alpha Asp-Phe and alpha Asp-PheNH2. Two other decomposition products of aspartame, beta Asp-PheMe and cyclo-Asp-Phe, were essentially resistant to hydrolysis by both the human and pig intestinal microvillar membrane preparations and the purified preparations of aminopeptidases A and W. Although the relatively selective inhibitor of aminopeptidase N (EC 3.4.11.2), actinonin, partially inhibited the metabolism of aspartame, alpha Asp-Phe and alpha Asp-PheNH2 by the human and pig intestinal microvillar membrane preparations, these peptides were not hydrolysed by a purified preparation of aminopeptidase N. Membrane dipeptidase (EC 3.4.13.19) only hydrolysed the unblocked dipeptide, alpha Asp-Phe, but the selective inhibitor of this enzyme, cilastatin, did not block the metabolism of alpha Asp-Phe by the microvillar membrane preparations. PMID:8141778

Preparation of imprinted cryogel cartridge for chiral separation of l-phenylalanine.

PubMed

Akgönüllü, Semra; Yavuz, Handan; Denizli, Adil

2017-06-01

l-Phe-imprinted cryogel cartridge was prepared for the chiral separation of l-Phe. N-Methacryloyl l-phenylalanine (MAPA) was used as a functional monomer for complexing with l-Phe. The selectivity of the membranes was investigated by using d-Phe, l-Trp, and d-Trp as competitor molecules. The PHEMAPA-l-Trp membranes were 6.4, 4.3, and 5.5 times more selective for l-Phe than d-Phe, l-Trp, and d-Trp, respectively. The PHEMAPA-l-Phe cryogel cartridge was incorporated into the fast protein liquid chromatography (FPLC) equipment and was able to separate D,l-Phe racemic mixture efficiently. The PHEMAPA-l-Phe membranes were shown to be reusable many times without significant loss of the adsorption capacity.

Inhibition of biofilm development and spoilage potential of Shewanella baltica by quorum sensing signal in cell-free supernatant from Pseudomonas fluorescens.

PubMed

Zhao, Aifei; Zhu, Junli; Ye, Xiaofeng; Ge, Yangyang; Li, Jianrong

2016-08-02

The objective of this study was to in vitro evaluate the effect of a cell-free supernatant (CFS) containing quorum sensing (QS) signal of Pseudomonas fluorescens on the growth, biofilm development and spoilage potential of Shewanella baltica, and preliminarily assess the interactive influences of various chemically synthesized autoinducers on spoilage phenotypes of S. baltica. PF01 strain isolated from spoiled Pseudosciaen crocea was identified P. fluorescens. The addition of 25% and 50% CFS to S. baltica culture had no effect on the growth rate during the lag and exponential phase, however, caused cell decline during the stationary phase. The presence of CFS from P. fluorescens significantly inhibited biofilm development, and greatly decreased the production of trimethylamine (TMA) and biogenic amino in S. baltica. Various signal molecules of QS in the CFS of P. fluorescens culture were detected, including seven N-acyl-l-homoserine lactones (AHLs), autoinducer-2 (AI-2) and two diketopiperazines (DKPs). Exogenous supplement of synthesized seven AHLs containing in the CFS decreased biofilm formation and TMA production in S. baltica, while exposure to exogenous cyclo-(l-Pro-l-Leu) was showed to promote spoilage potential, which revealed that S. baltica also sense the two QS molecules. Furthermore, the stimulating effect of cyclo-(l-Pro-l-Leu) was affected when AHL was simultaneously added, suggesting that the inhibitory activity of spoilage phenotypes in S. baltica might be attributed to a competitive effect of these QS compounds in the CFS of P. fluorescens. The present studies provide a good basis for future research on the role of QS in the regulation of spoilage microbial flora. Copyright © 2016 Elsevier B.V. All rights reserved.

Modification of ginseng flavors by bitter compounds found in chocolate and coffee.

PubMed

Sook Chung, Hee; Lee, Soo-Yeun

2012-06-01

Ginseng is not widely accepted by U.S. consumers due to its unfamiliar flavors, despite its numerous health benefits. Previous studies have suggested that the bitter compounds in chocolate and coffee may mask the off-flavors of ginseng. The objectives of this study were to: (1) profile sensory characteristics of ginseng extract solution, caffeine solution, cyclo (L-Pro-L-Val) solution, theobromine solution, and 2 model solutions simulating chocolate bitterness; and (2) determine the changes in the sensory characteristics of ginseng extract solution by the addition of the bitter compounds found in chocolate and coffee. Thirteen solutions were prepared in concentrations similar to the levels of the bitter compounds found in coffee and chocolate products. Twelve panelists participated in a descriptive analysis panel which included time-intensity ratings. Ginseng extract was characterized as sweeter, starchier, and more green tea than the other sample solutions. Those characteristics of ginseng extract were effectively modified by the addition of caffeine, cyclo (L-Pro-L-Val), and 2 model solutions. A model solution simulating dark chocolate bitterness was the least influenced in intensities of bitterness by the addition of ginseng extract. Results from time-intensity ratings show that the addition of ginseng extract increased duration time in certain bitterness of the 2 model solutions. Bitter compounds found in dark chocolate could be proposed to effectively mask the unique flavors of ginseng. Future studies blending aroma compounds of chocolate and coffee into such model solutions may be conducted to investigate the influence on the perception of the unique flavors through the congruent flavors. © 2012 Institute of Food Technologists®

Cyclo(His-Pro) promotes cytoprotection by activating Nrf2-mediated up-regulation of antioxidant defence

PubMed Central

Minelli, Alba; Conte, Carmela; Grottelli, Silvia; Bellezza, Maria; Cacciatore, Ivana; Bolaños, Juan P

2009-01-01

Hystidyl-proline [cyclo(His-Pro)] is an endogenous cyclic dipeptide produced by the cleavage of thyrotropin releasing hormone. Previous studies have shown that cyclo(His-Pro) protects against oxidative stress, although the underlying mechanism has remained elusive. Here, we addressed this issue and found that cyclo(His-Pro) triggered nuclear accumulation of NF-E2-related factor-2 (Nrf2), a transcription factor that up-regulates antioxidant-/electrophile-responsive element (ARE-EpRE)-related genes, in PC12 cells. Cyclo(His-Pro) attenuated reactive oxygen species production, and prevented glutathione depletion caused by glutamate, rotenone, paraquat and β-amyloid treatment. Moreover, real-time PCR analyses revealed that cyclo(His-Pro) induced the expression of a number of ARE-related genes and protected cells against hydrogen peroxide-mediated apoptotic death. Furthermore, these effects were abolished by RNA interference-mediated Nrf2 knockdown. Finally, pharmacological inhibition of p-38 MAPK partially prevented both cyclo(His-Pro)-mediated Nrf2 activation and cellular protection. These results suggest that the signalling mechanism responsible for the cytoprotective actions of cyclo(His-Pro) would involve p-38 MAPK activation leading to Nrf2-mediated up-regulation of antioxidant cellular defence. PMID:18373731

Terahertz spectra of L-phenylalanine and its monohydrate

NASA Astrophysics Data System (ADS)

Pan, Tingting; Li, Shaoping; Zou, Tao; Yu, Zheng; Zhang, Bo; Wang, Chenyang; Zhang, Jianbing; He, Mingxia; Zhao, Hongwei

2017-05-01

The low-frequency vibrational property of L-phenylalanine (L-Phe) and L-phenylalanine monohydrate (L-Phe·H2O) has been investigated by terahertz time-domain spectroscopy (THz-TDS) at room and low temperature ranging from 0.5 to 4.5 THz. Distinctive THz absorption spectra of the two compounds were observed. Density functional theory (DFT) calculations based on the crystal structures have been performed to simulate the vibrational modes of L-Phe and L-Phe·H2O and the results agree well with the experimental observations. The study indicates that the characterized features of L-Phe mainly originate from the collective vibration of molecules. And the characterized features of L-Phe·H2O mainly come from hydrogen bond interactions between L-Phe and water molecules. L-Phe and L-Phe·H2O were also verified by differential scanning calorimetry and thermogravimetry (DSC-TG) and powder X-ray diffraction (PXRD) examinations.

[Effect of the novel dipeptide nootropic agent noopept and its metabolite cyclo-L-prolylglycine on the transcallosal evoked potential in the rat brain].

PubMed

Molodavkin, G M; Borlikova, G G; Voronina, T A; Gudasheva, T A; Ostrovskaia, R U; Tushmalova, N A; Seredenin, S B

2002-01-01

The effect of new nootropic dipeptides--noopept (N-phenylacetyl-L-prolylglycine, GVS-111) and its metabolite (cyclo-L-prolylglycine)--and a standard nootrope piracetam on the transcallosal evoked potential (TEP) in rat brain was studied. In the dose range from 150 to 300 mg/kg, piracetam increased the TEP amplitude, which exhibited a maximum after 1.5-2 h and then gradually decreased. Both noopept and cyclo-L-prolylglycine also increased the TEP amplitude, which attained a plateau and retained this level over the entire observation time (above 3.5 h). All the nootropes studied increased both components of the evoked potential. Piracetam and cyclo-L-prolylglycine led to an approximately equal increase in both waves, while noopept induced a somewhat greater increase in the negative TEP wave amplitude. It is suggested that the positive effect of noopept and cyclo-L-prolylglycine upon the interhemispheric signal transfer (indicated by the improved transcallosal response) can be considered as a potential neurophysiological basis for a positive drug influence on the behavioral level.

Eco-Friendly Insecticide Discovery via Peptidomimetics: Design, Synthesis, and Aphicidal Activity of Novel Insect Kinin Analogues.

PubMed

Zhang, Chuanliang; Qu, Yanyan; Wu, Xiaoqing; Song, Dunlun; Ling, Yun; Yang, Xinling

2015-05-13

Insect kinin neuropeptides are pleiotropic peptides that are involved in the regulation of hindgut contraction, diuresis, and digestive enzyme release. They share a common C-terminal pentapeptide sequence of Phe(1)-Xaa(2)-Yaa(3)-Trp(4)-Gly(5)-NH2 (where Xaa(2) = His, Asn, Phe, Ser, or Tyr; Yaa(3) = Pro, Ser, or Ala). Recently, the aphicidal activity of insect kinin analogues has attracted the attention of researchers. Our previous work demonstrated that the sequence-simplified insect kinin pentapeptide analogue Phe-Phe-[Aib]-Trp-Gly-NH2 could retain good aphicidal activity and be the lead compound for the further discovery of eco-friendly insecticides which encompassed a broad array of biochemicals derived from micro-organisms and other natural sources. Using the peptidomimetics strategy, we chose Phe-Phe-[Aib]-Trp-Gly-NH2 as the lead compound, and we designed and synthesized three series, including 31 novel insect kinin analogues. The aphicidal activity of the new analogues against soybean aphid was determined. The results showed that all of the analogues exhibited aphicidal activity. Of particular interest was the analogue II-1, which exhibited improved aphicidal activity with an LC50 of 0.019 mmol/L compared with the lead compound (LC50 = 0.045 mmol/L) or the commercial insecticide pymetrozine (LC50 = 0.034 mmol/L). This suggests that the analogue II-1 could be used as a new lead for the discovery of potential eco-friendly insecticides.

Subsite specificity of trypanosomal cathepsin L-like cysteine proteases. Probing the S2 pocket with phenylalanine-derived amino acids.

PubMed

Lecaille, F; Authié, E; Moreau, T; Serveau, C; Gauthier, F; Lalmanach, G

2001-05-01

The S2 subsite of mammalian cysteine proteinases of the papain family is essential for specificity. Among natural amino acids, all these enzymes prefer bulky hydrophobic residues such as phenylalanine at P2. This holds true for their trypanosomal counterparts: cruzain from Trypanosoma cruzi and congopain from T. congolense. A detailed analysis of the S2 specificity of parasitic proteases was performed to gain information that might be of interest for the design of more selective pseudopeptidyl inhibitors. Nonproteogenic phenylalanyl analogs (Xaa) have been introduced into position P2 of fluorogenic substrates dansyl-Xaa-Arg-Ala-Pro-Trp, and their kinetic constants (Km, kcat/Km) have been determined with congopain and cruzain, and related host cathepsins B and L. Trypanosomal cysteine proteases are poorly stereoselective towards D/L-Phe, the inversion of chirality modifying the efficiency of the reaction but not the Km. Congopain binds cyclohexylalanine better than aromatic Phe derivatives. Another characteristic feature of congopain compared to cruzain and cathepsins B and L was that it could accomodate a phenylglycyl residue (kcat/Km = 1300 mM-1.s-1), while lengthening of the side chain by a methylene group only slightly impaired the specificity constant towards trypanosomal cysteine proteases. Mono- and di-halogenation or nitration of Phe did not affect Km for cathepsin L-like enzymes, but the presence of constrained Phe derivatives prevented a correct fitting into the S2 subsite. A model of congopain has been built to study the fit of Phe analogs within the S2 pocket. Phe analogs adopted a positioning within the S2 pocket similar to that of the Tyr of the cruzain/Z-Tyr-Ala-fluoromethylketone complex. However, cyclohexylalanine has an energetically favorable chair-like conformation and can penetrate deeper into the subsite. Fitting of modeled Phe analogs were in good agreement with kinetic parameters. Furthermore, a linear relationship could be established with logP, supporting the suggestion that fitting into the S2 pocket of trypanosomal cysteine proteases depends on the hydrophobicity of Phe analogs.

Terahertz spectra of l-phenylalanine and its monohydrate.

PubMed

Pan, Tingting; Li, Shaoping; Zou, Tao; Yu, Zheng; Zhang, Bo; Wang, Chenyang; Zhang, Jianbing; He, Mingxia; Zhao, Hongwei

2017-05-05

The low-frequency vibrational property of l-phenylalanine (l-Phe) and l-phenylalanine monohydrate (l-Phe·H 2 O) has been investigated by terahertz time-domain spectroscopy (THz-TDS) at room and low temperature ranging from 0.5 to 4.5THz. Distinctive THz absorption spectra of the two compounds were observed. Density functional theory (DFT) calculations based on the crystal structures have been performed to simulate the vibrational modes of l-Phe and l-Phe·H 2 O and the results agree well with the experimental observations. The study indicates that the characterized features of l-Phe mainly originate from the collective vibration of molecules. And the characterized features of l-Phe·H 2 O mainly come from hydrogen bond interactions between l-Phe and water molecules. l-Phe and l-Phe·H 2 O were also verified by differential scanning calorimetry and thermogravimetry (DSC-TG) and powder X-ray diffraction (PXRD) examinations. Copyright © 2017. Published by Elsevier B.V.

Taste, umami-enhance effect and amino acid sequence of peptides separated from silkworm pupa hydrolysate.

PubMed

Yu, Zilin; Jiang, Hongrui; Guo, Rongcan; Yang, Bo; You, Gang; Zhao, Mouming; Liu, Xiaoling

2018-06-01

Four umami peptides were separated and purified by ultrafiltration, gel filtration chromatography and identified by ultra-performance liquid chromatography tandem mass-spectrometry (UPLC-MS/MS), the amino acid sequences of four peptides are Val-Pro-Tyr (VPY), Thr-Ala-Tyr (TAY), Ala-Ala-Pro-Tyr (AAPY) and Gly-Phe-Pro (GFP). The result illustrates that the umami amino acids are not the content of umami peptides, but bitter amino acids are included. The threshold of VPY, TAY, AAPY and GFP were 1.65 mmol/L, 1.76 mmol/L, 2.97 mmol/L and 6.26 mmol/L, respectively. The peptide TAY, VPY and AAPY had an umami-enhancement effect on the monosodium glutamate (MSG) + sodium chloride (NaCl) solution, their concentrations were 2.5 g/L, 5 g/L and 5 g/L, respectively, while GFP has no significant umami-enhancement effect in solution. In addition, the peptides have better taste than its composing amino acids, which indicates that the taste of peptide does not depend on its composing amino acids. Copyright © 2018. Published by Elsevier Ltd.

Arg-Phe-Phe D-Amino Acid Stereochemistry Scan in the Macrocyclic Agouti-Related Protein Antagonist Scaffold c[Pro-Arg-Phe-Phe-Xaa-Ala-Phe-DPro] Results in Unanticipated Melanocortin-1 Receptor Agonist Profiles.

PubMed

Ericson, Mark D; Koerperich, Zoe M; Freeman, Katie T; Fleming, Katlyn A; Haskell-Luevano, Carrie

2018-06-20

The melanocortin-3 and melanocortin-4 receptors (MC3R and MC4R), endogenous agonists derived from the proopiomelanocortin gene transcript, and naturally-occurring antagonists agouti and agouti-related protein (AGRP) have been linked to biological pathways associated with energy homeostasis. The active tripeptide sequence of AGRP, Arg111-Phe112-Phe113, is located on a hypothesized β-hairpin loop. Herein, stereochemical modifications of the Arg-Phe-Phe sequence were examined in the octapeptide AGRP-derived macrocyclic scaffold c[Pro-Arg-Phe-Phe-Xxx-Ala-Phe-DPro], where Xxx was Asn or diaminopropionic acid (Dap). Macrocyclic peptides were synthesized with one, two, or three residues of the Arg-Phe-Phe sequence substituted with the corresponding D-isomer(s), generating a 14 compound library. While L-to-D inversions of the Arg-Phe-Phe sequence in a 20-residue AGRP-derived ligand previously resulted in agonist activity at the MC1R, MC3R, MC4R, and MC5R, only the MC1R was consistently stimulated by the macrocyclic ligands in the present study, with varying ligand potencies and efficacies observed at the MC1R. A general trend of increased MC4R antagonist potency was observed for Dap-containing compounds, while MC5R inverse agonist activity was observed for select ligands. It was observed that stereochemical modification of the Arg-Phe-Phe active tripeptide sequence was insufficient to convert melanocortin antagonist into agonists. Overall, these observations are important in the design of melanocortin ligands possessing potent and selective agonist and antagonist activities.

Computational investigation of the conformational profile of the four stereomers of Ac-L-Pro-c3Phe-NHMe (c3Phe= 2,3-methanophenylalanine).

PubMed

Rodriguez, Alejandro; Canto, Josep; Corcho, Francesc J; Perez, Juan J

2009-01-01

The present report regards a computational study aimed at assessing the conformational profile of the four stereoisomers of the peptide Ace-Pro-c3Phe-NMe, previously reported to exhibit beta-turn structures in dichloromethane with different type I/type II beta-turn profiles. Molecular systems were represented at the molecular mechanics level using the parm96 parameterization of the AMBER force field. Calculations were carried out in dichloromethane using an implicit solvent approach. Characterization of the conformational features of the peptide analogs was carried out using simulated annealing (SA), molecular dynamics (MD) and replica exchange molecular dynamics (REMD). Present results show that MD calculations do not provide a reasonable sampling after 300 ns. In contrast, both SA and REMD provide similar results and agree well with experimental observations. Copyright 2009 Wiley Periodicals, Inc.

Detection of hydroxyl radicals by D-phenylalanine hydroxylation: a specific assay for hydroxyl radical generation in biological systems.

PubMed

Biondi, R; Xia, Y; Rossi, R; Paolocci, N; Ambrosio, G; Zweier, J L

2001-03-01

Hydroxylation of l-phenylalanine (Phe) by hydroxyl radical (*OH) yields 4-, 3-, and 2-hydroxyl-Phe (para-, meta-, and ortho-tyrosine, respectively). Phe derivative measurements have been employed to detect *OH formation in cells and tissues, however, the specificity of this assay is limited since Phe derivatives also arise from intracellular Phe hydroxylase. d-Phe, the d-type enantiomer, is not hydroxylated by Phe hydroxylase. We evaluate whether d-Phe reacts with *OH as well as l-Phe, providing a more reliable probe for *OH generation in biological systems. With *OH generated by a Fenton reaction or xanthine oxidase, d- and l-Phe equally gave rise to p, m, o-tyr and this could be prevented by *OH scavengers. Resting human neutrophils (PMNs) markedly converted l-Phe to p-tyr, through non-oxidant-mediated reactions, whereas d-Phe was unaffected. In contrast, when PMNs were stimulated in the presence of redox cycling iron the *OH formed resulted in more significant rise of p-tyr from d-Phe (9.4-fold) than l-Phe (3.6-fold) due to the significant background formation of p-tyr from l-Phe. Together, these data indicated that d- and l-Phe were equally hydroxylated by *OH. Using d-Phe instead of l-Phe can eliminate the formation of Phe derivatives from Phe hydroxylase and achieve more specific, sensitive measurement of *OH in biological systems.

Rational engineering of multiple module pathways for the production of L-phenylalanine in Corynebacterium glutamicum.

PubMed

Zhang, Chuanzhi; Zhang, Junli; Kang, Zhen; Du, Guocheng; Chen, Jian

2015-05-01

Microbial production of L-phenylalanine (L-Phe) from renewable sources has attracted much attention recently. In the present study, Corynebacterium glutamicum 13032 was rationally engineered to produce L-Phe from inexpensive glucose. First, all the L-Phe biosynthesis pathway genes were investigated and the results demonstrated that in addition to AroF and PheA, the native PpsA, TktA, AroE and AroA, and the heterologous AroL and TyrB were also the key enzymes for L-Phe biosynthesis. Through combinational expression of these key enzymes, the L-Phe production was increased to 6.33 ± 0.13 g l(-1) which was about 1.48-fold of that of the parent strain C. glutamicum (pXM-pheA (fbr)-aroF (fbr)) (fbr, feedback-inhibition resistance). Furthermore, the production of L-Phe was improved to 9.14 ± 0.21 g l(-1) by modifying the glucose and L-Phe transport systems and blocking the acetate and lactate biosynthesis pathways. Eventually, the titer of L-Phe was enhanced to 15.76 ± 0.23 g l(-1) with a fed-batch fermentation strategy. To the best of our knowledge, this was the highest value reported in rationally engineered C. glutamicum 13032 strains. The results obtained will also contribute to rational engineering of C. glutamicum for production of other valuable aromatic compounds.

Reversible inhibition of cathepsin L-like proteases by 4-mer pseudopeptides.

PubMed

Lecaille, F; Cotton, J; McKerrow, J H; Ferrer-Di Martino, M; Boll-Bataillé, E; Gauthier, F; Lalmanach, G

2001-11-02

A library of 121 pseudopeptides was designed to develop reversible inhibitors of trypanosomal enzymes (cruzain from Trypanosoma cruzi and congopain from Trypanosoma congolense). The peptides share the framework: Cha-X1-X2-Pro (Cha=cyclohexyl-alanine, X1 and X2 were phenylalanyl analogs), based on a previous report [Lecaille, F., Authié, E., Moreau, T., Serveau, C., Gauthier, F. and Lalmanach, G. (2001) Eur. J. Biochem. 268, 2733-2741]. Five peptides containing a nitro-substituted aromatic residue (Tyr/Phe) and one a 4-chloro-phenylalanine at the X1 position, and 3-(2-naphthyl)-alanine, homocyclohexylalanine or 3-nitro-tyrosine (3-NO(2)-Tyr) at the X2 position, were selected. They inhibited congopain more effectively than cruzain, except Cha-4-NO(2)-Phe-3-NO(2)-Tyr-Pro which bound the two parasitic enzymes similarly. Among this series, Cha-3-NO(2)-Tyr-HoCha-Pro and Cha-4-NO(2)-Phe-3-NO(2)-Tyr-Pro are the most selective for congopain relative to host cathepsins. No hydrolysis occurred upon prolonged incubation time with purified enzymes. In addition introduction of non-proteogenic residues in the peptidyl backbone greatly enhanced resistance to proteolysis by mammalian sera.

Cyclopropyl glycine and proline-containing preparation noopept evoke two types of membrane potential responses in synaptoneurosomes.

PubMed

Lutsenko, V K; Vukolova, M N; Gudasheva, T A

2003-06-01

Proline, cyclo(Pro-Gly), and acyl-prolyl-containing dipeptide GVS-111 decreased synaptoneurosome membrane potential in a Ca2+-free medium. The efficiency of these preparations decreased in the following order: GVS>cyclo(Pro-Gly)>proline. Depolarization responses induced by endogenous nootropic agent cyclo(Pro-Gly) was dose-dependent and saturable; the threshold concentration of cyclo(Pro-Gly) was 10(-9) M. In a Ca2+-containing medium GVS and cyclo(Pro-Gly) induced both hyperpolarizing and depolarizing membrane responses of synaptoneurosomes. Possible mechanisms underlying changes in the membrane potential of synaptoneurosomes induced by nootropic agents are discussed. It was interesting whether modulation of electrogenesis can improve memory and potentiate the neuroprotective effect of the test nootropic agents.

Maculosin, a host-specific phytotoxin for spotted knapweed from Alternaria alternata

PubMed Central

Stierle, Andrea C.; Cardellina, John H.; Strobel, Gary A.

1988-01-01

Several diketopiperazines have been isolated from liquid cultures of Alternaria alternata, the causal agent of black leaf blight of spotted knapweed, Centaurea maculosa Lam. One of these compounds, maculosin [the diketopiperazine cyclo(-L-Pro-L-Tyr-)], was active in the nicked-leaf bioassay at 10-5 M; synthetic maculosin possessed chemical and biological activities identical to those of the natural product. Other diketopiperazines isolated from the fungus possessed either less activity or none at all. In tests against 19 plant species, maculosin was phytotoxic only to spotted knapweed. Thus maculosin is a host-specific phytotoxin from a weed pathogen. PMID:16593989

Computational and experimental investigations into the conformations of cyclic tetra-α/β-peptides.

PubMed

Oakley, Mark T; Oheix, Emmanuel; Peacock, Anna F A; Johnston, Roy L

2013-07-11

We present a combined computational and experimental study of the energy landscapes of cyclic tetra-α/β-peptides. We have performed discrete path sampling calculations on a series of cyclic tetra-α/β-peptides to obtain the relative free energies and barriers to interconversion of their conformers. The most stable conformers of cyclo-[(β-Ala-Gly)2] contain all-trans peptide groups. The relative energies of the cis isomers and the cis-trans barriers are lower than in acyclic peptides but not as low as in the highly strained cyclic α-peptides. For cyclic tetra-α/β-peptides containing a single proline residue, of the type cyclo-[β-Ala-Xaa-β-Ala-Pro], the energy landscapes show that the most stable isomers containing cis and trans β-Ala-Pro have similar free energies and are separated by barriers of approximately 15 kcal mol(-1). We show that the underlying energy landscapes of cyclo-[β-Ala-Lys-β-Ala-Pro] and cyclo-[β-Ala-Ala-β-Ala-Pro] are similar, allowing the substitution of the flexible side chain of Lys with Ala to reduce the computational demand of our calculations. However, the steric bulk of the Val side chain in cyclo-[β-Ala-Val-β-Ala-Pro] affects the conformations of the ring, leading to significant differences between its energy landscape and that of cyclo-[β-Ala-Ala-β-Ala-Pro]. We have synthesized the cyclic peptide cyclo-[β-Ala-Lys-β-Ala-Pro], and NMR spectroscopy shows the presence of conformers that interconvert slowly on the NMR time scale at temperatures up to 80 °C. Calculated circular dichroism (CD) spectra for the proposed major isomer of cyclo-[β-Ala-Ala-β-Ala-Pro] are in good agreement with the experimental spectra of cyclo-[β-Ala-Lys-β-Ala-Pro], suggesting that the Ala cyclic tetrapeptide is a viable model for the Lys analogue.

Inhibition of Sendai virus fusion with phospholipid vesicles and human erythrocyte membranes by hydrophobic peptides

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

Kelsey, D.R.; Flanagan, T.D.; Young, J.E.

1991-06-01

Hydrophobic di- and tripeptides which are capable of inhibiting enveloped virus infection of cells are also capable of inhibiting at least three different types of membrane fusion events. Large unilamellar vesicles (LUV) of N-methyl dioleoylphosphatidylethanolamine (N-methyl DOPE), containing encapsulated 1-aminonaphthalene-3,6,8-trisulfonic acid (ANTS) and/or p-xylene bis(pyridinium bromide) (DPX), were formed by extrusion. Vesicle fusion and leakage were then monitored with the ANTS/DPX fluorescence assay. Sendai virus fusion with lipid vesicles and Sendai virus fusion with human erythrocyte membranes were measured by following the relief of fluorescence quenching of virus labeled with octadecylrhodamine B chloride (R18). This study found that the effectivenessmore » of the peptides carbobenzoxy-L-Phe-L-Phe (Z-L-Phe-L-Phe), Z-L-Phe, Z-D-Phe, and Z-Gly-L-Phe-L-Phe in inhibiting N-methyl DOPE LUV fusion or fusion of virus with N-methyl DOPE LUV also paralleled their reported ability to block viral infectivity. Furthermore, Z-D-Phe-L-PheGly and Z-Gly-L-Phe inhibited Sendai virus fusion with human erythrocyte membranes with the same relative potency with which they inhibited vesicle-vesicle and virus-vesicle fusion. The evidence suggests a mechanism by which these peptides exert their inhibition of plaque formation by enveloped viruses. This class of inhibitors apparently acts by inhibiting fusion of the viral envelope with the target cell membrane, thereby preventing viral infection. The physical pathway by which these peptides inhibit membrane fusion was investigated. {sup 31}P nuclear magnetic resonance (NMR) of proposed intermediates in the pathway for membrane fusion in LUV revealed that the potent fusion inhibitor Z-D-Phe-L-PheGly selectively altered the structure (or dynamics) of the hypothesized fusion intermediates and that the poor inhibitor Z-Gly-L-Phe did not.« less

The immunologic and antioxidant effects of L-phenylalanine on the uterine implantation of mice embryos during early pregnancy.

PubMed

Dong, Yulan; Bai, Yongping; Liu, Guanhui; Wang, Zixu; Cao, Jing; Chen, Yaoxing; Yang, Hongliang

2014-10-01

L-phenylalanine (L-PHE) is a synthetic precursor of catecholamines. Because it cannot be synthesised by an organism, it must be absorbed from the environment. Despite the wide use of L-PHE, whether L-PHE has a negative impact on embryo implantation and development is poorly understood. This study attempted to determine the roles of L-PHE in embryo implantation and development and in the immune response and antioxidant status of the uterus in early pregnancy mice injected intraperitoneally with 320 mg/kg L-PHE. The embryo number of treated mice decreased by 57.6%, and the size of their embryos was reduced by 2.8% (P⟩0.05) along the long diameter and 11.9% (P⟨0.05) along the short diameter at E9 compared with control mice. In addition, L-PHE significantly suppressed B lymphocyte proliferation. L-PHE increased IL-2 secretion but decreased the IL-4 concentration, thereby up-regulating the ratio of IL-2/IL-4 to 1.37-8.45. An analysis of the oxidant and antioxidant status showed that, compared with the control mice, the level of superoxide dismutase activity decreased by 21.54-39.94% and the glutathione peroxidase activity decreased by 15.27-18.96% among the L-PHE-treated mice at E1-E9. However, the malonaldehyde content increased by 14.29%-90.11% among the L-PHE-treated mice. Therefore, L-PHE impaired embryo implantation by disrupting cytokine-based immunity and oxidative stress in the uterus.

Reduction of L-phenylalanine in protein hydrolysates using L-phenylalanine ammonia-lyase from Rhodosporidium toruloides.

PubMed

Castañeda, María Teresita; Adachi, Osao; Hours, Roque Alberto

2015-10-01

L-Phenylalanine ammonia-lyase (PAL, EC 4.3.1.25) from Rhodosporidium toruloides was utilized to remove L-phenylalanine (L-Phe) from different commercial protein hydrolysates. A casein acid hydrolysate (CAH, L-Phe ~2.28 %) was employed as a model substrate. t-Cinnamic acid resulting from deamination of L-Phe was extracted, analyzed at λ = 290 nm, and used for PAL activity determination. Optimum reaction conditions, optimized using successive Doehlert design, were 35 mg mL(-1) of CAH and 800 mU mL(-1) of PAL, while temperature and pH were 42 °C and 8.7, respectively. Reaction kinetics of PAL with CAH was determined under optimized conditions. Then, removal of L-Phe from CAH was tested. Results showed that more than 92 % of initial L-Phe was eliminated. Similar results were obtained with other protein hydrolysates. These findings demonstrate that PAL is a useful biocatalyst for L-Phe removal from protein hydrolysates, which can be evaluated as potential ingredients in foodstuffs for PKU patients.

Gas chromatographic determination and mechanism of formation of D-amino acids occurring in fermented and roasted cocoa beans, cocoa powder, chocolate and cocoa shell.

PubMed

Pätzold, R; Brückner, H

2006-07-01

Fermented cocoa beans of various countries of origin (Ivory Coast, Ghana, Sulawesi), cocoa beans roasted under defined conditions (100-150 degrees C; 30-120 min), low and high fat cocoa powder, various brands of chocolate, and cocoa shells were analyzed for their contents of free L-and D-amino acids. Amino acids were isolated from defatted products using a cation exchanger and converted into volatile N(O)-pentafluoropropionyl amino acid 2-propyl esters which were analyzed by enantioselective gas chromatography mass spectrometry on a Chirasil-L-Val capillary column. Besides common protein L-amino acids low amounts of D-amino acids were detected in fermented cocoa beans. Quantities of D-amino acids increased on heating. On roasting cocoa beans of the Forastero type from the Ivory Coast at 150 degrees C for 2 h, relative quantities of D-amino acids approached 17.0% D-Ala, 11.7% D-Ile, 11.1% D-Asx (Asp + Asn), 7.9% D-Tyr, 5.8% D-Ser, 4.8% D-Leu, 4.3% D-Phe, 37.0% D-Pro, and 1.2% D-Val. In cocoa powder and chocolate relative quantities amounted to 14.5% D-Ala, 10.6% D-Tyr, 9.8% D-Phe, 8.1% L-Asx, and 7.2% D-Ile. Lower quantities of other D-amino acids were also detected. In order to corroborate our hypothesis that D-amino acids are generated from Amadori compounds (fructose amino acids) formed in the course of the Maillard reaction, fructose-L-phenylalanine and fructose-D-phenylalanine were synthesized and heated at 200 degrees C for 5-60 min. Already after 5 min release of 11.7% D-Phe and 11.8% L-Phe in the free form could be analyzed. Based on the data a racemization mechanism is presented founded on the intermediate and reversible formation of an amino acid carbanion in the Amadori compounds.

l-phenylalanine modulates gut hormone release and glucose tolerance, and suppresses food intake through the calcium-sensing receptor in rodents.

PubMed

Alamshah, A; Spreckley, E; Norton, M; Kinsey-Jones, J S; Amin, A; Ramgulam, A; Cao, Y; Johnson, R; Saleh, K; Akalestou, E; Malik, Z; Gonzalez-Abuin, N; Jomard, A; Amarsi, R; Moolla, A; Sargent, P R; Gray, G W; Bloom, S R; Murphy, K G

2017-11-01

High-protein diets (HPDs) are associated with greater satiety and weight loss than diets rich in other macronutrients. The exact mechanisms by which HPDs exert their effects are unclear. However, evidence suggests that the sensing of amino acids produced as a result of protein digestion may have a role in appetite regulation and satiety. We investigated the effects of l-phenylalanine (L-Phe) on food intake and glucose homeostasis in rodents. We investigated the effects of the aromatic amino-acid and calcium-sensing receptor (CaSR) agonist l-phenylalanine (L-Phe) on food intake and the release of the gastrointestinal (GI) hormones peptide YY (PYY), glucagon-like peptide-1 (GLP-1) and ghrelin in rodents, and the role of the CaSR in mediating these effects in vitro and in vivo. We also examined the effect of oral l-Phe administration on glucose tolerance in rats. Oral administration of l-Phe acutely reduced food intake in rats and mice, and chronically reduced food intake and body weight in diet-induced obese mice. Ileal l-Phe also reduced food intake in rats. l-Phe stimulated GLP-1 and PYY release, and reduced plasma ghrelin, and also stimulated insulin release and improved glucose tolerance in rats. Pharmacological blockade of the CaSR attenuated the anorectic effect of intra-ileal l-Phe in rats, and l-Phe-induced GLP-1 release from STC-1 and primary L cells was attenuated by CaSR blockade. l-Phe reduced food intake, stimulated GLP-1 and PYY release, and reduced plasma ghrelin in rodents. Our data provide evidence that the anorectic effects of l-Phe are mediated via the CaSR, and suggest that l-Phe and the CaSR system in the GI tract may have therapeutic utility in the treatment of obesity and diabetes. Further work is required to determine the physiological role of the CaSR in protein sensing in the gut, and the role of this system in humans.

Two groups of phenylalanine biosynthetic operon leader peptides genes: a high level of apparently incidental frameshifting in decoding Escherichia coli pheL

PubMed Central

Gurvich, Olga L.; Näsvall, S. Joakim; Baranov, Pavel V.; Björk, Glenn R.; Atkins, John F.

2011-01-01

The bacterial pheL gene encodes the leader peptide for the phenylalanine biosynthetic operon. Translation of pheL mRNA controls transcription attenuation and, consequently, expression of the downstream pheA gene. Fifty-three unique pheL genes have been identified in sequenced genomes of the gamma subdivision. There are two groups of pheL genes, both of which are short and contain a run(s) of phenylalanine codons at an internal position. One group is somewhat diverse and features different termination and 5′-flanking codons. The other group, mostly restricted to Enterobacteria and including Escherichia coli pheL, has a conserved nucleotide sequence that ends with UUC_CCC_UGA. When these three codons in E. coli pheL mRNA are in the ribosomal E-, P- and A-sites, there is an unusually high level, 15%, of +1 ribosomal frameshifting due to features of the nascent peptide sequence that include the penultimate phenylalanine. This level increases to 60% with a natural, heterologous, nascent peptide stimulator. Nevertheless, studies with different tRNAPro mutants in Salmonella enterica suggest that frameshifting at the end of pheL does not influence expression of the downstream pheA. This finding of incidental, rather than utilized, frameshifting is cautionary for other studies of programmed frameshifting. PMID:21177642

Design of a recombinant Escherichia coli for producing L-phenylalanine from glycerol.

PubMed

Thongchuang, Mayura; Pongsawasdi, Piamsook; Chisti, Yusuf; Packdibamrung, Kanoktip

2012-10-01

A recombinant Escherichia coli was engineered to produce the commercially important amino acid L-phenylalanine (L-Phe) using glycerol as the carbon source. Compared to the conventionally used glucose and sucrose, glycerol is a less expensive carbon source. As phenylalanine dehydrogenase (PheDH) activity is involved in the last step of L-Phe synthesis in E. coli, a phenylalanine dehydrogenase gene (phedh) from the thermotolerant Bacillus lentus was cloned into pRSFDuet-1 (pPheDH) and expressed in E. coli BL21(DE3). The resulting clone had a limited ability to produce L-Phe from glycerol, possibly because of a poor glycerol uptake by the cell, or an inability to excrete L-Phe, or both. Therefore, yddG gene encoding an aromatic amino acid exporter and glpF gene encoding a glycerol transport facilitator were coexpressed with the phedh in a reengineered E. coli. In a glycerol medium, the maximum L-Phe production rates of the clones pPY (phedh and yddG genes) and pPYF (phedh, yddG and glpF genes) were 1.4- and 1.8-fold higher than the maximum production rate of the pPheDH clone. The better producing pPYF clone was further evaluated in a 5 l stirred-tank fermenter (37 °C, an aeration rate of 1 vvm, an agitation speed of 400 rpm). In the fermenter, the maximum concentration of L-Phe (366 mg/l) was achieved in a much shorter period compared to in the shake flasks. In the latter, the highest titer of L-Phe was only 76 % of the maximum value attained in the fermenter.

Genetic engineering of Escherichia coli to improve L-phenylalanine production.

PubMed

Liu, Yongfei; Xu, Yiran; Ding, Dongqin; Wen, Jianping; Zhu, Beiwei; Zhang, Dawei

2018-01-30

L-phenylalanine (L-Phe) is an essential amino acid for mammals and applications expand into human health and nutritional products. In this study, a system level engineering was conducted to enhance L-Phe biosynthesis in Escherichia coli. We inactivated the PTS system and recruited glucose uptake via combinatorial modulation of galP and glk to increase PEP supply in the Xllp01 strain. In addition, the HTH domain of the transcription factor TyrR was engineered to decrease the repression on the transcriptional levels of L-Phe pathway enzymes. Finally, proteomics analysis demonstrated the third step of the SHIK pathway (catalyzed via AroD) as the rate-limiting step for L-Phe production. After optimization of the aroD promoter strength, the titer of L-Phe increased by 13.3%. Analysis of the transcriptional level of genes involved in the central metabolic pathways and L-Phe biosynthesis via RT-PCR showed that the recombinant L-Phe producer exhibited a great capability in the glucose utilization and precursor (PEP and E4P) generation. Via systems level engineering, the L-Phe titer of Xllp21 strain reached 72.9 g/L in a 5 L fermenter under the non-optimized fermentation conditions, which was 1.62-times that of the original strain Xllp01. The metabolic engineering strategy reported here can be broadly employed for developing genetically defined organisms for the efficient production of other aromatic amino acids and derived compounds.

Toxicity of a PAH photooxidation product to the bacteria Photobacterium phosphoreum and the duckweed Lemna gibba: Effects of phenanthrene and its primary photoproduct, phenanthrenequinone

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

McConkey, B.J.; Duxbury, C.L.; Dixon, D.G.

1997-05-01

Phenanthrene (PHE) undergoes a significant increase in toxicity after exposure to simulated or natural sunlight in aqueous media, coincident with the appearance of PHE photoproducts. To investigate whether the primary photoproduct of PHE, 9,10-phenanthrenequinone (PHEQ), contributes to the increased hazards of solutions containing photomodified PHE, toxicity assays were conducted using the marine bacteria Photobacterium phosphoreum and the aquatic plant Lemna gibba (duckweed). Photobacterium phosphoreum was exposed to PHE, PHEQ, a photomodified PHE mixture containing known amounts of PHE and PHEQ (pmPHE), and a mixture mimicking the amounts of PHE and PHEQ in the pmPHE mixture. The bacteria were found tomore » be equally sensitive to PHE in simulated solar radiation or darkness, with an EC50 of 0.53 mg/L. In both darkness or SSR, solutions containing PHEQ (with or without PHE) all exhibited an EC50 of 0.06 to 0.10 mg/L based on PHEQ concentrations, indicating that PHEQ was the primary active component of the pmPHE mixture. Lemna gibba was tested in SSR and visible light with PHE, PHEQ, and the pmPHE mixture. The calculated EC50 for PHE was 3.5 mg/L in SSR and 10.8 mg/L in visible light, showing that the presence of UV radiation in the SSR source increased the phytotoxicity of PHE. Strikingly, PHEQ was much more toxic to L. gibba than PHE in a light-independent manner. Thus, for both P. phosphoreum and L. gibba the major photooxidation product of PHE in SSR, PHEQ, is the more toxic of the two chemicals.« less

Novel strategy for phenyllactic acid biosynthesis from phenylalanine by whole cell recombinant Escherichia coli coexpressing L-phenylalanine oxidase and L-lactate dehydrogenase.

PubMed

Zhang, Jianzhi; Li, Xi

2018-01-01

To enhance the efficiency of phenyllactic acid (PLA) production from L-phenylalanine (L-Phe) by introducing a novel artificial pathway into Escherichia coli RESULTS: The production of PLA from L-Phe by recombinant E. coli (ldh-lpox) coexpressing L-phenylalanine oxidase and L-lactate dehydrogenase was studied. The new PLA synthesis pathway was confirmed to be efficient in recombinant E. coli. Subsequently, two different biocatalyst processes were carried out and optimized for PLA production. In the whole cell biosynthesis process at high cell density using collected recombinant cells as catalyst, at optimal conditions (L-Phe 6 g/l, pH 7.5, 35 °C, CDW 24.5 g/l and 200 rpm), the recombinant E. coli (ldh-lpox) produced 1.62 g PLA/l with a conversion of 28% from L-Phe. Similarly, during the two-temperature-stage fermentation process in flasks using IPTG-induced cells, the temperature in the second stage was increased to 35 °C to benefit the biocatalyst process, and comparable phenyllactic acid production of 1.47 g/l was obtained from 12 g L-Phe/l. Recombinant E. coli (ldh-lpox) was efficient in PLA production realizing a high titer of several folds compared with studies using L-Phe as substrate.

Fluorometric and theoretical studies on inclusion complexes of β-cyclodextrin and D-, L-phenylalanine

NASA Astrophysics Data System (ADS)

Aree, Thammarat; Arunchai, Rungthiwa; Koonrugsa, Narongsak; Intasiri, Amarawan

2012-10-01

Inclusion complexes of β-cyclodextrin (β-CD) with L- and D-phenylalanine (Phe) have been characterized in solution by fluorometry and in gas phase by semiempirical PM3 calculations. The unimolar stoichiometric ratio of both β-CD-L-Phe and β-CD-D-Phe complexes and the stability constants (K) were deduced from fluorometric titrations. The β-CD-L-Phe complex is more stable than the β-CD-D-Phe complex as indicated by the larger K values, 21.1 vs. 6.86 M-1. This is consistent with the stabilization energies (ΔEstb) and inclusion geometries obtained from PM3 calculations. The β-CD-L-Phe complex with L-Phe residing in the central β-CD cavity and pointing its COOH group downwards to the O6 end has ΔEstb = -62.7 kJ mol-1, whereas the β-CD-D-Phe complex with D-Phe placing at 3 Å beneath the β-CD O4-plane and pointing its COOH group upwards to the O2/O3 end has ΔEstb = -53.3 kJ mol-1. The unison of host-guest intermolecular hydrogen bonds, hydrophobic interactions and molecular deformations plays an essential role in forming and stabilizing the inclusion complexes. Our results show that the β-CD-L-Phe and β-CD-D-Phe inclusion complexes are relatively stable and differentiable, suggesting the applications of CDs in foods and drugs.

Carbonic anhydrase activators. Activation of isoforms I, II, IV, VA, VII, and XIV with L- and D-phenylalanine and crystallographic analysis of their adducts with isozyme II: stereospecific recognition within the active site of an enzyme and its consequences for the drug design.

PubMed

Temperini, Claudia; Scozzafava, Andrea; Vullo, Daniela; Supuran, Claudiu T

2006-05-18

Activation of six human brain carbonic anhydrases (hCAs, EC 4.2.1.1), hCA I, II, IV, VA, VII, and XIV, with l-/d-phenylalanine was investigated kinetically and by X-ray crystallography. l-Phe was a potent activator of isozymes I, II, and XIV (K(A)s of 13-240 nM), a weaker activator of hCA VA and VII (K(A)s of 9.8-10.9 microM), and a quite inefficient hCA IV activator (K(A) of 52 microM). d-Phe showed good hCA II activatory properties (K(A) of 35 nM), being a moderate hCA VA, VII, and XIV (K(A)s of 4.6-9.7 microM) and a weak hCA I and IV activator (K(A)s of 63-86 microM). X-ray crystallography of the hCA II-l-Phe/d-Phe adducts showed the activators to be anchored at the entrance of the active site, participating in numerous bonds and hydrophobic interactions with amino acid residues His64, Thr200, Trp5, and Pro201. This is the first study showing different binding modes of stereoisomeric activators within the hCA II active site, with consequences for overall proton transfer processes (rate-determining for the catalytic cycle). It also points out differences of activation efficiency between various isozymes with structurally related activators, exploitable for designing alternative proton transfer pathways. CA activators may lead to the design of pharmacologically useful derivatives for the enhancement of synaptic efficacy, which may represent a conceptually new approach for the treatment of Alzheimer's disease, aging, and other conditions in which spatial learning and memory therapy must be enhanced. As the blood and brain concentrations of l-Phe are quite variable (30-73 microM), activity of some brain CAs may strongly be influenced by the level of activator(s) present in such tissues.

Petroleum degradation by Pseudomonas sp. ZS1 is impeded in the presence of antagonist Alcaligenes sp. CT10.

PubMed

Liang, Jibei; Cheng, Tao; Huang, Yi; Liu, Jianhua

2018-05-28

Enhanced bioremediation is a favorable approach for petroleum pollutant cleanup, which depends on the growth of oil-eating microorganisms. In this study, we show that, by using the modified T-RFLP (mT-RFLP) methodology, one of the four major microbial populations derived from oil sludge has failed to propagate in MS medium supplemented with 2% yeast extract (YE). rDNA sequence-based analysis indicated that the four populations were Donghicola sp. CT5, Bacillus sp. CT6, Alcaligenes sp. CT10, and Pseudomonas sp. ZS1. Four purified strains grow well individually in MS medium supplemented with 2% YE, suggesting that ZS1 growth is antagonized by other strains. Co-growth analysis using mT-RFLP methodology and plate inhibitory assay indicated that ZS1 exhibited antagonistic effect against CT5 and CT6. On the other hand, co-growth analysis and plate inhibition assay showed that CT10 antagonized against ZS1. To investigate the potential compounds responsible for the antagonism, supernatant of CT10 culture was subjected to GC-MS analysis. Analysis indicated that CT10 produced a number of antimicrobial compounds including cyclodipeptide c-(L-Pro-L-Phe), which was known to inhibit the growth of Pseudomonas sp. Growth test using the purified c-(L-Pro-L-Phe) from CT10 confirmed its inhibitory activity. We further showed that, using both gravimetric and GC analysis, CT10 antagonism against the oil-eating ZS1 led to the diminishing of crude oil degradation. Together, our results indicate that bioremediation can be affected by environmental antagonists.

Improvement of L-phenylalanine production from glycerol by recombinant Escherichia coli strains: the role of extra copies of glpK, glpX, and tktA genes.

PubMed

Gottlieb, Katrin; Albermann, Christoph; Sprenger, Georg A

2014-07-11

For the production of L-phenylalanine (L-Phe), two molecules of phosphoenolpyruvate (PEP) and one molecule erythrose-4-phosphate (E4P) are necessary. PEP stems from glycolysis whereas E4P is formed in the pentose phosphate pathway (PPP). Glucose, commonly used for L-Phe production with recombinant E. coli, is taken up via the PEP-dependent phosphotransferase system which delivers glucose-6-phosphate (G6P). G6P enters either glycolysis or the PPP. In contrast, glycerol is phosphorylated by an ATP-dependent glycerol kinase (GlpK) thus saving one PEP. However, two gluconeogenic reactions (fructose-1,6-bisphosphate aldolase, fructose-1,6-bisphosphatase, FBPase) are necessary for growth and provision of E4P. Glycerol has become an important carbon source for biotechnology and reports on production of L-Phe from glycerol are available. However, the influence of FBPase and transketolase reactions on L-Phe production has not been reported. L-Phe productivity of parent strain FUS4/pF81 (plasmid-encoded genes for aroF, aroB, aroL, pheA) was compared on glucose and glycerol as C sources. On glucose, a maximal carbon recovery of 0.19 mM C(Phe)/C(Glucose) and a maximal space-time-yield (STY) of 0.13 g l(-1) h(-1) was found. With glycerol, the maximal carbon recovery was nearly the same (0.18 mM C(Phe)/C(Glycerol)), but the maximal STY was higher (0.21 g l(-1) h(-1)). We raised the chromosomal gene copy number of the genes glpK (encoding glycerol kinase), tktA (encoding transketolase), and glpX (encoding fructose-1,6-bisphosphatase) individually. Overexpression of glpK (or its feedback-resistant variant, glpK(G232D)) had little effect on growth rate; L-Phe production was about 30% lower than in FUS4/pF81. Whereas the overexpression of either glpX or tktA had minor effects on productivity (0.20 mM C(Phe)/C(Glycerol); 0.25 g l(-1) h(-1) and 0.21 mM C(Phe)/C(Glycerol); 0.23 g l(-1) h(-1), respectively), the combination of extra genes of glpX and tktA together led to an increase in maximal STY of about 80% (0.37 g l(-1) h(-1)) and a carbon recovery of 0.26 mM C(Phe)/C(Glycerol). Enhancing the gene copy numbers for glpX and tktA increased L-Phe productivity from glycerol without affecting growth rate. Engineering of glycerol metabolism towards L-Phe production in E. coli has to balance the pathways of gluconeogenesis, glycolysis, and PPP to improve the supply of the precursors, PEP and E4P.

[The pharmacokinetics of the dipeptide analog of piracetam with nootropic activity GVS-111 and of its basic metabolites].

PubMed

Boĭko, S S; Zherdev, V P; Dvorianinov, A A; Gudasheva, T A; Ostrovskaia, R U; Voronina, T A; Rozantsev, G G; Seredenin, S B

1997-01-01

The pharmacokinetics of a new nootropic dipeptide analog of piracetam-N-phenylacetyl-L-prolylglycine (GWS-111) and its main metabolites were studied in rats by means of high performance liquid chromatography and gas-liquid chromatography. The compound under study showed a greater resistance to an enzymatic effect than natural neuropeptides. In addition to an unchanged compound three of its metabolites were found in the blood plasma of the rats. One of them, cyclo-Pro-Gly was an active metabolite of GWS-111.

The Dirigible: A Catalyst for Resource Exploitation in Remote Areas?

DTIC Science & Technology

1985-04-01

ir’ship (26’.: 13). .. Hybrid . A he ,vi er than air vehiLile which Lomb nes.’-. s.tar~ it (gas) anrid cly ,mi L (pro~pel 1er s, jet e, g ines ) li ft...another test with a hybrid airship (8:.33). TABLE 1. - Airships Rejected p The three remaining airships will be discussed next. All arc? hybrid LTAs...heavy-lift markets. (Drawing at Appendix 3.) The Cyclo-Crane is a hybrid aircraft utilizing aerostatic lift from a helium filled centerbody to support

Potent inhibition of endopeptidase 24.16 and endopeptidase 24.15 by the phosphonamide peptide N-(phenylethylphosphonyl)-Gly-L-Pro-L-aminohexanoic acid.

PubMed Central

Barelli, H; Dive, V; Yiotakis, A; Vincent, J P; Checler, F

1992-01-01

A phosphonamide peptide, N-(phenylethylphosphonyl)-Gly-L-Pro-L-aminohexanoic acid, previously shown to block Clostridium histolyticum collagenases, was examined as a putative inhibitor of endopeptidase 24.16 and endopeptidase 24.15. Hydrolysis of two endopeptidase 24.16 substrates, i.e. 3-carboxy-7-methoxycoumarin (Mcc)-Pro-Leu-Gly-Pro-D-Lys-dinitrophenyl (Dnp) and neurotensin, were completely and dose-dependently inhibited by the phosphonamide inhibitor with KI values of 0.3 and 0.9 nM respectively. In addition, the phosphonamide peptide inhibited the hydrolysis of benzoyl (Bz)-Gly-Ala-Ala-Phe-(pAB) p-aminobenzoate and neurotensin by endopeptidase 24.15 with about a 10-fold lower potency (KI values of 5 and 7.5 nM respectively). The selectivity of this inhibitor towards several exo- and endo-peptidases belonging to the zinc-containing metallopeptidase family established that a 1 microM concentration of this inhibitor was unable to affect leucine aminopeptidase, carboxypeptidase A, angiotensin-converting enzyme and endopeptidase 24.11. The present paper therefore reports on the first hydrophilic highly potent endopeptidase 24.16 inhibitor and describes the most potent inhibitory agent directed towards endopeptidase 24.15 developed to date. These tools should allow one to assess the contribution of endopeptidase 24.16 and endopeptidase 24.15 to the physiological inactivation of neurotensin as well as other neuropeptides. PMID:1332678

Characterization of labelling and de-labelling reagents for detection and recovery of tyrosine residue in peptide.

PubMed

Toyo'oka, Toshimasa; Mantani, Tomomi; Kato, Masaru

2003-01-01

This paper characterized the labelling and de-labelling reagents for reversible labelling of tyrosine (Tyr)-containing peptide, which involves detection and recovery. The phenolic hydroxyl group (-OH) in Tyr structure reacted with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F), and 1-fluoro-2,4-dinitrobenzene (DNFB) under mild conditions at room temperature at pH 9.3. The labels in the resulting derivatives were removed with the treatment of nucleophiles, such as thiols (cysteine, N-acetyl-L-cysteine and dithiothreitol) and amines (dimethylamine, methylamine, diethylamine, ethylamine and pyrrolidine). The de-labelling reactions of NBD-labelled N-acetyl-L-tyrosine (N-AcTyr) with the nucleophiles produced N-AcTyr, accompanied by NBD-nucleophile. Although DBD-F and DNFB also successfully labeled the -OH group in N-AcTyr, the efficiency of Cbond;O bond cleavage and recovery of N-AcTyr by the nucleophiles was relatively low compared with NBD-label. Among the de-labelling reagents, N-acetyl-L-cysteine and dimethylamine were recommended for the elimination of NBD moiety, with respect to the reaction rate, the side reaction, and the yield of recovery. The proposed procedure, which includes the labelling with NBD-F and the removal of NBD moiety by the nucleophiles, was successfully applied to the reversible labelling of N-terminal amine-blocked peptides, i.e. N-AcTyr-Val-Gly, Z-Glu-Tyr, Z-Phe-Tyr, N-Formyl-Met-Leu-Tyr, and N-AcArg-Pro-Pro-Gly-Phe-Ser-Pro-Tyr-Arg. Copyright 2003 John Wiley & Sons, Ltd.

Cellular protein receptors of maculosin, a host specific phytotoxin of spotted knapweed (Centaurea maculosa L.).

PubMed

Park, S H; Strobel, G A

1994-01-05

Maculosin (the diketopiperazine, cyclo (L-Pro-L-Tyr)) is a host specific phytotoxin produced by Alternaria alternata on spotted knapweed (Centaurea maculosa L.). Receptors for this phytotoxin have been isolated from spotted knapweed. Knapweed leaves possess most of the maculosin-binding activity in the cytosolic fraction. However, activity was also observed in the whole membrane fraction of the leaf. The binding component of the cytosolic fraction was identified as a protein(s) because of its heat-lability and sensitivity to proteases. A 16-fold purification of a toxin-binding protein was carried out by ammonium sulfate fractionation, and Sephadex G-200, and maculosin-affinity column chromatography. The affinity column was prepared with epoxy activated Sepharose 6B to which the phenolic group of maculosin was attached. The receptor was estimated to contain more than one binding protein by native and SDS-PAGE. At least one of the maculosin-binding proteins was identified as ribulose-1,5-biphosphate carboxylase (RuBPcase).

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

PubMed

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.

Characterization of Gly-D-Phe, Gly-L-Leu, and D-Phe as affinity ligands to thermolysin.

PubMed

Yasukawa, Kiyoshi; Kusano, Masayuki; Nakamura, Koji; Inouye, Kuniyo

2006-04-01

In this study, glycyl-D-phenylalanine (Gly-D-Phe), glycyl-L-leucine (Gly-L-Leu), and D-phenylalanine (D-Phe) were characterized for their abilities as affinity ligands to thermolysin. Each of the ligands was immobilized to the resin. The optimum pH for adsorption of thermolysin is 5.0-6.0 for each of the ligands. By the affinity column chromatography in which 2mg thermolysin was applied onto 4 ml volume of the resins at pH 5.5, the adsorption ratios based on casein hydrolysis activity were 100% for each of the ligands. However, the adsorption ratios of the resins containing Gly-L-Leu and D-Phe, unlike that of Gly-D-Phe, were progressively decreased with increasing the amounts of thermolysin applied to the column. Measurement of adsorption isotherms showed that the association constant to thermolysin at pH 5.5 of the resins containing Gly-D-Phe was (3.3+/-0.8)x10(5)M(-1), while those of Gly-L-Leu and D-Phe were approximately ten times less. This result is coincident with the observations of performances in affinity column chromatography. On the other hand, maximum thermolysin binding capacities were almost the same among the resins examined. These results indicate that Gly-D-Phe is more suitable than Gly-L-Leu and D-Phe as an affinity ligand for purification of thermolysin.

Adsorption mechanism of physiologically active L-phenylalanine phosphonodipeptide analogues: Comparison of colloidal silver and macroscopic silver substrates

NASA Astrophysics Data System (ADS)

Podstawka, E.; Kudelski, A.; Proniewicz, L. M.

2007-11-01

Here we present SERS spectra of several L-phenylalanine (Phe) phosphonodipeptides, i.e., L-Phe- L-Ala-PO 3H 2 ( MD1), L-Phe- L-Va L-PO 3H 2 ( MD2), L-Phe-β-Ala-CH(OH)-PO 3H 2 ( MD3), L-Phe- L-Ala-CH(OH)-PO 3H 2 ( MD4), L-Ala-(3,4-dimethoxy)- L-Phe-PO 3H 2 ( MD5), and L-Ala-(3,4-dimethoxy)-(des-CH 2)- L-Phe-PO 3H 2 ( MD6), immobilized on electrochemically roughened silver electrodes. These spectra are analyzed by theoretical calculations using density functional theory (DFT) at the B3LYP level with 6-31++G∗∗ basis set. In addition, these spectra are compared with SERS spectra of these species adsorbed on a colloidal silver surface. We showed that on the macroscopic silver substrate, the Phe aromatic ring of MD3 and MD4 is oriented vertically, while for MD1 it almost "stands up" on this surface. In the other three cases, the Phe ring adopts a tilted orientation in regard to the substrate. We also find that the phosphonate (-PO32-), methyl/methane, or dimethoxy groups of MD1, MD2, MD3, MD5, and MD6 are involved in the interaction of these phosphonodipeptides with the electrochemically roughened surface. This phenomenon is clearly seen for -CH 2-/-CH 3/-OCH 3 moieties as well as for the PO32- group that adsorbs on the macroscopic silver substrates mainly via the P dbnd O fragment. We also showed that MD4 binds to the macroscopic silver substrate through the hydroxyl, amine, and phosphonate groups, while the methylene/methane moieties are remote from this surface. We found that studied phosphonodipeptides often adsorb differently on the macroscopic silver substrate and on the colloidal silver nanoparticles. For example, MD1 adopts an almost vertical orientation on the electrochemically roughened silver substrate and is tilted or close to flat on the silver nanoparticles.

[A new strategy of gene therapy for hyperphenylalaninemia rats].

PubMed

Jia, X; Liu, J; Xiang, H

2000-06-01

To construct a recombinant vector which expresses active phenylalanine-amonia-lyase (PAL) in Lactococcus lactis (L. L.), and to convert phe into cinnamic acid in small intestine by the engineering L. L. to decrease the phe level in the peripheral blood, and to cure hyperphenylalaninemia rats. PAL cDNA from Petroselinum crispum was subcloned into expression vector pMG36e and transformed L. L. The pMG36ePAL/L.L. was screened and characterized by using PCR and HPLC, and prepared as enteric-coated microcapsules and oral liquid type preparation that were given orally to hyperphenylalaninemia-rats. Engineering L. L. expressing PAL activity was obtained. The phe levels plasma of in the rats receiving preparations made from the engineering L. L. were significantly reduced compared with non-treated hyperphenylalaninemia rats. And the effects of different preparations were different from each other. The engineering L. L. expressing PAL activity can reduce the blood phe level of the hyperphenylalaninemia rats. This may be a potential way for PKU gene therapeutics.

Immunomodulatory constituents from an ascomycete, Microascus tardifaciens.

PubMed

Fujimoto, H; Fujimaki, T; Okuyama, E; Yamazaki, M

1999-10-01

Fractionation guided by the immunosuppressive activity of the defatted AcOEt extract of an Ascomycete, Microascus tardifaciens, afforded eight constituents, questin (emodin 8-O-methylether) (1), rubrocristin (2), 5,7-dihydroxy-4-methylphthalide (3), cladosporin (asperentin) (4), cladosporin 8-O-methylether (5), tradioxopiperazine A [cyclo-L-alanyl-5-isopentenyl-2-(1',1'-dimethylallyl)-L-tryptophan] (6), tradioxopiperazine B [cyclo-L-alanyl-7-isopentenyl-2-(1',1'-dimethylallyl)-L-tryptophan] (7), and asperflavin (8), among which 6 and 7 were new compounds. Compounds 1 and 2 showed considerably high immunosuppressive activity, 6 was moderate and, 3, 4, 5, 7 and 8 showed low activity.

Identification of Streptomyces sp. KH29, which produces an antibiotic substance processing an inhibitory activity against multidrug-resistant Acinetobacter baumannii.

PubMed

Lee, Keyong Ho; Kim, Kye-Woong; Rhee, Ki-Hyeong

2010-12-01

The Actinomycete strain KH29 is antagonistic to the multidrug-resistant Acinetobacter baumannii. Based on the diaminopimelic acid (DAP) type, and the morphological and physiological characteristics observed through the use of scanning electron microscopy (SEM), KH29 was confirmed as belonging to the genus Streptomyces. By way of its noted 16S rDNA nucleotide sequences, KH29 was found to have a relationship with Streptomyces cinnamonensis. The production of an antibiotic from this strain was found to be most favorable when cultured with glucose, polypeptone, and yeast extract (PY) medium for 6 days at 27 degrees C. The antibiotic produced was identified, through comparisons with reported spectral data including MS and NMR as a cyclo(L-tryptophanyl-L-tryptophanyl). Cyclo(L-Trp-L-Trp), from the PY cultures of KH29, was seen to be highly effective against 41 of 49 multidrugresistant Acinetobacter baumannii. Furthermore, cyclo(LTrp- L-Trp) had antimicrobial activity against Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, Saccharomyces cerevisiae, Aspergillus niger, and Candida albicans, However, it was ineffective against Streptomyces murinus.

Sensitive, site-specific, and stable vibrational probe of local protein environments: 4-azidomethyl-L-phenylalanine.

PubMed

Bazewicz, Christopher G; Liskov, Melanie T; Hines, Kevin J; Brewer, Scott H

2013-08-01

We have synthesized the unnatural amino acid (UAA), 4-azidomethyl-L-phenylalanine (pN₃CH₂Phe), to serve as an effective vibrational reporter of local protein environments. The position, extinction coefficient, and sensitivity to local environment of the azide asymmetric stretch vibration of pN₃CH₂Phe are compared to the vibrational reporters: 4-cyano-L-phenylalanine (pCNPhe) and 4-azido-L-phenylalanine (pN₃Phe). This UAA was genetically incorporated in a site-specific manner utilizing an engineered, orthogonal aminoacyl-tRNA synthetase in response to an amber codon with high efficiency and fidelity into two distinct sites in superfolder green fluorescent protein (sfGFP). This allowed for the dependence of the azide asymmetric stretch vibration of pN₃CH₂Phe to different protein environments to be measured. The photostability of pN₃CH₂Phe was also measured relative to the photoreactive UAA, pN₃Phe.

Tracing amino acid exchange during host-pathogen interaction by combined stable-isotope time-resolved Raman spectral imaging

NASA Astrophysics Data System (ADS)

Naemat, Abida; Elsheikha, Hany M.; Boitor, Radu A.; Notingher, Ioan

2016-02-01

This study investigates the temporal and spatial interchange of the aromatic amino acid phenylalanine (Phe) between human retinal pigment epithelial cell line (ARPE-19) and tachyzoites of the apicomplexan protozoan parasite Toxoplasma gondii (T. gondii). Stable isotope labelling by amino acids in cell culture (SILAC) is combined with Raman micro-spectroscopy to selectively monitor the incorporation of deuterium-labelled Phe into proteins in individual live tachyzoites. Our results show a very rapid uptake of L-Phe(D8) by the intracellular growing parasite. T. gondii tachyzoites are capable of extracting L-Phe(D8) from host cells as soon as it invades the cell. L-Phe(D8) from the host cell completely replaces the L-Phe within T. gondii tachyzoites 7-9 hours after infection. A quantitative model based on Raman spectra allowed an estimation of the exchange rate of Phe as 0.5-1.6 × 104 molecules/s. On the other hand, extracellular tachyzoites were not able to consume L-Phe(D8) after 24 hours of infection. These findings further our understanding of the amino acid trafficking between host cells and this strictly intracellular parasite. In particular, this study highlights new aspects of the metabolism of amino acid Phe operative during the interaction between T. gondii and its host cell.

GPR139, an Orphan Receptor Highly Enriched in the Habenula and Septum, Is Activated by the Essential Amino Acids L-Tryptophan and L-Phenylalanine.

PubMed

Liu, Changlu; Bonaventure, Pascal; Lee, Grace; Nepomuceno, Diane; Kuei, Chester; Wu, Jiejun; Li, Qingqin; Joseph, Victory; Sutton, Steven W; Eckert, William; Yao, Xiang; Yieh, Lynn; Dvorak, Curt; Carruthers, Nicholas; Coate, Heather; Yun, Sujin; Dugovic, Christine; Harrington, Anthony; Lovenberg, Timothy W

2015-11-01

GPR139 is an orphan G-protein-coupled receptor expressed in the central nervous system. To identify its physiologic ligand, we measured GPR139 receptor activity from recombinant cells after treatment with amino acids, orphan ligands, serum, and tissue extracts. GPR139 activity was measured using guanosine 5'-O-(3-[(35)S]thio)-triphosphate binding, calcium mobilization, and extracellular signal-regulated kinases phosphorylation assays. Amino acids L-tryptophan (L-Trp) and L-phenylalanine (L-Phe) activated GPR139, with EC50 values in the 30- to 300-μM range, consistent with the physiologic concentrations of L-Trp and L-Phe in tissues. Chromatography of rat brain, rat serum, and human serum extracts revealed two peaks of GPR139 activity, which corresponded to the elution peaks of L-Trp and L-Phe. With the purpose of identifying novel tools to study GPR139 function, a high-throughput screening campaign led to the identification of a selective small-molecule agonist [JNJ-63533054, (S)-3-chloro-N-(2-oxo-2-((1-phenylethyl)amino)ethyl) benzamide]. The tritium-labeled JNJ-63533054 bound to cell membranes expressing GPR139 and could be specifically displaced by L-Trp and L-Phe. Sequence alignment revealed that GPR139 is highly conserved across species, and RNA sequencing studies of rat and human tissues indicated its exclusive expression in the brain and pituitary gland. Immunohistochemical analysis showed specific expression of the receptor in circumventricular regions of the habenula and septum in mice. Together, these findings suggest that L-Trp and L-Phe are candidate physiologic ligands for GPR139, and we hypothesize that this receptor may act as a sensor to detect dynamic changes of L-Trp and L-Phe in the brain. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Elevated phenylalanine on newborn screening: follow-up testing may reveal undiagnosed galactosaemia.

PubMed

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 with one asymptomatic individual having a concentration <210 μmol/L. Adoption of the UKNSPC recommended cut-off (>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.

Amino acid ionic liquids as chiral ligands in ligand-exchange chiral separations.

PubMed

Liu, Qian; Wu, Kangkang; Tang, Fei; Yao, Lihua; Yang, Fei; Nie, Zhou; Yao, Shouzhuo

2009-09-28

Recently, amino acid ionic liquids (AAILs) have attracted much research interest. In this paper, we present the first application of AAILs in chiral separation based on the chiral ligand exchange principle. By using 1-alkyl-3-methylimidazolium L-proline (L-Pro) as a chiral ligand coordinated with copper(II), four pairs of underivatized amino acid enantiomers-dl-phenylalanine (dl-Phe), dl-histidine (dl-His), dl-tryptophane (dl-Trp), and dl-tyrosine (dl-Tyr)-were successfully separated in two major chiral separation techniques, HPLC and capillary electrophoresis (CE), with higher enantioselectivity than conventionally used amino acid ligands (resolution (R(s))=3.26-10.81 for HPLC; R(s)=1.34-4.27 for CE). Interestingly, increasing the alkyl chain length of the AAIL cation remarkably enhanced the enantioselectivity. It was inferred that the alkylmethylimidazolium cations and L-Pro form ion pairs on the surface of the stationary phase or on the inner surface of the capillary. The ternary copper complexes with L-Pro are consequently attached to the support surface, thus inducing an ion-exchange type of retention for the dl-enantiomers. Therefore, the AAIL cation plays an essential role in the separation. This work demonstrates that AAILs are good alternatives to conventional amino acid ligands for ligand-exchange-based chiral separation. It also reveals the tremendous application potential of this new type of task-specific ILs.

Efficacy of 3,5-dibromo-L-phenylalanine in rat models of stroke, seizures and sensorimotor gating deficit

PubMed Central

Cao, W; Shah, HP; Glushakov, AV; Mecca, AP; Shi, P; Sumners, C; Seubert, CN; Martynyuk, AE

2009-01-01

Background and purpose: Abnormal glutamatergic activity is implicated in neurologic and neuropsychiatric disorders. Selective glutamate receptor antagonists were highly effective in animal models of stroke and seizures but failed in further clinical development because of serious side effects, including an almost complete set of symptoms of schizophrenia. Therefore, the novel polyvalent glutamatergic agent 3,5-dibromo-L-phenylalanine (3,5-DBr-L-Phe) was studied in rat models of stroke, seizures and sensorimotor gating deficit. Experimental approach: 3,5-DBr-L-Phe was administered intraperitoneally as three boluses after intracerebral injection of endothelin-1 (ET-1) adjacent to the middle cerebral artery to cause brain injury (a model of stroke). 3,5-DBr-L-Phe was also given as a single bolus prior to pentylenetetrazole (PTZ) injection to induce seizures or prior to the administration of the N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine (MK-801) to cause disruption of prepulse inhibition (PPI) of startle (sensorimotor gating deficit). Key results: Brain damage caused by ET-1 was reduced by 52%, which is comparable with the effects of MK-801 in this model as reported by others. 3,5-DBr-L-Phe significantly reduced seizures induced by PTZ without the significant effects on arterial blood pressure and heart rate normally caused by NMDA antagonists. 3,5-DBr-L-Phe prevented the disruption of PPI measured 3 days after the administration of ET-1. 3,5-DBr-L-Phe also eliminated sensorimotor gating deficit caused by MK-801. Conclusion and implications: The pharmacological profile of 3,5-DBr-L-Phe might be beneficial not only for developing a therapy for the neurological and cognitive symptoms of stroke and seizures but also for some neuropsychiatric disorders. PMID:20050189

Efficacy of 3,5-dibromo-L-phenylalanine in rat models of stroke, seizures and sensorimotor gating deficit.

PubMed

Cao, W; Shah, H P; Glushakov, A V; Mecca, A P; Shi, P; Sumners, C; Seubert, C N; Martynyuk, A E

2009-12-01

Abnormal glutamatergic activity is implicated in neurologic and neuropsychiatric disorders. Selective glutamate receptor antagonists were highly effective in animal models of stroke and seizures but failed in further clinical development because of serious side effects, including an almost complete set of symptoms of schizophrenia. Therefore, the novel polyvalent glutamatergic agent 3,5-dibromo-L-phenylalanine (3,5-DBr-L-Phe) was studied in rat models of stroke, seizures and sensorimotor gating deficit. 3,5-DBr-L-Phe was administered intraperitoneally as three boluses after intracerebral injection of endothelin-1 (ET-1) adjacent to the middle cerebral artery to cause brain injury (a model of stroke). 3,5-DBr-L-Phe was also given as a single bolus prior to pentylenetetrazole (PTZ) injection to induce seizures or prior to the administration of the N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine (MK-801) to cause disruption of prepulse inhibition (PPI) of startle (sensorimotor gating deficit). Brain damage caused by ET-1 was reduced by 52%, which is comparable with the effects of MK-801 in this model as reported by others. 3,5-DBr-L-Phe significantly reduced seizures induced by PTZ without the significant effects on arterial blood pressure and heart rate normally caused by NMDA antagonists. 3,5-DBr-L-Phe prevented the disruption of PPI measured 3 days after the administration of ET-1. 3,5-DBr-L-Phe also eliminated sensorimotor gating deficit caused by MK-801. The pharmacological profile of 3,5-DBr-L-Phe might be beneficial not only for developing a therapy for the neurological and cognitive symptoms of stroke and seizures but also for some neuropsychiatric disorders.

The major metabolite of dipeptide piracetam analogue GVS-111 in rat brain and its similarity to endogenous neuropeptide cyclo-L-prolylglycine.

PubMed

Gudasheva, T A; Boyko, S S; Ostrovskaya, R U; Voronina, T A; Akparov, V K; Trofimov, S S; Rozantsev, G G; Skoldinov, A P; Zherdev, V P; Seredenin, S B

1997-01-01

The metabolism of a new piracetam analogue, the dipeptide cognitive enhancer N-phenylacetyl-L-prolylglycine ethyl ester (GVS-111) was studied in vivo. GVS-111 itself was not found in rat brain 1 h after 5 mg/kg i.p. administration up to limit of detection (LOD) under high performance liquid chromatography (HPLC) conditions. Three substances corresponding to the three possible GVS-111 metabolites, namely phenylacetic acid, prolylglycine and cyclo-prolylglycine, were found in experimental rat brain samples as well as in controls using HPLC, gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) methods. Only cyclo-prolylglycine concentration increased (2.5-fold) 1 h after GVS-111 administration. Cyclo-prolylglycine formation from GVS-111 in the presence of plasma and brain enzymes was shown in vitro. These data could be considered as evidence that GVS-111 is prodrug which converts in the body to cyclo-prolylglycine, and which is identical to the endogenous cyclopeptide that produces the nootropic activity.

Biochemical characterization of an L-tryptophan dehydrogenase from the photoautotrophic cyanobacterium Nostoc punctiforme.

PubMed

Ogura, Ryutaro; Wakamatsu, Taisuke; Mutaguchi, Yuta; Doi, Katsumi; Ohshima, Toshihisa

2014-06-10

An NAD(+)-dependent l-tryptophan dehydrogenase from Nostoc punctiforme NIES-2108 (NpTrpDH) was cloned and overexpressed in Escherichia coli. The recombinant NpTrpDH with a C-terminal His6-tag was purified to homogeneity using a Ni-NTA agarose column, and was found to be a homodimer with a molecular mass of 76.1kDa. The enzyme required NAD(+) and NADH as cofactors for oxidative deamination and reductive amination, respectively, but not NADP(+) or NADPH. l-Trp was the preferred substrate for deamination, though l-Phe was deaminated at a much lower rate. The enzyme exclusively aminated 3-indolepyruvate; phenylpyruvate was inert. The pH optima for the deamination of l-Trp and amination of 3-indolpyruvate were 11.0 and 7.5, respectively. For deamination of l-Trp, maximum enzymatic activity was observed at 45°C. NpTrpDH retained more than 80% of its activity after incubation for 30min at pHs ranging from 5.0 to 11.5 or incubation for 10min at temperatures up to 40°C. Unlike l-Trp dehydrogenases from higher plants, NpTrpDH activity was not activated by metal ions. Typical Michaelis-Menten kinetics were observed for NAD(+) and l-Trp for oxidative deamination, but with reductive amination there was marked substrate inhibition by 3-indolepyruvate. NMR analysis of the hydrogen transfer from the C4 position of the nicotinamide moiety of NADH showed that NpTrpDH has a pro-S (B-type) stereospecificity similar to the Glu/Leu/Phe/Val dehydrogenase family. Copyright © 2014 Elsevier Inc. All rights reserved.

Induction of immune-related gene expression in Ctenopharyngodon idella kidney cells by secondary metabolites from immunostimulatory Alcaligenes faecalis FY-3.

PubMed

Wu, Z-F; Liu, G-L; Zhou, Z; Wang, G-X; Xia, L; Liu, J-L

2012-08-01

This study was undertaken to isolate active secondary metabolites from immunostimulatory Alcaligenes faecalis FY-3 and evaluate their activities using grass carp Ctenopharyngodon idella kidney (CIK) cells. By applying chromatography techniques and successive recrystallization, three purified metabolites were obtained and identified by spectral data (mass spectrometry and nuclear magnetic resonance) as: (1) phenylacetic acid, (2) p-hydroxyphenylacetylamide and (3) cyclo-(Gly-(L)-Pro). CIK cells were stimulated by different concentrations (1, 10 and 100 μg/ml) of the isolated compounds, and expression of MyD88, IL-1β, TNF-α, type I-IFN and IL-8 genes at different time points (2, 8 and 24 h) post-stimulation was quantified by real-time PCR. The known immunostimulatory agent lipopolysaccharide (LPS) was used as a positive control. To analyse whether these compounds are toxic to the cells, the methyl tetrazolium assay was employed to measure changes in cell viability. The obtained results revealed that transcribing level of MyD88, an important adaptor molecule in toll-like receptor signalling pathway, was augmented remarkably by all the three isolated compounds and LPS as early as 2-h exposure. These compounds also induced gene expression of cytokines such as IL-1β, TNF-α and type I-IFN. Under the experimental conditions, none of the test compounds is toxic to the CIK cells. These findings demonstrate that the immunostimulatory properties of the three metabolites [phenylacetic acid, p-hydroxyphenylacetylamide and cyclo-(Gly-(L)-Pro)] from A. faecalis FY-3 in CIK cells and highlight the potential of using these metabolites as immunostimulants in fish aquaculture. © 2012 The Authors. Scandinavian Journal of Immunology © 2012 Blackwell Publishing Ltd.

Comparison of L-selectin blood level and gene polymorphism in tuberculosis patients with healthy individuals.

PubMed

Eini, Peyman; Shirvani, Maria; Hajilooi, Mehrdad; Esna-Ashari, Farzaneh

2018-02-12

The inflammatory response to Mycobacterium tuberculosis bacilli influences tuberculosis (TB) progression. In this study, we aimed to identify the Phe206Leu polymorphism and serum L-selectin level in TB patients, compared to healthy individuals. Ninety patients with a diagnosis of TB and 90 healthy controls were selected in this study. The serum L-selectin level was determined, using ELISA. L-selectin polymorphism was also evaluated using PCR. For data analysis, SPSS was used at a significance level of 0.05. According to the findings, the mean±SD age of the participants was 57.5 ± 18.4 and 56.5 ± 17.5 years in the TB and healthy groups, respectively. The TB group showed a significantly higher serum L-selectin level (1721.1 ± 330.9) versus the healthy controls (1624 ± 279). The L-selectin Phe allele frequencies were higher than the Leu allele frequencies in the main population, whereas the patients and controls were not significantly different. Eight (0.04%) subjects had Leu/Leu genotypes, 84 (46.6%) carried Phe/Leu genotypes, and 88 (48.8%) had Phe/Phe genotypes. Our results showed that the groups were not significantly different regarding L-selectin genotypes. TB patients had a significantly higher serum L-selectin level, compared to the controls. Based on the findings, the incidence of TB and L-selectin polymorphism in the Phe206Leu gene had no significant association. © 2018 Wiley Periodicals, Inc.

Evaluation of sewage sludge and slow pyrolyzed sewage sludge-derived biochar for adsorption of phenanthrene and pyrene.

PubMed

Zielińska, Anna; Oleszczuk, Patryk

2015-09-01

The present study investigated the sorption of phenanthrene (PHE) and pyrene (PYR) by sewage sludges and sewage sludge-derived biochars. The organic carbon normalized distribution coefficient (log K(OC) for C(w) = 0.01 S(w)) for the sewage sludges ranged from 5.62 L kg(-1) to 5.64 L kg(-1) for PHE and from 5.72 L kg(-1) to 5.75 L kg(-1) for PYR. The conversion of sewage sludges into biochar significantly increased their sorption capacity. The value of log K(OC) for the biochars ranged from 5.54 L kg(-1) to 6.23 L kg(-1) for PHE and from 5.95 L kg(-1) to 6.52 L kg(-1) for PYR depending on temperature of pyrolysis. The dominant process was monolayer adsorption in the micropores and/or multilayer surface adsorption (in the mesopores), which was indicated by the significant correlations between log K(OC) and surface properties of biochars. PYR was sorbed better on the tested materials than PHE. Copyright © 2015 Elsevier Ltd. All rights reserved.

Urinary naphthalene and phenanthrene as biomarkers of occupational exposure to polycyclic aromatic hydrocarbons

PubMed Central

Sobus, Jon R.; Waidyanatha, Suramya; McClean, Michael D.; Herrick, Robert F.; Smith, Thomas J.; Garshick, Eric; Laden, Francine; Hart, Jaime E.; Zheng, Yuxin; Rappaport, Stephen M.

2009-01-01

Objectives We investigated the utility of unmetabolized naphthalene (Nap) and phenanthrene (Phe) in urine as surrogates for exposures to mixtures of polycyclic aromatic hydrocarbons (PAHs). Methods Our study included workers exposed to diesel exhausts (low PAH exposure level, n = 39) as well as those exposed to emissions from asphalt (medium PAH exposure level, n = 26) and coke ovens (high PAH exposure level, n = 28). Levels of Nap and Phe were measured in urine from each subject using head space-solid phase microextraction and gas chromatography-mass spectrometry. Published levels of airborne Nap, Phe, and other PAHs in the coke-producing and aluminum industries were also investigated. Results In post-shift urine, the highest estimated geometric mean concentrations of Nap and Phe were observed in coke-oven workers (Nap: 2,490 ng/l; Phe: 975 ng/l), followed by asphalt workers (Nap: 71.5 ng/l; Phe: 54.3 ng/l), and by diesel-exposed workers (Nap: 17.7 ng/l; Phe: 3.60 ng/l). After subtracting logged background levels of Nap and Phe from the logged post-shift levels of these PAHs in urine, the resulting values [referred to as ln(adjNap) and ln(adjPhe), respectively] were significantly correlated in each group of workers (0.71 ≤ Pearson r ≤ 0.89), suggesting a common exposure source in each case. Surprisingly, multiple linear regression analysis of ln(adjNap) on ln(adjPhe) showed no significant effect of the source of exposure (coke ovens, asphalt, and diesel exhaust) and further suggested that the ratio of urinary Nap/Phe (in natural scale) decreased with increasing exposure levels. These results were corroborated with published data for airborne Nap and Phe in the coke-producing and aluminum industries. The published air measurements also indicated that Nap and Phe levels were proportional to the levels of all combined PAHs in those industries. Conclusion Levels of Nap and Phe in urine reflect airborne exposures to these compounds and are promising surrogates for occupational exposures to PAH mixtures. Main Messages Urinary naphthalene and phenanthrene are promising surrogates for occupational exposures to PAHs. Policy Implications Measurement of urinary naphthalene and phenanthrene could simplify the assessment of occupational exposures to PAHs. PMID:19017700

The in vivo anti-fibrotic function of calcium sensitive receptor (CaSR) modulating poly(p-dioxanone-co-l-phenylalanine) prodrug.

PubMed

Wang, Bing; Wen, Aiping; Feng, Chengmin; Niu, Lijing; Xiao, Xin; Luo, Le; Shen, Chengyi; Zhu, Jiang; Lei, Jun; Zhang, Xiaoming

2018-06-01

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. The released Ca 2+ can simultaneously activate Ca 2+ /calcineurin and then trigger apoptosis and G1 arrest of fibroblast. Hence, l-phenylalanine could be seen as anti-fibrosis drug and PDPA which conjugate l-phenylalanine by hydrolytic covalent bonds could be seen as l-phenylalanine polymer prodrug. Based above, the in vivo anti-fibrotic function of PDPA were verified in three different animal models. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

A new approach for quantitative analysis of L-phenylalanine using a novel semi-sandwich immunometric assay.

PubMed

Kubota, Kazuyuki; Mizukoshi, Toshimi; Miyano, Hiroshi

2013-10-01

Here, we describe a novel method for L-phenylalanine analysis using a sandwich-type immunometric assay approach for use as a new method for amino acid analysis. To overcome difficulties of the preparation of high-affinity and selectivity monoclonal antibodies against L-phenylalanine and the inability to use sandwich-type immunometric assays due to their small molecular weight, three procedures were examined. First, amino groups of L-phenylalanine were modified by "N-Fmoc-L-cysteine" (FC) residues and the derivative (FC-Phe) was used as a hapten. Immunization of mice with bovine serum albumin/FC-Phe conjugate successfully yielded specific monoclonal anti-FC-Phe antibodies. Second, a new derivatization reagent, "biotin linker conjugate of FC-Phe N-succinimidyl ester" (FC(Biotin)-NHS), was synthesized to convert L-phenylalanine to FC-(Biotin)-Phe as a hapten structure. The biotin moiety linked to the thiol group of cysteine formed a second binding site for streptavidin/horseradish peroxidase (HRP) conjugates for optical detection. Third, a new semi-sandwich-type immunometric assay was established using pre-derivatized L-phenylalanine, the monoclonal anti-FC-Phe antibody, and streptavidin/HRP conjugate (without second antibody). Using the new "semi-sandwich" immunometric assay system, a detection limit of 35 nM (60 amol per analysis) and a detection range of 0.1-20 μM were attained using a standard L-phenylalanine solution. Rat plasma samples were analyzed to test reliability. Intra-day assay precision was within 6% of the coefficient of variation; inter-day variation was 0.1%. The recovery rates were from 92.4 to 123.7%. This is the first report of the quantitative determination of L-phenylalanine using a reliable semi-sandwich immunometric assay approach and will be applicable to the quantitative determination of other amino acids.

Purification and characterization of angiotensin I converting enzyme inhibition peptides from sandworm Sipunculus nudus

NASA Astrophysics Data System (ADS)

Sun, Xueping; Wang, Man; Liu, Buming; Sun, Zhenliang

2017-10-01

Three angiotensin I converting enzyme (ACE) inhibition peptides were isolated from sandworm Sipunculus nudus protein hydrolysate prepared using protamex. Consecutive purification methods, including size exclusion chromatography and reverse-phase high performance liquid chromatography (RP-HPLC), were used to isolate the ACE inhibition peptides. The amino acid sequences of the peptides were identified as Ile-Asn-Asp, Val-Glu-Pro-Gly and Leu-Ala-Asp-Glu-Phe. The IC50 values of the purified peptides for ACE inhibition activity were 34.72 μmol L-1, 20.55 μmol L-1 and 22.77 μmol L-1, respectively. These results suggested that S. nudus proteins contain specific peptides that can be released by enzymatic hydrolysis. This study may provide an experimental basis for further systematic research, rational development and clinical utilization of sandworm resources.

Construction and application of novel feedback-resistant 3-deoxy-d-arabino-heptulosonate-7-phosphate synthases by engineering the N-terminal domain for L-phenylalanine synthesis.

PubMed

Zhang, Chuanzhi; Kang, Zhen; Zhang, Junli; Du, Guocheng; Chen, Jian; Yu, Xiaobin

2014-04-01

3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAHP synthase) encoded by aroF is the first enzyme of the shikimate pathway. In the present study, an AroF variant with a deficiency in residue Ile11 (named AroF*) was shown to be insensitive to l-tyrosine. According to three-dimensional structure analysis, nine AroF variants were constructed with truncation of different N-terminal fragments, and overexpression of the variants AroF(Δ(1-9)) , AroF(Δ(1-10)) , AroF(Δ(1-12)) and, in particular, AroF(Δ(1-11)) significantly increased the accumulation of l-phenylalanine (l-Phe). However, the AroG and AroH variants with similar truncations of the N-terminal fragments decreased the production of l-Phe. By co-overexpressing AroF(Δ(1-11)) and PheA(fbr) , the production of l-Phe was increased from 2.36 ± 0.07 g L(-1) (co-overexpression of the wild-type AroF and PheA(fbr) ) to 4.29 ± 0.06 g L(-1) . The novel variant AroF(Δ(1-11)) showed great potential for the production of aromatic amino acids and their derivatives. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Ethylene glycol ethers induce apoptosis and disturb glucose metabolism in the rat brain.

PubMed

Pomierny, Bartosz; Krzyżanowska, Weronika; Niedzielska, Ewa; Broniowska, Żaneta; Budziszewska, Bogusława

2016-02-01

Ethylene glycol ethers (EGEs) are compounds widely used in industry and household products, but their potential, adverse effect on brain is poorly understood, so far. The aim of the present study was to determine whether 4-week administration of 2-buthoxyethanol (BE), 2-phenoxyethanol (PHE), and 2-ethoxyethanol (EE) induces apoptotic process in the rat hippocampus and frontal cortex, and whether their adverse effect on the brain cells can result from disturbances in the glucose metabolism. Experiments were conducted on 40 rats, exposed to BE, PHE, EE, saline or sunflower oil for 4 weeks. Markers of apoptosis and glucose metabolism were determined in frontal cortex and hippocampus by western blot, ELISA, and fluorescent-based assays. BE and PHE, but not EE, increased expression of the active form of caspase-3 in the examined brain regions. BE and PHE increased caspase-9 level in the cortex and PHE also in the hippocampus. BE and PHE increased the level of pro-apoptotic proteins (Bax, Bak) and/or reduced the concentration of anti-apoptotic proteins (Bcl-2, Bcl-xL); whereas, the effect of BE was observed mainly in the cortex and that of PHE in the hippocampus. It has also been found that PHE increased brain glucose level, and both BE and PHE elevated pyruvate and lactate concentration. It can be concluded that chronic treatment with BE and PHE induced mitochondrial pathway of apoptosis, and disturbed glucose metabolism in the rat brain. Copyright © 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Molecularly imprinted polymer based on chemiluminescence imaging for the chiral recognition of dansyl-phenylalanine.

PubMed

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.

Cyclo(dehydroala-L-Leu), an alpha-glucosidase inhibitor from Penicillium sp. F70614.

PubMed

Kwon, O S; Park, S H; Yun, B S; Pyun, Y R; Kim, C J

2000-09-01

A diketopiperazine (1) has been isolated from the culture broth of Penicillium sp. F70614 and its structure has been determined to be cyclo(dehydroala-L-Leu) by various spectroscopic analyses. This compound selectively inhibited yeast alpha-glucosidase and porcine intestinal alpha-glucosidase with IC50 values of 35 and 50 microg/ml, respectively. However, it did not show significant inhibitory effects against almond beta3-glucosidase, Aspergillus alpha-galactosidase, Escherichia coli beta-galactosidase and jack bean alpha-mannosidase.

An Easy and Effective Demonstration of Enzyme Stereospecificity and Equilibrium Thermodynamics

ERIC Educational Resources Information Center

Herdman, Chelsea; Dickman, Michael

2011-01-01

Enzyme stereospecificity and equilibrium thermodynamics can be demonstrated using the coupling of two amino acid derivatives by Thermoase C160. This protease will catalyze peptide bond formation between Z-L-AspOH and L-PheOMe to form the Aspartame precursor Z-L-Asp-L-PheOMe. Reaction completion manifests itself by precipitation of the product. As…

The extracellular calcium-sensing receptor is required for cholecystokinin secretion in response to l-phenylalanine in acutely isolated intestinal I cells

PubMed Central

Liou, Alice P.; Sei, Yoshitatsu; Zhao, Xilin; Feng, Jianying; Lu, Xinping; Thomas, Craig; Pechhold, Susanne; Raybould, Helen E.

2011-01-01

The extracellular calcium-sensing receptor (CaSR) has recently been recognized as an l-amino acid sensor and has been implicated in mediating cholecystokinin (CCK) secretion in response to aromatic amino acids. We investigated whether direct detection of l-phenylalanine (l-Phe) by CaSR results in CCK secretion in the native I cell. Fluorescence-activated cell sorting of duodenal I cells from CCK-enhanced green fluorescent protein (eGFP) transgenic mice demonstrated CaSR gene expression. Immunostaining of fixed and fresh duodenal tissue sections confirmed CaSR protein expression. Intracellular calcium fluxes were CaSR dependent, stereoselective for l-Phe over d-Phe, and responsive to type II calcimimetic cinacalcet in CCK-eGFP cells. Additionally, CCK secretion by an isolated I cell population was increased by 30 and 62% in response to l-Phe in the presence of physiological (1.26 mM) and superphysiological (2.5 mM) extracellular calcium concentrations, respectively. While the deletion of CaSR from CCK-eGFP cells did not affect basal CCK secretion, the effect of l-Phe or cinacalcet on intracellular calcium flux was lost. In fact, both secretagogues, as well as superphysiological Ca2+, evoked an unexpected 20–30% decrease in CCK secretion compared with basal secretion in CaSR−/− CCK-eGFP cells. CCK secretion in response to KCl or tryptone was unaffected by the absence of CaSR. The present data suggest that CaSR is required for hormone secretion in the specific response to l-Phe by the native I cell, and that a receptor-mediated mechanism may inhibit hormone secretion in the absence of a fully functional CaSR. PMID:21252045

Thermodynamics of the complex formation of copper(II) with L-phenylalanine in aqueous ethanol solutions

NASA Astrophysics Data System (ADS)

Burov, D. M.; Ledenkov, S. F.; Vandyshev, V. N.

2013-05-01

Constants of the acid dissociation and complexation of L-phenylalanine (HPhe) with copper(II) ions are determined by potentiometry in aqueous ethanol solutions containing 0 to 0.7 molar fraction of alcohol. Changes in the Gibbs energy for the transfer from water to a binary solvent of L-phenylalanine, Phe- anion, and [CuPhe]+ complex are calculated. It is found that the weakening of solvation of the ligand donor groups in solvents with high ethanol contents is accompanied by an increase in the stability of [CuPhe]+ complex.

The inhibitory effect of somatostatin peptides on the rat anococcygeus muscle in vitro.

PubMed Central

Priestley, T.; Woodruff, G. N.

1988-01-01

1. Electrically evoked contractions of the rat anococcygeus muscle were inhibited in a concentration-dependent manner by somatostatin-14 (SS14), -28 (SS28) and two synthetic hexapeptide analogues: L-363,301 (Pro-Phe-D-Trp-Lys-Thr-Phe) and L-363,586 (N-Me-Ala-Tyr-D-Trp-Lys-Val-Phe), with pIC50 values of 7.41, 7.38, 7.07 and 8.34, respectively. 2. The inhibitory effects of SS14 were dependent on stimulation frequency and external calcium ion concentration. Calcium behaved as a non-competitive antagonist of SS14, it reduced the maximal inhibitory effect of the peptide and at a concentration of 5.08 mM it significantly affected the pIC50 value. 3. SS14 (3 x 10(-7) M) did not affect the tonic actions of bath-applied noradrenaline in the absence of field stimulation. 4. The effects of SS14 persisted in naloxone (10(-5) M) and were, therefore, not due to an action at opiate receptors. Furthermore, experiments involving the lyophilization of bath contents, showed no evidence to support an indirect mechanism involving the release of an endogenous inhibitory substance. 5. High concentrations (10(-5) M) of SS14 or L-363,301 inhibited the relaxation response evoked by electrical stimulation of guanethidine (3 x 10(-4) M)-treated preparations. 6. These results are consistent with similar actions of SS14 on other smooth muscle preparations and are presumed to reflect a presynaptic inhibition of transmitter release by a direct action on somatostatin receptors. The antagonistic effect of calcium on this response is discussed with reference to a possible role in receptor desensitization. PMID:2900039

Preparation of amino acid-based polymer functionalized magnetic nanoparticles as adsorbents for analysis of plant growth regulators in bean sprouts.

PubMed

Ji, Shilei; Qi, Li; Li, Nan; Wang, Minglin

2016-09-01

A novel magnetic solid phase extraction (MSPE) adsorbent has been developed for enriching two plant growth regulators, including 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-chlorophenoxyacetic acid (4-CPA), in bean sprouts. For preparing the MSPE adsorbent, poly(N-methacryloyl-L-phenylalanine methyl ester (P(MA-L-Phe-OMe)), amino acid-based polymer, was modified onto the magnetic nanoparticles via "grafting to" method by free radical polymerization. The resultant P(MA-L-Phe-OMe)-functionalized magnetic nanoparticles (Fe3O4@P(MA-L-Phe-OMe)) were characterized by Fourier transform infrared (FT-IR) spectroscopy and elemental analysis. The adsorption amount of Fe3O4@P(MA-L-Phe-OMe) nanoparticles to 2,4-D and 4-CPA were 39.82mgg(-1) and 29.02mgg(-1), respectively. Moreover, the prepared MSPE adsorbents showed good selectivity towards 2,4-D and 4-CPA due to the hydrophobic interactions and electrostatic forces between the target analytes and Fe3O4@P(MA-L-Phe-OMe). The results demonstrated that the proposed MSPE adsorbents have high affinity to the targets 2,4-D and 4-CPA. Under the optimized conditions, the proposed materials were successfully applied to enrich 2,4-D and 4-CPA in bean sprouts samples. The recovery values of the bean sprouts solution spiked the targets were from 90.9% to 96.4% with the relative standard deviations of 2.3-3.9%. Our work proved that the novel Fe3O4@P(MA-L-Phe-OMe) nanoparticles were the good adsorbents of magnetic solid phase extraction (MSPE) and have good potential for the analysis of trace compound in real samples. Copyright © 2016 Elsevier B.V. All rights reserved.

L-Phenylalanine concentration in blood of phenylketonuria patients: a modified enzyme colorimetric assay compared with amino acid analysis, tandem mass spectrometry, and HPLC methods.

PubMed

De Silva, Veronica; Oldham, Charlie D; May, Sheldon W

2010-09-01

Phenylketonuria (PKU) is an autosomal recessive disorder caused by an impaired conversion of L-phenylalanine (Phe) to L-tyrosine, typically resulting from a deficiency in activity of a hepatic and renal enzyme L-phenylalanine hydroxylase. The disease is characterized by an increased concentration of Phe and its metabolites in body fluids. A modified assay based on an enzymatic-colorimetric methodology was developed for measuring blood Phe levels in PKU patients; this method is designed for use with undeproteinized samples and avoids the use of solvents or amphiphilic agents. Thus, the method could be suitable for incorporation into a simple home-monitoring device. We report here on a comparison of blood Phe concentrations in PKU patients measured in undeproteinized plasma using this enzyme colorimetric assay (ECA), with values determined by amino acid analysis (AAA) of deproteinized samples, and HPLC and tandem mass spectrometry (MS/MS) analyses of dried blood spot (DBS) eluates. Pearson correlation coefficients of 0.951, 0.976 and 0.988 were obtained when AAA-measured Phe concentrations were compared with the ECA-, HPLC- or MS/MS-measured values, respectively. A Bland-Altman analysis revealed that mean Phe concentrations determined using AAA were on average 65 μmol/L lower than values measured by our ECA. These results may be the result of minimizing the manipulations performed on the patient sample compared with AAA, HPLC, and MS/MS methods, which involve plasma deproteinization or DBS elution and derivatization. The results reported here confirm that Phe concentrations determined by our ECA method are comparable to those determined by other widely used methods for a broad range of plasma Phe concentrations.

Sensitive, Site-Specific, and Stable Vibrational Probe of Local Protein Environments: 4-Azidomethyl-L-Phenylalanine

PubMed Central

Bazewicz, Christopher G.; Liskov, Melanie T.; Hines, Kevin J.; Brewer, Scott H.

2013-01-01

We have synthesized the unnatural amino acid (UAA), 4-azidomethyl-Lphenylalanine (pN3CH2Phe), to serve as an effective vibrational reporter of local protein environments. The position, extinction coefficient, and sensitivity to local environment of the azide asymmetric stretch vibration of pN3CH2Phe are compared to the vibrational reporters: 4-cyano-L-phenylalanine (pCNPhe) and 4-azido-L-phenylalanine (pN3Phe). This UAA was genetically incorporated in a site-specific manner utilizing an engineered, orthogonal aminoacyl-tRNA synthetase in response to an amber codon with high efficiency and fidelity into two distinct sites in superfolder green fluorescent protein (sfGFP). This allowed for the dependence of the azide asymmetric stretch vibration of pN3CH2Phe to different protein environments to be measured. The photo-stability of pN3CH2Phe was also measured relative to the photoreactive UAA, pN3Phe. PMID:23865850

Properties of chymotrypsin bound covalently to dextran.

PubMed

Zlateva, T P; Krysteva, M; Balajthy, Z; Elödi, P

1988-01-01

The kinetic properties dextran-chymotrypsin conjugate were studied by means of low molecular weight substrates. It was found that KM, kcat and kcat/KM of dextran chymotrypsin for the hydrolysis of benzoyl-L-tyrosine-ethyl-ester did not differ substantially from those of the free enzyme. However, the data found for kcat of dextran-chymotrypsin and free chymotrypsin assayed for the hydrolysis of three tripeptidyl-p-nitroanilide D-Arg-Val-Trp-pNA, D-Arg-Val-Tyr-pNA, Z-Phe-Pro-Phe-pNA, were definitely different. The inhibition of the modified chymotrypsin with soybean trypsin inhibitor was found to be less pronounced than that with the free enzyme. The effect of potassium and magnesium salts on the inactivation of both enzymes was also studied. The effect of dextran matrix on the catalytic properties and the conformational stability of modified chymotrypsin is discussed.

Effect of aging on intestinal absorption of aromatic amino acids in vitro in the rat

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

Navab, F.; Winter, C.G.

Whole-thickness everted jejunal rings were used to measure uptake of L-tyrosine (L-Tyr), L-phenylalanine (L-Phe), and L-tryptophan (L-Trp) in 6-, 12-, and 24-mo-old rats. The rate of uptake of all tree amino acids (1 mM) was significantly reduced after 20 min of incubation in 24-mo-old compared with 6-mo-old rats. Results of influx (2 min) of 0.5-40.0 mL L-Phe and L-Trp suggested an increased affinity but decreased capacity for the transporter with age; these differences were significant for L-Trp. Respective values for apparent K{sub t} and V{sub max} are given.

Improvement of L-phenylalanine production from glycerol by recombinant Escherichia coli strains: The role of extra copies of glpK, glpX, and tktA genes

PubMed Central

2014-01-01

Background For the production of L-phenylalanine (L-Phe), two molecules of phosphoenolpyruvate (PEP) and one molecule erythrose-4-phosphate (E4P) are necessary. PEP stems from glycolysis whereas E4P is formed in the pentose phosphate pathway (PPP). Glucose, commonly used for L-Phe production with recombinant E. coli, is taken up via the PEP-dependent phosphotransferase system which delivers glucose-6-phosphate (G6P). G6P enters either glycolysis or the PPP. In contrast, glycerol is phosphorylated by an ATP-dependent glycerol kinase (GlpK) thus saving one PEP. However, two gluconeogenic reactions (fructose-1,6-bisphosphate aldolase, fructose-1,6-bisphosphatase, FBPase) are necessary for growth and provision of E4P. Glycerol has become an important carbon source for biotechnology and reports on production of L-Phe from glycerol are available. However, the influence of FBPase and transketolase reactions on L-Phe production has not been reported. Results L-Phe productivity of parent strain FUS4/pF81 (plasmid-encoded genes for aroF, aroB, aroL, pheA) was compared on glucose and glycerol as C sources. On glucose, a maximal carbon recovery of 0.19 mM CPhe/CGlucose and a maximal space-time-yield (STY) of 0.13 g l−1 h−1 was found. With glycerol, the maximal carbon recovery was nearly the same (0.18 mM CPhe/CGlycerol), but the maximal STY was higher (0.21 g l−1 h−1). We raised the chromosomal gene copy number of the genes glpK (encoding glycerol kinase), tktA (encoding transketolase), and glpX (encoding fructose-1,6-bisphosphatase) individually. Overexpression of glpK (or its feedback-resistant variant, glpKG232D) had little effect on growth rate; L-Phe production was about 30% lower than in FUS4/pF81. Whereas the overexpression of either glpX or tktA had minor effects on productivity (0.20 mM CPhe/CGlycerol; 0.25 g l−1 h−1 and 0.21 mM CPhe/CGlycerol; 0.23 g l−1 h−1, respectively), the combination of extra genes of glpX and tktA together led to an increase in maximal STY of about 80% (0.37 g l−1 h−1) and a carbon recovery of 0.26 mM CPhe/CGlycerol. Conclusions Enhancing the gene copy numbers for glpX and tktA increased L-Phe productivity from glycerol without affecting growth rate. Engineering of glycerol metabolism towards L-Phe production in E. coli has to balance the pathways of gluconeogenesis, glycolysis, and PPP to improve the supply of the precursors, PEP and E4P. PMID:25012491

Creating Prebiotic Sanctuary: Self-Assembling Supramolecular Peptide Structures Bind and Stabilize RNA

NASA Astrophysics Data System (ADS)

Carny, Ohad; Gazit, Ehud

2011-04-01

Any attempt to uncover the origins of life must tackle the known `blind watchmaker problem'. That is to demonstrate the likelihood of the emergence of a prebiotic system simple enough to be formed spontaneously and yet complex enough to allow natural selection that will lead to Darwinistic evolution. Studies of short aromatic peptides revealed their ability to self-assemble into ordered and stable structures. The unique physical and chemical characteristics of these peptide assemblies point out to their possible role in the origins of life. We have explored mechanisms by which self-assembling short peptides and RNA fragments could interact together and go through a molecular co-evolution, using diphenylalanine supramolecular assemblies as a model system. The spontaneous formation of these self-assembling peptides under prebiotic conditions, through the salt-induced peptide formation (SIPF) pathway was demonstrated. These peptide assemblies possess the ability to bind and stabilize ribonucleotides in a sequence-depended manner, thus increase their relative fitness. The formation of these peptide assemblies is dependent on the homochirality of the peptide monomers: while homochiral peptides (L-Phe-L-Phe and D-Phe-D-Phe) self-assemble rapidly in aqueous environment, heterochiral diastereoisomers (L-Phe-D-Phe and D-Phe-L-Phe) do not tend to self-assemble. This characteristic consists with the homochirality of all living matter. Finally, based on these findings, we propose a model for the role of short self-assembling peptides in the prebiotic molecular evolution and the origin of life.

Creating prebiotic sanctuary: self-assembling supramolecular Peptide structures bind and stabilize RNA.

PubMed

Carny, Ohad; Gazit, Ehud

2011-04-01

Any attempt to uncover the origins of life must tackle the known 'blind watchmaker problem'. That is to demonstrate the likelihood of the emergence of a prebiotic system simple enough to be formed spontaneously and yet complex enough to allow natural selection that will lead to Darwinistic evolution. Studies of short aromatic peptides revealed their ability to self-assemble into ordered and stable structures. The unique physical and chemical characteristics of these peptide assemblies point out to their possible role in the origins of life. We have explored mechanisms by which self-assembling short peptides and RNA fragments could interact together and go through a molecular co-evolution, using diphenylalanine supramolecular assemblies as a model system. The spontaneous formation of these self-assembling peptides under prebiotic conditions, through the salt-induced peptide formation (SIPF) pathway was demonstrated. These peptide assemblies possess the ability to bind and stabilize ribonucleotides in a sequence-depended manner, thus increase their relative fitness. The formation of these peptide assemblies is dependent on the homochirality of the peptide monomers: while homochiral peptides (L-Phe-L-Phe and D-Phe-D-Phe) self-assemble rapidly in aqueous environment, heterochiral diastereoisomers (L-Phe-D-Phe and D-Phe-L-Phe) do not tend to self-assemble. This characteristic consists with the homochirality of all living matter. Finally, based on these findings, we propose a model for the role of short self-assembling peptides in the prebiotic molecular evolution and the origin of life.

Nitrogen recycling during phenylpropanoid metabolism in sweet potato tubers

NASA Technical Reports Server (NTRS)

Singh, S.; Lewis, N. G.; Towers, G. H.

1998-01-01

In the first step of the phenylpropanoid metabolic pathway, L-phenylalanine (L-Phe) is deaminated to form E-cinnamate, in a conversion catalyzed by phenylalanine ammonia-lyase (PAL; EC 4.3.1.5). The metabolic fate of the ammonium ion (NH4+) produced in this reaction was investigated in sweet potato (Ipomoea batatas) tuber discs. [15N]-Labeled substrates including L-Phe, in the presence or absence of specific enzyme inhibitors, were administered to sweet potato discs in light under aseptic conditions. 15N-Nuclear magnetic resonance spectroscopic analyses revealed that the 15NH4+ liberated during the PAL reaction is first incorporated into the amide nitrogen of L-glutamine (L-Gln) and then into L-glutamate (L-Glu). These results extend our previous observations in pine and potato that PAL-generated NH4+ is assimilated by the glutamine synthetase (GS; EC 6.3.1.2)/glutamate synthase (GOGAT; EC 1.4.1.13) pathway, with the NH4+ so formed ultimately being recycled back to L-Phe via L-Glu as aminoreceptor and donor.

Secondary structure inducing potential of beta-amino acids: torsion angle clustering facilitates comparison and analysis of the conformation during MD trajectories.

PubMed

Guthöhrlein, E W; Malesević, M; Majer, Z; Sewald, N

2007-01-01

While numerous examples of beta-peptides--exclusively composed of beta-amino acids--have been investigated during the past decade, there are only few reports on the conformational preference of a single beta-amino acid when incorporated into a cyclopeptide. The conformational bias of beta-amino acids on the secondary structure of cyclopeptides has been investigated by NMR spectroscopy in combination with distance geometry (DG) and molecular dynamics (MD) calculations using experimental constraints. The atomic coordinate RMSD criterion usually employed for clustering of conformations after DG and MD calculations does not necessarily group similar peptide conformations, as there is an insufficient correlation between atomic coordinates and torsion angles. To improve on this shortcoming and to eliminate any arbitrary decisions during this process, a torsion angle clustering procedure has been implemented. For the cyclic pentapeptides cyclo-(-Val-beta-Hala-Phe-Leu-Ile-) 1 and cyclo-(-Ser-Pro-Leu-beta-Hasn-Asp-) 3, the beta-amino acid is found in the central position of an extended gamma-turn (pseudo gamma-turn, Psigamma-turn), while the beta-Hpro residue in the cyclic hexapeptide cyclo-(-Ser-beta-Hpro-Leu-Asn-Ile-Asp-) 5 preferentially occupies position i+1 of a pseudo beta-turn (Psibeta-turn). These results further corroborate the hypothesis of beta-amino acids being reliable inducers of secondary structure in cyclic penta- and hexapeptides. They can be employed in the de novo design of biologically active cyclopeptides in pharmaceutical research, since the three-dimensional presentation of pharmacophoric groups in the side chains can be tailored by incorporation of beta-amino acids in strategic sequential positions. (c) 2007 Wiley Periodicals, Inc.

Oxidation of L-phenylalanine by diperiodatoargentate(III) in aqueous alkaline medium. A Mechanistic approach

NASA Astrophysics Data System (ADS)

Lamani, S. D.; Veeresh, T. M.; Nandibewoor, S. T.

2009-12-01

The kinetics of oxidation of L-phenylalanine (L-Phe) by diperiodatoargentate(III) (DPA) in alkaline medium at a constant ionic strength of 0.25 mol/dm-3 has been studied spectrophotometrically. The reaction between DPA and L-phenylalanine in alkaline medium exhibits 1: 1 stoichiometry (L-phenylalanine: DPA). The reaction shows first order in [DPA] and has less than unit order dependence each in both [L-Phe] and [Alkali] and retarding effect of [IO{4/-}] under the reaction conditions. The active species of DPA is understood to be as monoperiodatoargentate(III) (MPA). The reaction is shown to proceed via a MPA-L-Phe complex, which decomposes in a rate-determining step to give intermediates followed by a fast step to give the products. The products were identified by spot and spectroscopic studies. The reaction constants involved in the different steps of the mechanisms were calculated. The activation parameters with respect to slow step of the mechanism were computed and discussed. The thermodynamic quantities were also determined for the reaction.

Aromatic dipeptides and their salts—Solid-state linear-dichroic infrared (IR-LD) spectral analysis and ab initio calculations

NASA Astrophysics Data System (ADS)

Ivanova, Bojidarka B.

2008-07-01

Stereo-structural analysis and IR-bands assignment of the aromatic dipeptides L-tryrosyl- L-phenylalanine ( Tyr-Phe), L-phenylalanyl- L-tyrosine ( Phe-Tyr) and their hydrochloride salts have been carried out by means of IR-LD spectroscopy of oriented as nematic liquid crystal suspension solid samples. The experimental data are compared with known crystallographic ones and theoretical predicted geometries at RHF/ and UHF/6-31G**.

Glycomacropeptide for nutritional management of phenylketonuria: a randomized, controlled, crossover trial12

PubMed Central

Stroup, Bridget M; Clayton, Murray K; Murali, Sangita G; Rice, Gregory M; Rohr, Frances; Levy, Harvey L

2016-01-01

Background: To prevent cognitive impairment, phenylketonuria requires lifelong management of blood phenylalanine (Phe) concentration with a low-Phe diet. The diet restricts intake of Phe from natural proteins in combination with traditional amino acid medical foods (AA-MFs) or glycomacropeptide medical foods (GMP-MFs) that contain primarily intact protein and a small amount of Phe. Objective: We investigated the efficacy and safety of a low-Phe diet combined with GMP-MFs or AA-MFs providing the same quantity of protein equivalents in free-living subjects with phenylketonuria. Design: This 2-stage, randomized crossover trial included 30 early-treated phenylketonuria subjects (aged 15–49 y), 20 with classical and 10 with variant phenylketonuria. Subjects consumed, in random order for 3 wk each, their usual low-Phe diet combined with AA-MFs or GMP-MFs. The treatments were separated by a 3-wk washout with AA-MFs. Fasting plasma amino acid profiles, blood Phe concentrations, food records, and neuropsychological tests were obtained. Results: The frequency of medical food intake was higher with GMP-MFs than with AA-MFs. Subjects rated GMP-MFs as more acceptable than AA-MFs and noted improved gastrointestinal symptoms and less hunger with GMP-MFs. ANCOVA indicated no significant mean ± SE increase in plasma Phe (62 ± 40 μmol/L, P = 0.136), despite a significant increase in Phe intake from GMP-MFs (88 ± 6 mg Phe/d, P = 0.026). AA-MFs decreased plasma Phe (−85 ± 40 μmol/L, P = 0.044) with stable Phe intake. Blood concentrations of Phe across time were not significantly different (AA-MFs = 444 ± 34 μmol/L, GMP-MFs = 497 ± 34 μmol/L), suggesting similar Phe control. Results of the Behavior Rating Inventory of Executive Function were not significantly different. Conclusions: GMP-MFs provide a safe and acceptable option for the nutritional management of phenylketonuria. The greater acceptability and fewer side effects noted with GMP-MFs than with AA-MFs may enhance dietary adherence for individuals with phenylketonuria. This trial was registered at www.clinicaltrials.gov as NCT01428258. PMID:27413125

Glycomacropeptide for nutritional management of phenylketonuria: a randomized, controlled, crossover trial.

PubMed

Ney, Denise M; Stroup, Bridget M; Clayton, Murray K; Murali, Sangita G; Rice, Gregory M; Rohr, Frances; Levy, Harvey L

2016-08-01

To prevent cognitive impairment, phenylketonuria requires lifelong management of blood phenylalanine (Phe) concentration with a low-Phe diet. The diet restricts intake of Phe from natural proteins in combination with traditional amino acid medical foods (AA-MFs) or glycomacropeptide medical foods (GMP-MFs) that contain primarily intact protein and a small amount of Phe. We investigated the efficacy and safety of a low-Phe diet combined with GMP-MFs or AA-MFs providing the same quantity of protein equivalents in free-living subjects with phenylketonuria. This 2-stage, randomized crossover trial included 30 early-treated phenylketonuria subjects (aged 15-49 y), 20 with classical and 10 with variant phenylketonuria. Subjects consumed, in random order for 3 wk each, their usual low-Phe diet combined with AA-MFs or GMP-MFs. The treatments were separated by a 3-wk washout with AA-MFs. Fasting plasma amino acid profiles, blood Phe concentrations, food records, and neuropsychological tests were obtained. The frequency of medical food intake was higher with GMP-MFs than with AA-MFs. Subjects rated GMP-MFs as more acceptable than AA-MFs and noted improved gastrointestinal symptoms and less hunger with GMP-MFs. ANCOVA indicated no significant mean ± SE increase in plasma Phe (62 ± 40 μmol/L, P = 0.136), despite a significant increase in Phe intake from GMP-MFs (88 ± 6 mg Phe/d, P = 0.026). AA-MFs decreased plasma Phe (-85 ± 40 μmol/L, P = 0.044) with stable Phe intake. Blood concentrations of Phe across time were not significantly different (AA-MFs = 444 ± 34 μmol/L, GMP-MFs = 497 ± 34 μmol/L), suggesting similar Phe control. Results of the Behavior Rating Inventory of Executive Function were not significantly different. GMP-MFs provide a safe and acceptable option for the nutritional management of phenylketonuria. The greater acceptability and fewer side effects noted with GMP-MFs than with AA-MFs may enhance dietary adherence for individuals with phenylketonuria. This trial was registered at www.clinicaltrials.gov as NCT01428258. © 2016 American Society for Nutrition.

Structure and dynamics of H2O vis-á-vis phenylalanine recognition at a DPPC lipid membrane via interfacial H-bond types: insights from polarized FT-IRRAS and ADMP simulations.

PubMed

Sarangi, Nirod Kumar; Ramesh, Nivarthi; Patnaik, Archita

2015-01-14

Preferential and enantioselective interactions of L-/D-Phenylalanine (L-Phe and D-Phe) and butoxycarbonyl-protected L-/D-Phenylalanine (LPA and DPA) as guest with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (L-DPPC) as host were tapped by using real time Fourier transform infrared reflection absorption spectroscopy (FT-IRRAS). Polarization-modulated FT-IRRAS of DPPC monolayers above the phenylalanine modified subphases depicted fine structure/conformation differences under considerations of controlled 2D surface pressure. Selective molecular recognition of D-enantiomer over L-enantiomer driven by the DPPC head group via H-bonding and electrostatic interactions was evident spectroscopically. Accordingly, binding constants (K) of 145, 346, 28, and 56 M(-1) for LPA, DPA, L-Phe, and D-Phe, respectively, were estimated. The real time FT-IRRAS water bands were strictly conformation sensitive. The effect of micro-solvation on the structure and stability of the 1:1 diastereomeric L-lipid⋯, LPA/DPA and L-lipid⋯, (L/D)-Phe adducts was investigated with the aid of Atom-centered Density Matrix Propagation (ADMP), a first principle quantum mechanical molecular dynamics approach. The phosphodiester fragment was the primary site of hydration where specific solvent interactions were simulated through single- and triple- "water-phosphate" interactions, as water cluster's "tetrahedral dice" to a "trimeric motif" transformation as a partial de-clusterization was evident. Under all the hydration patterns considered in both static and dynamic descriptions of density functional theory, L-lipid/D-amino acid enantiomer adducts continued to be stable structures while in dynamic systems, water rearranged without getting "squeezed-out" in the process of recognition. In spite of the challenging computational realm of this multiscale problem, the ADMP simulated molecular interactions complying with polarized vibrational spectroscopy unraveled a novel route to chiral recognition and interfacial water structure.

Structure and dynamics of H2O vis-á-vis phenylalanine recognition at a DPPC lipid membrane via interfacial H-bond types: Insights from polarized FT-IRRAS and ADMP simulations

NASA Astrophysics Data System (ADS)

Sarangi, Nirod Kumar; Ramesh, Nivarthi; Patnaik, Archita

2015-01-01

Preferential and enantioselective interactions of l-/d-Phenylalanine (l-Phe and d-Phe) and butoxycarbonyl-protected l-/d-Phenylalanine (LPA and DPA) as guest with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (l-DPPC) as host were tapped by using real time Fourier transform infrared reflection absorption spectroscopy (FT-IRRAS). Polarization-modulated FT-IRRAS of DPPC monolayers above the phenylalanine modified subphases depicted fine structure/conformation differences under considerations of controlled 2D surface pressure. Selective molecular recognition of d-enantiomer over l-enantiomer driven by the DPPC head group via H-bonding and electrostatic interactions was evident spectroscopically. Accordingly, binding constants (K) of 145, 346, 28, and 56 M-1 for LPA, DPA, l-Phe, and d-Phe, respectively, were estimated. The real time FT-IRRAS water bands were strictly conformation sensitive. The effect of micro-solvation on the structure and stability of the 1:1 diastereomeric l-lipid⋯, LPA/DPA and l-lipid⋯, (l/d)-Phe adducts was investigated with the aid of Atom-centered Density Matrix Propagation (ADMP), a first principle quantum mechanical molecular dynamics approach. The phosphodiester fragment was the primary site of hydration where specific solvent interactions were simulated through single- and triple- "water-phosphate" interactions, as water cluster's "tetrahedral dice" to a "trimeric motif" transformation as a partial de-clusterization was evident. Under all the hydration patterns considered in both static and dynamic descriptions of density functional theory, l-lipid/d-amino acid enantiomer adducts continued to be stable structures while in dynamic systems, water rearranged without getting "squeezed-out" in the process of recognition. In spite of the challenging computational realm of this multiscale problem, the ADMP simulated molecular interactions complying with polarized vibrational spectroscopy unraveled a novel route to chiral recognition and interfacial water structure.

Structure and dynamics of H{sub 2}O vis-á-vis phenylalanine recognition at a DPPC lipid membrane via interfacial H-bond types: Insights from polarized FT-IRRAS and ADMP simulations

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

Sarangi, Nirod Kumar; Ramesh, Nivarthi; Patnaik, Archita

Preferential and enantioselective interactions of L-/D-Phenylalanine (L-Phe and D-Phe) and butoxycarbonyl-protected L-/D-Phenylalanine (LPA and DPA) as guest with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (L-DPPC) as host were tapped by using real time Fourier transform infrared reflection absorption spectroscopy (FT-IRRAS). Polarization-modulated FT-IRRAS of DPPC monolayers above the phenylalanine modified subphases depicted fine structure/conformation differences under considerations of controlled 2D surface pressure. Selective molecular recognition of D-enantiomer over L-enantiomer driven by the DPPC head group via H-bonding and electrostatic interactions was evident spectroscopically. Accordingly, binding constants (K) of 145, 346, 28, and 56 M{sup −1} for LPA, DPA, L-Phe, and D-Phe, respectively, were estimated. The realmore » time FT-IRRAS water bands were strictly conformation sensitive. The effect of micro-solvation on the structure and stability of the 1:1 diastereomeric L-lipid⋯, LPA/DPA and L-lipid⋯, (L/D)-Phe adducts was investigated with the aid of Atom-centered Density Matrix Propagation (ADMP), a first principle quantum mechanical molecular dynamics approach. The phosphodiester fragment was the primary site of hydration where specific solvent interactions were simulated through single- and triple- “water-phosphate” interactions, as water cluster’s “tetrahedral dice” to a “trimeric motif” transformation as a partial de-clusterization was evident. Under all the hydration patterns considered in both static and dynamic descriptions of density functional theory, L-lipid/D-amino acid enantiomer adducts continued to be stable structures while in dynamic systems, water rearranged without getting “squeezed-out” in the process of recognition. In spite of the challenging computational realm of this multiscale problem, the ADMP simulated molecular interactions complying with polarized vibrational spectroscopy unraveled a novel route to chiral recognition and interfacial water structure.« less

Mechanism behind Resistance against the Organophosphate Azamethiphos in Salmon Lice (Lepeophtheirus salmonis)

PubMed Central

Kaur, Kiranpreet; Helgesen, Kari Olli; Bakke, Marit Jørgensen; Horsberg, Tor Einar

2015-01-01

Acetylcholinesterase (AChE) is the primary target for organophosphates (OP). Several mutations have been reported in AChE to be associated with the reduced sensitivity against OP in various arthropods. However, to the best of our knowledge, no such reports are available for Lepeophtheirus salmonis. Hence, in the present study, we aimed to determine the association of AChE(s) gene(s) with resistance against OP. We screened the AChE genes (L. salmonis ace1a and ace1b) in two salmon lice populations: one sensitive (n=5) and the other resistant (n=5) for azamethiphos, a commonly used OP in salmon farming. The screening led to the identification of a missense mutation Phe362Tyr in L. salmonis ace1a, (corresponding to Phe331 in Torpedo californica AChE) in all the samples of the resistant population. We confirmed the potential role of the mutation, with reduced sensitivity against azamethiphos in L. salmonis, by screening for Phe362Tyr in 2 sensitive and 5 resistant strains. The significantly higher frequency of the mutant allele (362Tyr) in the resistant strains clearly indicated the possible association of Phe362Tyr mutation in L. salmonis ace1a with resistance towards azamethiphos. The 3D modelling, short term survival experiments and enzymatic assays further supported the imperative role of Phe362Tyr in reduced sensitivity of L. salmonis for azamethiphos. Based on all these observations, the present study, for the first time, presents the mechanism of resistance in L. salmonis against azamethiphos. In addition, we developed a rapid diagnostic tool for the high throughput screening of Phe362Tyr mutation using High Resolution Melt analysis. PMID:25893248

Improving in vivo conversion of oleuropein into hydroxytyrosol by oral granules containing probiotic Lactobacillus plantarum 299v and an Olea europaea standardized extract.

PubMed

Aponte, Maria; Ungaro, Francesca; d'Angelo, Ivana; De Caro, Carmen; Russo, Roberto; Blaiotta, Giuseppe; Dal Piaz, Fabrizio; Calignano, Antonio; Miro, Agnese

2018-05-30

This study reports novel food-grade granules for co-delivery of L. plantarum 299v and a standardized extract of Olea europaea leaves (Phenolea®) as oral carrier of probiotics and hydroxytyrosol. Different granule formulations containing either L. plantarum 299v (Lac), or the olive leave extract (Phe) or their combination (Lac-Phe) have been successfully produced through wet granulation employing excipients generally regarded as safe as granulating/binding agents. L. plantarum cells withstood the manufacturing process and were stable upon storage at 4 °C for more than 6 months. In vitro dissolution studies in simulated gastro-intestinal fluids showed the capability of the granules to rapidly dissolve and deliver both olive leave phenols and living L. plantarum cells. In simulated digestion conditions, Lac and Lac-Phe granules protected L. plantarum against the harsh environment of the gastro-intestinal tract. Co-administration of Lac and Phe oral granules to healthy mice provided for higher amounts of hydroxytyrosol in urines as compared to Phe granules alone, suggesting that L. plantarum 299v boosted in vivo conversion of oleuropein to hydroxytyrosol. On the other hand, PCR-assisted profiling of the Lactobacillus population in faeces obtained from mice treated with Lac or Lac plus Phe confirmed that the probiotic arrived alive to colon and was there able to exert a sort of perturbing effect on the climax colonic microflora. Overall, these results pave the way towards the development of a nutraceutical useful for combined delivery of bioactive hydroxytyrosol and probiotics to colon site. Copyright © 2018 Elsevier B.V. All rights reserved.

Versatile Picklocks To Access All Opioid Receptors: Tuning the Selectivity and Functional Profile of the Cyclotetrapeptide c[Phe-d-Pro-Phe-Trp] (CJ-15,208).

PubMed

De Marco, Rossella; Bedini, Andrea; Spampinato, Santi; Cavina, Lorenzo; Pirazzoli, Edoardo; Gentilucci, Luca

2016-10-13

Recently, the tryptophan-containing noncationizable opioid peptides emerged with atypical structure and unexpected in vivo activity. Herein, we describe analogs of the naturally occurring mixed κ/μ-ligand c[Phe-d-Pro-Phe-Trp] 1 (CJ-15,208). Receptor affinity, selectivity, and agonism/antagonism varied upon enlarging macrocycle size, giving the μ-agonist 9 or the δ-antagonist 10 characterized by low nanomolar affinity. In particular, the μ-agonist c[β-Ala-d-Pro-Phe-Trp] 9 was shown to elicit potent antinociception in a mouse model of visceral pain upon systemic administration.

Production of the angiotensin I converting enzyme inhibitory peptides and isolation of four novel peptides from jellyfish (Rhopilema esculentum) protein hydrolysate.

PubMed

Liu, Xin; Zhang, Miansong; Shi, Yaping; Qiao, Ruojin; Tang, Wei; Sun, Zhenliang

2016-07-01

Angiotensin I converting enzyme (ACE) plays an important role in regulating blood pressure in the human body. ACE inhibitory peptides derived from food proteins could exert antihypertensive effects without side effects. Jellyfish (Rhopilema esculentum) is an important fishery resource suitable for production of ACE inhibitory peptides. The objective of this study was to optimize the hydrolysis conditions for production of protein hydrolysate from R. esculentum (RPH) with ACE inhibitory activity, and to isolate and identify the ACE inhibitory peptides from RPH. Rhopilema esculentum protein was hydrolyzed with Compound proteinase AQ to produce protein hydrolysate with ACE inhibitory activity, and the hydrolysis conditions were optimized using response surface methodology. The optimum parameters for producing peptides with the highest ACE inhibitory activity were as follows: hydrolysis time 3.90 h, hydrolysis temperature 58 °C, enzyme:substrate ratio 2.8% and pH 7.60. Under these conditions, the ACE inhibitory rate reached 32.21%. In addition, four novel ACE inhibitory peptides were isolated, and their amino acids sequences were identified as Val-Gly-Pro-Tyr, Phe-Thr-Tyr-Val-Pro-Gly, Phe-Thr-Tyr-Val-Pro-Gly-Ala and Phe-Gln-Ala-Val-Trp-Ala-Gly, respectively. The IC50 value of the purified peptides for ACE inhibitory activity was 8.40, 23.42, 21.15 and 19.11 µmol L(-1) . These results indicate that the protein hydrolysate prepared from R. esculentum might be a commercial competitive source of ACE inhibitory ingredients to be used in functional foods. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

Determination of L-phenylalanine on-line based on molecularly imprinted polymeric microspheres and flow injection chemiluminescence

NASA Astrophysics Data System (ADS)

Qiu, Huamin; Xi, Yulei; Lu, Fuguang; Fan, Lulu; Luo, Chuannan

2012-02-01

A novel molecular imprinting-chemiluminescence (MIP-CL) sensor for the determination of L-phenylalanine (Phe) using molecularly imprinted polymer (MIP) as recognition element is reported. The Phe-MIP was synthesized using acrylamide (AM) as functional monomer and ethylene glycol dimethacrylate (EGDMA) as cross-linker, 2,2-azobisisobutyronitrile (AIBN) as initiator and the polymers' properties were characterized. Then the synthesized MIP was employed as recognition element by packing into flow cell to establish a novel flow injection CL sensor. The CL intensity responded linearly to the concentration of Phe in the range 1.3 × 10 -6 to 5.44 × 10 -4 mol/L with a detection limit of 6.23 × 10 -7 mol/L (3 σ), which is lower than that of conventional methods. The sensor is reusable and has a great improvement in sensitivity and selectivity for CL analysis. As a result, the new MIP-CL sensor had been successfully applied to the determination of Phe in samples.

[Singleton and twin pregnancies of PKU patients - individual variability of phenylalanine tolerance: experience of a single treatment center (Preliminary report)].

PubMed

Żółkowska, Joanna; Hozyasz, Kamil K; Nowacka, Maria

2017-01-01

Phenylketonuria (PKU) is the autosomal recessive deficiency of phenylalanine hydroxylase resulting in the accumulation of phenylalanine (Phe) in blood and in the brain. Phe restriction in a patient's diet is determined depending on the amount of Phe intake which allows for stable blood Phe levels within the therapeutic range of 120-360µmol/L. In clinical practice the empirical determination of Phe tolerance relies on frequent assessment of blood Phe concentrations in relation to Phe intake from food records. Untreated maternal PKU may lead to maternal PKU syndrome in offspring. The objective of the study was to compare Phe tolerance during the course of singleton and multiple pregnancies of PKU patients. Case subjects and methods: The cases reviewed included three sets of classical PKU-affected Polish women on a low-phenylalanine diet during the course of singleton and twin pregnancies and their PKU-unaffected newborns. All the patients were under regular supervision of a metabolic dietitian to stabilize blood Phe levels and determine Phe tolerance. Data on pregnancy weight gain, the gestational age when the diet initiated, the percent of Phe assessments < 120 µmol/L and > 360 µmol/L, as well as offspring birth measurements were analyzed. The total increase in Phe tolerance and its pattern during the course of singleton and twin pregnancies differed remarkably in each patient. Three PKU women (Q383X/R408W, EX3DEL/EX3DEL, R281L/R408W) increased their Phe tolerance in singleton and twin pregnancies by 579%/468%, 674%/261%, and 427%/236%, respectively. During the last 10 weeks of singleton and twin pregnancy Phe tolerance showed an increase by 62%/149%, 33%/64%, and 37%/40%, respectively. The analysis of predictors for Phe tolerance showed that an individual's weight gain and the fetal weight gain as estimated from liveborn birth-weight data had no predictive capacity. Individual Phe tolerance in singleton pregnancies of PKU patients does not predict tolerance in twin pregnancy. Further research on the growing population of multiple pregnancy PKU patients is necessary to provide evidence-based guidelines to optimize the treatment of PKU in females of childbearing age.

L-phenylalanine binding and domain organization in human phenylalanine hydroxylase: a differential scanning calorimetry study.

PubMed

Thórólfsson, Matthías; Ibarra-Molero, Beatriz; Fojan, Peter; Petersen, Steffen B; Sanchez-Ruiz, Jose M; Martínez, Aurora

2002-06-18

Human phenylalanine hydroxylase (hPAH) is a tetrameric enzyme that catalyzes the hydroxylation of L-phenylalanine (L-Phe) to L-tyrosine; a dysfunction of this enzyme causes phenylketonuria. Each subunit in hPAH contains an N-terminal regulatory domain (Ser2-Ser110), a catalytic domain (Asp112-Arg410), and an oligomerization domain (Ser411-Lys452) including dimerization and tetramerization motifs. Two partially overlapping transitions are seen in differential scanning calorimetry (DSC) thermograms for wild-type hPAH in 0.1 M Na-Hepes buffer, 0.1 M NaCl, pH 7.0. Although these transitions are irreversible, studies on their scan-rate dependence support that the equilibrium thermodynamics analysis is permissible in this case. Comparison with the DSC thermograms for truncated forms of the enzyme, studies on the protein and L-Phe concentration effects on the transitions, and structure-energetic calculations based on a modeled structure support that the thermal denaturation of hPAH occurs in three stages: (i) unfolding of the four regulatory domains, which is responsible for the low-temperature calorimetric transition; (ii) unfolding of two (out of the four) catalytic domains, which is responsible for the high-temperature transition; and (iii) irreversible protein denaturation, which is likely responsible for the observed exothermic distortion in the high-temperature side of the high-temperature transition. Stages 1 and 2 do not appear to be two-state processes. We present an approach to the analysis of ligand effects on DSC transition temperatures, which is based on the general binding polynomial formalism and is not restricted to two-state transitions. Application of this approach to the L-Phe effect on the DSC thermograms for hPAH suggests that (i) there are no binding sites for L-Phe in the regulatory domains; therefore, contrary to the common belief, the activation of PAH by L-Phe seems to be the result of its homotropic cooperative binding to the active sites. (ii) The regulatory domain appears to be involved in cooperativity through its interactions with the catalytic and oligomerization domains; thus, upon regulatory domain unfolding, the cooperativity in the binding of L-Phe to the catalytic domains seems to be lost and the value of the L-Phe concentration corresponding to half-saturation is increased. Overall, our results contribute to the understanding of the conformational stability and the substrate-induced cooperative activation of this important enzyme.

Surface-enhanced Raman scattering studies on bombesin, its selected fragments and related peptides adsorbed at the silver colloidal surface

NASA Astrophysics Data System (ADS)

Podstawka-Proniewicz, Edyta; Ozaki, Yukihiro; Kim, Younkyoo; Xu, Yizhuang; Proniewicz, Leonard M.

2011-07-01

SERS studies presented in this work on BN8-14, [ D-Phe 6,β-Ala11,Phe13,Nle14]BN6-14, [ D-Tyr 6, β-Ala11,Phe13,Nle14]BN6-14, BN and its modified analogues, as well as NMB, NMC, and PG-L show that these molecules at pH 8.3 bind to a colloidal silver surface mainly through Trp 8 and Met 14 residues. Trp 8 adsorbs at the surface almost perpendicularly. Met 14 appears on the surface mainly as a P C-G conformer. His 12, as is evident from the spectra, practically does not take part in the adsorption process. Substitution of L-leucine at the 13 position of amino acid sequence with L-phenylalanine does not change substantially the pattern of the adsorption mechanism; however, substitution of phenylalanine at the 12 position (instead of L-histidine) causes changes in the SERS spectra that show that Phe 12 takes parallel orientation to the surface upon adsorption of [ D-Phe 12]BN, while in the case of [Tyr4, D-Phe 12]BN this residue is perpendicular to the surface and influences the orientation of the bound Trp 8. On the other hand, substitution of Asn with Tyr in the 6 position in nonapeptide fragment causes changes in the adsorption mechanism. In this case, the discussed fragment binds to the silver colloidal surface by Tyr 6, Trp 8, and Met 14. The SERS spectrum of NMC is very similar to that of BN; although it differs by the binding orientation of the amide bond towards the surface. Appearance of Phe 13 in NMB and PG-L causes that this residue competes successfully with Trp 8 forcing it to take tilted orientation. As seen from the enhancement of the characteristic Phe vibrations this moiety in NMB and PG-L adsorbs on the silver surface in a tilted fashion. This arrangements cause that the 8-14 peptide chain in all these studied compounds takes almost a parallel orientation to the surface while the 1-5 fragment of the peptide chain is removed from the silver surface vicinity.

Sequence inversion and phenylalanine surrogates at the beta-turn enhance the antibiotic activity of gramicidin S.

PubMed

Solanas, Concepción; de la Torre, Beatriz G; Fernández-Reyes, María; Santiveri, Clara M; Jiménez, M Angeles; Rivas, Luis; Jiménez, Ana I; Andreu, David; Cativiela, Carlos

2010-05-27

A series of gramicidin S (GS) analogues have been synthesized where the Phe (i + 1) and Pro (i + 2) residues of the beta-turn have been swapped while the respective chiralities (D-, L-) at each position are preserved, and Phe is replaced by surrogates with aromatic side chains of diverse size, orientation, and flexibility. Although most analogues preserve the beta-sheet structure, as assessed by NMR, their antibiotic activities turn out to be highly dependent on the bulkiness and spatial arrangement of the aromatic side chain. Significant increases in microbicidal potency against both Gram-positive and Gram-negative pathogens are observed for several analogues, resulting in improved therapeutic profiles. Data indicate that seemingly minor replacements at the GS beta-turn can have significant impact on antibiotic activity, highlighting this region as a hot spot for modulating GS plasticity and activity.

Sequence inversion and phenylalanine surrogates at the β-turn enhance the antibiotic activity of gramicidin S

PubMed Central

Solanas, Concepción; de la Torre, Beatriz G.; Fernández-Reyes, María; Santiveri, Clara M.; Jiménez, M. Ángeles; Rivas, Luis; Jiménez, Ana I.; Andreu, David; Cativiela, Carlos

2010-01-01

A series of gramicidin S (GS) analogs have been synthesized where the Phe (i+1) and Pro (i+2) residues of the β-turn have been swapped while the respective chiralities (D-, L-) at each position are preserved, and Phe is replaced by surrogates with aromatic side chains of diverse size, orientation and flexibility. Although most analogs preserve the β-sheet structure, as assessed by NMR, their antibiotic activities turn out to be highly dependent on the bulkiness and spatial arrangement of the aromatic side chain. Significant increases in microbicidal potency against both Gram-positive and Gram-negative pathogens are observed for several analogs, resulting in improved therapeutic profiles. Data indicate that seemingly minor replacements at the GS β-turn can have significant impact on antibiotic activity, highlighting this region as a hot spot for modulating GS plasticity and activity. PMID:20411945

Time-of-Day Dictates Transcriptional Inflammatory Responses to Cytotoxic Chemotherapy

PubMed Central

Borniger, Jeremy C.; Walker II, William H.; Gaudier-Diaz, Monica M.; Stegman, Curtis J.; Zhang, Ning; Hollyfield, Jennifer L.; Nelson, Randy J.; DeVries, A. Courtney

2017-01-01

Many cytotoxic chemotherapeutics elicit a proinflammatory response which is often associated with chemotherapy-induced behavioral alterations. The immune system is under circadian influence; time-of-day may alter inflammatory responses to chemotherapeutics. We tested this hypothesis by administering cyclophosphamide and doxorubicin (Cyclo/Dox), a common treatment for breast cancer, to female BALB/c mice near the beginning of the light or dark phase. Mice were injected intravenously with Cyclo/Dox or the vehicle two hours after lights on (zeitgeber time (ZT2), or two hours after lights off (ZT14). Tissue was collected 1, 3, 9, and 24 hours later. Mice injected with Cyclo/Dox at ZT2 lost more body mass than mice injected at ZT14. Cyclo/Dox injected at ZT2 increased the expression of several pro-inflammatory genes within the spleen; this was not evident among mice treated at ZT14. Transcription of enzymes within the liver responsible for converting Cyclo/Dox into their toxic metabolites increased among mice injected at ZT2; furthermore, transcription of these enzymes correlated with splenic pro-inflammatory gene expression when treatment occurred at ZT2 but not ZT14. The pattern was reversed in the brain; pro-inflammatory gene expression increased among mice injected at ZT14. These data suggest that inflammatory responses to chemotherapy depend on time-of-day and are tissue specific. PMID:28117419

Angiotensin converting enzyme (ACE) inhibitory and antihypertensive activities of protein hydrolysate from meat of Kacang goat (Capra aegagrus hircus).

PubMed

Mirdhayati, Irdha; Hermanianto, Joko; Wijaya, Christofora H; Sajuthi, Dondin; Arihara, Keizo

2016-08-01

The meat of Kacang goat has potential for production of a protein hydrolysate. Functional ingredients from protein hydrolysate of Kacang goat meat were determined by the consistency of angiotensin-converting enzyme (ACE) inhibitory activity and antihypertensive effect. This study examined the potency of Kacang goat protein hydrolysate in ACE inhibition and antihypertensive activity. Protein hydrolysates of Kacang goat meat were prepared using sequential digestion of endo-proteinase and protease complex at several concentrations and hydrolysis times. The highest ACE inhibitory activity resulted from a hydrolysate that was digested for 4 h with 5 g kg(-1) of both enzymes. An ACE inhibitory peptide was purified and a novel peptide found with a sequence of Phe-Gln-Pro-Ser (IC50 value of 27.0 µmol L(-1) ). Both protein hydrolysates and a synthesised peptide (Phe-Gln-Pro-Ser) demonstrated potent antihypertensive activities in spontaneously hypertensive rats. Protein hydrolysate of Kacang goat meat produced by sequential digestion with endo-proteinase and protease complex has great potential as a functional ingredient, particularly as an antihypertensive agent. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

Role of Intramolecular Aromatic π-π Interactions in the Self-Assembly of Di-l-Phenylalanine Dipeptide Driven by Intermolecular Interactions: Effect of Alanine Substitution.

PubMed

Reddy, Samala Murali Mohan; Shanmugam, Ganesh

2016-09-19

Although the role of intermolecular aromatic π-π interactions in the self-assembly of di-l-phenylalanine (l-Phe-l-Phe, FF), a peptide that is known for hierarchical structure, is well established, the influence of intramolecular π-π interactions on the morphology of the self-assembled structure of FF has not been studied. Herein, the role of intramolecular aromatic π-π interactions is investigated for FF and analogous alanine (Ala)-containing dipeptides, namely, l-Phe-l-Ala (FA) and l-Ala-l-Phe (AF). The results reveal that these dipeptides not only form self-assemblies, but also exhibit remarkable differences in structural morphology. The morphological differences between FF and the analogues indicate the importance of intramolecular π-π interactions, and the structural difference between FA and AF demonstrates the crucial role of the nature of intramolecular side-chain interactions (aromatic-aliphatic or aliphatic-aromatic), in addition to intermolecular interactions, in deciding the final morphology of the self-assembled structure. The current results emphasise that intramolecular aromatic π-π interaction may not be essential to induce self-assembly in smaller peptides, and π (aromatic)-alkyl or alkyl-π (aromatic) interactions may be sufficient. This work also illustrates the versatility of aromatic and a combination of aromatic and aliphatic residues in dipeptides in the formation of structurally diverse self-assembled structures. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effects of a common worldwide drink (Beer) on L-Phenylalanine and L-Tyrosine fibrillar assemblies

NASA Astrophysics Data System (ADS)

Banik, Debasis; Banerjee, Pavel; Sabeehuddin, Ghazi; Sarkar, Nilmoni

2017-11-01

In this letter, small amount of beer [0.42-2.08% (v/v)] is employed to investigate the fibril inhibition kinetics of 1 mM L-Phenylalanine and L-Tyrosine (relevant to disease condition) using Fluorescence Lifetime imaging Microscopy (FLIM), Field Emission Scanning Electron Microscopy (FESEM) and High Resolution Transmission Electron Microscopic (HR-TEM) techniques. Our results indicate that 1.67 and 0.42% of beer is sufficient for effective breakdown of L-Phe and L-Tyr assemblies, respectively. Quantitative information about fibril inhibition is obtained from Fluorescence Correlation Spectroscopic (FCS) measurements. We have shown that the morphology of L-Phe changes to L-Tyr in presence of 2,2‧-Bipyridine-3,3‧-diol (BP(OH)2).

Lactobacillus ixorae sp. nov., isolated from a flower (West-Indian jasmine).

PubMed

Techo, Sujitra; Miyashita, Mika; Shibata, Chiyo; Tanaka, Naoto; Wisetkhan, Preeyarach; Visessanguan, Wonnop; Tanasupawat, Somboon

2016-12-01

A Gram-stain-positive, lactic acid bacterium, strain Ru20-1T, was isolated from a flower (West-Indian jasmine) collected from Kalasin province, Thailand. A polyphasic approach was used to determine the taxonomic position of this strain. Studies of morphological and biochemical characteristics revealed that strain Ru20-1T belonged to the genus Lactobacillus. The strain was heterofermentative, non-spore-forming and rod-shaped. It produced dl-lactic acid. Based on 16S rRNA gene sequence similarity, this strain was closely related to Lactobacillus lindneri LMG 14528T (96.8 %), Lactobacillus sanfranciscensis NRIC 1548T (95.4 %) and Lactobacillus florum NRIC 0771T (95.2 %), respectively. In addition, the pheS gene sequence of strain Ru20-1T was closely related to those of L. sanfranciscensis NRIC 1548T (92.0 %), L. lindneri LMG 14528T (89.0 %) and L. florum NRIC 0771T(85.0 %). Phylogenetic analysis indicated that strain Ru20-1T was clearly separated from closely related species of the genus Lactobacillus. The DNA G+C content of strain Ru20-1T was 47.8 mol %. The cell-wall peptidoglycan type was l-Lys-d-Asp. The major cellular fatty acids were C18 : 1ω9c, C20 : 0, C20 : 1ω9c and summed feature 7 (unknown 18.846 and/or C19 : 1ω6c and/or C19 : 0 cyclo). On the basis of the data provided, strain Ru20-1T represents a novel species of the genus Lactobacillus, for which the name Lactobacillus ixorae sp. nov. is proposed. The type strain is Ru20-1T (=LMG 29008T=NBRC 111239T=PCU 346T=TISTR 2381T).

Purification and synergistic antibacterial activity of arginine derived cyclic dipeptides, from Achromobacter sp. associated with a rhabditid entomopathogenic nematode against major clinically relevant biofilm forming wound bacteria

PubMed Central

Deepa, Indira; Kumar, Sasidharan N.; Sreerag, Ravikumar S.; Nath, Vishnu S.; Mohandas, Chellapan

2015-01-01

Skin and chronic wound infections caused by various pathogenic bacteria are an increasing and urgent health problem worldwide. In the present investigation ethyl acetate extract of an Achromobacter sp. associated with a Rhabditis entomopathogenic nematode (EPN), displayed promising antibacterial property and was further purified by silica gel column chromatography to get three different cyclic dipeptides (CDPs). Based on the spectral data and Marfey's analyses, the CDPs were identified as cyclo(D-Leu-D-Arg) (1), cyclo(L-Trp-L-Arg) (2), and cyclo(D-Trp-D-Arg) (3), respectively. Three CDPs were active against all the 10 wound associated bacteria tested. The significant antibacterial activity was recorded by CDP 3, and highest activity of 0.5 μg/ml was recorded against Staphylococcus aureus and Pseudomonas aeruginosa. The synergistic antibacterial activities of CDPs and ampicillin were assessed using the checkerboard microdilution method. The results of the current study recorded that the combined effects of CDPs and ampicillin principally recorded synergistic activity. Interestingly, the combination of CDPs and ampicillin also recorded enhanced inhibition of biofilm formation by bacteria. Moreover, CDPs significantly stimulate the production of IL-10 and IL-4 (anti-inflammatory cytokines) by human peripheral blood mononuclear cells. CDPs do not make any significant effect on the production of pro-inflammatory cytokines like TNF-α. The three CDPs have been studied for their effect on intracellular S. aureus in murine macrophages (J774) using 24 h exposure to 0.5X, 1X, and 2X MIC concentrations. Significant decrease in intracellular S. aureus burden was recorded by CDPs. CDPs also recorded no cytotoxicity toward FS normal fibroblast, VERO, and L231 normal lung epithelial cell lines. Antimicrobial activity of the arginine containing CDPs against the wound associated bacteria is reported here for the first. Moreover, this is also the first report on the production of CDPs by Achromobacter sp. Finally, we conclude that the Achromobacter sp. is an incredibly promising source of natural bioactive secondary metabolites especially against wound pathogenic bacteria that may receive significant benefit in the field of human medicine in near future as topical agents. PMID:26379651

Influence of aliphatic spacer group on adsorption mechanisms of phosphonate derivatives of L-phenylalanine: Surface-enhanced Raman, Raman, and infrared studies

NASA Astrophysics Data System (ADS)

Podstawka, E.; Kudelski, A.; Kafarski, P.; Proniewicz, L. M.

2007-10-01

The nature of phosphonopeptides containing N-terminal L-phenylalanine ( L-Phe), namely L-Phe- DL-NH-CH(CH(CH 3) 2)-PO 3H 2 ( A), L-Phe- L-NH-CH(CH 3)-PO 3H 2 ( B), and L-Phe- DL-NH-CH(CH 2CH 2COOH)-PO 3H 2 ( C) ( Fig. 1 presents molecular structure of these molecules), adsorbed on electrochemically roughened and colloidal silver surfaces has been explored by surface-enhanced Raman spectroscopy (SERS). To reveal adsorption mechanism of these species on the basis of their SERS spectra at first Fourier-transform Raman (FT-RS) and absorption infrared (FT-IR) spectra of non-adsorbed molecules were measured. Examination of enhancement, frequency shifts, and changes in relative intensities of SERS bands due to adsorption and surface roughens variation reveals that the tilted compounds adsorb on the electrochemically roughened silver substrate in similar way, while they behave differently on the colloidal silver surface. A stronger enhancement of in-plane ring vibrations of the L-Phe ring, i.e., ν3 and ν18b (B 2), over these of the A 2 symmetry in all SERS spectra on the electrochemically roughened silver substrate suggests that the ring interacts with this surface adopting slightly deflect orientation from the perpendicular one. Also, enhancement of P dbnd O and -CH 2-/-CH 3 fragments vibrations points out that they are involved in adsorption process on this substrate. This conclusion was drawn on the basis of the enhancement of 1274-1279 and 1138-1152 ( ν(P dbnd O)), 1393-1400 ( δ(CH) + ρb(CNH 2) + ν(C-C dbnd O ) + δ(CH 3)), ˜1455 ( δ(CCH 3/CCH 2) + ρb(CH 3/CH 2), and 1505-1512 cm -1 ( δ(CH 2) + Phe( ν19a)) bands. Although a relative intensity ratio of these bands in the presented SERS spectra is different. On the other hand, on the colloidal silver nanoparticles, the aromatic ring of all molecules is lying flat or takes almost parallel orientation to this surface. Besides, A interacts also via P-terminal group (568, 765, 827, 1040, and 1150 cm -1), whereas B mainly through NH 2-C-(C dbnd O)-CNH-(712 and 1255 cm -1). In the case of C, it adsorbs on the silver colloidal surface mainly through the aromatic ring of L-Phe, while other fragments of the molecule are in close proximity to this surface as comes off the weak enhancement of bands due to the aliphatic vibrations.

Synthesis, characterization and in vitro biocompatibility assessment of a novel tripeptide hydrogelator, as a promising scaffold for tissue engineering applications.

PubMed

Pospišil, Tihomir; Ferhatović Hamzić, Lejla; Brkić Ahmed, Lada; Lovrić, Marija; Gajović, Srećko; Frkanec, Leo

2016-10-20

We have synthesized and characterized a self-assembling tripeptide hydrogelator Ac-l-Phe-l-Phe-l-Ala-NH2. A series of experiments showed that the hydrogel material could serve as a stabile and biocompatible physical support as it improves the survival of HEK293T cells in vitro, thus being a promising biomaterial for use in tissue engineering applications.

Rhamnolipid influences biosorption and biodegradation of phenanthrene by phenanthrene-degrading strain Pseudomonas sp. Ph6.

PubMed

Ma, Zhao; Liu, Juan; Dick, Richard P; Li, Hui; Shen, Di; Gao, Yanzheng; Waigi, Michael Gatheru; Ling, Wanting

2018-05-08

Given the sub-lethal risks of synthetic surfactants, rhamnolipid is a promising class of biosurfactants with the potential to promote the bioavailability of polycyclic aromatic hydrocarbons (PAHs), to provide a favorable substitute for synthetic surfactants. However, few previous studies have integrated the behavior and mechanism behind rhamnolipid-influenced PAH biosorption and biodegradation. This is, to our knowledge, the first report of a bacterial envelope regulated link between phenanthrene (PHE) biosorption and biodegradation by rhamnolipid-induced PHE-degrading strain Pseudomonas sp. Ph6. Rhamnolipid (0─400 mg L -1 ) can change the cell-surface zeta potential, cell surface hydrophobicity (CSH), cell ultra-microstructure and functional groups, and then alter PHE biosorption and biodegradation of Ph6. Greater amounts of PHE sorbed on cell envelopes results in more PHE diffusing into cytochylema, thus favoring PHE intracellular biodegradation of Ph6. Rhamnolipid (≤100 mg L -1 ) could change the microstructures and functional groups of cell envelopes of Ph6, enhance the cell-surface zeta potential and CSH, thus consequently favor PHE biosorption and biodegradation by strain Ph6. By contrast, rhamnolipid at higher concentrations (≥200 mg L -1 ) hindered PHE biosorption and biodegradation. Rhamnolipid, as a biosurfactant, can be successfully utilized as an additive to improve the microbial biodegradation of PAHs in the environments. Copyright © 2018 Elsevier Ltd. All rights reserved.

The adsorption of L-phenylalanine on oxidized single-walled carbon nanotubes.

PubMed

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.

N-Acetyl-D- and L-esters of 5'-AMP hydrolyze at different rates

NASA Technical Reports Server (NTRS)

Wickramasinghe, N. S.; Lacey, J. C. Jr; Lacey JC, J. r. (Principal Investigator)

1993-01-01

Studies of the properties of aminoacyl derivatives of 5'-AMP are aimed at understanding the origin of the process of protein synthesis. Aminoacyl (2',3') esters of 5'-AMP can serve as models of the 3'-terminus of aminoacyl tRNA. We report here on the relative rates of hydrolysis of Ac-D- and L-Phe AMP esters as a function of pH. At all pHs above 3, the rate constant of hydrolysis of the Ac-L-Phe ester is 1.7 to 2.1 times that of Ac-D-Phe ester. The D-isomer seems partially protected from hydrolysis by a stronger association with the adenine ring of the 5'-AMP.

Precise structural analysis of α-helical polypeptide by quantum-chemical calculation related to reciprocal side-chain combination of two L-phenylalanine residues

NASA Astrophysics Data System (ADS)

Niimura, Subaru; Kurosu, Hiromichi; Shoji, Akira

2010-04-01

To clarify the positive role of side-chain conformation in the stability of protein secondary structure (main-chain conformation), we successfully calculated the optimization structure of a series of well-defined α-helical octadecapeptides composed of two L-phenylalanine (Phe) and 16 L-alanine (Ala) residues, based on the molecular orbital calculation with density functional theory (DFT/B3LYP/6-31G(d)). From the total energy calculation and the precise secondary structural analysis, we found that the conformational stability of the α-helix is closely related to the reciprocal side-chain combinations (such as positional relation and side-chain conformation) of two Phe residues in this system. Furthermore, we demonstrated that the 1H, 13C, 15N and 17O isotropic chemical shifts of each Phe residue depend on the respective side-chain conformations of the Phe residue.

Derivative emission spectrofluorimetry for the simultaneous determination of guaifenesin and phenylephrine hydrochloride in pharmaceutical tablets.

PubMed

Maher, Hadir M; Alshehri, Mona M; Al-taweel, Shorog M

2015-05-01

Rapid, simple and sensitive derivative emission spectrofluorimetric methods have been developed for the simultaneous analysis of binary mixtures of guaifenesin (GUA) and phenylephrine hydrochloride (PHE). The methods are based upon measurement of the native fluorescence intensity of the two drugs at λex = 275 nm in methanolic solutions, followed by differentiation using first (D1) and second (D2) derivative techniques. The derivative fluorescence intensity-concentration plots were rectilinear over a range of 0.1-2 µg/mL for both GUA and PHE. The limits of detection were 0.027 (D1, GUA), 0.025 (D2, GUA), 0.031 (D1, PHE) and 0.033 (D2, PHE) µg/mL and limits of quantitation were 0.089 (D1, GUA), 0.083 (D2, GUA), 0.095 (D1, PHE) and 0.097 (D2, PHE) µg/mL. The proposed derivative emission spectrofluorimetric methods (D1 and D2) were successfully applied for the determination of the two compounds in binary mixtures and tablets with high precision and accuracy. The proposed methods were fully validated as per ICH guidelines. Copyright © 2014 John Wiley & Sons, Ltd.

Effect of a novel selective and potent phosphinic peptide inhibitor of endopeptidase 3.4.24.16 on neurotensin-induced analgesia and neuronal inactivation.

PubMed

Vincent, B; Jiracek, J; Noble, F; Loog, M; Roques, B; Dive, V; Vincent, J P; Checler, F

1997-06-01

1. We have examined a series of novel phosphinic peptides as putative potent and selective inhibitors of endopeptidase 3.4.24.16. 2. The most selective inhibitor, Pro-Phe-psi(PO2CH2)-Leu-Pro-NH2 displayed a Ki value of 12 nM towards endopeptidase 3.4.24.16 and was 5540 fold less potent on its related peptidase endopeptidase 3.4.24.15. Furthermore, this inhibitor was 12.5 less potent on angiotensin-converting enzyme and was unable to block endopeptidase 3.4.24.11, aminopeptidases B and M, dipeptidylaminopeptidase IV and proline endopeptidase. 3. The effect of Pro-Phe-psi(PO2CH2)-Leu-Pro-NH2, in vitro and in vivo, on neurotensin metabolism in the central nervous system was examined. 4. Pro-Phe-psi(PO2CHH2)-Leu-Pro-NH2 dose-dependently inhibited the formation of neurotensin 1-10 and concomittantly protected neurotensin from degradation by primary cultured neurones from mouse embryos. 5. Intracerebroventricular administration of Pro-Phe-psi(PO2CH2)-Leu-Pro-NH2 significantly potentiated the neurotensin-induced antinociception of mice in the hot plate test. 6. Altogether, our study has established Pro-Phe-psi(PO2CH2)-Leu-Pro-NH2 as a fully selective and highly potent inhibitor of endopeptidase 3.4.24.16 and demonstrates, for the first time, the contribution of this enzyme in the central metabolism of neurotensin.

Effect of a novel selective and potent phosphinic peptide inhibitor of endopeptidase 3.4.24.16 on neurotensin-induced analgesia and neuronal inactivation

PubMed Central

Vincent, Bruno; Jiracek, Jirì; Noble, Florence; Loog, Mart; Roques, Bernard; Dive, Vincent; Vincent, Jean-Pierre; Checler, Frédéric

1997-01-01

We have examined a series of novel phosphinic peptides as putative potent and selective inhibitors of endopeptidase 3.4.24.16. The most selective inhibitor, Pro-Phe-Ψ(PO2CH2)-Leu-Pro-NH2 displayed a Ki value of 12 nM towards endopeptidase 3.4.24.16 and was 5540 fold less potent on its related peptidase endopeptidase 3.4.24.15. Furthermore, this inhibitor was 12.5 less potent on angiotensin-converting enzyme and was unable to block endopeptidase 3.4.24.11, aminopeptidases B and M, dipeptidylaminopeptidase IV and proline endopeptidase. The effect of Pro-Phe-Ψ(PO2CH2)-Leu-Pro-NH2, in vitro and in vivo, on neurotensin metabolism in the central nervous system was examined. Pro-Phe-Ψ(PO2CHH2)-Leu-Pro-NH2 dose-dependently inhibited the formation of neurotensin 1-10 and concomittantly protected neurotensin from degradation by primary cultured neurones from mouse embryos. Intracerebroventricular administration of Pro-Phe-Ψ(PO2CH2)-Leu-Pro-NH2 significantly potentiated the neurotensin-induced antinociception of mice in the hot plate test. Altogether, our study has established Pro-Phe-Ψ(PO2CH2)-Leu-Pro-NH2 as a fully selective and highly potent inhibitor of endopeptidase 3.4.24.16 and demonstrates, for the first time, the contribution of this enzyme in the central metabolism of neurotensin. PMID:9208137

Penicyrones A and B, an epimeric pair of α-pyrone-type polyketides produced by the marine-derived Penicillium sp.

PubMed

Bu, Ying-Yue; Yamazaki, Hiroyuki; Takahashi, Ohgi; Kirikoshi, Ryota; Ukai, Kazuyo; Namikoshi, Michio

2016-01-01

Two polyketides containing an α-pyrone unit, named penicyrones A (1) and B (2), were isolated from a culture broth of the marine-derived Penicillium sp. TPU1271 together with nine known compounds: verrucosidin (3), fructigenine A (4), verrucofortine (5), cyclo-(L-Trp-L-Phe) (6), cyclopenol (7), cyclopenin (8), penipratynolene (9), aspterric acid (10) and viridicatol (11). The structures of 1 and 2 were elucidated by analyzing the spectroscopic data of 1, 2 and their O-acetyl derivatives (1a and 2a). Compounds 1 and 2 were epimers of each other at the C-9 position. The absolute configurations of 1 and 2 were assigned on the basis of NOESY data for 1, 2, 1a and 2a, a conformational analysis and the identity of the biogenetic pathway with verrucosidin (3). The planar structure of penicyrones was found in the SciFinder as a compound in the commercial chemical libraries; however, the stereostructure and spectroscopic data were not available. Therefore, this is the first study on the isolation and structure elucidation, including the absolute configurations, of penicyrones A (1) and B (2) as fungal metabolites. Compound 3 exhibited growth inhibitory activity against Mycobacterium smegmatis at 40 μg per disc (inhibition zone of 11 mm). This is the first study to demonstrate that verrucosidin (3) exhibited anti-mycobacterial activity.

Post-Translational Incorporation of L-Phenylalanine into the C-Terminus of α-Tubulin as a Possible Cause of Neuronal Dysfunction.

PubMed

Ditamo, Yanina; Dentesano, Yanela M; Purro, Silvia A; Arce, Carlos A; Bisig, C Gastón

2016-12-01

α-Tubulin C-terminus undergoes post-translational, cyclic tyrosination/detyrosination, and L-Phenylalanine (Phe) can be incorporated in place of tyrosine. Using cultured mouse brain-derived cells and an antibody specific to Phe-tubulin, we showed that: (i) Phe incorporation into tubulin is reversible; (ii) such incorporation is not due to de novo synthesis; (iii) the proportion of modified tubulin is significant; (iv) Phe incorporation reduces cell proliferation without affecting cell viability; (v) the rate of neurite retraction declines as level of C-terminal Phe incorporation increases; (vi) this inhibitory effect of Phe on neurite retraction is blocked by the co-presence of tyrosine; (vii) microtubule dynamics is reduced when Phe-tubulin level in cells is high as a result of exogenous Phe addition and returns to normal values when Phe is removed; moreover, microtubule dynamics is also reduced when Phe-tubulin is expressed (plasmid transfection). It is known that Phe levels are greatly elevated in blood of phenylketonuria (PKU) patients. The molecular mechanism underlying the brain dysfunction characteristic of PKU is unknown. Beyond the differences between human and mouse cells, it is conceivable the possibility that Phe incorporation into tubulin is the first event (or among the initial events) in the molecular pathways leading to brain dysfunctions that characterize PKU.

Molecular cloning, characterization and functional analysis of a novel juvenile-specific cathepsin L of Fasciola gigantica.

PubMed

Sansri, Veerawat; Changklungmoa, Narin; Chaichanasak, Pannigan; Sobhon, Prasert; Meemon, Krai

2013-10-01

Cathepsin L proteases are a major class of endopeptidases expressed at a high level in Fasciola parasites. Several isoforms of cathepsin L were detected and they may perform different functions during the parasite development. In this study, a complete cDNA encoding a cathepsin L protease was cloned from a newly excysted juvenile (NEJ) cDNA library of Fasciola gigantica and named FgCatL1H. It encoded a 326 amino acid preproenzyme which shared 62.8-83.1% and 39.5-42.9% identity to Fasciola spp. and mammalian cathepsins L, respectively. All functionally important residues previously described for cathepsin L were conserved in FgCatL1H. Phylogenetic analysis demonstrated that FgCatL1H belonged to a distinct group, clade 4, with respect to adult and other juvenile Fasciola cathepsin L genes. FgCatL1H expression was detected by RT-PCR, using gene specific primers, in metacercariae and NEJ, and the expression gradually decreased in advanced developmental stages. A recombinant proFgCatL1H (rproFgCatL1H) was expressed in the yeast Pichia pastoris, affinity purified, and found to migrate in SDS-PAGE at approximately 47.6 and 38.3kDa in glycosylated and deglycosylated forms, respectively. The molecular mass of the activated mature rFgCatL1H in glycosylated form was approximately 40.7kDa. Immunoblotting and immunohistochemistry using rabbit antibodies against rproFgCatL1H showed that FgCatL1H was predominantly expressed in epithelial cells of the digestive tract of metacercariae, NEJs and juveniles of F. gigantica. FgCatL1H could cleave the synthetic fluorogenic substrate Z-Phe-Arg-MCA preferentially over Z-Gly-Pro-Arg-MCA at an optimum pH of 6.5. It also showed hydrolytic activity against native substrates, including type I collagen, laminin, and immunoglobulin G (IgG) in vitro, suggesting possible roles in host tissue migration and immune evasion. Therefore, the FgCatL1H is a possible target for vaccine and chemotherapy for controlling F. gigantica infection. Copyright © 2013 Elsevier B.V. All rights reserved.

Substituting Tyr138 in the active site loop of human phenylalanine hydroxylase affects catalysis and substrate activation.

PubMed

Leandro, João; Stokka, Anne J; Teigen, Knut; Andersen, Ole A; Flatmark, Torgeir

2017-07-01

Mammalian phenylalanine hydroxylase (PAH) is a key enzyme in l-phenylalanine (l-Phe) metabolism and is active as a homotetramer. Biochemical and biophysical work has demonstrated that it cycles between two states with a variably low and a high activity, and that the substrate l-Phe is the key player in this transition. X-ray structures of the catalytic domain have shown mobility of a partially intrinsically disordered Tyr 138 -loop to the active site in the presence of l-Phe. The mechanism by which the loop dynamics are coupled to substrate binding at the active site in tetrameric PAH is not fully understood. We have here conducted functional studies of four Tyr 138 point mutants. A high linear correlation ( r 2 = 0.99) was observed between their effects on the catalytic efficiency of the catalytic domain dimers and the corresponding effect on the catalytic efficiency of substrate-activated full-length tetramers. In the tetramers, a correlation ( r 2 = 0.96) was also observed between the increase in catalytic efficiency (activation) and the global conformational change (surface plasmon resonance signal response) at the same l-Phe concentration. The new data support a similar functional importance of the Tyr 138 -loop in the catalytic domain and the full-length enzyme homotetramer.

Influence of magnetic fields on the hydration process of amino acids: vibrational spectroscopy study of L-phenylalanine and L-glutamine.

PubMed

De Ninno, Antonella; Congiu Castellano, Agostina

2014-02-01

Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy has been used to investigate the effect of weak electromagnetic fields on the structure of L-glutamine (L-Gln) and L-phenylalnine (L-Phe) in aqueous solution. It has been found that the exposure to a DC field or a 50 Hz AC field, for a short time induces modifications in the spectra of exposed samples in agreement with our preceding observations on glutamic acid. Furthermore, the acid-base equilibrium has been investigated by using the ratio of the intensity of the deprotonated on protonated species. In the case of L-Phe, the exposure induces a measurable shift of acid dissociation constant pKa1 out of the experimental errors, while in case of L-Gln, the effect is under the limit detectable with this method. The phenomenon of the shift of the acid-base equilibrium has been connected elsewhere to modification of the water-water hydrogen bonds in the water around both the backbone and the residue (R). Here we suggest that the magnetic field modifies the water structure around the molecules and changes the hydrophobic interactions allowing the molecules of amino acids to aggregate. The differences observed in the behavior of L-Phe and L-Gln may be related to the differences in the polarity of their residues. © 2013 Wiley Periodicals, Inc.

Chiral zinc phenylalanine nanofibers with fluorescence.

PubMed

Chen, Erdan; Guo, Beidou; Zhang, Baohong; Gan, Li-Hua; Gong, Jian Ru

2011-09-01

Chiral Zn(II)/D-,L-phenylalanine (Phe) bio-coordination polymer nanofibers with fluorescence were prepared by fast coordination-assisted assembly. The synthetic strategy is based on the fact that the Zn2+ ions were linked to oxygen atoms from carboxylate groups of the D- or L-amino acid by coordination interactions to form the chiral polymers. The Zn(II)/D-,L-Phe nanofibers had homogeneous diameters in the range of 700-900 nm and ultra-long length in several hundred micrometers, and the surface of the fiber was extremely smooth. In addition, the enantiomers of Zn(II)/Phe nanofibers exhibited both optical activity and fluorescent property in the solid state, which has great potential for application in the field of biomimetic nanofabrication and micro-/nano-optoelectronics.

Binding Interactions of Agents That Alter α-Synuclein Aggregation

PubMed Central

Sivanesam, K.; Byrne, A.; Bisaglia, M.; Bubacco, L.

2015-01-01

Further examination of peptides with well-folded antiparallel β strands as inhibitors of amyloid formation from α-synuclein has resulted in more potent inhibitors. Several of these had multiple Tyr residues and represent a new lead for inhibitor design by small peptides that do not divert α-synuclein to non-amyloid aggregate formation. The most potent inhibitor obtained in this study is a backbone cyclized version of a previously studied β hairpin, designated as WW2, with a cross-strand Trp/Trp cluster. The cyclization was accomplished by adding a d-Pro-l-Pro turn locus across strand termini. At a 2:1 peptide to α-synuclein ratio, cyclo-WW2 displays complete inhibition of β-structure formation. Trp-bearing antiparallel β-sheets held together by a disulphide bond are also potent inhibitors. 15N HSQC spectra of α-synuclein provided new mechanistic details. The time course of 15N HSQC spectral changes observed during β-oligomer formation has revealed which segments of the structure become part of the rigid core of an oligomer at early stages of amyloidogenesis and that the C-terminus remains fully flexible throughout the process. All of the effective peptide inhibitors display binding-associated titration shifts in 15N HSQC spectra of α-synuclein in the C-terminal Q109-E137 segment. Cyclo-WW2, the most potent inhibitor, also displays titration shifts in the G41-T54 span of α-synuclein, an additional binding site. The earliest aggregation event appears to be centered about H50 which is also a binding site for our most potent inhibitor. PMID:25705374

Binding Interactions of Agents That Alter α-Synuclein Aggregation.

PubMed

Sivanesam, K; Byrne, A; Bisaglia, M; Bubacco, L; Andersen, N

Further examination of peptides with well-folded antiparallel β strands as inhibitors of amyloid formation from α-synuclein has resulted in more potent inhibitors. Several of these had multiple Tyr residues and represent a new lead for inhibitor design by small peptides that do not divert α-synuclein to non-amyloid aggregate formation. The most potent inhibitor obtained in this study is a backbone cyclized version of a previously studied β hairpin, designated as WW2, with a cross-strand Trp/Trp cluster. The cyclization was accomplished by adding a d-Pro-l-Pro turn locus across strand termini. At a 2:1 peptide to α-synuclein ratio, cyclo-WW2 displays complete inhibition of β-structure formation. Trp-bearing antiparallel β-sheets held together by a disulphide bond are also potent inhibitors. 15 N HSQC spectra of α-synuclein provided new mechanistic details. The time course of 15 N HSQC spectral changes observed during β-oligomer formation has revealed which segments of the structure become part of the rigid core of an oligomer at early stages of amyloidogenesis and that the C-terminus remains fully flexible throughout the process. All of the effective peptide inhibitors display binding-associated titration shifts in 15 N HSQC spectra of α-synuclein in the C-terminal Q109-E137 segment. Cyclo-WW2, the most potent inhibitor, also displays titration shifts in the G41-T54 span of α-synuclein, an additional binding site. The earliest aggregation event appears to be centered about H50 which is also a binding site for our most potent inhibitor.

A periplasmic D-alanyl-D-alanine dipeptidase in the gram-negative bacterium Salmonella enterica.

PubMed

Hilbert, F; García-del Portillo, F; Groisman, E A

1999-04-01

The VanX protein is a D-alanyl-D-alanine (D-Ala-D-Ala) dipeptidase essential for resistance to the glycopeptide antibiotic vancomycin. While this enzymatic activity has been typically associated with vancomycin- and teicoplainin-resistant enterococci, we now report the identification of a D-Ala-D-Ala dipeptidase in the gram-negative species Salmonella enterica. The Salmonella enzyme is only 36% identical to VanX but exhibits a similar substrate specificity: it hydrolyzes D-Ala-D-Ala, DL-Ala-DL-Phe, and D-Ala-Gly but not the tripeptides D-Ala-D-Ala-D-Ala and DL-Ala-DL-Lys-Gly or the dipeptides L-Ala-L-Ala, N-acetyl-D-Ala-D-Ala, and L-Leu-Pro. The Salmonella dipeptidase gene, designated pcgL, appears to have been acquired by horizontal gene transfer because pcgL-hybridizing sequences were not detected in related bacterial species and the G+C content of the pcgL-containing region (41%) is much lower than the overall G+C content of the Salmonella chromosome (52%). In contrast to wild-type Salmonella, a pcgL mutant was unable to use D-Ala-D-Ala as a sole carbon source. The pcgL gene conferred D-Ala-D-Ala dipeptidase activity upon Escherichia coli K-12 but did not allow growth on D-Ala-D-Ala. The PcgL protein localizes to the periplasmic space of Salmonella, suggesting that this dipeptidase participates in peptidoglycan metabolism.

Weed growth inhibitors from Aspergillus fischeri TISTR 3272.

PubMed

Phattanawasin, P; Pojchanakom, K; Sotanaphun, U; Piyapolrungroj, N; Zungsontiporn, S

2007-12-01

Chloroform and ethyl acetate extracts of Aspergillus fischeri TISTR 3272 showed good growth inhibitory activity on Mimosa pigra and Echinochloa crus-galli. Bioassay-directed fractionation of the active extracts led to the isolation of five known compounds, (+)-terrein (1), (-)-6-hydroxymellein (2), two diketopiperazines (cyclo-(S-Pro-S-Leu) (3) and cyclo-(S-Pro-S-Val) (4)) and butyrolactone I (5). Compounds 2-5 were reported for the first time in this fungus. Their structural determinations were based on analyses of spectroscopic data and their weed growth inhibitory effects were assessed.

Bioactive metabolites produced by Penicillium sp. 1 and sp. 2, two endophytes associated with Alibertia macrophylla (Rubiaceae).

PubMed

Oliveira, Camila M; Silva, Geraldo H; Regasini, Luis O; Zanardi, Lisinéia M; Evangelista, Alana H; Young, Maria C M; Bolzani, Vanderlan S; Araujo, Angela R

2009-01-01

In the course of our continuous search for bioactive metabolites from endophytic fungi living in plants from the Brazilian flora, leaves of Alibertia macrophylla (Rubiaceae) were submitted to isolation of endophytes, and two species of Penicillium were isolated. The acetonitrile fraction obtained in corn from a culture of Penicillium sp. 1 afforded orcinol (1). On the other hand, Penicillium sp. 1 cultivated in potato-dextrose-broth furnished two different compounds, cyclo-(L-Pro-L-Val) (2) and uracil (3). The chromatographic fractionation of the acetonitrile fraction obtained from Penicillium sp. 2 led to three dihydroisocoumarins, 4-hydroxymellein (4), 8-methoxymellein (5) and 5-hydroxymellein (6). Compounds 5 and 6 were obtained from the Penicillium genus for the first time. Additionally, metabolites 1-6 were evaluated for their antifungal and acetylcholinesterase (AChE) inhibitory activities. The most active compounds 1 and 4 exhibited detection limits of 5.00 and 10.0 microg against Cladosporium cladosporioides and C. sphaerospermum, respectively. Compound 2 showed a detection limit of 10.0 microg, displaying potent AChE inhibitory activity.

Investigation of biotechnological potential of sponge-associated bacteria collected in Brazilian coast.

PubMed

Santos, O C S; Soares, A R; Machado, F L S; Romanos, M T V; Muricy, G; Giambiagi-deMarval, M; Laport, M S

2015-02-01

Marine bacteria are a rich source of structurally unique natural compounds, several of which have shown a wide variety of biological activities. In this study, the metabolites present in the culture supernatants of the eight sponge-associated bacteria were extracted using ethyl acetate, and all extracts showed activity against Staphylococcus aureus. Subsequently, the extracts of the Pseudomonas fluorescens H40 and H41, and Pseudomonas aeruginosa H51 were subjected to solvent partitioning, and the active fractions were submitted to chromatographic separation. Three different active fractions were obtained, one of which was identified as diketopiperazine cyclo-(L-Leu-L-Pro). This substance was bactericidal for Staph. aureus and Ps. aeruginosa and showed cytotoxic activity against HEp-2 tumour cells. Putative gene fragments coding for the type I polyketide synthase (PKS-I) and nonribosomal peptide synthetase (NRPS) domains were PCR-amplified from five and three strains, respectively. The results suggest that sponge-associated bacteria analysed in this study may represent a potential source for production of antimicrobial substances against bacterial pathogens of medical importance. © 2014 The Society for Applied Microbiology.

Cathepsin D immobilized capillary reactors for on-flow screening assays.

PubMed

Cornelio, Vivian Estevam; de Moraes, Marcela Cristina; Domingues, Vanessa de Cassia; Fernandes, João Batista; da Silva, Maria Fátima das Gracas Fernandes; Cass, Quezia Bezerra; Vieira, Paulo Cezar

2018-03-20

The treatment of diseases using enzymes as targets has called for the development of new and reliable methods for screening. The protease cathepsin D is one such target involved in several diseases such as tumors, degenerative processes, and vital processes of parasites causing schistosomiasis. Herein, we describe the preparation of a fused silica capillary, cathepsin D (CatD)-immobilized enzyme reactor (IMER) using in a multidimensional High Performance Liquid Chromatography-based method (2D-HPLC) and zonal affinity chromatography as an alternative in the search for new ligands. The activity and kinetic parameters of CatD-IMER were evaluated by monitoring the product MOCAc-Gly-Lys-Pro-Ile-Leu-Phe (P-MOCAc) (K M  = 81.9 ± 7.49 μmol/L) generated by cleavage of the fluorogenic substrate MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(DNP)-d-Arg-NH2 (S-MOCAc). Stability studies have indicated that CatD-IMER retained 20% of activity after 5 months, a relevant result, because proteases are susceptible to autoproteolysis in solution assays with free enzyme. In the search for inhibitors, 12 crude natural product extracts were analyzed using CatD-IMER as the target, resulting in the isolation of different classes of natural products. In addition, 26 compounds obtained from different species of plants were also screened, demonstrating the efficiency and reproducibility of the herein reported assay even in the case of complex matrices such as plant crude extracts. Copyright © 2018 Elsevier B.V. All rights reserved.

Bifunctional [2’,6’-Dimethyl-l-tyrosine1]Endomorphin-2 Analogues Substituted at Position 3 with Alkylated Phenylalanine Derivatives Yield Potent Mixed μ-Agonist/δ-Antagonist and Dual μ-/δ-Agonist Opioid Ligands

PubMed Central

Li, Tingyou; Shiotani, Kimitaka; Miyazaki, Anna; Tsuda, Yuko; Ambo, Akihiro; Sasaki, Yusuke; Jinsmaa, Yunden; Marczak, Ewa; Bryant, Sharon D.; Lazarus, Lawrence H.; Okada, Yoshio

2009-01-01

Endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH2) and [Dmt1]EM-2 (Dmt = 2’,6’-dimethyl-l-tyrosine) analogues were synthesized containing alkylated Phe3 derivatives, 2’-monomethyl (2, 2’), 3’,5’- and 2’,6’-dimethyl (3, 3’, and 4’, respectively), 2’,4’,6’-trimethyl (6, 6’), 2’-ethyl-6’-methyl (7, 7’) and 2’-isopropyl-6’-methyl (8, 8’) groups or Dmt (5, 5’). They had the following characteristics: (i) [Xaa3]EM-2 analogues improved μ- and δ-opioid receptor affinities, the latter were inconsequential (Kiδ= 491–3,451 nM); (ii) [Dmt1,Xaa3]EM-2 analogues enhanced μ- and δ-opioid receptor affinities (Kiμ = 0.069–0.32 nM; Kiδ = 1.83–99.8 nM) and lacked interaction with κ-opioid receptors, and (iii) elevated μ-bioactivity (IC50 = 0.12–14.4 nM) and abolished δ-agonism (IC50 > 10 µM; 2’, 3’, 4’, 5’, 6’); however, 4’ and 6’ exhibited mixed μ-agonism/δ-antagonism (4’: IC50μ = 0.12, pA2 = 8.15; 6’: IC50μ = 0.21 nM, pA2 = 9.05), and 7’ was a dual μ-/δ -agonist (IC50μ = 0.17 nM; IC50δ = 0.51 nM). Alteration of EM-2 activity by Dmt1 and alkylated Phe3 residues retained μ-receptor bioactivity and formed dual μ-/δ -agonists and mixed μ-agonists/δ-antagonists. PMID:17497839

Method of treating tumors

DOEpatents

DeNardo, Sally J.; Burke, Patricia A.; DeNardo, Gerald L.; Goodman, Simon; Matzku, legal representative, Kerstin; Matzku, Siegfried

2006-04-18

A method of treating tumors, such as prostate tumors, breast tumors, non-Hodgkin's lymphoma, and the like, includes the sequential steps of administering to the patient at least one dose of an antiangiogenic cyclo-arginine-glycine-aspartic acid-containing pentapeptide (cRGD pentapeptide); administering to the patient an anti-tumor effective amount of a radioimmunotherapeutic agent (RIT); and then administering to the patient at least one additional dose of cRGD pentapeptide. The cRGD pentapeptide is preferably cyclo-(Arg-Gly-Asp-D-Phe-[N-Me]-Val), and the RIT is preferably a radionuclide-labeled chelating agent-ligand complex in which chelating agent is chemically bonded to a tumor-targeting molecule, such as a monoclonal antibody.

Post-Translational Incorporation of L-Phenylalanine into the C-Terminus of α-Tubulin as a Possible Cause of Neuronal Dysfunction

PubMed Central

Ditamo, Yanina; Dentesano, Yanela M.; Purro, Silvia A.; Arce, Carlos A.; Bisig, C. Gastón

2016-01-01

α-Tubulin C-terminus undergoes post-translational, cyclic tyrosination/detyrosination, and L-Phenylalanine (Phe) can be incorporated in place of tyrosine. Using cultured mouse brain-derived cells and an antibody specific to Phe-tubulin, we showed that: (i) Phe incorporation into tubulin is reversible; (ii) such incorporation is not due to de novo synthesis; (iii) the proportion of modified tubulin is significant; (iv) Phe incorporation reduces cell proliferation without affecting cell viability; (v) the rate of neurite retraction declines as level of C-terminal Phe incorporation increases; (vi) this inhibitory effect of Phe on neurite retraction is blocked by the co-presence of tyrosine; (vii) microtubule dynamics is reduced when Phe-tubulin level in cells is high as a result of exogenous Phe addition and returns to normal values when Phe is removed; moreover, microtubule dynamics is also reduced when Phe-tubulin is expressed (plasmid transfection). It is known that Phe levels are greatly elevated in blood of phenylketonuria (PKU) patients. The molecular mechanism underlying the brain dysfunction characteristic of PKU is unknown. Beyond the differences between human and mouse cells, it is conceivable the possibility that Phe incorporation into tubulin is the first event (or among the initial events) in the molecular pathways leading to brain dysfunctions that characterize PKU. PMID:27905536

Removal efficiency of polycyclic aromatic hydrocarbons and phthalate esters by surface flow wetland in Shunyi district, Beijing

NASA Astrophysics Data System (ADS)

Li, Binghua; Liu, Licai; Han, Li; Yang, Yong

2017-03-01

The surface flow wetland (SFW) system was located on Shunyi district, Beijing. It was built to treat industrial wastewater and domestic sewage, which were looked as its influent. Here sixteen polycyclic aromatic hydrocarbons (PAHs) and six phthalate esters (PAEs) were detected by gas chromatography-mass spectrometry (GC-MS).To determine treatment effect of SFW system, concentrations of targeted compounds in the influent were compared with those in the effluent. Results showed typical compounds of industrial wastewater were naphthalene (NAP), phenanthrene (PHE), dibutyl phthalate (DBP), di-(2-ethylhexyl) phthalate (DEHP), and their concentrations were ranged from 122.6 ng.L-1 to 760.6 ng.L-1. However typical compounds of domestic sewage were NAP, anthracene (ANT), PHE, DBP, diethyl phthalate (DEP), DEHP, and their concentrations were ranged from 280 ng.L-1 to 7998.1 ng.L-1. Typical compounds of effluent were NAP, PHE, DBP, DEHP, and their concentrations changed between 4.2 ng.L-1 and 1430.74 ng.L-1. The removal rate of those compounds were 10% ~ 99%, and nineteen compounds removal rate reached above 70%.Therefore, it can be concluded that SFW system had a strong effect on the removal of these compounds.

Preparation of catalytically active, covalent α-polylysine-enzyme conjugates via UV/vis-quantifiable bis-aryl hydrazone bond formation.

PubMed

Grotzky, Andrea; Manaka, Yuichi; Kojima, Taisuke; Walde, Peter

2011-01-10

Covalent UV/vis-quantifiable bis-aryl hydrazone bond formation was investigated for the preparation of conjugates between α-poly-d-lysine (PDL) and either α-chymotrypsin (α-CT) or horseradish peroxidase (HRP). PDL and the enzymes were first modified via free amino groups with the linking reagents succinimidyl 6-hydrazinonicotinate acetone hydrazone (S-HyNic, at pH 7.6) and succinimidyl 4-formylbenzoate (S-4FB, at pH 7.2), respectively. The modified PDL and enzymes were then conjugated at pH 4.7, whereby polymer chains carrying several enzymes were obtained. Kinetics of the bis-aryl hydrazone bond formation was investigated spectrophotometrically at 354 nm. Retention of the enzymatic activity after conjugate formation was confirmed by using the substrates N-succinimidyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide (for α-CT) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS, for HRP). Thus, not only a mild and efficient preparation and convenient quantification of a conjugate between the polycationic α-polylysine and enzymes could be shown, but also the complete preservation of the enzymatic activity.

Effect of dietary aspartame on plasma concentrations of phenylalanine and tyrosine in normal and homozygous phenylketonuric patients.

PubMed

Mackey, S A; Berlin, C M

1992-07-01

Six normal subjects each ingested a single 12-oz can of a diet cola (Diet Coke) providing 184 mg aspartame (APM), of which 104 mg is phenylalanine (Phe), and, on another occasion, a single 12-oz can of regular cola (Coke Classic). Neither cola significantly affected plasma concentrations of Phe or tyrosine over the three-hour postingestion study period. Each of five homozygous phenylketonuric (PKU) subjects (ages 11, 16, 17, 21, and 23 years) ingested a single 12-oz can of the same diet cola. In these five subjects (three with classic PKU and two with hyperphenylalinemia), the increase in plasma Phe concentrations varied from 0.26 mg/dL to 1.77 mg/dL two or three hours after ingestion (baseline levels, 5.04 to 17.2 mg/dL). Tyrosine concentrations did not differ significantly from baseline levels. The data indicate that ingestion of dietary Phe, as supplied in a single can of diet cola, is readily handled in both normal and PKU subjects. The small increases in plasma Phe concentrations in the homozygous PKU patients are not considered clinically significant.

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

PubMed

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.

/sup 2/H NMR demonstration of amino acid - nucleotide interactions

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

Khaled, M.A.; Watkins, C.L.; Lacey, J.C. Jr.

Deuterium (/sup 2/H) NMR was used to investigate the interaction of L-Phenylalanine (with aromatic protons replaced by deuterons (Phe-D/sub 5/)), with 5'-AMP and polyadenylic acid (poly A). A considerable change in line width of the aromatic deuteron signals of Phe was observed. These data were plotted using a Scatchard-type equation, and yielded apparent binding constants for L-Phe to 5'-AMP and poly A of 7 and 11 M/sup -1/, respectively. Future applications of /sup 2/H-NMR in the study of nucleic acid-protein interactions are discussed.

Novel selective agonists and antagonists confirm neurokinin NK1 receptors in guinea-pig vas deferens.

PubMed Central

Hall, J. M.; Morton, I. K.

1991-01-01

1. This study investigated the recognition characteristics of neurokinin receptors mediating potentiation of the contractile response to field stimulation in the guinea-pig vas deferens. 2. A predominant NK1 receptor population is strongly suggested by the relative activities of the common naturally-occurring tachykinin agonists, which fall within less than one order of magnitude. This conclusion is supported by the relative activities of the synthetic NK1 selective agonists substance P methyl ester, [Glp6,L-Pro9]-SP(6-11) and delta-aminovaleryl-[L-Pro9,N-MeLeu10]- SP(7-11) (GR73632) which were 0.78, 9.3 and 120 as active as substance P, respectively. Furthermore, the NK2 selective agonist [Lys3, Gly8,-R-gamma-lactam-Leu9]-NKA(3-10) (GR64349) was active only at the highest concentrations tested (greater than 10 microM), and the NK3 selective agonist, succ-[Asp6,N-MePhe8]-SP(6-11) (senktide) was essentially inactive (10 nM-32 microM). 3. [D-Arg1,D-Pro2,D-Trp7,9,Leu11]-SP(1-11) antagonized responses to neurokinin A, neurokinin B, physalaemin, eledoisin, [Glp6,D-Pro9]-SP(6-11), GR73632 and GR64349 (apparent pKB s 5.6-6.2), but was less potent in antagonizing responses to substance P, substance P methyl ester and [Glp6,L-Pro9]-SP(6-11) (apparent pKB s less than or equal to 5.0-5.0). 4. In contrast, the recently developed NK1-selective receptor antagonist [D-Pro9[Spiro-gamma-lactam]Leu10,Trp11]-SP(1-11) (GR71251) did not produce agonist-dependent pKB estimates. Schild plot analysis indicated a competitive interaction with a single receptor population where the antagonist had an estimated overall pKB of 7.58 +/- 0.13 for the four agonists of differing subtype selectivity tested (GR73632, GR64349, substance P methyl ester and neurokinin B).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1707714

Efficient biodegradation of phenanthrene by a novel strain Massilia sp. WF1 isolated from a PAH-contaminated soil.

PubMed

Wang, Haizhen; Lou, Jun; Gu, Haiping; Luo, Xiaoyan; Yang, Li; Wu, Laosheng; Liu, Yong; Wu, Jianjun; Xu, Jianming

2016-07-01

A novel phenanthrene (PHE)-degrading strain Massilia sp. WF1, isolated from PAH-contaminated soil, was capable of degrading PHE by using it as the sole carbon source and energy in a range of pH (5.0-8.0), temperatures (20-35 °C), and PHE concentrations (25-400 mg L(-1)). Massilia sp. WF1 exhibited highly effective PHE-degrading ability that completely degraded 100 mg L(-1) of PHE over 2 days at optimal conditions (pH 6.0, 28 °C). The kinetics of PHE biodegradation by Massilia sp. WF1 was well represented by the Gompertz model. Results indicated that PHE biodegradation was inhibited by the supplied lactic acid but was promoted by the supplied carbon sources of glucose, citric acid, and succinic acid. Salicylic acid (SALA) and phthalic acid (PHTA) were not utilized by Massilia sp. WF1 and had no obvious effect on PHE biodegradation. Only two metabolites, 1-hydroxy-2-naphthoic acid (1H2N) and PHTA, were identified in PHE biodegradation process. Quantitatively, nearly 27.7 % of PHE was converted to 1H2N and 30.3 % of 1H2N was further metabolized to PHTA. However, the PHTA pathway was broken and the SALA pathway was ruled out in PHE biodegradation process by Massilia sp. WF1.

In vitro quantitative study of the degradation of endomorphins.

PubMed

Tömböly, Csaba; Péter, Antal; Tóth, Géza

2002-09-01

The catabolism of the endomorphins was investigated in detail. The endomorphins were degraded relatively slowly in the rat brain homogenate (t1/2(endomorphin-1)=4.94 min; t1/2(endomorphin-2)=3.81 min). The inhibition of metalloproteases and aminopeptidases stabilised the endomorphins to the greatest extent. The digestion of endomorphins tritiated specifically on Tyr(1), Pro(2) or Phe(3) established also that only the aminopeptidase pathways were essential for inactivation of the endomorphins, and that the tetrapeptides were degraded by cleavage of the Pro(2)-Trp(3) or Pro(2)-Phe(3) bond. The end-products of the catabolism were amino acids; the fragments Tyr-Pro-OH and Pro-Trp-Phe-NH2 were present as intermediates. Metabolites produced by brain carboxypeptidases were not detected.

Lactobacillus cerevisiae sp. nov., isolated from a spoiled brewery sample.

PubMed

Koob, Jennifer; Jacob, Fritz; Wenning, Mareike; Hutzler, Mathias

2017-09-01

A Gram-stain-positive, non-motile, rod-shaped bacterium, designated TUM BP 140423000-2250T (=DSM 100836T=LMG 29073T), was isolated from spoiled beer. This bacterium did not form spores, and was catalase-negative and facultatively anaerobic. Its taxonomic position was determined in a polyphasic study. The 16S rRNA gene sequence similarity data showed that the strain belonged to the Lactobacillus genus with the nearest neighbours being Lactobacillus koreensis DCY50T (sequence similarity 99.5 %), Lactobacillus yonginensis THK-V8T (99.2 %) and Lactobacillus parabrevis LMG 11984T (98.7 %). Sequence comparisons of additional phylogenetic markers, pheS and rpoA, confirmed the 16S rRNA gene sequence tree topology. The maximum rpoA sequence similarity was 92.3 % with L. yonginensis THK-V8T. The DNA G+C content of the isolate was 50.0 mol%. The DNA-DNA relatedness showed that strain TUM BP 140423000-2250T could be clearly distinguished from L. koreensis DCY 50T (30.8±0.4 %) and L. yonginensis THK-V8T (23.6±5.9 %). The major fatty acids were C18 : 1ω9c, summed feature 7 (comprised of C19 : 0 cyclo ω10c/C19 : 1ω6c) and C16 : 0. Based on phenotypic and genotypic studies, the authors propose classifying the new isolate as a representative of a novel species of the genus Lactobacillus, Lactobacillus cerevisiae sp. nov. The type strain is deposited at the Research Centre Weihenstephan for Brewing and Food Quality as TUM BP 140423000-2250T (=DSM 100836T=LMG 29073T).

Determination of adenine based on the fluorescence recovery of the L-Tryptophan-Cu(2+) complex.

PubMed

Duan, Ruilin; Li, Chunyan; Liu, Shaopu; Liu, Zhongfang; Li, Yuanfang; Yuan, Yusheng; Hu, Xiaoli

2016-01-05

A simple and sensitive method for determination of adenine was developed based on fluorescence quenching and recovery of L-Tryptophan (L-Trp). The fluorescence of L-Trp could efficiently quenched by copper ion compared with other common metal ions. Upon addition of adenine (Ade) in L-Trp-Cu(II) system, the fluorescence was reoccurred. Under the optimum conditions, the recovery fluorescence intensity was linearly correlated with the concentration of adenine in the range from 0.34 to 25.0μmolL(-1), with a correlation coefficient (R(2)) of 0.9994. The detection limit (3σ/k) was 0.046μmolL(-1), indicating that this method could applied to detect trace adenine. In this study, amino acids including L-Trp, D-Trp, L-Tyr, D-Tyr, L-Phe, D-Phe were investigated and only L-Trp could well chelated copper ion. Additionally, the mechanism of quench and recovery also were discussed and the method was successfully applied to detect the adenine in DNA with satisfactory results. Copyright © 2015 Elsevier B.V. All rights reserved.

Pegvaliase for the treatment of phenylketonuria: Results of a long-term phase 3 clinical trial program (PRISM).

PubMed

Thomas, Janet; Levy, Harvey; Amato, Stephen; Vockley, Jerry; Zori, Roberto; Dimmock, David; Harding, Cary O; Bilder, Deborah A; Weng, Haoling H; Olbertz, Joy; Merilainen, Markus; Jiang, Joy; Larimore, Kevin; Gupta, Soumi; Gu, Zhonghua; Northrup, Hope

2018-05-01

Phenylketonuria (PKU) is caused by phenylalanine hydroxylase (PAH) deficiency that results in phenylalanine (Phe) accumulation. Pegvaliase, PEGylated recombinant Anabaena variabilis phenylalanine ammonia lyase (PAL), converts Phe to trans-cinnamic acid and ammonia, and is a potential enzyme substitution therapy to lower blood Phe in adults with PKU. Two Phase 3 studies, PRISM-1 and PRISM-2, evaluated the efficacy and safety of pegvaliase treatment using an induction, titration, and maintenance dosing regimen in adults with PKU. In PRISM-1, pegvaliase-naïve participants with blood Phe >600 μmol/L were randomized 1:1 to a maintenance dose of 20 mg/day or 40 mg/day of pegvaliase. Participants in PRISM-1 continued pegvaliase treatment in PRISM-2, a 4-part clinical trial that includes an ongoing, open-label, long-term extension study of pegvaliase doses of 5 mg/day to 60 mg/day. Of 261 participants who received pegvaliase treatment, 72.0% and 32.6% reached ≥12 months and ≥ 24 months of study treatment, respectively, and 65% are still actively receiving treatment. Mean (SD) blood Phe was 1232.7 (386.4) μmol/L at baseline, 564.5 (531.2) μmol/L at 12 months, and 311.4 (427) μmol/L at 24 months, a decrease from baseline of 51.1% and 68.7%, respectively. Within 24 months, 68.4% of participants achieved blood Phe ≤600 μmol/L, 60.7% of participants achieved blood Phe ≤360 μmol/L, below the upper limit recommended in the American College of Medical Genetics and Genomics PKU management guidelines, and 51.2% achieved blood Phe ≤120 μmol/L, below the upper limit of normal in the unaffected population. Improvements in neuropsychiatric outcomes were associated with reductions in blood Phe and were sustained with long-term pegvaliase treatment. Adverse events (AEs) were more frequent in the first 6 months of exposure (early treatment phase) than after 6 months of exposure (late treatment phase); 99% of AEs were mild or moderate in severity and 96% resolved without dose interruption or reduction. The most common AEs were arthralgia (70.5%), injection-site reaction (62.1%), injection-site erythema (47.9%), and headache (47.1%). Acute systemic hypersensitivity events consistent with clinical National Institute of Allergy and Infectious Diseases and the Food Allergy and Anaphylaxis Network anaphylaxis criteria were observed in 12 participants (17 events); of these, 6 participants remained on treatment. Acute systemic hypersensitivity events including potential events of anaphylaxis were not associated with immunoglobulin E, and all events resolved without sequelae. Results from the PRISM Phase 3 program support the efficacy of pegvaliase for the treatment of adults with PKU, with a manageable safety profile in most participants. The PRISM-2 extension study will continue to assess the long-term effects of pegvaliase treatment. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Integration of E. coli aroG-pheA tandem genes into Corynebacterium glutamicum tyrA locus and its effect on L-phenylalanine biosynthesis

PubMed Central

Liu, Dong-Xin; Fan, Chang-Sheng; Tao, Ju-Hong; Liang, Guo-Xin; Gao, Shan-E; Wang, Hai-Jiao; Li, Xin; Song, Da-Xin

2004-01-01

AIM: To study the effect of integration of tandem aroG-pheA genes into the tyrA locus of Corynebacterium glutamicum (C. glutamicum) on the production of L-phenylalanine. METHODS: By nitrosoguanidine mutagenesis, five p-fluorophenylalanine (FP)-resistant mutants of C.glutamicum FP were selected. The tyrA gene encoding prephenate dehydrogenase (PDH) of C.glutamicum was amplified by polymerase chain reaction (PCR) and cloned on the plasmid pPR. Kanamycin resistance gene (Km) and the PBF-aroG-pheA-T (GA) fragment of pGA were inserted into tyrA gene to form targeting vectors pTK and pTGAK, respectively. Then, they were transformed into C.glutamicum FP respectively by electroporation. Cultures were screened by a medium containing kanamycin and detected by PCR and phenotype analysis. The transformed strains were used for L-phenylalanine fermentation and enzyme assays. RESULTS: Engineering strains of C.glutamicum (Tyr-) were obtained. Compared with the original strain, the transformed strain C. glutamicum GAK was observed to have the highest elevation of L-phenylalanine production by a 1.71-fold, and 2.9-, 3.36-, and 3.0-fold in enzyme activities of chorismate mutase, prephenate dehydratase and 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase, respectively. CONCLUSION: Integration of tandem aroG-pheA genes into tyrA locus of C. glutamicum chromosome can disrupt tyrA gene and increase the yield of L-phenylalanine production. PMID:15534933

Endophytic Streptomyces in the traditional medicinal plant Arnica montana L.: secondary metabolites and biological activity.

PubMed

Wardecki, Tina; Brötz, Elke; De Ford, Christian; von Loewenich, Friederike D; Rebets, Yuriy; Tokovenko, Bogdan; Luzhetskyy, Andriy; Merfort, Irmgard

2015-08-01

Arnica montana L. is a medical plant of the Asteraceae family and grows preferably on nutrient poor soils in mountainous environments. Such surroundings are known to make plants dependent on symbiosis with other organisms. Up to now only arbuscular mycorrhizal fungi were found to act as endophytic symbiosis partners for A. montana. Here we identified five Streptomyces strains, microorganisms also known to occur as endophytes in plants and to produce a huge variety of active secondary metabolites, as inhabitants of A. montana. The secondary metabolite spectrum of these strains does not contain sesquiterpene lactones, but consists of the glutarimide antibiotics cycloheximide and actiphenol as well as the diketopiperazines cyclo-prolyl-valyl, cyclo-prolyl-isoleucyl, cyclo-prolyl-leucyl and cyclo-prolyl-phenylalanyl. Notably, genome analysis of one strain was performed and indicated a huge genome size with a high number of natural products gene clusters among which genes for cycloheximide production were detected. Only weak activity against the Gram-positive bacterium Staphylococcus aureus was revealed, but the extracts showed a marked cytotoxic activity as well as an antifungal activity against Candida parapsilosis and Fusarium verticillioides. Altogether, our results provide evidence that A. montana and its endophytic Streptomyces benefit from each other by completing their protection against competitors and pathogens and by exchanging plant growth promoting signals with nutrients.

Characterization of two key enzymes for aromatic amino acid biosynthesis in symbiotic archaea.

PubMed

Shlaifer, Irina; Turnbull, Joanne L

2016-07-01

Biosynthesis of L-tyrosine (L-Tyr) and L-phenylalanine (L-Phe) is directed by the interplay of three enzymes. Chorismate mutase (CM) catalyzes the rearrangement of chorismate to prephenate, which can be either converted to hydroxyphenylpyruvate by prephenate dehydrogenase (PD) or to phenylpyruvate by prephenate dehydratase (PDT). This work reports the first characterization of a trifunctional PD-CM-PDT from the smallest hyperthermophilic archaeon Nanoarchaeum equitans and a bifunctional CM-PD from its host, the crenarchaeon Ignicoccus hospitalis. Hexa-histidine tagged proteins were expressed in Escherichia coli and purified by affinity chromatography. Specific activities determined for the trifunctional enzyme were 21, 80, and 30 U/mg for CM, PD, and PDT, respectively, and 47 and 21 U/mg for bifunctional CM and PD, respectively. Unlike most PDs, these two archaeal enzymes were insensitive to regulation by L-Tyr and preferred NADP(+) to NAD(+) as a cofactor. Both the enzymes were highly thermally stable and exhibited maximal activity at 90 °C. N. equitans PDT was feedback inhibited by L-Phe (Ki = 0.8 µM) in a non-competitive fashion consistent with L-Phe's combination at a site separate from that of prephenate. Our results suggest that PD from the unique symbiotic archaeal pair encompass a distinct subfamily of prephenate dehydrogenases with regard to their regulation and co-substrate specificity.

Enhancement of  l-phenylalanine production in Escherichia coli by heterologous expression of Vitreoscilla hemoglobin.

PubMed

Wu, Wei-Bin; Guo, Xiao-Lei; Zhang, Ming-Liang; Huang, Qing-Gen; Qi, Feng; Huang, Jian-Zhong

2018-05-01

l-Phenylalanine is an important amino acid that is widely used in the production of food flavors and pharmaceuticals. Generally, l-phenylalanine production by engineered Escherichia coli requires a high rate of oxygen supply. However, the coexpression of Vitreoscilla hemoglobin gene (vgb), driven bya tac promoter, with the genes encoding 3-deoxy-d-arabinoheptulosonate-7-phosphate synthetase (aroF) and feedback-resistant chorismate mutase/prephenate dehydratase (pheA fbr ), led to increased productivity and decreased demand for aeration by E. coli CICC10245. Shake-flask studies showed that vgb-expressing strains displayed higher rates of oxygen uptake, and l-phenylalanine production under standard aeration conditions was increased. In the aerobic fermentation process, cell growth, l-phenylalanine production, and glucose consumption by the recombinant E. coli strain PAPV, which harbored aroF, pheA fbr , and tac-vgb genes, were increased compared to that in the strain harboring only aroF and pheA fbr (E. coli strain PAP), especially under oxygen-limited conditions. The vgb-expressing strain PAPV produced 21.9% more biomass and 16.6% more l-phenylalanine, while consuming only approximately 5% more glucose after 48 H of fermentation. This study demonstrates a method to enhance the l-phenylalanine production by E. coli using less intensive and thus more economical aeration conditions. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

Structural Insights into l-Tryptophan Dehydrogenase from a Photoautotrophic Cyanobacterium, Nostoc punctiforme.

PubMed

Wakamatsu, Taisuke; Sakuraba, Haruhiko; Kitamura, Megumi; Hakumai, Yuichi; Fukui, Kenji; Ohnishi, Kouhei; Ashiuchi, Makoto; Ohshima, Toshihisa

2017-01-15

l-Tryptophan dehydrogenase from Nostoc punctiforme NIES-2108 (NpTrpDH), despite exhibiting high amino acid sequence identity (>30%)/homology (>50%) with NAD(P) + -dependent l-Glu/l-Leu/l-Phe/l-Val dehydrogenases, exclusively catalyzes reversible oxidative deamination of l-Trp to 3-indolepyruvate in the presence of NAD + Here, we determined the crystal structure of the apo form of NpTrpDH. The structure of the NpTrpDH monomer, which exhibited high similarity to that of l-Glu/l-Leu/l-Phe dehydrogenases, consisted of a substrate-binding domain (domain I, residues 3 to 133 and 328 to 343) and an NAD + /NADH-binding domain (domain II, residues 142 to 327) separated by a deep cleft. The apo-NpTrpDH existed in an open conformation, where domains I and II were apart from each other. The subunits dimerized themselves mainly through interactions between amino acid residues around the β-1 strand of each subunit, as was observed in the case of l-Phe dehydrogenase. The binding site for the substrate l-Trp was predicted by a molecular docking simulation and validated by site-directed mutagenesis. Several hydrophobic residues, which were located in the active site of NpTrpDH and possibly interacted with the side chain of the substrate l-Trp, were arranged similarly to that found in l-Leu/l-Phe dehydrogenases but fairly different from that of an l-Glu dehydrogenase. Our crystal structure revealed that Met-40, Ala-69, Ile-74, Ile-110, Leu-288, Ile-289, and Tyr-292 formed a hydrophobic cluster around the active site. The results of the site-directed mutagenesis experiments suggested that the hydrophobic cluster plays critical roles in protein folding, l-Trp recognition, and catalysis. Our results provide critical information for further characterization and engineering of this enzyme. In this study, we determined the three-dimensional structure of l-Trp dehydrogenase, analyzed its various site-directed substitution mutants at residues located in the active site, and obtained the following informative results. Several residues in the active site form a hydrophobic cluster, which may be a part of the hydrophobic core essential for protein folding. To our knowledge, there is no previous report demonstrating that a hydrophobic cluster in the active site of any l-amino acid dehydrogenase may have a critical impact on protein folding. Furthermore, our results suggest that this hydrophobic cluster could strictly accommodate l-Trp. These studies show the structural characteristics of l-Trp dehydrogenase and hence would facilitate novel applications of l-Trp dehydrogenase. Copyright © 2016 American Society for Microbiology.

Investigation of Fibril Forming Mechanisms of l-Phenylalanine and l-Tyrosine: Microscopic Insight toward Phenylketonuria and Tyrosinemia Type II.

PubMed

Banik, Debasis; Kundu, Sangita; Banerjee, Pavel; Dutta, Rupam; Sarkar, Nilmoni

2017-02-23

Phenylketonuria and tyrosinemia type II, the two metabolic disorders, are originated due to the complications in metabolism of phenylalanine (Phe) and tyrosine (Tyr), respectively. Several neurological injuries, involving microcephaly, mental retardation, epilepsy, motor disease, and skin problems etc., are the symptoms of these two diseases. It has been reported that toxic amyloid fibrils are formed at high concentrations of Phe and Tyr. Our study indicates that the fibril forming mechanisms of Phe and Tyr are completely different. In the case of Phe, -NH 3 + and -COO - groups of neighboring molecules interact via hydrogen bonding and polar interactions. On the other hand, there is no role of - NH 3 + group in the fibril forming mechanism of Tyr. In Tyr fibril, the two hydrogen bonding partners are -OH and -COO - groups. In addition, we have also investigated the effect of three lanthanide cations on the fibrillar assemblies of Phe. It has been observed that the efficiencies of three lanthanides to inhibit the fibrillar assemblies of Phe follow the order Tb 3+ < Sm 3+ < Eu 3+ .

Oligopeptides and copeptides of homochiral sequence, via beta-sheets, from mixtures of racemic alpha-amino acids, in a one-pot reaction in water; relevance to biochirogenesis.

PubMed

Illos, Roni A; Bisogno, Fabricio R; Clodic, Gilles; Bolbach, Gerard; Weissbuch, Isabelle; Lahav, Meir

2008-07-09

As part of our studies on the biochirogenesis of peptides of homochiral sequence during early evolution, the formation of oligopeptides composed of 14-24 residues of the same handedness in the polymerization of dl-leucine (Leu), dl-phenylalanine (Phe), and dl-valine (Val) in aqueous solutions, by activation with N, N'-carbonyldiimidazole and then initiation with a primary amine, in a one-pot reaction, was demonstrated by MALDI-TOF MS using deuterium enantio-labeled alpha-amino acids. The formation of long isotactic peptides is rationalized by the following steps occurring in tandem: (i) creation of a library of short diasteroisomeric oligopeptides containing isotactic peptides in excess in comparison to a binomial kinetics, as a result of an asymmetric induction exerted by the N-terminal residue of a given handedness; (ii) precipitation of the less soluble racemic isotactic penta- and hexapeptides in the form of beta-sheets that are delineated by homochiral rims; (iii) regio-enantiospecific chain elongation occurring heterogeneously at the beta-sheets/solution interface. Polymerization of l-Leu with l-isoleucine (Ile) or l-Phe with l- (1) N-Me-histidine yielded mixtures of copeptides containing both residues. In contrast, in the polymerization of the corresponding mixtures of l- + d-alpha-amino acids, the long oligopeptides were composed mainly from oligo- l-Leu and oligo- d-Ile in the first system and oligo- d-Phe in the second. Furthermore, in the polymerization of mixtures of hydrophobic racemic alpha-amino acids dl-Leu, dl-Val, and dl-Phe and with added racemic dl-alanine and dl-tyrosine, copeptides of homochiral sequences are most dominantly represented. Possible routes for a spontaneous "mirror-symmetry breaking" process of the racemic mixtures of homochiral peptides are presented.

Optical resolution of phenylthiohydantoin-amino acids by capillary electrophoresis and identification of the phenylthiohydantoin-D-amino acid residue of [D-Ala2]-methionine enkephalin.

PubMed

Kurosu, Y; Murayama, K; Shindo, N; Shisa, Y; Ishioka, N

1996-11-01

This is an initial report to propose a protein sequence analysis system with DL differentiation using capillary electrophoresis (CE). This system consists of a protein sequencer and a CE system. After fractionation of phenyl-thiohydantoin (PTH)-amino acids using a protein sequencer, optical resolution for each PTH-amino acid is performed by CE using some chiral selectors such as digitonin, beta-escin and others. As a model peptide, [D-Ala2]-methionine enkephalin (L-Tyr-D-Ala-Gly-L-Phe-L-Met), was used and the sequence with DL differentiation was determined, with the exception of the fourth amino acid, L-Phe, using our proposed system.

Oral and intravenous l-[1-13 C]phenylalanine delivery measure similar rates of elimination when gastric emptying and splanchnic extraction are accounted for in adult mixed hounds.

PubMed

Gooding, M A; Cant, J P; Pencharz, P B; Davenport, G M; Atkinson, J L; Shoveller, A K

2013-02-01

There are few reported estimates of amino acid (AA) kinetics in adult mammals and none exist in adult dogs. The study objectives were to evaluate the use of oral isotope delivery in contrast to the more commonly used intravenous (IV) delivery to estimate AA kinetics in adult dogs and to estimate splanchnic extraction and gastric emptying using a commonly accepted mathematical model. Dogs received 25 × 1/2-hourly meals (13 g/kg BW/day) and either an oral or IV bolus of l-[1-(13) C]Phe (12 mg/kg BW). Blood samples were taken immediately before each feeding. Concentrations of plasma Phe were measured using liquid chromatography-tandem mass spectrometry. There were no differences in baseline plasma Phe concentrations (34 μm ± 0.61), Phe distribution volume, Phe pool size and rate constants between dogs when the tracer was administered IV or orally (p > 0.25). Decay curve for plasma l-[1-(13) C]Phe differed between IV and oral dosing protocols with IV dosing fit best using a two-compartment model. Phe disappeared from plasma at a mean rate of 2.8%/min. Estimates of gastric emptying and splanchnic extraction did not differ based on oral or IV tracer dosing when the decay curves were fit with the two-compartment model (p > 0.40). The half-life for gastric emptying was 18 min, and first-pass Phe extraction by the splanchnic bed was 24% of the dietary Phe. These results suggest that oral isotope dosing can be used as an alternative to IV isotope dosing in studies that utilize a primed, constant dosing approach to measure protein and amino acid kinetics. © 2011 Blackwell Verlag GmbH.

The conformation of cyclo(-D-Pro-Ala4-) as a model for cyclic pentapeptides of the DL4 type.

PubMed

Heller, Markus; Sukopp, Martin; Tsomaia, Natia; John, Michael; Mierke, Dale F; Reif, Bernd; Kessler, Horst

2006-10-25

The conformation of the cyclic pentapeptide cyclo(-D-Pro-Ala(4)-) in solution and in the solid state was reinvestigated using modern NMR techniques. To allow unequivocal characterization of hydrogen bonds, relaxation behavior, and intramolecular distances, differently labeled isotopomers were synthesized. The NMR results, supported by extensive MD simulations, demonstrate unambiguously that the preferred conformation previously described by us, but recently questioned, is indeed correct. The validation of the conformational preferences of this cyclic peptide is important given that this system is a template for several bioactive compounds and for controlled "spatial screening" for the search of bioactive conformations.

Antioxidant properties of salmon (Salmo salar L.) protein fraction hydrolysates revealed following their ex vivo digestion and in vitro hydrolysis.

PubMed

Borawska, Justyna; Darewicz, Małgorzata; Pliszka, Monika; Vegarud, Gerd E

2016-06-01

Salmon (Salmo salar L.) myofibryllar protein (MP) and sarcoplasmic protein (SP) were digested with human gastric and duodenal juices and hydrolysed in vitro with commercial pepsin and Corolase PP. The digestion after duodenal juice/Corolase PP caused almost complete breakdown of peptide bonds in MP and SP. The DPPH(•) scavenging activity of proteins decreased during both ex vivo digestion and in vitro hydrolysis. The highest value of DPPH(•) scavenging activity was shown for the gastric digest of SP (8.88 ± 0.87%). The ABTS(+•) scavenging activity of MP and SP increased during digestion/hydrolysis. The duodenal digest of SP was characterised by the highest value of ABTS(+•) scavenging activity (72.7 ± 1.2%). In turn, the highest value of ferric-reducing power was determined for the gastric digest of SP (84.8 ± 0.2%). Salmon antioxidant peptides Phe-Ile-Lys-Lys, His-Leu, Ile-Tyr, Pro-His-Leu, Pro-Trp, Val-Pro-Trp were identified in both ex vivo digested and in vitro hydrolysed MP and SP. An antioxidant peptide, Val-Tyr, was additionally detected in the in vitro hydrolysate of SP. The results indicate the salmon myofibrillar and sarcoplasmic protein fractions as potential sources of antioxidant peptides that could be released in the gastrointestinal tract but their amino acid sequence and quantification vary. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

Assessment of the impact of phenylketonuria and its treatment on quality of life of patients and parents from seven European countries.

PubMed

Bosch, Annet M; Burlina, Alberto; Cunningham, Amy; Bettiol, Esther; Moreau-Stucker, Flavie; Koledova, Ekaterina; Benmedjahed, Khadra; Regnault, Antoine

2015-06-18

The strict and demanding dietary treatment and mild cognitive abnormalities seen in PKU treated from a young age can be expected to affect the health-related quality of life (HRQoL) of patients and their families. Our aim was to describe the HRQoL of patients with PKU from a large international study, using generic HRQoL measures and an innovative PKU-specific HRQoL questionnaire (PKU-QOL). Analyses were exploratory, performed post-hoc on data collected primarily to validate the PKU-QOL. A multicentre, prospective, non-interventional, observational study conducted in France, Germany, Italy, The Netherlands, Spain, Turkey and the UK. Patients diagnosed with PKU aged ≥9 years old and treated with a Phe-restricted diet and/or Phe-free amino acid protein supplements and/or pharmacological therapy were included in the study; parents of at least one patient with PKU aged <18 years were also included. HRQoL was assessed by generic measures (Pediatric Quality-of-Life Inventory; Medical Outcome Survey 36 item Short Form; Child Health Questionnaire 28 item Parent Form) and the newly developed PKU-QOL. Mean generic domain scores were interpreted using published reference values from the general population. PKU-QOL domain scores were described overall and in different subgroups of patients defined according to severity of PKU, overall assessment of patient's health status by the investigator and treatment with tetrahydrobiopterin (BH4). Data from 559 subjects were analysed: 306 patients (92 children, 110 adolescents, 104 adults) and 253 parents. Mean domain scores of generic measures in the study were comparable to the general population. The highest PKU-QOL impact scores (indicating greater impact) were for emotional impact of PKU, anxiety about blood Phe levels, guilt regarding poor adherence to dietary restrictions or Phe-free amino acid supplement intake and anxiety regarding blood Phe levels during pregnancy. Patients with mild/moderate PKU and those receiving BH4 reported lower practical and emotional impacts of the diet and Phe-free amino acid supplement intake. Patients with PKU showed good HRQoL in the study, both with the generic and PKU-specific measures. Negative impacts of PKU on a patient's life, including the emotional impact of PKU and its management, was delineated by the PKU-QOLs across all age groups.

Metabolic transcription analysis of engineered Escherichia coli strains that overproduce L-phenylalanine

PubMed Central

Báez-Viveros, José Luis; Flores, Noemí; Juárez, Katy; Castillo-España, Patricia; Bolivar, Francisco; Gosset, Guillermo

2007-01-01

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 both PTS inactivation and the presence of plasmids. For instance, acs-ackA, sucABCD, and sdhABCD operons were up-regulated in PB12 (PTS mutant that carries an arcB- mutation). The induction of these operons was further increased by the presence of plasmids in PB12-ev2. Some genes involved in the shikimate and specific aromatic amino acid pathways showed down-regulation in the L-Phe overproducing strains, might cause possible metabolic limitations in the shikimate pathway. Conclusion The identification of potential rate-limiting steps and the detection of transcriptional responses in overproducing microorganisms may suggest "reverse engineering" strategies for the further improvement of L-Phe production strains. PMID:17880710

A Macrocyclic Agouti-Related Protein/[Nle4,DPhe7]α-Melanocyte Stimulating Hormone Chimeric Scaffold Produces Subnanomolar Melanocortin Receptor Ligands.

PubMed

Ericson, Mark D; Freeman, Katie T; Schnell, Sathya M; Haskell-Luevano, Carrie

2017-01-26

The melanocortin system consists of five receptor subtypes, endogenous agonists, and naturally occurring antagonists. These receptors and ligands have been implicated in numerous biological pathways including processes linked to obesity and food intake. Herein, a truncation structure-activity relationship study of chimeric agouti-related protein (AGRP)/[Nle4,DPhe7]α-melanocyte stimulating hormone (NDP-MSH) ligands is reported. The tetrapeptide His-DPhe-Arg-Trp or tripeptide DPhe-Arg-Trp replaced the Arg-Phe-Phe sequence in the AGRP active loop derivative c[Pro-Arg-Phe-Phe-Xxx-Ala-Phe-DPro], where Xxx was the native Asn of AGRP or a diaminopropionic (Dap) acid residue previously shown to increase antagonist potency at the mMC4R. The Phe, Ala, and Dap/Asn residues were successively removed to generate a 14-member library that was assayed for agonist activity at the mouse MC1R, MC3R, MC4R, and MC5R. Two compounds possessed nanomolar agonist potency at the mMC4R, c[Pro-His-DPhe-Arg-Trp-Asn-Ala-Phe-DPro] and c[Pro-His-DPhe-Arg-Trp-Dap-Ala-DPro], and may be further developed to generate novel melanocortin probes and ligands for understanding and treating obesity.

Specialist gelator for ionic liquids.

PubMed

Hanabusa, Kenji; Fukui, Hiroaki; Suzuki, Masahiro; Shirai, Hirofusa

2005-11-08

Cyclo(l-beta-3,7-dimethyloctylasparaginyl-L-phenylalanyl) (1) and cyclo(L-beta-2-ethylhexylasparaginyl-L-phenylalanyl) (2), prepared from L-asparaginyl-L-phenylalanine methyl ester, have been found to be specialist gelators for ionic liquids. They can gel a wide variety of ionic liquids, including imizazolium, pyridinium, pyrazolidinium, piperidinium, morpholinium, and ammonium salts. The mean minimum gel concentrations (MGCs) necessary to make gels at 25 degrees C were determined for ionic liquids. The gel strength increased at a rate nearly proportional to the concentration of added gelator. The strength of the transparent gel of 1-butylpyridinium tetrafluoroborate ([C(4)py]BF(4)), prepared at a concentration of 60 g L(-1) (gelator 1/[C(4)py]BF(4)), was ca. 1500 g cm(-2). FT-IR spectroscopy indicated that a driving force for gelation was intermolecular hydrogen bonding between amides and that the phase transition from gel to liquid upon heating was brought about by the collapse of hydrogen bonding. The gels formed from ionic liquids were very thermally stable; no melting occurs up to 140 degrees C when the gels were prepared at a concentration of 70 g L(-1) (gelator/ionic liquid). The ionic conductivities of the gels were nearly the same as those of pure ionic liquids. The gelator had electrochemical stability and a wide electrochemical window. When the gels were prepared from ionic liquids containing propylene carbonate, the ionic conductivities of the resulting gels increased to levels rather higher than those of pure ionic liquids. The gelators also gelled ionic liquids containing supporting electrolytes.

Advanced Catalytic Combustors for Low Pollutant Emissions

DTIC Science & Technology

1979-11-01

liner materials in both designs are sheet or forged HS188 . This alloy was designed for stability of the microstructure and properties during heat...625 Inco 718 I: Inca 625 HS188 L605 Hast X Figure 37. Catalytic Combustor Materials. 111 &I ~ 4 L. ,.Add. R service. The hot corrosion resistance of... HS188 is similar to L605 and somewhat F better than Hastelloy X, another common liner material. HS188 exhibits good low cyclo fatigue resistance up to

Daily Profiles of Fibrinogen Metabolism for 5 Days Following Hemorrhage and Lactated Ringer’s Resuscitation in Pigs

DTIC Science & Technology

2012-01-01

Sterile stable isotope solutions of 1 13C phenylalanine (1 13C phe, 100 2mol/mL) and d5 phe (100 2mol/mL) were made in 0.45% saline and infused via the left...described by Stein et al. (15). The enrichments of phenylalanine from isolated fibrinogen were determined by gas chromatography mass spectrometry (GC MS...baseline of 5.5 T 0.3 to 4.3 T 0.3 g/ dL (P G 0.05) on day 1 but returned to baseline on day 2 and thereafter. Changes in plasma fibrinogen

Introduction of antigenic determining 2,4-dinitrophenyl residues into 4-thiouridine, N3-(3-L-amino-3-carboxypropyl) uridine and tRNA-Phe from E. coli.

PubMed Central

Seela, F; Hansske, F; Watanabe, K; Cramer, F

1977-01-01

The introduction of antigenic determining 2,4-dinitrophenyl residues into the rare ribonucleosides 4-thiouridine (1a), and N3-(3-L-amino-3-carboxypropyl) uridine (2) as well as into tRNA-Phe from E. coli has been investigated. Alkylation of 1a with omega-bromo-2,4-dinitroacetophenone (3b) gives S-(2,4-dinitrophenacyl)-4-thiouridine (5A). Applying the reaction to the 5'-monophosphate of 1a, 5b is formed, but this product decomposes at pH 7. However, acylation of 2 with 2,4-dinitrobenzoic acid N-hydroxysuccinimide ester (4b) leads to N3-[3-carboxy-3-L-(2,4-dinitrobenzamido)propyl]uridine (6) which is stable in aqueous solution. The latter reaction was used for the introduction of an antigenic determining 2,4-dinitrophenyl residue into tRNA-Phe from E. coli. The modified tRNA-Phe was isolated and by degradation of the molecule with RNase T2 and alkaline phosphatase the nucleoside derivative 6 was obtained and found to be identical with the synthetic product. PMID:68463

L-Phenylalanine functionalized silver nanoparticles: Photocatalytic and nonlinear optical applications

NASA Astrophysics Data System (ADS)

Nidya, M.; Umadevi, M.; Sankar, Pranitha; Philip, Reji; Rajkumar, Beulah J. M.

2015-04-01

An extensive study on the behavior of L-Phenylalanine capped silver nanoparticles (Phe-Ag NPs) in the aqueous phase and in a sol-gel thin film showed different UV/Vis, Transmission Electron Microscope (TEM), Dynamic Light Scattering and Zeta potential profiles. Scanning Electron Microscope (SEM) images of the samples in the sol gel film showed Ag embedded in the SiO2 matrix. Surface Enhanced Raman Spectra (SERS) confirmed that both in the aqueous media and in the sol gel film, the attachment of Phe to the Ag NP surface was through the benzene ring, with the sol-gel film showing a better enhancement. Photocatalytic degradation of crystal violet was measured spectrophotometrically using Phe-Ag NPs as a nanocatalyst under visible light illumination. Intensity-dependent nonlinear optical absorption of Phe-Ag measured using the open aperture Z-scan technique revealed that the material is an efficient optical limiter with potential applications.

SARS-CoV 3CL protease cleaves its C-terminal autoprocessing site by novel subsite cooperativity.

PubMed

Muramatsu, Tomonari; Takemoto, Chie; Kim, Yong-Tae; Wang, Hongfei; Nishii, Wataru; Terada, Takaho; Shirouzu, Mikako; Yokoyama, Shigeyuki

2016-11-15

The 3C-like protease (3CL pro ) of severe acute respiratory syndrome coronavirus (SARS-CoV) cleaves 11 sites in the polyproteins, including its own N- and C-terminal autoprocessing sites, by recognizing P4-P1 and P1'. In this study, we determined the crystal structure of 3CL pro with the C-terminal prosequence and the catalytic-site C145A mutation, in which the enzyme binds the C-terminal prosequence of another molecule. Surprisingly, Phe at the P3' position [Phe(P3')] is snugly accommodated in the S3' pocket. Mutations of Phe(P3') impaired the C-terminal autoprocessing, but did not affect N-terminal autoprocessing. This difference was ascribed to the P2 residue, Phe(P2) and Leu(P2), in the C- and N-terminal sites, as follows. The S3' subsite is formed by Phe(P2)-induced conformational changes of 3CL pro and the direct involvement of Phe(P2) itself. In contrast, the N-terminal prosequence with Leu(P2) does not cause such conformational changes for the S3' subsite formation. In fact, the mutation of Phe(P2) to Leu in the C-terminal autoprocessing site abolishes the dependence on Phe(P3'). These mechanisms explain why Phe is required at the P3' position when the P2 position is occupied by Phe rather than Leu, which reveals a type of subsite cooperativity. Moreover, the peptide consisting of P4-P1 with Leu(P2) inhibits protease activity, whereas that with Phe(P2) exhibits a much smaller inhibitory effect, because Phe(P3') is missing. Thus, this subsite cooperativity likely exists to avoid the autoinhibition of the enzyme by its mature C-terminal sequence, and to retain the efficient C-terminal autoprocessing by the use of Phe(P2).

Development of a counterselectable seamless mutagenesis system in lactic acid bacteria.

PubMed

Xin, Yongping; Guo, Tingting; Mu, Yingli; Kong, Jian

2017-07-05

Lactic acid bacteria (LAB) are receiving more attention to act as cell factories for the production of high-value metabolites. However, the molecular tools for genetic modifying these strains are mainly vector-based double-crossover strategies, which are laborious and inefficient. To address this problem, several counterselectable markers have been developed, while few of them could be used in the wild-type host cells without pretreatment. The pheS gene encoding phenylalanyl-tRNA synthetase alpha subunit was identified in Lactococcus lactis NZ9000 genome. When mutant pheS gene (pheS*) under the control of the Lc. lactis NZ9000 L-lactate dehydrogenase promoter (P ldh ) was expressed from a plasmid, the resulted PheS* with an A312G substitution rendered cells sensitive to the phenylalanine analog p-chloro-phenylalanine (p-Cl-Phe). This result suggested pheS* was suitable to be used as a counterselectable marker in Lc. lactis. However, the expression level of pheS* from a chromosomal copy was too low to confer p-Cl-Phe sensitivity. Therefore, a strategy of cascading promoters was attempted for strengthening the expression level of pheS*. Expectedly, a cassette 5Pldh-pheS* with five tandem repetitive promoters P ldh resulted in a sensitivity to 15 mM p-Cl-Phe. Subsequently, a counterselectable seamless mutagenesis system PheS*/pG + host9 based on a temperature-sensitive plasmid pG + host9 harboring a 5Pldh-pheS* cassette was developed in Lc. lactis. We also demonstrated the possibility of applying pheS* to be a counterselectable marker in Lactobacillus casei BL23. As reported in E. coli, pheS* as a counterselectable marker has been demonstrated to be functional in targeted gene(s) deletion in Lc. lactis as well as in L. casei. Moreover, the efficiency and timesaving counterselectable seamless mutagenesis system PheS*/pG + host9 could be used in the wild-type host cells without pretreatment.

Recombinant Brucella abortus gene expressing immunogenic protein

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

Mayfield, J.E.; Tabatabai, L.B.

This patent describes a synthetic recombinant DNA molecule containing a DNA sequence. It comprises a gene of Brucella abortus encoding an immunogenic protein having a molecular weight of approximately 31,000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis under denaturing conditions, the protein having an isoelectric point around 4.9, and containing a twenty-five amino acid sequence from its amino terminal end consisting of Gln-Ala-Pro-Thr-Phe-Phe-Arg-Ile-Gly-Thr-Gly-Gly-Thr-Ala-Gly-Thr-Tyr-Tyr-Pro-Ile-Gly-Gly-Leu-Ile-Ala, wherein Gln, Ala, Pro, Thr, Phe, Arg, Ile, Gly, Tyr, and Leu, respectively, represent glutamine, alanine, proline, threonine, phenylalanine, arginine, isolecuine, glycine, tyrosine, and leucine.

Synthetic Helizyme Enzymes.

DTIC Science & Technology

1987-08-18

NOTATION 17. COSATI CODES 18. SUBJECT TERMS (Continue on reverse if necessary and identify by block number) FIELD GROUP SUB-GROUP I Synthetic enzymes...chymotrypsin; molecular modeling; 03 peptide synthesis 19. ABSTRACT (Continue on reverse if necessary and identify by block number) The object of this...for AChE. Additionally, synthetic models ofcL- chymotrypsin built using cyclo- dextrins show catalytic activity over a limited pH range.2 Using L

Controlled self-assembling structures of ferrocene-dipeptide conjugates composed of Ala-Pro-NHCH2CH2SH chain.

PubMed

Moriuchi, Toshiyuki; Nishiyama, Taiki; Tayano, Yoshiki; Hirao, Toshikazu

2017-12-01

Bioorganometallic ferrocene-dipeptide conjugates with the Ala-Pro-cysteamine chain, Fc-L-Ala-L-Pro-NHCH 2 CH 2 SH (2) and Fc-L-Ala-D-Pro-NHCH 2 CH 2 SH (4) (Fc=ferrocenoyl), were prepared by the reduction of the ferrocene-dipeptide conjugates, Fc-L-Ala-L-Pro-cystamine-L-Pro-L-Ala-Fc (1) or Fc-L-Ala-D-Pro-cystamine-D-Pro-L-Ala-Fc (3), respectively. Control of the self-assembling structures of the ferrocene-dipeptide conjugates was demonstrated by changing the chirality of the amino acid. The molecular structure of 2 composed of the L-Ala-L-Pro-NHCH 2 CH 2 SH chain confirmed the formation of intramolecular hydrogen bond of N-H⋯N pattern between the NH of cysteamine moiety and the nitrogen of Pro moiety. Furthermore, intermolecular hydrogen bonds between NH (Ala) and CO (Pro of another molecule) and between NH (cysteamine) and CO (the ferrocenoyl moiety of another molecule) were formed, wherein each molecule is connected to four neighboring molecules by continuous intermolecular hydrogen bonds to form the hydrogen-bonded molecular assembling structure. On the contrary, the left-handed helical assembly through an intermolecular hydrogen-bonding network of 15-membered intermolecularly hydrogen-bonded ring between NH (Ala) and CO (the ferrocenoyl moiety of another molecule) and between NH (the cysteamine moiety of another molecule) and CO (Ala) was observed in the crystal packing of 4 composed of the L-Ala-D-Pro-NHCH 2 CH 2 SH chain. Copyright © 2017 Elsevier Inc. All rights reserved.

Proline cis-trans isomerization and its implications for the dimerization of analogues of cyclopeptide stylostatin 1: a combined computational and experimental study.

PubMed

López-Martínez, C; Flores-Morales, P; Cruz, M; González, T; Feliz, M; Diez, A; Campanera, Josep M

2016-05-14

Cis and trans proline conformers are often associated with dramatic changes in the biological function of peptides. A slow equilibrium between cis and trans Ile-Pro amide bond conformers occurs in constrained derivatives of the native marine cyclic heptapeptide stylostatin 1 (cyclo-(NSLAIPF)), a potential anticancer agent. In this work, four cyclopeptides, cyclo-(NSTAIPF), cyclo-(KSTAIPF), cyclo-(RSTAIPF) and cyclo-(DSTAIPF), which are structurally related to stylostatin 1, are experimentally and computationally examined in order to assess the effect of residue mutations on the cis-trans conformational ratio and the apparent capacity to form dimeric aggregates. Primarily, cyclo-(KSTAIPF) and cyclo-(RSTAIPF) showed specific trends in circular dichroism, MALDI-TOF and HPLC purification experiments, which suggests the occurrence of peptide dimerization. Meanwhile, the NMR spectrum of cyclo-(KSTAIPF) indicates that this cyclopeptide exists in the two slow-exchange families of conformations mentioned above. Molecular dynamics simulations combined with quantum mechanical calculations have shed light on the factors governing the cis/trans conformational ratio. In particular, we have found that residue mutations affect the internal hydrogen bond pattern which ultimately tunes the cis/trans conformational ratio and that only trans conformers are capable of aggregating due to the shape complementarity of the two subunits.

Potential of Endophytic Bacterium Paenibacillus sp. PHE-3 Isolated from Plantago asiatica L. for Reduction of PAH Contamination in Plant Tissues

PubMed Central

Zhu, Xuezhu; Jin, Li; Sun, Kai; Li, Shuang; Ling, Wanting; Li, Xuelin

2016-01-01

Endophytes are ubiquitous in plants, and they may have a natural capacity to biodegrade polycyclic aromatic hydrocarbons (PAHs). In our study, a phenanthrene-degrading endophytic Paenibacillus sp. PHE-3 was isolated from P. asiatica L. grown in a PAH-contaminated site. The effects of environmental variables on phenanthrene biodegradation by strain PHE-3 were studied, and the ability of strain PHE-3 to use high molecular weight PAH (HMW-PAH) as a sole carbon source was also evaluated. Our results indicated that pH value of 4.0–8.0, temperature of 30 °C–42 °C, initial phenanthrene concentration less than 100 mg·L−1, and some additional nutrients are favorable for the biodegradation of phenanthrene by strain PHE-3. The maximum biodegradation efficiency of phenanthrene was achieved at 99.9% after 84 h cultivation with additional glutamate. Moreover, the phenanthrene biodegradation by strain PHE-3 was positively correlated with the catechol 2,3-dioxygenase activity (ρ = 0.981, p < 0.05), suggesting that strain PHE-3 had the capability of degrading HMW-PAHs. In the presence of other 2-, 3-ringed PAHs, strain PHE-3 effectively degraded HMW-PAHs through co-metabolism. The results of this study are beneficial in that the re-colonization potential and PAH degradation performance of endophytic Paenibacillus sp. PHE-3 may be applied towards reducing PAH contamination in plants. PMID:27347988

Carbonic anhydrase activators: Activation of the β-carbonic anhydrase from Malassezia globosa with amines and amino acids.

PubMed

Vullo, Daniela; Del Prete, Sonia; Capasso, Clemente; Supuran, Claudiu T

2016-03-01

The β-carbonic anhydrase (CA, EC 4.2.1.1) from the dandruff producing fungus Malassezia globosa, MgCA, was investigated for its activation with amines and amino acids. MgCA was weakly activated by amino acids such as L-/D-His, L-Phe, D-DOPA, D-Trp, L-/D-Tyr and by the amine serotonin (KAs of 12.5-29.3μM) but more effectively activated by d-Phe, l-DOPA, l-Trp, histamine, dopamine, pyridyl-alkylamines, and 4-(2-aminoethyl)-morpholine, with KAs of 5.82-10.9μM. The best activators were l-adrenaline and 1-(2-aminoethyl)piperazine, with activation constants of 0.72-0.81μM. This study may help a better understanding of the activation mechanisms of β-CAs from pathogenic fungi as well as the design of tighter binding ligands for this enzyme which is a drug target for novel types of anti-dandruff agents. Copyright © 2016 Elsevier Ltd. All rights reserved.

The tripeptide aldehyde, Boc-DPhe-Phe-Lysinal, is a novel Ca2+ channel inhibitor in pituitary cells.

PubMed

Makara, G B; Rappay, G; Garamvölgyi, V; Nagy, I; Dankó, S; Bajusz, S

1988-06-22

The effect of Boc-DPhe-Phe-Lysinal (Boc-DPPL) on the 45Ca2+ uptake of rat anterior pituitary monolayer cultures was investigated. The compound decreased the basal Ca2+ uptake at 3 x 10(-4) mol/l. The 45Ca2+ uptake stimulated by potassium-induced depolarization was more sensitive to Boc-DPPL inhibition, a slight decrease was seen with 3 x 10(-6) mol/l and there was a half maximal inhibition at 3 x 10(-5) mol/l. Boc-DPPL is known to inhibit pituitary hormone release in similar concentrations, an effect might also be due to its calcium antagonist property.

Libraries of 2β-(N-substituted piperazino)-5α-androstane-3α, 17β-diols: chemical synthesis and cytotoxic effects on human leukemia HL-60 cells and on normal lymphocytes.

PubMed

Roy, Jenny; Maltais, René; Jegham, Hajer; Poirier, Donald

2011-05-01

Libraries of steroid derivatives with two levels of molecular diversity were prepared to optimize the antiproliferative activity on leukemia HL-60 cells by first varying the amino acid (AA) at R(1) (libraries A, B, C, and D: with 45, 45, 20, and 20 members, respectively) and, subsequently, the capping group at R(2) (library E: 168 members). The screening of these aminosteroids revealed interesting structure-activity relationships. In library A, the compounds bearing a tetrahydroisoquinolone residue as the first element of diversity showed potent cytotoxicity, principally when isovaleric or cyclohexyl acetic acid was used as a capping group (>40% of cell growth inhibition at 1 μM). In library B, the phenylalanine (Phe) derivatives bearing a cyano group induced a higher growth inhibition than the other Phe derivatives. The screening of library C indicated the increase of hydrophobicity of proline (Pro) seems to preserve the cytotoxic effect achieved by the lead compound. However, the synthesis of structural Pro variants (library D) clearly shows weaker activities when compared to L-Pro building blocks. Finally, by incorporating some of the most active AA of libraries A-D in library E, we observed that the amide coupling functionality gave stronger cytotoxic activity compared to the corresponding sulfonamides or benzylamines. Six of the most active amide derivatives (E-37P, E-41P, E-42P, E-46P, E-48F, and E-12T) were selected and IC(50) determined on HL-60 cells as well as on normal human lymphocytes. Among this series of new anticancer agents, good to high selectivity indices (SI = IC(50) (lymphocytes)/IC(50) (HL-60 cells) = 5 - 55) were obtained.

Production and Characterization of a New α-Glucosidase Inhibitory Peptide from Aspergillus oryzae N159-1

PubMed Central

Kang, Min-Gu; Yi, Sung-Hun

2013-01-01

An α-glucosidase inhibitor was developed from Aspergillus oryzae N159-1, which was screened from traditional fermented Korean foods. The intracellular concentration of the inhibitor reached its highest level when the fungus was cultured in tryptic soy broth medium at 27℃ for five days. The inhibitor was purified using a series of purification steps involving ultrafiltration, Sephadex G-25 gel permeation chromatography, strong cation exchange solid phase extraction, reverse-phase high performance liquid chromatography, and size exclusion chromatography. The final yield of the purification was 1.9%. Results of the liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis indicated that the purified α-glucosidase inhibitor was a tri-peptide, Pro-Phe-Pro, with the molecular weight of 360.1 Da. The IC50 value of the peptide against α-glucosidase activity was 3.1 mg/mL. Using Lineweaver-Burk plot analysis, the inhibition pattern indicated that the inhibitor acts as a mixed type inhibitor. PMID:24198670

[Multicomponent antithrombotic effect of the neuroprotective prolyl dipeptide GVS-111 and its major metabolite cyclo-L-prolylglycine].

PubMed

Ostrovskaia, R U; Liapina, L A; Pastorova, V E; Mirzoev, T Kh; Gudasheva, T A; Seredenin, S B; Ashmarin, I P

2002-01-01

The experiments in vivo showed that the new nootropic prolyl-containing GVS-111 produces an antithrombotic effect, influencing various stages of the blood coagulation process. GVS-111 exhibits anticoagulant and fibrinolytic properties and enhances fibrin destabilization by reducing the XIIIa factor activity. These effects are manifested upon both intraperitoneal (1 mg/kg) and peroral (10 mg/kg) administration of GVS-111 (in both cases, a single daily treatment over a period of 10 days). The same effects (anticoagulant, fibrinolytic, antifibrin-stabilizing) were observed in in vitro experiments with both GVS-111 (10(-3)-10(-6) M) and its main metabolite cyclo-L-prolylglycine (up to 10(-10) M). In addition, the latter metabolite exhibited an antiaggregant effect. The antithrombotic activity of GVS-111, together with previously established neuroprotector properties, low toxicity, and the absence of complications, makes this compound a promising antistroke drug.

Synthesis, structure, luminescence, and magnetic properties of a single-ion magnet "mer"-[tris(N-[(imidazol-4-yl)-methylidene]-DL-phenylalaninato)terbium(III) and related "fac"-DL-alaninato derivative.

PubMed

Yamauchi, Suguru; Fujinami, Takeshi; Matsumoto, Naohide; Mochida, Naotaka; Ishida, Takayuki; Sunatsuki, Yukinari; Watanabe, Masayuki; Tsuchimoto, Masanobu; Coletti, Cecilia; Re, Nazzareno

2014-06-16

Two Tb(III) complexes with the same N6O3 donor atoms but different coordination geometries, "fac"-[Tb(III)(HL(DL-ala))3]·7H2O (1) and "mer"-[Tb(III)(HL(DL-phe))3]·7H2O (2), were synthesized, where H2L(DL-ala) and H2L(DL-phe) are N-[(imidazol-4-yl)methylidene]-DL-alanine and -DL-phenylalanine, respectively. Each Tb(III) ion is coordinated by three electronically mononegative NNO tridentate ligands to form a coordination geometry of a tricapped trigonal prism. Compound 1 consists of enantiomers "fac"-[Tb(III)(HL(D-ala))3] and "fac"-[Tb(III)(HL(L-ala))3], while 2 consists of "mer"-[Tb(III)(HL(D-phe))2(HL(L-phe))] and "mer"-[Tb(III)(HL(D-phe))(HL(L-phe))2]. Magnetic data were analyzed by a spin Hamiltonian including the crystal field effect on the Tb(III) ion (4f(8), J = 6, S = 3, L = 3, gJ = 3/2, (7)F6). The Stark splitting of the ground state (7)F6 was evaluated from magnetic analysis, and the energy diagram pattern indicated easy-plane and easy-axis (Ising type) magnetic anisotropies for 1 and 2, respectively. Highly efficient luminescences with Φ = 0.50 and 0.61 for 1 and 2, respectively, were observed, and the luminescence fine structure due to the (5)D4 → (7)F6 transition is in good accordance with the energy diagram determined from magnetic analysis. The energy diagram of 1 shows an approximate single-well potential curve, whereas that of 2 shows a double- or quadruple-well potential within the (7)F6 multiplets. Complex 2 displayed an onset of the out-of-phase signal in alternating current (ac) susceptibility at a direct current bias field of 1000 Oe on cooling down to 1.9 K. A slight frequency dependence was recorded around 2 K. On the other hand, 1 did not show any meaningful out-of-phase ac susceptibility. Pulsed-field magnetizations of 1 and 2 were measured below 1.6 K, and only 2 exhibited magnetic hysteresis. This finding agrees well with the energy diagram pattern from crystal field calculation on 1 and 2. DFT calculation allowed us to estimate the negative charge distribution around the Tb(III) ion, giving a rationale to the different magnetic anisotropies of 1 and 2.

Characterization of an intracellular oligopeptidase from Lactobacillus paracasei.

PubMed Central

Tobiassen, R O; Sørhaug, T; Stepaniak, L

1997-01-01

An intracellular oligopeptidase from Lactobacillus paracasei Lc-01 has been purified to homogeneity by Fast Flow Q Sepharose, hydroxyapatite, and Mono Q chromatography. The molecular mass of the enzyme was determined to be 140 kDa by gel filtration and approximately 30 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and SDS-capillary electrophoresis. The pI of the enzyme was at pH 4.5. The enzyme expressed maximum activity at pH 8.0 and 40 degrees C. Oligopeptidase activity on bradykinin was inhibited strongly by 1,10-phenantroline and EDTA and partly by p-chloromercuribenzoic acid but not by phosphoramidon or phenylmethylsulfonyl fluoride. Marked inhibition by beta-casein fragment 58 to 72 was demonstrated. The enzyme showed neither general aminopeptidase nor caseinolytic activity, and it degraded only oligopeptides between 8 and 13 amino acids. The enzyme readily hydrolyzed the Phe-Ser and Pro-Phe bonds of bradykinin; the Phe-His bond of angiotensin I; the Pro-Gln, Gln-Phe, and Phe-Gly bonds of substance P; and the Pro-Tyr bond of neurotensin. Weak activity toward the Ala-Tyr and Pro-Ser bonds of alpha(s1)-casein fragment 157 to 164, was observed. The N-terminal amino acid sequence of the oligopeptidase showed a high degree of homology to the lactacin B inducer from Lactobacillus acidophilus. PMID:9097425

Hydrolysis of substance P in the presence of the osteosarcoma cell line SaOS-2: release of free amino acids.

PubMed

Cavazza, Antonella; Marini, Mario; Roda, L Giorgio; Tarantino, Umberto; Valenti, Angela

2011-12-01

The possible hydrolysis of substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met) in presence of the osteoblastic cell line SaOS-2 was measured by capillary electrophoresis coupled to mass detection. The results obtained indicate that a very rapid disappearance of the intact undecapeptide was associated to a slower appearance of seven of its eight component amino acids. These results can be interpreted as indicating that an extremely fast hydrolysis of substance P by endopeptidases, which released peptidic by-products, was followed by a noticeably slower secondary degradation which released free amino acids. In decreasing quantitative importance, these phenomena appear to originate by the hydrolysis of the Pro(4)-Gln(5) bond, followed by C-terminal sequential degradation of the Arg(1)-Pro(4) tetrapeptide; by the hydrolysis of or Phe(7)-Phe(8) bond (or, possibly, of Gln(6)-Phe(7)) leading to release of free Phe and Gln; by hydrolysis of the Gly(9)-Leu(10) bond with subsequent release of Met and Leu. Results obtained appear to be compatible with the expression by SaOS-2 cells of enzymes already known to catalyze substance P hydrolysis, together with an apparent low efficiency of aminopeptidases. Because of the activity of C-terminal fragments on NK1 receptors, the delay between primary hydrolysis of substance P and secondary hydrolysis of its peptidic fragments indicated by the data shown implies a possible persistence of substance P physiological effects even after degradation of the intact peptide.

Processing of an anglerfish somatostatin precursor to a hydroxylysine-containing somatostatin 28.

PubMed Central

Spiess, J; Noe, B D

1985-01-01

A novel 28-residue somatostatin (SS) has been isolated from anglerfish pancreatic islets and characterized by complete Edman degradation, peptide mapping, and amino acid analysis. The primary structure of this anglerfish SS-28 (aSS-28) containing hydroxylysine (Hyl) was established to be H-Ser-Val-Asp-Ser-Thr-Asn-Asn-Leu-Pro-Pro-Arg-Glu-Arg-Lys-Ala-Gly-Cys- Lys-Asn-Phe-Tyr-Trp-Hyl-Gly-Phe-Thr-Ser-Cys-OH. This sequence (with the exception of hydroxylysine-23, which is replaced by lysine) is identical to the sequence of the COOH-terminal 28 residues of prepro-SS II predicted on the basis of cDNA analysis [Hobart, P., Crawford, R., Shen, L., Pictet, R. & Rutter, W. J. (1980) Nature (London) 288, 137-141]. This is the first instance in which hydroxylysine (to date characteristically observed in collagen or collagen-like structures) has been found in a potential regulatory peptide. Chromatographic characterization of peptides, radiolabeled in islet culture, revealed that aSS-28 contained 10-12% of the radioactivity incorporated into the 8000- to 1000-dalton SS-like polypeptides, whereas 88-90% of this radioactivity was detected in anglerfish SS-14. It appears probable that aSS-28 represents the predominant primary cleavage product derived from prepro-SS II by cleavage at the COOH-terminal side of a single arginine. Based on knowledge of the collagen biosynthesis, it is speculated that hydroxylation may take place as an early post-translational event. Images PMID:2857489

Isolation and structural analysis of antihypertensive peptides that exist naturally in Gouda cheese.

PubMed

Saito, T; Nakamura, T; Kitazawa, H; Kawai, Y; Itoh, T

2000-07-01

Seven kinds of ripened cheeses (8-mo-aged and 24-mo-aged Gouda, Emmental, Blue, Camembert, Edam, and Havarti) were homogenized with distilled water, and water-soluble peptides were prepared by C-18 hydrophobic chromatography. The inhibitory activity to angiotensin I-converting enzyme and decrease in the systolic blood pressure in spontaneously hypertensive rats were measured before and after oral administration of each peptide sample. The strongest depressive effect in the systolic blood pressure (-24.7 mm Hg) and intensive inhibitory activity to angiotensin I-converting enzyme (75.7%) were detected in the peptides from 8-mo-aged Gouda cheese. Four peptides were isolated by HPLC with reverse-phase and gel filtration modes. Their chemical structures and origins, clarified by combination analyses of protein sequencing, amino acid composition, and mass spectrometry, were as follows: peptide A, Arg-Pro-Lys-His-Pro-Ile-Lys-His-Gln [alpha(s1)-casein (CN), B-8P; f 1-9]; peptide B, Arg-Pro-Lys-His-Pro-Ile-Lys-His-Gln-Gly-Leu-Pro-Gln (alpha(s1)-CN, B-8P; f 1-13); peptide F, Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn (beta-CN, A2-5P; f 60-68); and peptide G, Met-Pro-Phe-Pro-Lys-Tyr-Pro-Val-Gln-Pro-Phe (beta-CN, A2-5P; f 109-119). Peptides A and F, which were chemically synthesized, showed potent angiotensin I-converting enzyme inhibitory activity with little antihypertensive effects.

Aspartame and sucrose produce a similar increase in the plasma phenylalanine to large neutral amino acid ratio in healthy subjects.

PubMed

Burns, T S; Stargel, W W; Tschanz, C; Kotsonis, F N; Hurwitz, A

1991-01-01

Aspartame (L-aspartyl-L-phenylalanine methyl ester) consumption has been postulated to increase brain phenylalanine levels by increasing the molar ratio of the plasma phenylalanine concentration to the sum of the plasma concentrations of the other large neutral amino acids (Phe/LNAA). Dietary manipulations with carbohydrate or protein can also produce changes in the Phe/LNAA value. To compare the effects of aspartame and carbohydrate on Phe/LNAA, beverages sweetened with aspartame, sucrose, and aspartame plus sucrose, and unsweetened beverage were ingested by 8 healthy, fasted subjects in a randomized, four-way crossover design. The beverages were sweetened with an amount of aspartame (500 mg) and/or sucrose (100 g) approximately equivalent to that used to sweeten 1 liter of soft drink. The baseline-corrected plasma Phe/LNAA values did not differ significantly following ingestion of aspartame or sucrose. Following aspartame alone, the high mean ratio increased 26% over baseline 1 h after ingestion. Following sucrose alone, the high mean ratio increased 19% at 2.5 h. Sucrose increased the Phe/LNAA value due to an insulin-mediated decrease in the plasma LNAA, while aspartame increased the ratio by increasing the plasma Phe concentration. These findings indicate that similar increases in plasma Phe/LNAA occur when healthy, fasting subjects ingest amounts of equivalent sweetness of sucrose or aspartame.

Ruthenium-Nitrosyl Complexes with Glycine, l-Alanine, l-Valine, l-Proline, d-Proline, l-Serine, l-Threonine, and l-Tyrosine: Synthesis, X-ray Diffraction Structures, Spectroscopic and Electrochemical Properties, and Antiproliferative Activity

PubMed Central

2014-01-01

The reactions of [Ru(NO)Cl5]2– with glycine (Gly), l-alanine (l-Ala), l-valine (l-Val), l-proline (l-Pro), d-proline (d-Pro), l-serine (l-Ser), l-threonine (l-Thr), and l-tyrosine (l-Tyr) in n-butanol or n-propanol afforded eight new complexes (1–8) of the general formula [RuCl3(AA–H)(NO)]−, where AA = Gly, l-Ala, l-Val, l-Pro, d-Pro, l-Ser, l-Thr, and l-Tyr, respectively. The compounds were characterized by elemental analysis, electrospray ionization mass spectrometry (ESI-MS), 1H NMR, UV–visible and ATR IR spectroscopy, cyclic voltammetry, and X-ray crystallography. X-ray crystallography studies have revealed that in all cases the same isomer type (from three theoretically possible) was isolated, namely mer(Cl),trans(NO,O)-[RuCl3(AA–H)(NO)], as was also recently reported for osmium analogues with Gly, l-Pro, and d-Pro (see Z. Anorg. Allg. Chem.2013, 639, 1590–1597). Compounds 1, 4, 5, and 8 were investigated by ESI-MS with regard to their stability in aqueous solution and reactivity toward sodium ascorbate. In addition, cell culture experiments in three human cancer cell lines, namely, A549 (nonsmall cell lung carcinoma), CH1 (ovarian carcinoma), and SW480 (colon carcinoma), were performed, and the results are discussed in conjunction with the lipophilicity of compounds. PMID:24555845

Multifunctional roles of leader protein of foot-and-mouth disease viruses in suppressing host antiviral responses.

PubMed

Liu, Yingqi; Zhu, Zixiang; Zhang, Miaotao; Zheng, Haixue

2015-10-28

Foot-and-mouth disease virus (FMDV) leader protein (L(pro)) is a papain-like proteinase, which plays an important role in FMDV pathogenesis. L(pro) exists as two forms, Lab and Lb, due to translation being initiated from two different start codons separated by 84 nucleotides. L(pro) self-cleaves from the nascent viral polyprotein precursor as the first mature viral protein. In addition to its role as a viral proteinase, L(pro) also has the ability to antagonize host antiviral effects. To promote FMDV replication, L(pro) can suppress host antiviral responses by three different mechanisms: (1) cleavage of eukaryotic translation initiation factor 4 γ (eIF4G) to shut off host protein synthesis; (2) inhibition of host innate immune responses through restriction of interferon-α/β production; and (3) L(pro) can also act as a deubiquitinase and catalyze deubiquitination of innate immune signaling molecules. In the light of recent functional and biochemical findings regarding L(pro), this review introduces the basic properties of L(pro) and the mechanisms by which it antagonizes host antiviral responses.

Influence of functional groups on the C α-C β chain of L-phenylalanine and its derivatives

NASA Astrophysics Data System (ADS)

Ganesan, Aravindhan; Brunger, Michael; Wang, Feng

2010-07-01

L-phenylalanine ( L-phe) consists of three different functional groups, i.e., phenyl, carboxyl (-COOH) and amino (-NH 2), joining through the C α-C β bridge. Substitution of these groups produces 2-phenethylamine (PEA) and 3-phenylpropionic acid (PPA). Electronic structures of L-phe, PEA and PPA together with smaller "fragments" L-alanine and benzene were determined using density functional theory (DFT), from which core and valence shell ionization spectra were simulated. Comparison of the spectra reveals that core shell ionization energies clearly indicate that the carbon bridge is significantly affected by their functional group substitutions particularly at the C α site. In the valence space, quite unexpectedly, the frontier orbitals are concentrated on the benzene group although some energy splitting is observed. The orbitals which significantly affect the C α-C β carbon backbone are from the inner valence shell in the ionization energy region of 20-26 eV of the molecules.

An Effective Counterselection System for Listeria monocytogenes and Its Use To Characterize the Monocin Genomic Region of Strain 10403S.

PubMed

Argov, Tal; Rabinovich, Lev; Sigal, Nadejda; Herskovits, Anat A

2017-03-15

Construction of Listeria monocytogenes mutants by allelic exchange has been laborious and time-consuming due to lack of proficient selection markers for the final recombination event, that is, a marker conveying substance sensitivity to the bacteria bearing it, enabling the exclusion of merodiploids and selection for plasmid loss. In order to address this issue, we engineered a counterselection marker based on a mutated phenylalanyl-tRNA synthetase gene ( pheS* ). This mutation renders the phenylalanine-binding site of the enzyme more promiscuous and allows the binding of the toxic p -chloro-phenylalanine analog ( p -Cl-phe) as a substrate. When pheS* is introduced into L. monocytogenes and highly expressed under control of a constitutively active promoter, the bacteria become sensitive to p -Cl-phe supplemented in the medium. This enabled us to utilize pheS* as a negative selection marker and generate a novel, efficient suicide vector for allelic exchange in L. monocytogenes We used this vector to investigate the monocin genomic region in L. monocytogenes strain 10403S by constructing deletion mutants of the region. We have found this region to be active and to cause bacterial lysis upon mitomycin C treatment. The future applications of such an effective counterselection system, which does not require any background genomic alterations, are vast, as it can be modularly used in various selection systems (e.g., genetic screens). We expect this counterselection marker to be a valuable genetic tool in research on L. monocytogenes IMPORTANCE L. monocytogenes is an opportunistic intracellular pathogen and a widely studied model organism. An efficient counterselection marker is a long-standing need in Listeria research for improving the ability to design and perform various genetic manipulations and screening systems for different purposes. We report the construction and utilization of an efficient suicide vector for allelic exchange which can be conjugated, leaves no marker in the bacterial chromosome, and does not require the use of sometimes leaky inducible promoters. This highly efficient genome editing tool for L. monocytogenes will allow for rapid sequential mutagenesis, introduction of point mutations, and design of screening systems. We anticipate that it will be extensively used by the research community and yield novel insights into the diverse fields studied using this model organism. Copyright © 2017 American Society for Microbiology.

Development of Novel PD1/PD-L1 Antagonists Using Circular Cys-Knotted Micro Proteins

DTIC Science & Technology

2017-06-01

positive bacte- ria.[67] Similar antibacterial activities have been found in cyclo- tides isolated from Hedyota biflora (Rubiaceae family)[68] and C...immune control. Hence, reversing the inhibition of the adaptive immunity can lead to the activation of a patient’s immunity. For example, inhibition of...receptor and PD-L1, to block immune checkpoints, and facilitate antitumor activity . These checkpoint-blocking antibodies have demonstrated clinical

Substrate specificity of platypus venom L-to-D-peptide isomerase.

PubMed

Bansal, Paramjit S; Torres, Allan M; Crossett, Ben; Wong, Karen K Y; Koh, Jennifer M S; Geraghty, Dominic P; Vandenberg, Jamie I; Kuchel, Philip W

2008-04-04

The L-to-D-peptide isomerase from the venom of the platypus (Ornithorhyncus anatinus) is the first such enzyme to be reported for a mammal. In delineating its catalytic mechanism and broader roles in the animal, its substrate specificity was explored. We used N-terminal segments of defensin-like peptides DLP-2 and DLP-4 and natriuretic peptide OvCNP from the venom as substrates. The DLP analogues IMFsrs and ImFsrs (srs is a solubilizing chain; lowercase letters denote D-amino acid) were effective substrates for the isomerase; it appears to recognize the N-terminal tripeptide sequence Ile-Xaa-Phe-. A suite of 26 mutants of these hexapeptides was synthesized by replacing the second residue (Met) with another amino acid, viz. Ala, alpha-aminobutyric acid, Ile, Leu, Lys, norleucine, Phe, Tyr, and Val. It was shown that mutant peptides incorporating norleucine and Phe are substrates and exhibit L- or D-amino acid isomerization, but mutant peptides that contain residues with shorter, beta-branched or long side chains with polar terminal groups, viz. Ala, alpha-aminobutyric acid, Ile, Val, Leu, Lys, and Tyr, respectively, are not substrates. It was demonstrated that at least three N-terminal amino acid residues are absolutely essential for L-to-D-isomerization; furthermore, the third amino acid must be a Phe residue. None of the hexapeptides based on LLH, the first three residues of OvCNP, were substrates. A consistent 2-base mechanism is proposed for the isomerization; abstraction of a proton by 1 base is concomitant with delivery of a proton by the conjugate acid of a second base.

Identification of Collagen-Derived Hydroxyproline (Hyp)-Containing Cyclic Dipeptides with High Oral Bioavailability: Efficient Formation of Cyclo(X-Hyp) from X-Hyp-Gly-Type Tripeptides by Heating.

PubMed

Taga, Yuki; Kusubata, Masashi; Ogawa-Goto, Kiyoko; Hattori, Shunji

2017-11-01

Cyclic dipeptides (2,5-diketopiperazines) are present in a variety of foods and are reported to demonstrate antioxidant, antidepressant, and other beneficial effects. We recently developed a novel collagen hydrolysate characterized by a high content of X-hydroxyproline (Hyp)-Gly-type tripeptides using ginger protease. In the present study, we found that, through heating, X-Hyp-Gly can be easily converted into Hyp-containing cyclic dipeptides. After heating for 3 h at 85 °C and pH 4.8, Ala-Hyp-Gly was almost completely cyclized to cyclo(Ala-Hyp), in contrast to a slight cyclization of Ala-Hyp. The contents of cyclo(Ala-Hyp) and cyclo(Leu-Hyp) reached 0.5-1% (w/w) each in the ginger-degraded collagen hydrolysate under the heating conditions. Oral administration experiments using mice revealed that cyclo(Ala-Hyp) and cyclo(Leu-Hyp) were absorbed into the blood at markedly higher efficiencies compared to collagenous oligopeptides, including Pro-Hyp. The high productivity and oral bioavailability of the collagen-specific cyclic dipeptides suggest significant health benefits of the heat-treated ginger-degraded collagen hydrolysate.

CYP2C9 Amino Acid Residues Influencing Phenytoin Turnover and Metabolite Regio- and Stereochemistry

PubMed Central

Mosher, Carrie M.; Tai, Guoying; Rettie, Allan E.

2009-01-01

Phenytoin has been an effective anticonvulsant agent for over 60 years, although its clinical use is complicated by nonlinear pharmacokinetics, a narrow therapeutic index, and metabolically based drug-drug interactions. Although it is well established that CYP2C9 is the major cytochrome P450 enzyme controlling metabolic elimination of phenytoin through its oxidative conversion to (S)-5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH), nothing is known about the amino acid binding determinants within the CYP2C9 active site that promote metabolism and maintain the tight stereocontrol of hydroxy metabolite formation. This knowledge gap was addressed here through the construction of nine active site mutants at amino acid positions Phe100, Arg108, Phe114, Leu208, and Phe476 and in vitro analysis of the steady-state kinetics and stereochemistry of p-HPPH formation. The F100L and F114W mutants exhibited 4- to 5-fold increases in catalytic efficiency, whereas the F100W, F114L, F476L, and F476W mutants lost >90% of their phenytoin hydroxylation capacity. This pattern of effects differs substantially from that found previously for (S)-warfarin and (S)-flurbiprofen metabolism, suggesting that these three ligands bind within discrete locations in the CYP2C9 active site. Only the F114L, F476L, and L208V mutants altered phenytoin's orientation during catalytic turnover. The L208V mutant also uniquely demonstrated enhanced 6-hydroxylation of (S)-warfarin. These latter data provide the first experimental evidence for a role of the F-G loop region in dictating the catalytic orientation of substrates within the CYP2C9 active site. PMID:19258521

Precise side-chain conformation analysis of L-phenylalanine in α-helical polypeptide by quantum-chemical calculation and 13C CP-MAS NMR measurement

NASA Astrophysics Data System (ADS)

Niimura, Subaru; Suzuki, Junya; Kurosu, Hiromichi; Yamanobe, Takeshi; Shoji, Akira

2010-04-01

To clarify the positive role of side-chain conformation in the stability of protein secondary structure (main-chain conformation), we successfully calculated the optimization structure of a well-defined α-helical octadecapeptide composed of L-alanine (Ala) and L-phenylalanine (Phe) residues, H-(Ala) 8-Phe-(Ala) 9-OH, based on the molecular orbital calculation with density functional theory (DFT/B3LYP/6-31G(d)). From the total energy and the precise secondary structural parameters such as main-chain dihedral angles and hydrogen-bond parameters of the optimized structure, we confirmed that the conformational stability of an α-helix is affected dominantly by the side-chain conformation ( χ1) of the Phe residue in this system: model A ( T form: around 180° of χ1) is most stable in α-helix and model B ( G + form: around -60° of χ1) is next stable, but model C ( G - form: around 60° of χ1) is less stable. In addition, we demonstrate that the stable conformation of poly( L-phenylalanine) is an α-helix with the side-chain T form, by comparison of the carbonyl 13C chemical shift measured by 13C CP-MAS NMR and the calculated one.

Interaction of Gramicidin S and its Aromatic Amino-Acid Analog with Phospholipid Membranes

PubMed Central

Jelokhani-Niaraki, Masoud; Hodges, Robert S.; Meissner, Joseph E.; Hassenstein, Una E.; Wheaton, Laura

2008-01-01

To investigate the mechanism of interaction of gramicidin S-like antimicrobial peptides with biological membranes, a series of five decameric cyclic cationic β-sheet-β-turn peptides with all possible combinations of aromatic D-amino acids, Cyclo(Val-Lys-Leu-D-Ar1-Pro-Val-Lys-Leu-D-Ar2-Pro) (Ar ≡ Phe, Tyr, Trp), were synthesized. Conformations of these cyclic peptides were comparable in aqueous solutions and lipid vesicles. Isothermal titration calorimetry measurements revealed entropy-driven binding of cyclic peptides to POPC and POPE/POPG lipid vesicles. Binding of peptides to both vesicle systems was endothermic—exceptions were peptides containing the Trp-Trp and Tyr-Trp pairs with exothermic binding to POPC vesicles. Application of one- and two-site binding (partitioning) models to binding isotherms of exothermic and endothermic binding processes, respectively, resulted in determination of peptide-lipid membrane binding constants (Kb). The Kb1 and Kb2 values for endothermic two-step binding processes corresponded to high and low binding affinities (Kb1 ≥ 100 Kb2). Conformational change of cyclic peptides in transferring from buffer to lipid bilayer surfaces was estimated using fluorescence resonance energy transfer between the Tyr-Trp pair in one of the peptide constructs. The cyclic peptide conformation expands upon adsorption on lipid bilayer surface and interacts more deeply with the outer monolayer causing bilayer deformation, which may lead to formation of nonspecific transient peptide-lipid porelike zones causing membrane lysis. PMID:18621820

Determination of polycyclic aromatic hydrocarbons in urine of coke oven workers by headspace solid phase microextraction and gas chromatography-mass spectrometry.

PubMed

Waidyanatha, Suramya; Zheng, Yuxin; Rappaport, Stephen M

2003-05-06

Polycyclic aromatic hydrocarbons (PAHs) represent a complex mixture of toxic compounds that are ubiquitous in the environment. We investigated the utility of head space-solid phase microextraction (HS-SPME) to measure the following surrogate PAHs in urine: naphthalene (NAP), phenanthrene (PHE), pyrene (PYR), and benzo(a)pyrene (BAP), representing classes of 2-, 3-, 4- and 5-ring compounds, respectively. We then applied the method to urine from 28 coke oven workers (median levels (microg/l) were: NAP=3.65, PHE=1.51, PYR=0.003, BAP not detected) and 22 controls (median (microg/l) NAP=0.859, PHE=0.062, PYR=0.001, BAP not detected). Urinary levels of NAP, PHE, and PYR were all associated with exposure category (controls, side- and bottom-workers, and top-workers) but not with smoking status. Strong correlations were observed between urinary levels of NAP, PHE, and PYR in coke-oven workers. Our results indicate that unmetabolized 2-, 3- and 4-ring PAHs can be measured in urine by HS-SPME. Such measurements can be used to investigate the uptake and metabolism of complex PAH mixtures in humans.

Co-expression of five genes in E coli for L-phenylalanine in Brevibacterium flavum

PubMed Central

Wu, Yong-Qing; Jiang, Pei-Hong; Fan, Chang-Sheng; Wang, Jian-Gang; Shang, Liang; Huang, Wei-Da

2003-01-01

AIM: To study the effect of co-expression of ppsA, pckA, aroG, pheA and tyrB genes on the production of L-phenylalanine, and to construct a genetic engineering strain for L-phenylalanine. METHODS: ppsA and pckA genes were amplified from genomic DNA of E. coli by polymerase chain reaction, and then introduced into shuttle vectors between E coli and Brevibacterium flavum to generate constructs pJN2 and pJN5. pJN2 was generated by inserting ppsA and pckA genes into vector pCZ; whereas pJN5 was obtained by introducing ppsA and pckA genes into pCZ-GAB, which was originally constructed for co-expression of aroG, pheA and tyrB genes. The recombinant plasmids were then introduced into B. flavum by electroporation and the transformants were used for L-phenylalanine fermentation. RESULTS: Compared with the original B. flavum cells, all the transformants were showed to have increased five enzyme activities specifically, and have enhanced L-phenylalanine biosynthesis ability variably. pJN5 transformant was observed to have the highest elevation of L-phenylalanine production by a 3.4-fold. Co-expression of ppsA and pckA increased activity of DAHP synthetase significantly. CONCLUSION: Co-expression of ppsA and pckA genes in B. flavum could remarkably increase the expression of DAHP synthetase; Co-expression of ppsA, pckA, aroG, pheA and tyrB of E. coli in B. flavum was a feasible approach to construct a strain for phenylalanine production. PMID:12532463

Pomegranate Extract Enhances Endothelium-Dependent Coronary Relaxation in Isolated Perfused Hearts from Spontaneously Hypertensive Ovariectomized Rats

PubMed Central

Delgado, Nathalie T. B.; Rouver, Wender do N.; Freitas-Lima, Leandro C.; de Paula, Tiago D.-C.; Duarte, Andressa; Silva, Josiane F.; Lemos, Virgínia S.; Santos, Alexandre M. C.; Mauad, Helder; Santos, Roger L.; Moysés, Margareth R.

2017-01-01

Decline in estrogen levels promotes endothelial dysfunction and, consequently, the most prevalent cardiovascular diseases in menopausal women. The use of natural therapies such as pomegranate can change these results. Pomegranate [Punica granatum L. (Punicaceae)] is widely used as a phytotherapeutic agent worldwide, including in Brazil. We hypothesized that treatment with pomegranate hydroalcoholic extract (PHE) would improve coronary vascular reactivity and cardiovascular parameters. At the beginning of treatment, spontaneously hypertensive female rats were divided into Sham and ovariectomized (OVX) groups, which received pomegranate extract (PHE) (250 mg/kg) or filtered water (V) for 30 days by gavage. Systolic blood pressure was measured by tail plethysmography. After euthanasia, the heart was removed and coronary vascular reactivity was assessed by Langendorff retrograde perfusion technique. A dose-response curve for bradykinin was performed, followed by L-NAME inhibition. The protein expression of p-eNOS Ser1177, p-eNOS Thr495, total eNOS, p-AKT Ser473, total AKT, SOD-2, and catalase was quantified by Western blotting. The detection of coronary superoxide was performed using the protocol of dihydroethidium (DHE) staining Plasma nitrite measurement was analyzed by Griess method. Systolic blood pressure increased in both Sham-V and OVX-V groups, whereas it was reduced after treatment in Sham-PHE and OVX-PHE groups. The baseline coronary perfusion pressure was reduced in the Sham-PHE group. The relaxation was significantly higher in the treated group, and L-NAME attenuated the relaxation in all groups. The treatment has not changed p-eNOS (Ser1177), total eNOS, p-AKT (Ser473) and total AKT in any groups. However, in Sham and OVX group the treatment reduced the p-eNOS (Thr495) and SOD-2. The ovariectomy promoted an increasing in the superoxide anion levels and the treatment was able to prevent this elevation and reducing oxidative stress. Moreover, the treatment prevented the decreasing in plasmatic nitrite. We observed a reduction in total cholesterol and LDL in the Sham-PHE group. The treatment with PHE enhances the endothelium-dependent coronary relaxation and improves cardiovascular parameters, which suggests a therapeutic role of PHE. PMID:28101057

Functional Specialization and Evolution of Leader Proteinases in the Family Closteroviridae

PubMed Central

Peng, Chih-Wen; Peremyslov, Valera V.; Mushegian, Arcady R.; Dawson, William O.; Dolja, Valerian V.

2001-01-01

Members of the Closteroviridae and Potyviridae families of the plant positive-strand RNA viruses encode one or two papain-like leader proteinases. In addition to a C-terminal proteolytic domain, each of these proteinases possesses a nonproteolytic N-terminal domain. We compared functions of the several leader proteinases using a gene swapping approach. The leader proteinase (L-Pro) of Beet yellows virus (BYV; a closterovirus) was replaced with L1 or L2 proteinases of Citrus tristeza virus (CTV; another closterovirus), P-Pro proteinase of Lettuce infectious yellows virus (LIYV; a crinivirus), and HC-Pro proteinase of Tobacco etch virus (a potyvirus). Each foreign proteinase efficiently processed the chimeric BYV polyprotein in vitro. However, only L1 and P-Pro, not L2 and HC-Pro, were able to rescue the amplification of the chimeric BYV variants. The combined expression of L1 and L2 resulted in an increased RNA accumulation compared to that of the parental BYV. Remarkably, this L1-L2 chimera exhibited reduced invasiveness and inability to move from cell to cell. Similar analyses of the BYV hybrids, in which only the papain-like domain of L-Pro was replaced with those derived from L1, L2, P-Pro, and HC-Pro, also revealed functional specialization of these domains. In subcellular-localization experiments, distinct patterns were observed for the leader proteinases of BYV, CTV, and LIYV. Taken together, these results demonstrated that, in addition to a common proteolytic activity, the leader proteinases of closteroviruses possess specialized functions in virus RNA amplification, virus invasion, and cell-to-cell movement. The phylogenetic analysis suggested that functionally distinct L1 and L2 of CTV originated by a gene duplication event. PMID:11711606

Functional specialization and evolution of leader proteinases in the family Closteroviridae.

PubMed

Peng, C W; Peremyslov, V V; Mushegian, A R; Dawson, W O; Dolja, V V

2001-12-01

Members of the Closteroviridae and Potyviridae families of the plant positive-strand RNA viruses encode one or two papain-like leader proteinases. In addition to a C-terminal proteolytic domain, each of these proteinases possesses a nonproteolytic N-terminal domain. We compared functions of the several leader proteinases using a gene swapping approach. The leader proteinase (L-Pro) of Beet yellows virus (BYV; a closterovirus) was replaced with L1 or L2 proteinases of Citrus tristeza virus (CTV; another closterovirus), P-Pro proteinase of Lettuce infectious yellows virus (LIYV; a crinivirus), and HC-Pro proteinase of Tobacco etch virus (a potyvirus). Each foreign proteinase efficiently processed the chimeric BYV polyprotein in vitro. However, only L1 and P-Pro, not L2 and HC-Pro, were able to rescue the amplification of the chimeric BYV variants. The combined expression of L1 and L2 resulted in an increased RNA accumulation compared to that of the parental BYV. Remarkably, this L1-L2 chimera exhibited reduced invasiveness and inability to move from cell to cell. Similar analyses of the BYV hybrids, in which only the papain-like domain of L-Pro was replaced with those derived from L1, L2, P-Pro, and HC-Pro, also revealed functional specialization of these domains. In subcellular-localization experiments, distinct patterns were observed for the leader proteinases of BYV, CTV, and LIYV. Taken together, these results demonstrated that, in addition to a common proteolytic activity, the leader proteinases of closteroviruses possess specialized functions in virus RNA amplification, virus invasion, and cell-to-cell movement. The phylogenetic analysis suggested that functionally distinct L1 and L2 of CTV originated by a gene duplication event.

Isotropic C6, C8 and C10 interaction coefficients for CH 4, C 2H 6, C 3H 8, n-C 4H 10 and cyclo- C3H 6

NASA Astrophysics Data System (ADS)

Thomas, Gerald F.; Mulder, Fred; Meath, William J.

1980-12-01

The non-empirical generalized Kirkwood, Unsöld, and the single-Δ Unsöld methods (with double-zeta quality SCF wave-functions) are used to calculate isotropic dispersion (and induction) energy coefficients C2n, with n ⩽ 5, for interactions involving ground state CH 4, C 2H 6, C 3H 8, n-C 4H 10 and cyclo-C 3H 6. Results are also given for the related multipole polarizabilities α l, multipole sums S1/(0) and S1(-1) which are evaluated using sum rules, and the permanent multipole moments. for l = 1 (dipole) to l = 3 (octupole). Estimates of the reliability of the non-empirical methods, for the type of molecules considered, are obtained by a comparison with accurate literature values of α 1S1(-1) and C6. This, and the asymptotic properties of the multipolar expansion of the dispersion energy, the use to discuss recommended representation for the isotropic long range interaction energies through R-10 where R is the intermolecular separation.

Correction: β-Amyrin synthase from Euphorbia tirucalli L. functional analyses of the highly conserved aromatic residues Phe413, Tyr259 and Trp257 disclose the importance of the appropriate steric bulk, and cation-π and CH-π interactions for the efficient catalytic action of the polyolefin cyclization cascade.

PubMed

Ito, Ryousuke; Nakada, Chika; Hoshino, Tsutomu

2017-01-18

Correction for 'β-Amyrin synthase from Euphorbia tirucalli L. functional analyses of the highly conserved aromatic residues Phe413, Tyr259 and Trp257 disclose the importance of the appropriate steric bulk, and cation-π and CH-π interactions for the efficient catalytic action of the polyolefin cyclization cascade' by Ryousuke Ito et al., Org. Biomol. Chem., 2017, 15, 177-188.

L-cysteine-capped core/shell/shell quantum dot-graphene oxide nanocomposite fluorescence probe for polycyclic aromatic hydrocarbon detection.

PubMed

Adegoke, Oluwasesan; Forbes, Patricia B C

2016-01-01

Environmental pollutants, such as the polycyclic aromatic hydrocarbons (PAHs), become widely distributed in the environment after emission from a range of sources, and they have potential biological effects, including toxicity and carcinogenity. In this work, we have demonstrated the analytical potential of a covalently linked L-cysteine-capped CdSeTe/ZnSe/ZnS core/shell/shell quantum dot (QD)-graphene oxide (GO) nanocomposite fluorescence probe to detect PAH compounds in aqueous solution. Water-soluble L-cysteine-capped CdSeTe/ZnSe/ZnS QDs were synthesized for the first time and were covalently bonded to GO. The fluorescence of the QD-GO nanocomposite was enhanced relative to the unconjugated QDs. Various techniques including TEM, SEM, HRSEM, XRD, Raman, FT-IR, UV/vis and fluorescence spectrophotometry were employed to characterize both the QDs and the QD-GO nanocomposite. Four commonly found priority PAH analytes namely; phenanthrene (Phe), anthracene (Ant), pyrene (Py) and naphthalene (Naph), were tested and it was found that each of the PAH analytes enhanced the fluorescence of the QD-GO probe. Phe was selected for further studies as the PL enhancement was significantly greater for this PAH. A limit of detection (LOD) of 0.19 µg/L was obtained for Phe under optimum conditions, whilst the LOD of Ant, Py and Naph were estimated to be ~0.26 µg/L. The fluorescence detection mechanism is proposed. Copyright © 2015 Elsevier B.V. All rights reserved.

Foot-and-mouth disease virus leader proteinase inhibits dsRNA-induced type I interferon transcription by decreasing interferon regulatory factor 3/7 in protein levels

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

Wang, Dang; Fang, Liurong; Luo, Rui

2010-08-13

Research highlights: {yields} FMDV L{sup pro} inhibits poly(I:C)-induced IFN-{alpha}1/{beta} mRNA expression. {yields} L{sup pro} inhibits MDA5-mediated activation of the IFN-{alpha}1/{beta} promoter. {yields} L{sup pro} significantly reduced the transcription of multiple IRF-responsive genes. {yields} L{sup pro} inhibits IFN-{alpha}1/{beta} promoter activation by decreasing IRF-3/7 in protein levels. {yields} The ability to process eIF-4G of L{sup pro} is not necessary to inhibit IFN-{alpha}1/{beta} activation. -- Abstract: The leader proteinase (L{sup pro}) of foot-and-mouth disease virus (FMDV) has been identified as an interferon-{beta} (IFN-{beta}) antagonist that disrupts the integrity of transcription factor nuclear factor {kappa}B (NF-{kappa}B). In this study, we showed that the reductionmore » of double stranded RNA (dsRNA)-induced IFN-{alpha}1/{beta} expression caused by L{sup pro} was also associated with a decrease of interferon regulatory factor 3/7 (IRF-3/7) in protein levels, two critical transcription factors for activation of IFN-{alpha}/{beta}. Furthermore, overexpression of L{sup pro} significantly reduced the transcription of multiple IRF-responsive genes including 2',5'-OAS, ISG54, IP-10, and RANTES. Screening L{sup pro} mutants indicated that the ability to process eIF-4G of L{sup pro} is not required for suppressing dsRNA-induced activation of the IFN-{alpha}1/{beta} promoter and decreasing IRF-3/7 expression. Taken together, our results demonstrate that, in addition to disrupting NF-{kappa}B, L{sup pro} also decreases IRF-3/7 expression to suppress dsRNA-induced type I IFN production, suggesting multiple strategies used by FMDV to counteract the immune response to viral infection.« less

Experiments of the origins of optical activity.

PubMed

Bonner, W A; Flores, J J

1975-01-01

Two recent reports claim that (1) aqueous L-aspartic acid polymerizes faster than D-Asp in the presence of kaolin at 90 degrees, and (2) L-phenylalanine is adsorbed by kaolin more extensively than D-Phe at pH 4(the reverse being true at pH2). The novelty of these observations and their potential significance for the origin of optical activity has prompted us to duplicate these experiments using more sensitive methods. L- and D, L-Asp in 0.01 M solution were incubated with kaolin at 90 degrees for 8 days. Careful examination of the aqueous residues from such experiments failed to demonstrate any preferential polymerization of L-Asp over D-Asp, or indeed any significant gross polymerization of Asp at all. In other experiments 0.001 M solutions of D, L-Phe at pH 6 and pH 2 were stirred with large excesses of kaolin for 24 hr, and the aqueous extracts from these mixtures were examined for gross adsorption using the amino acid analyzer. No significant gross adsorption was noted. We then looked for asymmetric adsorption in the aqueous residues using optical rotatory dispersion, gas chromatography and thin layer chromatography. By none of these analytical criteria could we find any evidence whatsoever for the preferential adsorption of D- versus L-Phe from either pH 6 or pH 2 solutions. Finally, in experiments bearing on the origin of optical activity by parity violation during beta-decay, we have irradiated solid samples of D-, L- and D,L-leucine in a 61700 Ci Sr-90 source at Oak Ridge National Lab. for 1.34 yr (total dose: 4.2 x 10(8) rad). Gas chromatographic examination of the (appropriately derivitized) recovered samples showed that the L-Leu was 16.7% decomposed, the D-Leu 11.4% and theD,L-Leu 13.8% decomposed. The recovered D,L-Leu sample had a gas-chromatographically determined enantiomeric composition of 50.8% D-leu and 49.2% L-Leu. These data, though very close to experimental error, may indicate a slight preferential radiolysis of L-Leu compared to D-Leu by the Bremsstrahlung from Sr-90 beta-decay. These high intensity irradiation experiments are being continued on a prolonged basis in order to reach more definitive conclusions.

Reversal of defective lysosomal transport in NPC disease ameliorates liver dysfunction and neurodegeneration in the npc1-/- mouse.

PubMed

Liu, Benny; Turley, Stephen D; Burns, Dennis K; Miller, Anna M; Repa, Joyce J; Dietschy, John M

2009-02-17

Niemann-Pick type C disease is largely attributable to an inactivating mutation of NPC1 protein, which normally aids movement of unesterified cholesterol (C) from the endosomal/lysosomal (E/L) compartment to the cytosolic compartment of cells throughout the body. This defect results in activation of macrophages in many tissues, progressive liver disease, and neurodegeneration. In the npc1(-/-) mouse, a model of this disease, the whole-animal C pool expands from 2,082 to 4,925 mg/kg body weight (bw) and the hepatic C pool increases from 132 to 1,485 mg/kg bw between birth and 49 days of age. A single dose of 2-hydroxypropyl-beta-cyclodextrin (CYCLO) administered at 7 days of age immediately caused this sequestered C to flow from the lysosomes to the cytosolic pool in many organs, resulting in a marked increase in cholesteryl esters, suppression of C but not fatty acid synthesis, down-regulation of genes controlled by sterol regulatory element 2, and up-regulation of many liver X receptor target genes. There was also decreased expression of proinflammatory proteins in the liver and brain. In the liver, where the rate of C sequestration equaled 79 mg x d(-1) x kg(-1), treatment with CYCLO within 24 h increased C movement out of the E/L compartment from near 0 to 233 mg x d(-1) x kg(-1). By 49 days of age, this single injection of CYCLO resulted in a reduction in whole-body C burden of >900 mg/kg, marked improvement in liver function tests, much less neurodegeneration, and, ultimately, significant prolongation of life. These findings suggest that CYCLO acutely reverses the lysosomal transport defect seen in NPC disease.

Insulin-sensitizing and cholesterol-lowering effects of chromium (D-Phenylalanine)3.

PubMed

Yang, Xiaoping; Li, Shi-Yan; Dong, Feng; Ren, Jun; Sreejayan, Nair

2006-07-01

Low-molecular weight organic chromium complexes are thought to play a key role in carbohydrate and lipid metabolism and therefore have been gaining popularity as nutritional supplement for patients with diabetes and concomitant lipid disorders. The aim of the present study was to evaluate the effects of a novel synthetic chromium (d-phenylalanine)(3) complex on insulin-sensitivity, plasma lipid-profile and oxidant stress in a mouse model of type II diabetes. Plasma glucose levels following intraperitoneal insulin-challenge (1U/kg) to obese ob/ob(+/+) mice treated with Cr(d-Phe)(3) (150 microg/kg/day for 6 weeks) were significantly lower compared to vehicle-control (control: 175.8+/-43.2mg/dL versus Cr(d-Phe)(3) 115.3+/-18.0mg/dL, p<0.01, n=12). Total serum cholesterol to high-density lipoprotein ratio was significantly reduced following Cr(d-Phe)(3)-treatment (control: 2.19+/-0.08 versus Cr(d-Phe)(3) 1.63+/-0.05; p<0.05). Hepatic oxidant stress, assessed as malondialdehyde equivalents and protein-carbonyl content were significantly attenuated following Cr(d-Phe)(3) treatment. The complex also inhibited lipid-peroxidation in vitro, in a concentration dependent manner. Taken together, these data suggest that Cr(d-Phe)(3) may be of potential value in the therapy or prophylaxis of insulin-resistance and dyslipidemia associated with obesity.

Multidirectional testing of one- and two-level ProDisc-L versus simulated fusions.

PubMed

Panjabi, Manohar; Henderson, Gweneth; Abjornson, Celeste; Yue, James

2007-05-20

An in vitro human cadaveric biomechanical study. To evaluate intervertebral rotation changes due to lumbar ProDisc-L compared with simulated fusion, using follower load and multidirectional testing. Artificial discs, as opposed to the fusions, are thought to decrease the long-term accelerated degeneration at adjacent levels. A biomechanical assessment can be helpful, as the long-term clinical evaluation is impractical. Six fresh human cadaveric lumbar specimens (T12-S1) underwent multidirectional testing in flexion-extension, bilateral lateral bending, and bilateral torsion using the Hybrid test method. First, intact specimen total range of rotation (T12-S1) was determined. Second, using pure moments again, this range of rotation was achieved in each of the 5 constructs: A) ProDisc-L at L5-S1; B) fusion at L5-S1; C) ProDisc-L at L4-L5 and fusion at L5-S1; D) ProDisc-L at L4-L5 and L5-S1; and E) 2-level fusion at L4-L5 to L5-S1. Significant changes in the intervertebral rotations due to each construct were determined at the operated and nonoperated levels using repeated measures single factor ANOVA and Bonferroni statistical tests (P < 0.05). Adjacent-level effects (ALEs) were defined as the percentage changes in intervertebral rotations at the nonoperated levels due to the constructs. One- and 2-level ProDisc-L constructs showed only small ALE in any of the 3 rotations. In contrast, 1- and 2-level fusions showed increased ALE in all 3 directions (average, 7.8% and 35.3%, respectively, for 1 and 2 levels). In the disc plus fusion combination (construct C), the ALEs were similar to the 1-level fusion alone. In general, ProDisc-L preserved physiologic motions at all spinal levels, while the fusion simulations resulted in significant ALE.

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

PubMed

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

2015-02-01

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

Pharmaceutical optimization of lipid-based dosage forms for the improvement of taste-masking, chemical stability and solubilizing capacity of phenobarbital.

PubMed

Monteagudo, Ezequiel; Langenheim, Mariana; Salerno, Claudia; Buontempo, Fabián; Bregni, Carlos; Carlucci, Adriana

2014-06-01

Microemulsions (MEs) and self-emulsifying drug delivery systems (SEEDS) containing phenobarbital (Phe) were developed to improve its chemical stability, solubilizing capacity and taste-masking in oral liquid dosage forms. Cremophor® RH40 and Labrasol® were used as surfactants for the screening of ME regions, Capmul® MCM L, Captex® 355, Imwitor® 408, Myglyol® 840 and Isopropyl myristate were the oil phases assayed; Transcutol® P, Polyethylene-glycol 400, glycerol, Propylene-glycol and ethanol the cosurfactants. Phe stability assay was carried out (20:4:20:56% and 20:4:35:41% (w/w); surfactant:oily phase:cosurfactant:water) for both surfactants; only one containing ethanol showed significant dismissing in its drug content. Solubility capacity for these selected formulations were also evaluated, an amount between 17 and 58 mg/mL of Phe could be loaded. At last, an optimized ME formulation with Cremophor® RH40 20%, Capmul® MCM L 4%, PEG 400 35% and sucralose 2% (w/w) was chosen in order to optimize taste-masking using an electronic tongue. Strawberry along with banana and tutti-frutti flavors plus mint flavor proved to be the best ones. Labrasol-based pre-concentrates were tested for (micro)emulsifying properties; all of them resulted to behave as SEDDS. In summary, a rationale experimental design conducted to an optimized ME for Phe oral pediatric administration which was able to load 5-fold times the currently used dose (4 mg/mL), with no sign of physical or chemical instability and with improved taste; SEDDS for capsule filling were also obtained. The biopharmaceutical advantages described for these dosage forms encourage furthering in vivo evaluation.

[NT-proBNP as a diagnostic marker in dogs with dyspnea and in asymptomatic dogs with heart murmur].

PubMed

Haßdenteufel, E; Kresken, J-G; Henrich, E; Hildebrandt, N; Schneider, C; Stosic, A; Schneider, M

2012-01-01

The cardiac biomarker NT-proBNP indicates cardiac load in terms of myocardial wall stress. The objective of the study was to compare the results of NT-proBNP measurements in healthy dogs and dogs with dyspnea as well as asymptomatic dogs with heart murmur with the literature. Between April 2007 and December 2007 dogs with dyspnea of non-cardiac origin (n=11), dogs with dyspnea of cardiac origin (n=18) and asymptomatic dogs with heart murmur (n=22) were included. Twelve clinically healthy dogs served as a control group. All animals underwent cardiologic examination including echocardiography and measurement of serum NT-proBNP concentration. Serum was centrifuged and frozen within 30 minutes and was stored frozen until analysis was performed. Median NT-proBNP concentration in healthy dogs was 240 pmol/l (range 131-546 pmol/l). Dogs with dyspnea and primary respiratory disease displayed a median NT-proBNP concentration of 876 pmol/l (range 97-2614 pmol/l). In patients with dyspnea of non-cardiac origin, there was a difference in the values of NT-proBNP of dogs with and without pulmonary hypertension diagnosed by echocardiography. Dogs with dyspnea of cardiac origin displayed a median NT-proBNP concentration of 2000 pmol/l (range 137-2614 pmol/l). Low normal NT-proBNP values were only found in patients with pericardial effusion. Median NT-proBNP concentration in asymptomatic dogs with heart murmur was 698.5 pmol/l (range 121-2614 pmol/l). Considerably increased values were particularly measured in asymptomatic patients with severe congenital heart disease. NT-proBNP represents a useful additional diagnostic parameter in veterinary clinical cardiology to assess the severity of cardiac disease. Interpretation must take into consideration the clinical picture of the patient, as dogs with severe arrhythmias, sepsis and pulmonary thromboembolism may display high NT-proBNP levels without congestive heart failure. Our results indicate the following cut-off values: <500 pmol/l: no relevant cardiac load; 500-900 pmol/l: moderate cardiac load; >900 pmol/l: severe cardiac load.

The novel endomorphin degradation blockers Tyr-Pro-DClPhe-Phe-NH (EMDB-1) and Tyr-Pro-Ala-NH (EMDB-2) prolong endomorphin-2 action in rat ileum in vitro.

PubMed

Fichna, Jakub; Perlikowska, Renata; Gach, Katarzyna; do-Rego, Jean-Claude; Cravezic, Aurore; Janecka, Anna; Storr, Martin A

2010-07-01

The endogenous opioid system is involved in the control of gastrointestinal (GI) motility. The potential use of endogenous MOR ligands, endomorphins (EMs), as therapeutics is limited because of their rapid enzymatic degradation and short duration of action. Targeting enzymatic degradation is an approach to prolong EM activity. In the present study, we characterized the effects of novel blockers of EM degradation in GI tissue preparation in vitro. The effects of actinonin, diprotin A (DIP) and the novel peptide EM degradation blockers Tyr-Pro-DClPhe-Phe-NH(2) (EMDB-1), Tyr-Pro-Ala-NH(2) (EMDB-2) and Tyr-Pro-Ala-OH (EMDB-3) on EM-2-mediated inhibition of electrically induced cholinergic twitch contractions were compared in rat ileum in vitro using an organ bath. EMDB-1 and EMDB-2 significantly prolonged the inhibitory effect of EM-2 on smooth muscle contractility in rat ileum. EMDB-2 extended the EM-2 action for up to 60 min compared to 10 min in controls and was more potent than the conventional peptidase inhibitor DIP. EMDB-1 and EMDB-2 are potent EM degradation blockers, which prolong the inhibitory effects of EM-2 on smooth muscle contractility in rat ileum. These novel compounds may be of future use when targeting the endogenous opioid system in the treatment of GI motility disorders such as diarrhea.

Alanine analogues of [D-Trp]CJ-15,208: novel opioid activity profiles and prevention of drug- and stress-induced reinstatement of cocaine-seeking behaviour.

PubMed

Aldrich, J V; Senadheera, S N; Ross, N C; Reilley, K A; Ganno, M L; Eans, S E; Murray, T F; McLaughlin, J P

2014-07-01

The novel macrocyclic peptide cyclo[Phe-D-Pro-Phe-D-Trp] ([D-Trp]CJ-15,208) exhibits κ opioid (KOP) receptor antagonist activity in both in vitro and in vivo assays. The four alanine analogues of this peptide were synthesized and characterized both in vitro and in vivo to assess the contribution of different amino acid residues to the activity of [D-Trp]CJ-15,208. The peptides were synthesized by a combination of solid phase peptide synthesis and cyclization in solution. The analogues were evaluated in vitro in receptor binding and functional assays, and in vivo with mice using a tail-withdrawal assay for antinociceptive and opioid antagonist activity. Mice demonstrating extinction of cocaine conditioned-place preference (CPP) were pretreated with selected analogues to evaluate prevention of stress or cocaine-induced reinstatement of CPP. The alanine analogues displayed pharmacological profiles in vivo distinctly different from [D-Trp]CJ-15,208. While the analogues exhibited varying opioid receptor affinities and κ and μ opioid receptor antagonist activity in vitro, they produced potent opioid receptor-mediated antinociception (ED50 = 0.28-4.19 nmol, i.c.v.) in vivo. Three of the analogues also displayed KOP receptor antagonist activity in vivo. Pretreatment with an analogue exhibiting both KOP receptor agonist and antagonist activity in vivo prevented both cocaine- and stress-induced reinstatement of cocaine-seeking behaviour in the CPP assay in a time-dependent manner. These unusual macrocyclic peptides exhibit in vivo opioid activity profiles different from the parent compound and represent novel compounds for potential development as therapeutics for drug abuse and possibly as analgesics. © 2014 The British Pharmacological Society.

Alanine analogues of [D-Trp]CJ-15,208: novel opioid activity profiles and prevention of drug- and stress-induced reinstatement of cocaine-seeking behaviour

PubMed Central

Aldrich, J V; Senadheera, S N; Ross, N C; Reilley, K A; Ganno, M L; Eans, S E; Murray, T F; McLaughlin, J P

2014-01-01

BACKGROUND AND PURPOSE The novel macrocyclic peptide cyclo[Phe-D-Pro-Phe-D-Trp] ([D-Trp]CJ-15,208) exhibits κ opioid (KOP) receptor antagonist activity in both in vitro and in vivo assays. The four alanine analogues of this peptide were synthesized and characterized both in vitro and in vivo to assess the contribution of different amino acid residues to the activity of [D-Trp]CJ-15,208. EXPERIMENTAL APPROACH The peptides were synthesized by a combination of solid phase peptide synthesis and cyclization in solution. The analogues were evaluated in vitro in receptor binding and functional assays, and in vivo with mice using a tail-withdrawal assay for antinociceptive and opioid antagonist activity. Mice demonstrating extinction of cocaine conditioned-place preference (CPP) were pretreated with selected analogues to evaluate prevention of stress or cocaine-induced reinstatement of CPP. KEY RESULTS The alanine analogues displayed pharmacological profiles in vivo distinctly different from [D-Trp]CJ-15,208. While the analogues exhibited varying opioid receptor affinities and κ and μ opioid receptor antagonist activity in vitro, they produced potent opioid receptor-mediated antinociception (ED50 = 0.28–4.19 nmol, i.c.v.) in vivo. Three of the analogues also displayed KOP receptor antagonist activity in vivo. Pretreatment with an analogue exhibiting both KOP receptor agonist and antagonist activity in vivo prevented both cocaine- and stress-induced reinstatement of cocaine-seeking behaviour in the CPP assay in a time-dependent manner. CONCLUSIONS AND IMPLICATIONS These unusual macrocyclic peptides exhibit in vivo opioid activity profiles different from the parent compound and represent novel compounds for potential development as therapeutics for drug abuse and possibly as analgesics. PMID:24588614

Structural features of interfacial tyrosine residue in ROBO1 fibronectin domain-antibody complex: Crystallographic, thermodynamic, and molecular dynamic analyses

PubMed Central

Nakayama, Taisuke; Mizohata, Eiichi; Yamashita, Takefumi; Nagatoishi, Satoru; Nakakido, Makoto; Iwanari, Hiroko; Mochizuki, Yasuhiro; Kado, Yuji; Yokota, Yuki; Satoh, Reiko; Tsumoto, Kouhei; Fujitani, Hideaki; Kodama, Tatsuhiko; Hamakubo, Takao; Inoue, Tsuyoshi

2015-01-01

ROBO1, fibronectin Type-III domain (Fn)-containing protein, is a novel immunotherapeutic target for hepatocellular carcinoma in humans. The crystal structure of the antigen-binding fragment (Fab) of B2212A, the monoclonal antibody against the third Fn domain (Fn3) of ROBO1, was determined in pursuit of antibody drug for hepatocellular carcinoma. This effort was conducted in the presence or absence of the antigen, with the chemical features being investigated by determining the affinity of the antibody using molecular dynamics (MD) and thermodynamics. The structural comparison of B2212A Fab between the complex and the free form revealed that the interfacial TyrL50 (superscripts L, H, and F stand for the residues in the light chain, heavy chain, and Fn3, respectively) played important roles in Fn3 recognition. That is, the aromatic ring of TyrL50 pivoted toward PheF68, forming a CH/π interaction and a new hydrogen bond with the carbonyl O atom of PheF68. MD simulations predicted that the TyrL50-PheF68 interaction almost entirely dominated Fab-Fn3 binding, and Ala-substitution of TyrL50 led to a reduced binding of the resultant complex. On the contrary, isothermal titration calorimetry experiments underscored that Ala-substitution of TyrL50 caused an increase of the binding enthalpy between B2212A and Fn3, but importantly, it induced an increase of the binding entropy, resulting in a suppression of loss in the Gibbs free energy in total. These results suggest that mutation analysis considering the binding entropy as well as the binding enthalpy will aid in the development of novel antibody drugs for hepatocellular carcinoma. PMID:25492858

Nonribosomal biosynthesis of backbone-modified peptides

NASA Astrophysics Data System (ADS)

Niquille, David L.; Hansen, Douglas A.; Mori, Takahiro; Fercher, David; Kries, Hajo; Hilvert, Donald

2018-03-01

Biosynthetic modification of nonribosomal peptide backbones represents a potentially powerful strategy to modulate the structure and properties of an important class of therapeutics. Using a high-throughput assay for catalytic activity, we show here that an L-Phe-specific module of an archetypal nonribosomal peptide synthetase can be reprogrammed to accept and process the backbone-modified amino acid (S)-β-Phe with near-native specificity and efficiency. A co-crystal structure with a non-hydrolysable aminoacyl-AMP analogue reveals the origins of the 40,000-fold α/β-specificity switch, illuminating subtle but precise remodelling of the active site. When the engineered catalyst was paired with downstream module(s), (S)-β-Phe-containing peptides were produced at preparative scale in vitro (~1 mmol) and high titres in vivo (~100 mg l-1), highlighting the potential of biosynthetic pathway engineering for the construction of novel nonribosomal β-frameworks.

Use of spectroscopic technique to develop a reagent for Mo(VI) utilizing micellar effects on complex formation

NASA Astrophysics Data System (ADS)

Taşcioğlu, Sülin; Kaki, E.; Taşcioğlu, Senay

2012-09-01

Ultraviolet and visible spectral properties of aqueous solutions of molybdenum(VI) (Mo), gallic acid (GA), Lalanine (Ala), and L-Phenylalanine (Phe), and of their binary and ternary solutions were investigated in the absence and presence of anionic, cationic, and nonionic surfactant micelles. Evaluation of the spectra in a comparative way revealed that both Ala and Phe form ternary complexes with Mo and GA. The formation of a quaternary complex between Mo, GA, Phe, and cetyltrimethylammonium bromide at pH 4.5 provided a reagent system with a strikingly high sensitivity (1.2•106 l/(mol•cm)) for use in the spectrophotometric determination of Mo. A mechanism of micellar effects was discussed in terms of the substrate molecular charge and hydrophobicity, and rationalized on the basis of the spectral data obtained above and below the isoelectric pH of the amino acids.

Lactobacillus gorillae sp. nov., isolated from the faeces of captive and wild western lowland gorillas (Gorilla gorilla gorilla).

PubMed

Tsuchida, Sayaka; Kitahara, Maki; Nguema, Pierre Philippe Mbehang; Norimitsu, Saeko; Fujita, Shiho; Yamagiwa, Juichi; Ngomanda, Alfred; Ohkuma, Moriya; Ushida, Kazunari

2014-12-01

Four strains of Gram-staining-positive, anaerobic rods were isolated from the faeces of western lowland gorillas (Gorilla gorilla gorilla). Three strains, KZ01(T), KZ02 and KZ03, were isolated at the Kyoto City Zoo, Japan, and one strain, GG02, was isolated in the Moukalaba-Doudou National Park, Gabon. These strains were investigated taxonomically. These strains belonged to the Lactobacillus reuteri phylogenetic group according to phylogenetic analysis based on 16S rRNA gene sequences and specific phenotypic characteristics. Phylogenetic analysis of their 16S rRNA gene sequences revealed that strains KZ01(T), KZ02, KZ03 and GG02 formed a single monophyletic cluster and had a distinct line of descent. Based on sequence similarity of the 16S rRNA gene, Lactobacillus fermentum JCM 1173(T) (96.6 %) was the closest neighbour to these novel strains, although it was clear that these strains belonged to a different species. Partial pheS sequences also supported these relationships. DNA-DNA relatedness between strain KZ01(T) and L. fermentum JCM 1173(T) was less than 22 % and the DNA G+C content of strain KZ01(T) was 50.7 mol%. The cell-wall peptidoglycan type was A4β (l-Orn-d-Asp) and the major fatty acids were C16 : 0, C18 : 1ω9c and C19 : 1 cyclo 9,10. Therefore, based on phylogenetic, phenotypic and physiological evidence, these strains represent a novel species of the genus Lactobacillus, for which the name Lactobacillus gorillae sp. nov. is proposed. The type strain is KZ01(T) ( = JCM 19575(T) = DSM 28356(T)). © 2014 IUMS.

Burkholderia susongensis sp. nov., a mineral-weathering bacterium isolated from weathered rock surface.

PubMed

Gu, Jia-Yu; Zang, Sheng-Gang; Sheng, Xia-Fang; He, Lin-Yan; Huang, Zhi; Wang, Qi

2015-03-01

A novel type of mineral-weathering bacterium was isolated from the weathered surface of rock (mica schist) collected from Susong (Anhui, China). Cells of strain L226(T) were Gram-stain-negative. The strain grew optimally at 30 °C, with 1 % (w/v) NaCl and at pH 7.0 in trypticase soy broth. On the basis of 16S rRNA gene phylogeny, strain L226(T) was shown to belong to the genus Burkholderia and the closest phylogenetic relatives were Burkholderia sprentiae WSM5005(T) (98.3 %), Burkholderia acidipaludis NBRC 101816(T) (98.2 %), Burkholderia tuberum STM678(T) (97.2 %) and Burkholderia diazotrophica JPY461(T) (97.1 %). The DNA G+C content was 63.5 mol% and the respiratory quinone was Q-8. The major fatty acids were C16 : 0, C17 : 0 cyclo and C19 : 0 cyclo ω8c. The polar lipid profile of strain L226(T) consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, unknown lipids and unidentified aminophospholipids. Based on the low level of DNA-DNA relatedness (ranging from 25.8 % to 34.4 %) to the tested type strains of species of the genus Burkholderia and unique phenotypic characteristics, it is suggested that strain L226(T) represents a novel species of the genus Burkholderia, for which the name Burkholderia susongensis sp. nov., is proposed. The type strain is L226(T) ( = CCTCC AB2014142(T) = JCM 30231(T)). © 2015 IUMS.

Photoaffinity labeling the substance P receptor using a derivative of substance P containing para-benzoylphenylalanine

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

Boyd, N.D.; White, C.F.; Leeman, S.E.

A novel photoreactive substance P (SP) analogue has been synthesized by solid-phase peptide synthesis methodology to incorporate the amino acid p-benzoyl-L-phenylalanine (L-Phe(pBz)) in place of the Phe{sup 8} residue of SP. (Phe{sup 8}(OpBz))SP was equipotent with SP in competing for SP binding sites on rat submaxillary gland membranes and had potent sialagogic activity in vivo. In the absence of light, the {sup 125}I-labeled Bolton-Hunter conjugate of (Phe{sup 8}(pBz))SP bound in a saturable and reversible manner to an apparently homogeneous class of binding sites with an affinity K{sub D} = 0.4 nM. The binding of {sup 125}I-(Phe{sup 8}(pBz))SP was inhibited competitivelymore » by various tachykinin peptides and analogues with the appropriate specificity for SP/NK-1 receptors. Upon photolysis, up to 70{percent} of the specifically bound {sup 125}I-(Phe{sup 8}(pBz))SP underwent covalent linkage to two polypeptides of M{sub r} = 53 000 and 46 000, identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Quantitative analysis of the inhibitory effects of SP and related peptides on {sup 125}I-(Phe{sup 8}(pBz))SP photoincorporation indicated that the binding sites of the two photolabeled polypeptides have the same peptide specificity, namely, that typical of NK-1-type SP receptors. Further information on the relationship between the two labeled SP binding sites was provided by enzymatic digestion studies. The highly specific and remarkably efficient photolabeling achieved with {sup 125}I-(Phe{sup 8}(pBz))SP suggests that this photoaffinity probe will be of considerable value for the characterization of the molecular structure of the SP receptor.« less

Vibrational and chiroptical spectroscopic characterization of gamma-turn model cyclic tetrapeptides containing two beta-Ala residues.

PubMed

Vass, Elemér; Majer, Zsuzsa; Kohalmy, Krisztina; Hollósi, Miklós

2010-08-01

The optical spectroscopic characterization of gamma-turns in solution is uncertain and their distinction from beta-turns is often difficult. This work reports systematic ECD and vibrational circular dichroism (VCD) spectroscopic studies on gamma-turn model cyclic tetrapeptides cyclo(Ala-beta-Ala-Pro-beta-Ala) (1), cyclo(Pro-beta-Ala-Pro-beta-Ala) (2) and cyclo(Ala-beta-Ala-Ala-beta-Ala) (3). Conformational analysis performed at the 6-31G(d)/B3LYP level of theory using an adequate PCM solvent model predicted one predominant conformer for 1-3, featuring two inverse gamma-turns. The ECD spectra in ACN of 1 and 2 are characterized by a negative n-->pi* band near 230 nm and a positive pi-->pi* band below 200 nm with a long wavelength shoulder. The ECD spectra in TFE of 1-3 show similar spectra with blue-shifted bands. The VCD spectra in ACN-d(3) of 1 and 2 show a +/-/+/- amide I sign pattern resulting from four uncoupled vibrations in the case of 1 and a sequence of two positive couplets in the case of 2. A -/+/+/- amide I VCD pattern was measured for 3 in TFE-d(2). All three peptides give a positive couplet or couplet-like feature (+/-) in the amide II region. VCD spectroscopy, in agreement with theoretical calculations revealed that low frequency amide I vibrations (at approximately 1630 cm(-1) or below) are indicative of a C(7) H-bonded inverse gamma-turns with Pro in position 2, while gamma-turns encompassing Ala absorb at higher frequency (above 1645 cm(-1)). Copyright 2010 Wiley-Liss, Inc.

Mutations in the Fusion Protein of Measles Virus That Confer Resistance to the Membrane Fusion Inhibitors Carbobenzoxy-d-Phe-l-Phe-Gly and 4-Nitro-2-Phenylacetyl Amino-Benzamide

PubMed Central

Ha, Michael N.; Delpeut, Sébastien; Noyce, Ryan S.; Sisson, Gary; Black, Karen M.; Lin, Liang-Tzung; Bilimoria, Darius; Plemper, Richard K.; Privé, Gilbert G.

2017-01-01

ABSTRACT The inhibitors carbobenzoxy (Z)-d-Phe-l-Phe-Gly (fusion inhibitor peptide [FIP]) and 4-nitro-2-phenylacetyl amino-benzamide (AS-48) have similar efficacies in blocking membrane fusion and syncytium formation mediated by measles virus (MeV). Other homologues, such as Z-d-Phe, are less effective but may act through the same mechanism. In an attempt to map the site of action of these inhibitors, we generated mutant viruses that were resistant to the inhibitory effects of Z-d-Phe-l-Phe-Gly. These 10 mutations were localized to the heptad repeat B (HRB) region of the fusion protein, and no changes were observed in the viral hemagglutinin, which is the receptor attachment protein. Mutations were validated in a luciferase-based membrane fusion assay, using transfected fusion and hemagglutinin expression plasmids or with syncytium-based assays in Vero, Vero-SLAM, and Vero-Nectin 4 cell lines. The changes I452T, D458N, D458G/V459A, N462K, N462H, G464E, and I483R conferred resistance to both FIP and AS-48 without compromising membrane fusion. The inhibitors did not block hemagglutinin protein-mediated binding to the target cell. Edmonston vaccine/laboratory and IC323 wild-type strains were equally affected by the inhibitors. Escape mutations were mapped upon a three-dimensional (3D) structure modeled from the published crystal structure of parainfluenzavirus 5 fusion protein. The most effective mutations were situated in a region located near the base of the globular head and its junction with the alpha-helical stalk of the prefusion protein. We hypothesize that the fusion inhibitors could interfere with the structural changes that occur between the prefusion and postfusion conformations of the fusion protein. IMPORTANCE Due to lapses in vaccination worldwide that have caused localized outbreaks, measles virus (MeV) has regained importance as a pathogen. Antiviral agents against measles virus are not commercially available but could be useful in conjunction with MeV eradication vaccine programs and as a safeguard in oncolytic viral therapy. Three decades ago, the small hydrophobic peptide Z-d-Phe-l-Phe-Gly (FIP) was shown to block MeV infections and syncytium formation in monkey kidney cell lines. The exact mechanism of its action has yet to be determined, but it does appear to have properties similar to those of another chemical inhibitor, AS-48, which appears to interfere with the conformational change in the viral F protein that is required to elicit membrane fusion. Escape mutations were used to map the site of action for FIP. Knowledge gained from these studies could help in the design of new inhibitors against morbilliviruses and provide additional knowledge concerning the mechanism of virus-mediated membrane fusion. PMID:28904193

In Vivo Monitoring of pH, Redox Status, and Glutathione Using L-Band EPR for Assessment of Therapeutic Effectiveness in Solid Tumors

PubMed Central

Bobko, Andrey A.; Eubank, Timothy D.; Voorhees, Jeffrey L.; Efimova, Olga V.; Kirilyuk, Igor A.; Petryakov, Sergey; Trofimiov, Dmitrii G.; Marsh, Clay B.; Zweier, Jay L.; Grigor’ev, Igor A.; Samouilov, Alexandre; Khramtsov, Valery V.

2011-01-01

Approach for in vivo real-time assessment of tumor tissue extracellular pH (pHe), redox, and intracellular glutathione based on L-band EPR spectroscopy using dual function pH and redox nitroxide probe and disulfide nitroxide biradical, is described. These parameters were monitored in PyMT mice bearing breast cancer tumors during treatment with granulocyte macrophage colony-stimulating factor. It was observed that tumor pHe is about 0.4 pH units lower than that in normal mammary gland tissue. Treatment with granulocyte macrophage colony-stimulating factor decreased the value of pHe by 0.3 units compared with PBS control treatment. Tumor tissue reducing capacity and intracellular glutathione were elevated compared with normal mammary gland tissue. Granulocyte macrophage colony-stimulating factor treatment resulted in a decrease of the tumor tissue reducing capacity and intracellular glutathione content. In addition to spectroscopic studies, pHe mapping was performed using recently proposed variable frequency proton–electron double-resonance imaging. The pH mapping superimposed with MRI image supports probe localization in mammary gland/tumor tissue, shows high heterogeneity of tumor tissue pHe and a difference of about 0.4 pH units between average pHe values in tumor and normal mammary gland. In summary, the developed multifunctional approach allows for in vivo, noninvasive pHe, extracellular redox, and intracellular glutathione content monitoring during investigation of various therapeutic strategies for solid tumors. Magn Reson Med 000:000–000, 2011. PMID:22113626

The role of nitric oxide synthase in reduced vasocontractile responsiveness induced by prolonged α1-adrenergic receptor stimulation in rat thoracic aorta

PubMed Central

Gürdal, Hakan; Can, Alp; Uğur, Mehmet

2005-01-01

Prolonged exposure (6–12 h) of rat aorta to alpha1-adrenergic receptor (α1AR) agonist phenylephrine (Phe) leads to a decrease in α1AR-mediated vasoconstriction. This reduced responsiveness to α1AR stimulation was strongly dependent on the intactness of the endothelium. We examined the effect of Phe on nitric oxide synthase (NOS) activity by measuring the conversion of [3H]L-arginine to [3H]L-citrulline in rat aorta or in endothelial cells isolated from rat aorta. Phe stimulation increased NOS activity in control aortas. This response was antagonized by prazosin. However, Phe increased neither the activity of NOS nor intracellular Ca2+ in the isolated endothelial cells from the control aortas, whereas acetylcholine (Ach) was able to stimulate both responses in these cells. This result suggests that Phe stimulates α1AR on vascular smooth muscle cells and has an indirect influence on endothelial cells to increase NOS activity. In Phe-exposed aortic rings, basal NOS activity was found to have increased compared to vehicle-exposed control rings. Stimulation with Phe or Ach caused a small increase over basal NOS activity in these preparations. Prolonged exposure to Phe also caused an enhancement of Ach-mediated vasorelaxation in rat aorta. Immunoblot and reverse transcription–polymerase chain reaction experiments showed that prolonged exposure of rat aorta to Phe resulted in an increased expression of eNOS, but not iNOS. This increase was antagonized by nonselective antagonist prazosin. Immunohistochemical staining experiments also showed that expression of eNOS increased in endothelial cells after Phe exposure of the aortas. These results, all together, showed that prolonged exposure of rat aorta to α1AR agonist Phe enhanced the expression of eNOS and basal NOS activity, which probably causes a decreased vasocontractile response to Phe or to other agonists such as 5HT (5-hydroxytryptamine) in rat aorta. This phenomenon can be considered more as a functional antagonism of vasocontractile response to agonists mediated by endothelium than a specific desensitization of α1AR-mediated signalling in vascular smooth muscle cells. PMID:15753950

L-proline-based deep eutectic solvents (DESs) for deep catalytic oxidative desulfurization (ODS) of diesel.

PubMed

Hao, Lingwan; Wang, Meiri; Shan, Wenjuan; Deng, Changliang; Ren, Wanzhong; Shi, Zhouzhou; Lü, Hongying

2017-10-05

A series of L-proline-based DESs was prepared through an atom economic reaction between L-proline (L-Pro) and four different kinds of organic acids. The DESs were characterized by Fourier transform infrared spectroscopy (FT-IR), H nuclear magnetic resonance ( 1 HNMR), cyclic voltammogram (CV) and the Hammett method. The synthesized DESs were used for the oxidative desulfurization and the L-Pro/p-toluenesultonic acid (L-Pro/p-TsOH) system shows the highest catalytic activity that the removal of dibenzothiophene (DBT) reached 99% at 60°C in 2h, which may involve the dual activation of the L-Pro/p-TsOH. The acidity of four different L-proline-based DESs was measured and the results show that it could not simply conclude that the correlation between the acidity of DESs and desulfurization capability was positive or negative. The electrochemical measurements evidences and recycling experiment indicate a good stability performance of L-Pro/p-TsOH in desulfurization. This work will provide a novel and potential method for the deep oxidation desulfurization. Copyright © 2017 Elsevier B.V. All rights reserved.

Antagonists of substance P. Further modifications of substance P antagonists obtained by replacing either positions 7, 9 or 7, 8 and 11 of SP with D-amino acid residues.

PubMed

Dutta, A S; Gormley, J J; Graham, A S; Briggs, I; Growcott, J W; Jamieson, A

1986-07-01

Antagonists of SP and the C-terminal (6-11)-hexapeptide have been obtained by multiple D-amino acid substitutions in various positions of SP and by protecting the N alpha-Arg1 and N epsilon Lys3 amino groups with benzyloxycarbonyl groups. On the guinea pig ileum a number of these antagonized both SP and the hexapeptide. Except [N alpha-Z-Arg1,D-Pro2,N epsilon-Z-Lys3,Asn5,Arg6,D-Phe7,D-Trp9]-SP-OMe (4) and the corresponding amide 7, which were more potent antagonists of SP than the hexapeptide, all the others, e.g., [N alpha-Z-Arg1,D-Pro2,4,N epsilon-Z-Lys3,D-Phe7,8,Sar9,D-Met11]-SP-OMe (9), [N alpha-Z-Arg1,D-Pro2,4,N epsilon-Z-Lys3,D-Phe7,8,Sar9,MeLeu10,D-Met11]-SP -OMe (11), were more potent antagonists of the hexapeptide. On the rat spinal cord preparation, most of the antagonists were only active against the hexapeptide. A few antagonized SP, but these also reduced carbachol or both carbachol and glutamate responses. Two of the antagonists, [D-Pro2,Asn5,Lys6,D-Phe7,D-Trp9]-SP-OMe (2) and [Boc-D-Pro4,D-Phe7,8,Sar9,D-Met11]-SP(4-11)-OMe (10), were inactive on the ileum but still antagonized the hexapeptide on the spinal cord. The smallest peptides to antagonize SP and the hexapeptide were two heptapeptides, 6 and 21, [Z-Asn5,Arg6,D-Phe7,8,Gly9 psi (CH2S)D-Leu10,D-Met11]-SP(5-11)-OMe (21) being more potent than 6. None of the antagonists showed significant analgesic activity without side effects. Some of the antagonists were shown to release histamine from isolated rat peritoneal cells.

Antifouling Activity towards Mussel by Small-Molecule Compounds from a Strain of Vibrio alginolyticus Bacterium Associated with Sea Anemone Haliplanella sp.

PubMed

Wang, Xiang; Huang, Yanqiu; Sheng, Yanqing; Su, Pei; Qiu, Yan; Ke, Caihuan; Feng, Danqing

2017-03-28

Mussels are major fouling organisms causing serious technical and economic problems. In this study, antifouling activity towards mussel was found in three compounds isolated from a marine bacterium associated with the sea anemone Haliplanella sp. This bacterial strain, called PE2, was identified as Vibrio alginolyticus using morphology, biochemical tests, and phylogenetic analysis based on sequences of 16S rRNA and four housekeeping genes ( rpoD, gyrB, rctB, and toxR ). Three small-molecule compounds (indole, 3-formylindole, and cyclo (Pro-Leu)) were purified from the ethyl acetate extract of V. alginolyticus PE2 using column chromatography techniques. They all significantly inhibited byssal thread production of the green mussel Perna viridis , with EC 50 values of 24.45 μg/ml for indole, 50.07 μg/ml for 3-formylindole, and 49.24 μg/ml for cyclo (Pro-Leu). Previous research on the antifouling activity of metabolites from marine bacteria towards mussels is scarce. Indole, 3-formylindole and cyclo (Pro-Leu) also exhibited antifouling activity against settlement of the barnacle Balanus albicostatus (EC 50 values of 8.84, 0.43, and 11.35 μg/ml, respectively) and the marine bacterium Pseudomonas sp. (EC 50 values of 42.68, 69.68, and 39.05 μg/ml, respectively). These results suggested that the three compounds are potentially useful for environmentally friendly mussel control and/or the development of new antifouling additives that are effective against several biofoulers.

Accumulation of phenanthrene by roots of intact wheat (Triticum acstivnm L.) seedlings: passive or active uptake?

PubMed Central

2010-01-01

Background Polycyclic aromatic hydrocarbons (PAHs) are of particular concern due to their hydrophobic, recalcitrant, persistent, potentially carcinogenic, mutagenic and toxic properties, and their ubiquitous occurrence in the environment. Most of the PAHs in the environment are present in surface soil. Plants grown in PAH-contaminated soils or water can become contaminated with PAHs because of their uptake. Therefore, they may threaten human and animal health. However, the mechanism for PAHs uptake by crop roots is little understood. It is important to understand exactly how PAHs are transported into the plant root system and into the human food chain, since it is beneficial in governing crop contamination by PAHs, remedying soils or waters polluted by PAHs with plants, and modeling potential uptake for risk assessment. Results The possibility that plant roots may take up phenanthrene (PHE), a representative of PAHs, via active process was investigated using intact wheat (Triticum acstivnm L.) seedlings in a series of hydroponic experiments. The time course for PHE uptake into wheat roots grown in Hoagland solution containing 5.62 μM PHE for 36 h could be separated into two periods: a fast uptake process during the initial 2 h and a slow uptake component thereafter. Concentration-dependent PHE uptake was characterized by a smooth, saturable curve with an apparent Km of 23.7 μM and a Vmax of 208 nmol g-1 fresh weight h-1, suggesting a carrier-mediated uptake system. Competition between PHE and naphthalene for their uptake by the roots further supported the carrier-mediated uptake system. Low temperature and 2,4-dinitrophenol (DNP) could inhibit PHE uptake equally, indicating that metabolism plays a role in PHE uptake. The inhibitions by low temperature and DNP were strengthened with increasing concentration of PHE in external solution within PHE water solubility (7.3 μM). The contribution of active uptake to total absorption was almost 40% within PHE water solubility. PHE uptake by wheat roots caused an increase in external solution pH, implying that wheat roots take up PHE via a PHE/nH+ symport system. Conclusion It is concluded that an active, carrier-mediated and energy-consuming influx process is involved in the uptake of PHE by plant roots. PMID:20307286

Structural and computational analysis of peptide recognition mechanism of class-C type penicillin binding protein, alkaline D-peptidase from Bacillus cereus DF4-B

PubMed Central

Nakano, Shogo; Okazaki, Seiji; Ishitsubo, Erika; Kawahara, Nobuhiro; Komeda, Hidenobu; Tokiwa, Hiroaki; Asano, Yasuhisa

2015-01-01

Alkaline D-peptidase from Bacillus cereus DF4-B, called ADP, is a D-stereospecific endopeptidase reacting with oligopeptides containing D-phenylalanine (D-Phe) at N-terminal penultimate residue. ADP has attracted increasing attention because it is useful as a catalyst for synthesis of D-Phe oligopeptides or, with the help of substrate mimetics, L-amino acid peptides and proteins. Structure and functional analysis of ADP is expected to elucidate molecular mechanism of ADP. In this study, the crystal structure of ADP (apo) form was determined at 2.1 Å resolution. The fold of ADP is similar to that of the class C penicillin-binding proteins of type-AmpH. Docking simulations and fragment molecular orbital analyses of two peptides, (D-Phe)4 and (D-Phe)2-(L-Phe)2, with the putative substrate binding sites of ADP indicated that the P1 residue of the peptide interacts with hydrophobic residues at the S1 site of ADP. Furthermore, molecular dynamics simulation of ADP for 50 nsec suggested that the ADP forms large cavity at the active site. Formation of the cavity suggested that the ADP has open state in the solution. For the ADP, having the open state is convenient to bind the peptides having bulky side chain, such as (D-Phe)4. Taken together, we predicted peptide recognition mechanism of ADP. PMID:26370172

Crystal structures of resorcin[4]arene and pyrogallol[4]arene complexes with proline: A model for proline recognition through Csbnd H···π interaction

NASA Astrophysics Data System (ADS)

Fujisawa, Ikuhide; Kitamura, Yuji; Kato, Ryo; Aoki, Katsuyuki

2018-07-01

Resorcin[4]arene (resorcinol cyclic tetramer, abbreviated as RCT) or pyrogallol[4]arene (pyrogallol cyclic tetramer, PCT) form host-guest 1:1 complexes with DL-proline (DL-Pro) or L-proline (L-Pro), [RCT·DL-Pro]·2MeOH·3.5H2O (1) and 2[PCT·L-Pro]·2EtOH·10H2O (2), whose crystal structures have been determined. In each complex, the proline ligand is incorporated into the bowl-shaped cavity of RCT or PCT host molecules through Csbnd H … π interactions between alkyl protons of the pyrrolidine ring of proline and π-rings of RCT or PCT, forming an [RCT/PCT·Pro] structural fragment. In the crystal lattice, two [RCT/PCT·Pro] fragments self-associate to form a ligand-mediated dimeric structure, [RCT·D-Pro·L-Pro·RCT] in 1 or [PCT·L-Pro·L-Pro·PCT] in 2. A 1H NMR solution study gave the host‒ligand binding constants of 10.0 ± 1.1 M-1 for the RCT-DL-Pro system and 17.3 ± 1.3 M-1 for the PCT-L-Pro system. These complexes provide a synthetic model for the recognition of the proline residue in proline-containing substrates or inhibitors by enzymes through Csbnd H … π interaction. The CSD survey revealed that the absolute value of the torsion angle N-Cα-Csbnd O1 (O1 is cis to N) about the carboxyl Cα-C bond of proline is significantly smaller than that of the Cβ-Cα-Csbnd O2 (O2 is cis to Cβ) torsion angle.

Effect of protopine on cytosolic Ca2+ in rabbit platelets.

PubMed

Shen, Z Q; Chen, Z H; Duan, L

1999-04-01

To study the influence of protopine (Pro) on the cytoplasmic free Ca2+ concentration ([Ca2+]i) in rabbit platelets. Measurement of [Ca2+]i of platelets in vitro by Fura 2-AM fluorescence technique. In the presence of CaCl2 1 mmol.L-1, Pro 10, 20, and 40 mumol.L-1 attenuated the rise in [Ca2+]i evoked by ADP from (420 +/- 57) to (320 +/- 26), (264 +/- 21), and (180 +/- 14) nmol.L-1, respectively, by arachidonic acid (AA) from (280 +/- 36) to (210 +/- 17), (184 +/- 21), and (143 +/- 16) nmol.L-1, respectively, and by platelet-activating factor (PAF) from (350 +/- 42) to (282 +/- 31), (223 +/- 30), and (165 +/- 15) nmol.L-1, respectively. In the presence of egtazic acid 1 mmol.L-1, Pro 10, 20, and 40 mumol.L-1 reduced the Ca2+ release induced by ADP, AA, and PAF, respectively. Pro 10, 20, and 40 mumol.L-1 also decreased ADP-, AA-, and PAF-induced Ca2+ influx. Pro inhibited not only Ca2+ release but also the influx of Ca2+.

Bradykinin-related compounds as new drugs for cancer and inflammation.

PubMed

Stewart, John M; Gera, Lajos; Chan, Daniel C; Bunn, Paul A; York, Eunice J; Simkeviciene, Vitalija; Helfrich, Barbara

2002-04-01

Bradykinin (BK) (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) is an important growth factor for small-cell lung cancer (SCLC) and prostate cancer (PC). These cancers have cells of neuroendocrine origin and express receptors for a variety of neuropeptides. BK receptors are expressed on almost all lung cancer cell lines and on many PC cells. Our very potent BK antagonist B9430 (D-Arg-Arg-Pro-Hyp-Gly-lgl-Ser-D-Igl-Oic-Arg) (Hyp, trans-4-hydroxy-L-proline; Ig1, alpha-2-indanylglycine; Oic, octahydroindole-2-carboxylic acid) is a candidate anti-inflammatory drug but does not inhibit growth of SCLC or PC. When B9430 is dimerized by N-terminal cross-linking with a suberimide linker, the product B9870 is a potent growth inhibitor for SCLC both in vitro and in vivo in athymic nude mice. Daily i.p. injection at 5 mg x kg(-1) day(-1) beginning on day 8 after SCLC SHP-77 cell implantation gave 65% inhibition of tumor growth. B9870 stimulates apoptosis in SCLC by a novel "biased agonist" action. We have also developed new small mimetic antagonists. BKM-570 (F5C-OC2Y-Atmp) (F5C, pentafluorocinnamic acid; OC2Y, O-2,6-dichlorobenzyl tyrosine; Atmp, 4-amino-2,2,6,6-tetramethylpiperidine) is very potent for inhibition of SHP-77 growth in nude mice. When injected daily i.p. at 5 mg x kg(-1), M-570 gave 90% suppression of tumor growth. M-570 is more potent than the well-known anticancer drug cisPlatin (60% inhibition) or the recently developed SU5416 (40% inhibition) in this model. M-570 also showed activity against various other cancer cell lines in vitro (SCLC, non-SCLC, lung, prostate, colon, cervix) and inhibited growth of prostate cell line PC3 in nude mice. M-570 and related compounds evidently act in vivo through pathways other than BK receptors. These compounds have clinical potential for treatment of human lung and prostate cancers.

Changes in N-terminal pro-B-type natriuretic peptide and incidence of diabetes: The Multi-Ethnic Study of Atherosclerosis (MESA)

PubMed Central

Sanchez, O.A.; Duprez, D.A.; Bahrami, H.; Peralta, C.A.; Daniels, L.B.; Lima, J.A.; Maisel, A.; Folsom, A.R.; Jacobs, D.R.

2016-01-01

Aims This study looked at whether the inverse association of circulating N-terminal pro-B-type natriuretic peptide (NT-proBNP) with incident diabetes is modified by changes in NT-proBNP (ΔNT-proBNP) levels. Methods lasma NT-proBNP was assayed at baseline and 3.2 years later (visit 3) in the Multi-Ethnic Study of Atherosclerosis (MESA).ΔNT-proBNP was calculated as NT-proBNPvisit3 − NT-proBNPbaseline. A Poisson distribution was fitted to determine the incidence density of diabetes, adjusted for age, race, gender, educational attainment, antihypertensive medication, total intentional exercise and plasma IL-6 levels. In the primary analysis (n = 3236 without diabetes up to visit 3, followed for a mean of 6.3 years), incidence density was regressed for the following categories of baseline NT-proBNP: (1) <54.4 pg/mL; (2) 54.4–85.9 pg/mL; and (3) 86–54.2 pg/mL. This was crossed with categories of ΔNT-proBNP as medians (ranges): (1) −6.2 (−131–11.7) pg/mL; (2) 19.8 (11.8–30.1) pg/mL; (3) 44.0 (30.2–67.9) pg/mL; and (4) 111.2 (68.0–3749.9) pg/mL. Results The incidence density of diabetes followed a U-shaped association across categories of ΔNT-proBNP within categories of baseline NT-proBNP after adjusting for other covariates (P = 0.02). At each level of baseline NT-proBNP, the incidence density of diabetes was lowest for small-to-moderate increases in NT-proBNP. Conclusion This analysis suggests that NT-proBNP has a biphasic association with diabetes in which the risk of incident diabetes decreases within a ‘physiological range’ of ΔNT-proBNP, and plateaus or increases as NT-proBNP concentrations increase, probably in response to pathophysiological conditions leading to high levels of NT-proBNP. PMID:26047677

Exploration and Pharmacokinetic Profiling of Phenylalanine Based Carbamates as Novel Substance P 1–7 Analogues

PubMed Central

2014-01-01

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. PMID:25516784

Exploration and pharmacokinetic profiling of phenylalanine based carbamates as novel substance p 1-7 analogues.

PubMed

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.

Tyrocidine A Analogues Bearing the Planar d-Phe-2-Abz Turn Motif: How Conformation Impacts Bioactivity.

PubMed

Cameron, Alan J; Edwards, Patrick J B; Harjes, Elena; Sarojini, Vijayalekshmi

2017-12-14

The d-Phe-Pro β-turn of the cyclic β-hairpin antimicrobial decapeptide tyrocidine A, (Tyrc A) was substituted with the d-Phe-2-aminobenzoic acid (2-Abz) motif in a synthetic analogue (1). The NMR structure of 1 demonstrated that compound 1 retained the β-hairpin structure of Tyrc A with additional planarity, resulting in approximately 30-fold reduced hemolysis than Tyrc A. Although antibacterial activity was partially compromised, a single Gln to Lys substitution (2) restored activity equivalent to Tyrc A against S. aureus, enhanced activity against two Gram negative strains and maintained the reduced hemeloysis of 1. Analysis by transmission electron microscopy (TEM) suggested a membrane lytic mechanism of action for these peptides. Compound 2 also exhibits nanomolar antifungal activity in synergy with amphotericin B. The d-Phe-2-Abz turn may serve as a tool for the synthesis of structurally predictable β-hairpin libraries. Unlike traditional β-turn motifs such as d-Pro-Gly, both the 2-Abz and d-Phe rings may be further functionalized.

Novel direct factor Xa inhibitory compounds from Tenebrio molitor with anti-platelet aggregation activity.

PubMed

Lee, Wonhwa; Kim, Mi-Ae; Park, InWha; Hwang, Jae Sam; Na, MinKyun; Bae, Jong-Sup

2017-11-01

Tenebrio molitor is an edible insect that has antimicrobial, anticancer, and antihypertensive effects. The aim of this study was to identify the unreported bioactive compounds from T. molitor larvae with inhibitory activities against factor Xa (FXa) and platelet aggregation. Isolated compounds were evaluated for their anti-FXa and anti-platelet aggregation properties by monitoring clotting time, platelet aggregation, FXa activity, and thrombus formation. A diketopiperazine (1, cyclo( L -Pro- L -Tyr)) and a phenylethanoid (2, N-acetyltyramine) were isolated and inhibited the catalytic activity of FXa in a mixed inhibition model and inhibited platelet aggregation induced by adenosine diphosphate (ADP) and U46619. They inhibited ADP- and U46619-induced phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) and the expression of P-selectin and PAC-1 in platelets. They also improved the production of nitric oxide and inhibited the oversecretion of endothelin-1 compared to that of the ADP- or U46619-treated group. In an animal model of arterial and pulmonary thrombosis, the isolated compounds showed enhanced antithrombotic effects. They also elicited anticoagulant effects in mice. Compounds 1-2 inhibited ADP-, collagen-, or U46619-induced platelet aggregation and showed similar anti-thrombotic efficacy to rivaroxaban, a positive control. Therefore, 1-2 could serve as candidates and provide scaffolds for the development of new anti-FXa and anti-platelet drugs. Copyright © 2017 Elsevier Ltd. All rights reserved.

A peptidomimetic with a chiral switch is an inhibitor of epidermal growth factor receptor heterodimerization

PubMed Central

Kanthala, Shanthi P.; Liu, Yong-Yu; Singh, Sitanshu; Sable, Rushikesh; Pallerla, Sandeep; Jois, Seetharama D.

2017-01-01

Among different types of EGFR dimers, EGFR-HER2 and HER2-HER3 are well known in different types of cancers. Targeting dimerization of EGFR will have a significant impact on cancer therapies. A symmetric peptidomimetic was designed to inhibit the protein-protein interaction of EGFR. The peptidomimetic (Cyclo(1,10)PpR (R) Anapa-FDDF-(R)-Anapa)R, compound 18) was shown to exhibit antiproliferative activity with an IC50 of 194 nM in HER2-expressing breast cancer cell lines and 18 nM in lung cancer cell lines. The peptidomimetic has a Pro-Pro sequence in the structure to stabilize the β-turn and a β-amino acid, amino napthyl propionic acid. To investigate the effect of the chirality of β-amino acid on the structure of the peptide and its antiproliferative activity, diastereoisomers of compound 18 were designed and synthesized. Structure-activity relationships of these compounds indicated that there is a chiral switch at β-amino acid in the designed compound. The peptidomimetic with R configuration at β-amino acid and with a L-Pro-D-Pro sequence was the most active compound (18). Using enzyme complement fragmentation assay and proximity ligation assay, we show that compound 18 inhibits HER2:HER3 and EGFR:HER2 dimerization. Surface plasmon resonance studies suggested that compound 18 binds to the HER2 extracellular domain and in particular to domain IV. The anticancer activity of compound 18 was evaluated using a xenograft model of breast cancer in mice; compound 18 suppressed the tumor growth in mice compared to control. Compound 18 was also shown to have a synergistic effect with erlotinib on EGFR mutated lung cancer cell lines. PMID:29088782

Improving the Production of L-Phenylalanine by Identifying Key Enzymes Through Multi-Enzyme Reaction System in Vitro

PubMed Central

Ding, Dongqin; Liu, Yongfei; Xu, Yiran; Zheng, Ping; Li, Haixing; Zhang, Dawei; Sun, Jibin

2016-01-01

L-Phenylalanine (L-Phe) is an important amino acid used in both food and medicinal applications. We developed an in vitro system that allowed a direct, quantitative investigation of phenylalanine biosynthesis in E. coli. Here, the absolute concentrations of six enzymes (AroK, AroL, AroA, AroC, PheA and TyrB) involved in the shikimate (SHIK) pathway were determined by a quantitative proteomics approach and in vitro enzyme titration experiments. The reconstitution of an in vitro reaction system for these six enzymes was established and their effects on the phenylalanine production were tested. The results showed that the yield of phenylalanine increased 3.0 and 2.1 times when the concentrations of shikimate kinase (AroL) and 5-enolpyruvoyl shikimate 3-phosphate (EPSP) synthase (AroA) were increased 2.5 times. Consistent results were obtained from in vivo via the overexpression of AroA in a phenylalanine-producing strain, and the titer of phenylalanine reached 62.47 g/l after 48 h cultivation in a 5-liter jar fermentor. Our quantitative findings provide a practical method to detect the potential bottleneck in a specific metabolic pathway to determine which gene products should be targeted to improve the yield of the desired product. PMID:27558633

Pseudomonas putida as a platform for the synthesis of aromatic compounds.

PubMed

Molina-Santiago, Carlos; Cordero, Baldo F; Daddaoua, Abdelali; Udaondo, Zulema; Manzano, Javier; Valdivia, Miguel; Segura, Ana; Ramos, Juan-Luis; Duque, Estrella

2016-09-01

Aromatic compounds such as l-phenylalanine, 2-phenylethanol and trans-cinnamate are aromatic compounds of industrial interest. Current trends support replacement of chemical synthesis of these compounds by 'green' alternatives produced in microbial cell factories. The solvent-tolerant Pseudomonas putida DOT-T1E strain was genetically modified to produce up to 1 g l-1 of l-phenylalanine. In order to engineer this strain, we carried out the following stepwise process: (1) we selected random mutants that are resistant to toxic phenylalanine analogues; (2) we then deleted up to five genes belonging to phenylalanine metabolism pathways, which greatly diminished the internal metabolism of phenylalanine; and (3) in these mutants, we overexpressed the pheAfbr gene, which encodes a recombinant variant of PheA that is insensitive to feedback inhibition by phenylalanine. Furthermore, by introducing new genes, we were able to further extend the diversity of compounds produced. Introduction of histidinol phosphate transferase (PP_0967), phenylpyruvate decarboxylase (kdc) and an alcohol dehydrogenase (adh) enabled the strain to produce up to 180 mg l-1 2-phenylethanol. When phenylalanine ammonia lyase (pal) was introduced, the resulting strain produced up to 200 mg l-1 of trans-cinnamate. These results demonstrate that P. putida can serve as a promising microbial cell factory for the production of l-phenylalanine and related compounds.

Development and Validation of Eco-Friendly Liquid Chromatographic and Spectrophotometric Methods for Simultaneous Determination of Coformulated Drugs: Phenylephrine Hydrochloride and Prednisolone Acetate.

PubMed

Mostafa, Nadia M; Elsayed, Ghada M; Hassan, Nagiba Y; El Mously, Dina A

2017-11-01

Five simple, sensitive, and eco-friendly LC and UV spectrophotometric methods have been developed for the simultaneous determination of phenylephrine hydrochloride (PHE) and prednisolone acetate (PRD) in their combined dosage form. The first method was reversed-phase (RP) LC using methanol-water-heptane-1-sulfonic acid sodium salt (75 + 25 + 0.1, v/v/w) as a mobile phase. Separation was achieved using an XSelect HSS reversed-phase C18 analytical column (250 × 4.6 mm, 5µm). The flow rate was 1.0 mL/min and UV detection was done at 230 nm. Quantification was achieved over the concentration ranges of 5-50 µg/mL for PHE and 2-90 µg/mL for PRD. Four spectrophotometric methods were proposed, namely dual wavelength, first derivative of ratio spectra, ratio difference, and mean-centering of ratio spectra. Linearity was observed in the concentration ranges of 10-120 and 5-35 µg/mL for PHE and PRD, respectively, for the spectrophotometric methods. Green solvents were used in the proposed methods because they play a vital role in the analytical methods' influence on the environment. The suggested methods were validated regarding linearity, accuracy, and precision according to the International Conference on Harmonization guidelines, with satisfactory results. These methods could be used as harmless substitutes for routine analysis of the mentioned drugs, with no interference from excipients.

Molecular polygamy: The promiscuity of l-phenylalanyl-tRNA-synthetase triggers misincorporation of meta- and ortho-tyrosine in monoclonal antibodies expressed by Chinese hamster ovary cells.

PubMed

Popp, Oliver; Larraillet, Vincent; Kettenberger, Hubert; Gorr, Ingo H; Hilger, Maximiliane; Lipsmeier, Florian; Zeck, Anne; Beaucamp, Nicola

2015-06-01

In-depth analytical characterization of biotherapeutics originating from different production batches is mandatory to ensure product safety and consistent molecule efficacy. Previously, we have shown unintended incorporation of tyrosine (Tyr) and leucine/isoleucine (Leu/Ile) at phenylalanine (Phe) positions in a recombinant produced monoclonal antibody (mAb) using an orthogonal MASCOT/SIEVE based approach for mass spectrometry data analysis. The misincorporation could be avoided by sufficient supply of phenylalanine throughout the process. Several non-annotated signals in the primarily chromatographic peptide separation step for apparently single Phe→Tyr sequence variants (SVs) suggest a role for isobar tyrosine isoforms. Meta- and ortho-Tyr are spontaneously generated during aerobic fed-batch production processes using Chinese hamster ovary (CHO) cell lines. Process induced meta- and ortho-Tyr but not proteinogenic para-Tyr are incorporated at Phe locations in Phe-starved CHO cultures expressing a recombinant mAb. Furthermore, meta- and ortho-Tyr are preferably misincorporated over Leu. Structural modeling of the l-phenylalanyl-tRNA-synthetase (PheRS) substrate activation site indicates a possible fit of non-cognate ortho-Tyr and meta-Tyr substrates. Dose-dependent misincorporations of Tyr isoforms support the hypothesis that meta- and ortho-Tyr are competing, alternative substrates for PheRS in CHO processes. Finally, easily accessible at-line surrogate markers for Phe→Tyr SV formation in biotherapeutic production were defined by the calculation of critical ratios for meta-Tyr/Phe and ortho-Tyr/Phe to support early prediction of SV probability, and finally, to allow for immediate process controlled Phe→Tyr SV prevention. © 2014 Wiley Periodicals, Inc.

Kinetics of PAH degradation by a new acid-metal-tolerant Trabulsiella isolated from the MGP site soil and identification of its potential to fix nitrogen and solubilize phosphorous.

PubMed

Kuppusamy, Saranya; Thavamani, Palanisami; Megharaj, Mallavarapu; Lee, Yong Bok; Naidu, Ravi

2016-04-15

Development of an efficient bioinoculum is considered as an appropriate remedial approach to treat the PAHs-metal mixed contaminated sites. Therefore, we aimed to isolate a degrader able to exert an outstanding PAH catabolic potential with added traits of pH-metal-resistance, N-fix or P-solubilization from a manufactured gas plant site soil. The identified strain (MTS-6) was a first low and high molecular weight (LMW and HMW) PAHs degrading Trabulsiella sp. tolerant to pH 5. MTS-6 completely degraded the model 3 [150mgL(-1) phenanthrene (Phe)], 4 [150mgL(-1) pyrene (Pyr)] and 5 [50mgL(-1) benzo[a]pyrene (BaP)] ring PAHs in 6, 25 and 90 days, respectively. Presence of co-substrate (100mgL(-1) Phe) increased the biodegradation rate constant (k) and decreased the half-life time (t1/2) of HMW PAHs (100mgL(-1) Pyr or 50mgL(-1) BaP). The strain fixed 47μgmL(-1)N and solubilized 58μgmL(-1)P during PAH metabolism and exhibited an EC50 value of 3-4mgL(-1) for Cu, Cd, Pb and Zn. Over 6mgL(-1) metal levels was lethal for the microbe. The identified bacterium (MTS-6) with exceptional multi-functional traits opens the way for its exploitation in the bioremediation of manufactured gas plant sites in a sustainable way by employing bioaugmentation strategy. Copyright © 2016 Elsevier B.V. All rights reserved.

Chiral speciation and determination of selenomethionine enantiomers in selenized yeast by ligand-exchange micellar electrokinetic capillary chromatography after solid phase extraction.

PubMed

Duan, Jiankun; He, Man; Hu, Bin

2012-12-14

A new phenylalanine derivative (L-N-(2-hydroxy-propyl)-phenylalanine, L-HP-Phe) was synthesized and its chelate with Cu(II) (Cu(II)-(L-HP-Phe)(2)) was used as the chiral selector for the ligand-exchange (LE) chiral separation of D,L-selenomethionine (SeMet) in selenized yeast samples by micelle electrokinetic capillary chromatography (MEKC). In order to improve the sensitivity of MEKC-UV, two-step preconcentration strategy was employed, off-line solid phase extraction (SPE) and on-line large volume sample stacking (LVSS). D,L-SeMet was first retained on the Cu(II) loaded mesoporous TiO(2), then eluted by 0.1 mL of 5 mol L(-1) ammonia, and finally introduced for MEKC-UV analysis by LVSS injection after evaporation of NH(3). With the enrichment factors of 1400 and 1378, the LODs of 0.44 and 0.60 ng mL(-1) for L-SeMet and D-SeMet was obtained, respectively. The developed method was applied to the analysis of D,L-SeMet in a certified reference material of SELM-1 and a commercial nutrition yeast, and the results showed that most of SeMet in the SELM-1 selenized yeast was l isomer and the recovery for L and D isomers in the spiked commercial nutrition yeast was 96.3% and 103%, respectively. This method is featured with low running cost, high sensitivity and selectivity, and exhibits application potential in chiral analysis of seleno amino acids in real world samples. Copyright © 2012 Elsevier B.V. All rights reserved.

Diversity Analysis of Dairy and Nondairy Lactococcus lactis Isolates, Using a Novel Multilocus Sequence Analysis Scheme and (GTG)5-PCR Fingerprinting▿

PubMed Central

Rademaker, Jan L. W.; Herbet, Hélène; Starrenburg, Marjo J. C.; Naser, Sabri M.; Gevers, Dirk; Kelly, William J.; Hugenholtz, Jeroen; Swings, Jean; van Hylckama Vlieg, Johan E. T.

2007-01-01

The diversity of a collection of 102 lactococcus isolates including 91 Lactococcus lactis isolates of dairy and nondairy origin was explored using partial small subunit rRNA gene sequence analysis and limited phenotypic analyses. A subset of 89 strains of L. lactis subsp. cremoris and L. lactis subsp. lactis isolates was further analyzed by (GTG)5-PCR fingerprinting and a novel multilocus sequence analysis (MLSA) scheme. Two major genomic lineages within L. lactis were found. The L. lactis subsp. cremoris type-strain-like genotype lineage included both L. lactis subsp. cremoris and L. lactis subsp. lactis isolates. The other major lineage, with a L. lactis subsp. lactis type-strain-like genotype, comprised L. lactis subsp. lactis isolates only. A novel third genomic lineage represented two L. lactis subsp. lactis isolates of nondairy origin. The genomic lineages deviate from the subspecific classification of L. lactis that is based on a few phenotypic traits only. MLSA of six partial genes (atpA, encoding ATP synthase alpha subunit; pheS, encoding phenylalanine tRNA synthetase; rpoA, encoding RNA polymerase alpha chain; bcaT, encoding branched chain amino acid aminotransferase; pepN, encoding aminopeptidase N; and pepX, encoding X-prolyl dipeptidyl peptidase) revealed 363 polymorphic sites (total length, 1,970 bases) among 89 L. lactis subsp. cremoris and L. lactis subsp. lactis isolates with unique sequence types for most isolates. This allowed high-resolution cluster analysis in which dairy isolates form subclusters of limited diversity within the genomic lineages. The pheS DNA sequence analysis yielded two genetic groups dissimilar to the other genotyping analysis-based lineages, indicating a disparate acquisition route for this gene. PMID:17890345

Diversity analysis of dairy and nondairy Lactococcus lactis isolates, using a novel multilocus sequence analysis scheme and (GTG)5-PCR fingerprinting.

PubMed

Rademaker, Jan L W; Herbet, Hélène; Starrenburg, Marjo J C; Naser, Sabri M; Gevers, Dirk; Kelly, William J; Hugenholtz, Jeroen; Swings, Jean; van Hylckama Vlieg, Johan E T

2007-11-01

The diversity of a collection of 102 lactococcus isolates including 91 Lactococcus lactis isolates of dairy and nondairy origin was explored using partial small subunit rRNA gene sequence analysis and limited phenotypic analyses. A subset of 89 strains of L. lactis subsp. cremoris and L. lactis subsp. lactis isolates was further analyzed by (GTG)(5)-PCR fingerprinting and a novel multilocus sequence analysis (MLSA) scheme. Two major genomic lineages within L. lactis were found. The L. lactis subsp. cremoris type-strain-like genotype lineage included both L. lactis subsp. cremoris and L. lactis subsp. lactis isolates. The other major lineage, with a L. lactis subsp. lactis type-strain-like genotype, comprised L. lactis subsp. lactis isolates only. A novel third genomic lineage represented two L. lactis subsp. lactis isolates of nondairy origin. The genomic lineages deviate from the subspecific classification of L. lactis that is based on a few phenotypic traits only. MLSA of six partial genes (atpA, encoding ATP synthase alpha subunit; pheS, encoding phenylalanine tRNA synthetase; rpoA, encoding RNA polymerase alpha chain; bcaT, encoding branched chain amino acid aminotransferase; pepN, encoding aminopeptidase N; and pepX, encoding X-prolyl dipeptidyl peptidase) revealed 363 polymorphic sites (total length, 1,970 bases) among 89 L. lactis subsp. cremoris and L. lactis subsp. lactis isolates with unique sequence types for most isolates. This allowed high-resolution cluster analysis in which dairy isolates form subclusters of limited diversity within the genomic lineages. The pheS DNA sequence analysis yielded two genetic groups dissimilar to the other genotyping analysis-based lineages, indicating a disparate acquisition route for this gene.

Long-term safety and efficacy of sapropterin: the PKUDOS registry experience.

PubMed

Longo, Nicola; Arnold, Georgianne L; Pridjian, Gabriella; Enns, Gregory M; Ficicioglu, Can; Parker, Susan; Cohen-Pfeffer, Jessica L

2015-04-01

The Phenylketonuria (PKU) Demographics, Outcomes and Safety (PKUDOS) registry is designed to provide longitudinal safety and efficacy data on subjects with PKU who are (or have been) treated with sapropterin dihydrochloride. The PKUDOS population consists of 1189 subjects with PKU: N = 504 who were continuously exposed to sapropterin from date of registry enrollment, N = 211 who had intermittent exposure to the drug, and N = 474 with some other duration of exposure. Subjects continuously exposed to sapropterin showed an average 34% decrease in blood phenylalanine (Phe)--from 591 ± 382 μmol/L at baseline to 392 ± 239 μmol/L (p = 0.0009) after 5 years. This drop in blood Phe was associated with an increase in dietary Phe tolerance [from 1000 ± 959 mg/day (pre-sapropterin baseline) to 1539 ± 840 mg/day after 6 years]. Drug-related adverse events (AEs) were reported in 6% of subjects, were mostly considered non-serious, and were identified in the gastrointestinal, respiratory, and nervous systems. Serious drug-related AEs were reported in ≤ 1% of subjects. Similar safety and efficacy data were observed for children<4 years. Long-term data from the PKUDOS registry suggest that sapropterin has a tolerable safety profile and that continuous use is associated with a significant and persistent decrease in blood Phe and improvements in dietary Phe tolerance. Copyright © 2015. Published by Elsevier Inc.

The hyperthermia mediated by 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) is sensitive to sex differences

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

Wyeth, Richard P.; Division of Physiology, Virginia College of Osteopathic Medicine, Blacksburg, VA 24060; Mills, Edward M.

Female subjects have been reported to be less sensitive to the hyperthermic effects of 3,4-methylenedioxymethamine (MDMA) than males. Studies were designed to examine the cellular mechanisms involved in these sex sensitive differences. Gonadectomized female and male rats were treated with a 200 {mu}g 100 {mu}L{sup -1} of estrogen or 100 {mu}g 100 {mu}L{sup -1} of testosterone respectively every 5 days for a total of three doses. Rats were then challenged with either saline or MDMA (20 mg kg{sup -1}, sc). Rats were then euthanized and aortas were constricted, in vitro, by serial phenylephrine (Phe) addition with or without the inhibitormore » of nitric oxide (NO) synthase, g-nitro-L-Arginine-Methyl Ester (L-NAME). Skeletal muscle uncoupling protein-3 (UCP3) expression was measured as well as plasma norepinephrine (NE) levels. All males but no females developed hyperthermia following MDMA treatment. The EC{sub 50} for Phe dose response curves increased only in the females treated with MDMA and T{sub max} for Phe increased following L-NAME only in the females. Both males and females demonstrated an increase in plasma NE following MDMA treatment; however, males displayed a significantly greater NE concentration. Skeletal muscle UCP3 expression was 80% less in females than in males. These results suggest that the inability of MDMA to induce a thermogenic response in the female subjects may be due to four sex-specific mechanisms: 1) Female subjects have reduced sympathetic activation following MDMA challenge; 2) Female vasculature is less sensitive to {alpha}{sub 1}-AR stimulation following MDMA challenge; 3) Female vasculature has an increased sensitivity to NO; 4) UCP3 expression in skeletal muscle is less in females.« less

Papyracillic acid and its derivatives as biting deterrents against Aedes aegypti(Diptera: Culicidae): structure–activity relationships

USDA-ARS?s Scientific Manuscript database

Aedes aegypti L. is the major vector of the arboviruses responsible for dengue fever, one of the most devastating human diseases. Papyracillic acid, the main phytotoxin produced by Ascochyta agropyrina var. nana, was evaluated in a preliminary screening together with other fungal phytotoxins, cyclo...

Identification of the Allosteric Site for Phenylalanine in Rat Phenylalanine Hydroxylase*

PubMed Central

Zhang, Shengnan; Fitzpatrick, Paul F.

2016-01-01

Liver phenylalanine hydroxylase (PheH) is an allosteric enzyme that requires activation by phenylalanine for full activity. The location of the allosteric site for phenylalanine has not been established. NMR spectroscopy of the isolated regulatory domain (RDPheH(25–117) is the regulatory domain of PheH lacking residues 1–24) of the rat enzyme in the presence of phenylalanine is consistent with formation of a side-by-side ACT dimer. Six residues in RDPheH(25–117) were identified as being in the phenylalanine-binding site on the basis of intermolecular NOEs between unlabeled phenylalanine and isotopically labeled protein. The location of these residues is consistent with two allosteric sites per dimer, with each site containing residues from both monomers. Site-specific variants of five of the residues (E44Q, A47G, L48V, L62V, and H64N) decreased the affinity of RDPheH(25–117) for phenylalanine based on the ability to stabilize the dimer. Incorporation of the A47G, L48V, and H64N mutations into the intact protein increased the concentration of phenylalanine required for activation. The results identify the location of the allosteric site as the interface of the regulatory domain dimer formed in activated PheH. PMID:26823465

Development of an ionic liquid-based ultrasonic-assisted liquid-liquid microextraction method for sensitive determination of biogenic amines: application to the analysis of octopamine, tyramine and phenethylamine in beer samples.

PubMed

Huang, Ke-Jing; Jin, Chun-Xue; Song, Shi-Lin; Wei, Cai-Yun; Liu, Yan-Ming; Li, Jing

2011-03-15

A simple and efficient method, ionic liquid-based ultrasound-assisted liquid-liquid microextraction, has been developed for the determination of three biogenic amines including octopamine (OCT), tyramine (TYR) and phenethylamine (PHE). Fluorescence probe 2,6-dimethyl-4-quinolinecarboxylic acid N-hydroxysuccinimide ester was applied for derivatization of biogenic amines and high-performance liquid chromatography coupled with fluorescence detection was used for the determination of the derivatives. The factors affecting the extraction efficiency, such as the type and volume of ionic liquid, ultrasonication time and centrifugation time have been investigated in detail. Under the optimum conditions, linearity of the method was observed in the range of 0.5-50 μgmL(-1) for OCT and TYR, and 0.025-2.5 μgmL(-1) for PHE, respectively, with correlation coefficients (γ)>0.996. The limits of detection ranged from 0.25-50 ngmL(-1) (S/N=3). The spiked recoveries of three target compounds in beer samples were in the range of 90.2-114%. As a result, this method has been successfully applied for the sensitive determination of OCT, TYR and PHE in beer samples. Copyright © 2011 Elsevier B.V. All rights reserved.

Tributyltin contributes in reducing the vascular reactivity to phenylephrine in isolated aortic rings from female rats.

PubMed

Rodrigues, Samya Mere L; Ximenes, Carolina F; de Batista, Priscila R; Simões, Fabiana V; Coser, Pedro Henrique P; Sena, Gabriela C; Podratz, Priscila L; de Souza, Leticia N G; Vassallo, Dalton V; Graceli, Jones B; Stefanon, Ivanita

2014-03-21

Organotin compounds such as tributyltin (TBT) are used as antifouling paints by shipping companies. TBT inhibits the aromatase responsible for the transformation of testosterone into estrogen. Our hypothesis is that TBT modulates the vascular reactivity of female rats. Female Wistar rats were treated daily (Control; CONT) or TBT (100 ng/kg) for 15 days. Rings from thoracic aortas were incubated with phenylephrine (PHE, 10(-10)-10(-4) M) in the presence and absence of endothelium, and in the presence of N(G)-Nitro-L-Arginine Methyl Ester (L-NAME), tetraethylammonium (TEA) and apocynin. TBT decreased plasma levels of estrogen and the vascular response to PHE. In the TBT group, the vascular reactivity was increased in the absence of endothelium, L-NAME and TEA. The decrease in PHE reactivity during incubation with apocynin was more evident in the TBT group. The sensitivity to acetylcholine (ACh) and sodium nitroprusside (SNP) was reduced in the TBT group. TBT increased collagen, reduced α1-smooth muscle actin. Female rats treated with TBT for 15 days showed morphology alteration of the aorta and decreased their vascular reactivity, probably due to mechanisms dependent on nitric oxide (NO) bioavailability, K(+) channels and an increase in oxidative stress. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Polyaromatic hydrocarbon (PAH) degradation potential of a new acid tolerant, diazotrophic P-solubilizing and heavy metal resistant bacterium Cupriavidus sp. MTS-7 isolated from long-term mixed contaminated soil.

PubMed

Kuppusamy, Saranya; Thavamani, Palanisami; Megharaj, Mallavarapu; Lee, Yong Bok; Naidu, Ravi

2016-11-01

An isolate of Cupriavidus (strain MTS-7) was identified from a long-term PAHs and heavy metals mixed contaminated soil with the potential to biodegrade both LMW and HMW PAHs with added unique traits of acid and alkali tolerance, heavy metal tolerance, self-nutrient assimilation by N fixation and P solubilization. This strain completely degraded the model 3 (150 mg L(-1) Phe), 4 (150 mg L(-1) Pyr) and 5 (50 mg L(-1) BaP) ring PAHs in 4, 20 and 30 days, respectively. It could mineralize 90-100% of PAHs (200 mg L(-1) of Phe and Pyr) within 15 days across pH ranging from 5 to 8 and even in the presence of toxic metal contaminations. During biodegradation, the minimum inhibitory concentrations were 5 (Cu(2+)) and 3 (Cd(2+), Pb(2+), Zn(2+)) mg L(-1) of the potentially bioavailable metal ions and over 17 mg L(-1) metal levels was lethal for the microbe. Further, it could fix 217-274 μg mL(-1) of N and solubilize 79-135 μg mL(-1) of P while PAHs degradation. MTS-7 as a superior candidate could be thus used in the enhanced bioaugmentation and/or phytoremediation of long-term mixed contaminated sites. Copyright © 2016 Elsevier Ltd. All rights reserved.

Functional Identification and Structure Determination of Two Novel Prolidases from cog1228 in the Amidohydrolase Superfamily

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

Xiang, Dao Feng; Patskovsky, Yury; Xu, Chengfu

2010-12-07

Two uncharacterized enzymes from the amidohydrolase superfamily belonging to cog1228 were cloned, expressed, and purified to homogeneity. The two proteins, Sgx9260c (gi|44242006) and Sgx9260b (gi|44479596), were derived from environmental DNA samples originating from the Sargasso Sea. The catalytic function and substrate profiles for Sgx9260c and Sgx9260b were determined using a comprehensive library of dipeptides and N-acyl derivative of L-amino acids. Sgx9260c catalyzes the hydrolysis of Gly-L-Pro, L-Ala-L-Pro, and N-acyl derivatives of L-Pro. The best substrate identified to date is N-acetyl-L-Pro with a value of k{sub cat}/K{sub m} of 3 x 10{sup 5} M{sup -1} s{sup -1}. Sgx9260b catalyzes the hydrolysismore » of L-hydrophobic L-Pro dipeptides and N-acyl derivatives of L-Pro. The best substrate identified to date is N-propionyl-L-Pro with a value of k{sub cat}/K{sub m} of 1 x 10{sup 5} M{sup -1} s{sup -1}. Three-dimensional structures of both proteins were determined by X-ray diffraction methods (PDB codes 3MKV and 3FEQ). These proteins fold as distorted ({beta}/{alpha})8-barrels with two divalent cations in the active site. The structure of Sgx9260c was also determined as a complex with the N-methylphosphonate derivative of L-Pro (PDB code 3N2C). In this structure the phosphonate moiety bridges the binuclear metal center, and one oxygen atom interacts with His-140. The {alpha}-carboxylate of the inhibitor interacts with Tyr-231. The proline side chain occupies a small substrate binding cavity formed by residues contributed from the loop that follows {beta}-strand 7 within the ({beta}/{alpha})8-barrel. A total of 38 other proteins from cog1228 are predicted to have the same substrate profile based on conservation of the substrate binding residues. The structure of an evolutionarily related protein, Cc2672 from Caulobacter crecentus, was determined as a complex with the N-methylphosphonate derivative of L-arginine (PDB code 3MTW).« less

Executive function impairment in early-treated PKU subjects with normal mental development.

PubMed

Leuzzi, V; Pansini, M; Sechi, E; Chiarotti, F; Carducci, Cl; Levi, G; Antonozzi, I

2004-01-01

Executive functions were studied in 14 early and continuously treated PKU subjects (age 10.8 years, range 8-13) in comparison with controls matched for IQ, sex, age and socioeconomic status. Brain MRI examination was normal in all PKU patients. Neuropsychological evaluation included Wisconsin Card Sorting Test, Rey-Osterreith Complex Figure Test, Elithorn's Perceptual Maze Test, Weigl's Sorting Test, Tower of London, Visual Search and Motor Motor Learning Test. Whatever the IQ, PKU subjects performed worse than controls in tests exploring executive functions. Subgrouping the PKU subjects according to the quality of dietary control for the entire follow-up period (using 400 micromol/L as cut-off value for blood phenylalanine (Phe) concentration) showed that patients with worse dietary control performed more poorly than both the PKU group with the best dietary control and the control group. However, a mild impairment of executive functions was still found in PKU patients with a good dietary control (Phe <400 micromol/L) compared to controls. Concerning the PKU group as a whole, no linear correlation was found between neuropsychological performance and historical and concurrent biochemical parameters. We conclude that (a) PKU patients, even when treated early, rigorously and continuously, show an impairment of frontal lobe functions; (b) a protracted exposure to moderately high levels of Phe can affect frontal lobe functions independently of the possible effect of the same exposure on IQ; (c) in order to reduce the risk of frontal lobe dysfunction, the target of dietary therapy should be to maintain blood Phe concentration below 400 micromol/L.

Lactobacillus musae sp. nov., a novel lactic acid bacterium isolated from banana fruits.

PubMed

Chen, Yi-Sheng; Wang, Li-Ting; Liao, Yu-Jou; Lan, Yi-Shan; Chang, Chi-Huan; Chang, Yu-Chung; Wu, Hui-Chung; Lo, Huei-Yin; Otoguro, Misa; Yanagida, Fujitoshi

2017-12-01

Two Gram-stain-positive, catalase-negative, rod-shaped, bacterial strains (313 T and 311) were isolated from banana fruits in Taiwan. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the highest similarity to both strains corresponded to the type strain of Lactobacillus nantensis (99.19 %), followed by Lactobacillus crustorum (98.99 %), Lactobacillus heilongjiangensis (98.59 %) and Lactobacillus farciminis (98.52 %). Phylogenetic analysis based on the sequences of two housekeeping genes, pheS and rpoA, revealed that these two strains were well separated from the Lactobacillus reference strains. DNA-DNA relatedness values revealed genotype separation of the two strains from the above four species. The DNA G+C content of strain 313 T was 35.5 mol%. The strains were homofermentative and mainly produced l-lactic acid from glucose. The major cellular fatty acids of strain 313 T were 18 : 1ω6c and/or 18 : 1ω7c, 16 : 0, and 19 : 1ω6c and/or 19 : 0 cyclo ω10c. Based on their physiological and genotypic characteristics, the isolates represent a novel species of the genus Lactobacillus, for which the name Lactobacillusmusae sp. nov. is proposed. The type strain is 313 T =NBRC 112868 T =BCRC 81020 T ).

Lactobacillus cypricasei Lawson et al. 2001 is a later heterotypic synonym of Lactobacillus acidipiscis Tanasupawat et al. 2000.

PubMed

Naser, Sabri M; Vancanneyt, Marc; Hoste, Bart; Snauwaert, Cindy; Swings, Jean

2006-07-01

The applicability of a multilocus sequence analysis (MLSA)-based identification system for lactobacilli was evaluated. Two housekeeping genes that code for the phenylalanyl-tRNA synthase alpha-subunit (pheS) and RNA polymerase alpha-subunit (rpoA) were sequenced and analysed for members of the Lactobacillus salivarius species group. The type strains of Lactobacillus acidipiscis and Lactobacillus cypricasei were investigated further using a third gene that encodes the alpha-subunit of ATP synthase (atpA). The MLSA data revealed close relatedness between L. acidipiscis and L. cypricasei, with 99.8-100 % pheS, rpoA and atpA gene sequence similarities. Comparison of the 16S rRNA gene sequences of the type strains of the two species confirmed the close relatedness (99.8 % gene sequence similarity) between the two taxa. Similar phenotypes and high DNA-DNA binding values in the range of 84 to 97.5 % confirmed that L. acidipiscis and L. cypricasei are synonymous species. On the basis of the present study, it is proposed that Lactobacillus cypricasei is a later heterotypic synonym of Lactobacillus acidipiscis.

Detection of diastereomer peptides as the intermediates generating D-amino acids during acid hydrolysis of peptides.

PubMed

Miyamoto, Tetsuya; Sekine, Masae; Ogawa, Tetsuhiro; Hidaka, Makoto; Watanabe, Hidenori; Homma, Hiroshi; Masaki, Haruhiko

2016-11-01

In this study, we investigated whether the amino acid residues within peptides were isomerized (and the peptides converted to diastereomers) during the early stages of acid hydrolysis. We demonstrate that the model dipeptides L-Ala-L-Phe and L-Phe-L-Ala are epimerized to produce the corresponding diastereomers at a very early stage, prior to their acid hydrolytic cleavage to amino acids. Furthermore, the sequence-inverted dipeptides were generated via formation of a diketopiperazine during hydrolytic incubation, and these dipeptides were also epimerized. The proportion of diastereomers increased rapidly during incubation for 0.5-2 h. During acid hydrolysis, C-terminal residues of the model dipeptides were isomerized faster than N-terminal residues, consistent with the observation that the D-amino acid values of the C-terminal residues determined by the 0 h-extrapolating method were larger than those of the N-terminal residues. Thus, the artificial D-amino acid contents determined by the 0 h-extrapolating method appear to be products of the isomerization of amino acid residues during acid hydrolysis.

Chromium (D-phenylalanine)3 improves obesity-induced cardiac contractile defect in ob/ob mice.

PubMed

Dong, Feng; Yang, Xiaoping; Sreejayan, Nair; Ren, Jun

2007-11-01

Low-molecular weight chromium compounds, such as chromium picolinate [Cr(pic)(3)], improve insulin sensitivity, although toxicity is a concern. We synthesized a novel chromium complex, chromium (d-phenylalanine)(3) [Cr(d-phe)(3)], in an attempt to improve insulin sensitivity with reduced toxicity. The aim of this study was to compare the two chromium compounds on cardiac contractile function in ob/ob obese mice. C57BL lean and ob/ob obese mice were randomly divided into three groups: H(2)O, Cr(d-phe)(3), or Cr(pic)(3) (45 mug/kg per day orally for 6 months). The glucose tolerance test displayed improved glucose clearance by Cr(d-phe)(3) but not Cr(pic)(3). Myocytes from ob/ob mice exhibited depressed peak shortening (PS) and maximal velocity of shortening/relengthening (+/-dL/dt), prolonged time-to-PS and time-to-90% relengthening (TR90), reduced electrically stimulated rise in intracellular Ca(2+) (Deltafura-2 fluorescence intensity), and slowed intracellular Ca(2+) decay. Although a 3-month Cr(d-phe)(3) treatment for a separate group of ob/ob and lean 2-month-old mice only rectified reduced +/-dL/dt in ob/ob mice, all mechanical and intracellular Ca(2+) abnormalities were significantly attenuated or ablated by 6 months of Cr(d-phe)(3) but not Cr(pic)(3) treatment (except TR90). Sarco(endo)plasmic reticulum Ca(2+) ATPase activity and Na(+)-Ca(2+) exchanger expression were depressed in ob/ob mice, which were reversed by both Cr(d-phe)(3) and Cr(pic)(3), with a more pronounced effect from Cr(d-phe)(3). Cr(d-phe)(3) corrected reduced insulin-stimulated glucose uptake and improved basal phosphorylation of Akt and insulin receptor, as well as insulin-stimulated phosphorylation of Akt and insulin receptor in ob/ob myocytes. Heart homogenates from ob/ob mice had enhanced oxidative stress and protein carbonyl formation compared with the lean group, which were attenuated by both Cr(d-phe)(3) and Cr(pic)(3). Our data suggest that the new Cr(d-phe)(3) compound possesses better cardio-protective and insulin-sensitizing properties against obesity.

DNA-fiber EPR investigation of the influence of amino-terminal residue stereochemistry on the DNA binding orientation of Cu(II)•Gly-Gly-His-derived metallopeptides

PubMed Central

Hamada, Hirokazu; Abe, Yuko; Nagane, Ryoichi; Fang, Ya-Yin; Lewis, Mark A.; Long, Eric C.; Chikira, Makoto

2007-01-01

DNA fiber EPR was used to investigate the DNA binding stabilities and orientations of Cu(II)•Gly-Gly-His-derived metallopeptides containing d- vs. l-amino acid substitutions in the first peptide position. This examination included studies of Cu(II)•d-Arg-Gly-His and Cu(II)•d-Lys-Gly-His for comparison to metallopeptides containing l-Arg/Lys substitutions, and also the diastereoisomeric pairs Cu(II)•d/l-Pro-Gly-His and Cu(II)•d/l-Pro-Lys-His. Results indicated that l-Arg/Lys to d-Arg/Lys substitutions considerably randomized the orientation of the metallopeptides on DNA whereas the replacement of l-Pro by d-Pro in Cu(II)•l-Pro-Gly-His caused a decrease in randomness. The difference in the extent of randomness of d- vs. l-Pro-Gly-His complexes was diminished through the substitution of Gly for Lys in the middle peptide position, supporting the notion that the ε-amino group of Lys triggered further randomization, likely through hydrogen bonding or electrostatic interactions that disrupt binding of the metallopeptide equatorial plane and the DNA. The relationship between the stereochemistry of amino acid residues and the binding and reaction of M(II)•Xaa-Xaa’-His metallopeptides with DNA are also discussed. PMID:17706784

Synthesis of tritium labelled endomorphin II and its stability in the radioreceptor assay

NASA Astrophysics Data System (ADS)

Tömböly, Cs.; Spetea, M.; Borsodi, A.; Tóth, G.

1999-01-01

Endomorphine I (Tyr-Pro-Trp-Phe-NH2) and endomorphin II (Tyr-Pro-Phe-Phe-NH2) are recently isolated neuropeptides. They have the highest specificity and affinity for the μ-opiate receptor among all the endogenous substances so far described, and they may be natural ligands for this receptor [1]. We prepared the [3H] endomorphin II with high specific radioactivity (53.4 Ci/mmol) by catalytic dehalotritiation. The precursor [(3,5-I2)Tyr1]-endomorphin II was synthesized by solid phase peptide synthesis using Boc chemistry. Labelled endomorphin II was used to investigate its binding properties in rat brain membrane. The stability of [3H]endomorphin II toward enzymatic degradation in membrane preparation was examined by RP-HPLC and by using a radioactivity detector.

The complete mitochondrial genome of the black star fat minnow (Rhynchocypris semotilus), an endemic and endangered fish of Korea.

PubMed

Yu, Jeong-Nam; Kim, Byung-Jik; Kim, Changmu; Yeo, Joo-Hong; Kim, Soonok

2017-01-01

The Black star fat minnow (Rhynchocypris semotilus) is an endemic and critically endangered freshwater fish in Korea. Its genome was 16 605 bp long and consisted of 13 protein-coding genes (PCG), two rRNA genes, 22 tRNA genes, and a control region. The gene order and the composition of R. semotilus were similar to that of most other vertebrates. Four overlapping regions in ATP8/ATP6, ATP6/COX3, ND4L/ND4, and ND5/ND6, among the 13 PCGs were found. The control region was located between the tRNA-Pro and tRNA-Phe genes and was determined to be 935 bp in length with the 3' end containing a 12 TA-repeat sequence. Phylogenetic analysis suggested that R. semotilus is most closely related to R. oxycephalus.

Towards Understanding the Tandem Mass Spectra of Protonated Oligopeptides. 2: The Proline Effect in Collision-Induced Dissociation of Protonated Ala-Ala-Xxx-Pro-Ala (Xxx = Ala, Ser, Leu, Val, Phe, and Trp)

NASA Astrophysics Data System (ADS)

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

2011-06-01

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

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

PubMed

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

2011-06-01

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

Chimeric NDP-MSH and MTII melanocortin peptides with agouti-related protein (AGRP) Arg-Phe-Phe amino acids possess agonist melanocortin receptor activity.

PubMed

Joseph, Christine G; Wilczynski, Andrzej; Holder, Jerry R; Xiang, Zhimin; Bauzo, Rayna M; Scott, Joseph W; Haskell-Luevano, Carrie

2003-12-01

Agouti-related protein (AGRP) is one of only two known endogenous antagonists of G-protein coupled receptors (GPCRs). Specifically, AGRP antagonizes the brain melanocortin-3 and -4 receptors involved in energy homeostasis, regulation of feeding behavior, and obesity. Alpha-melanocyte stimulating hormone (alpha-MSH) is one of the known endogenous agonists for these receptors. It has been hypothesized that the Arg-Phe-Phe (111-113) human AGRP amino acids may be mimicking the melanocortin agonist Phe-Arg-Trp (7-9) residue interactions with the melanocortin receptors that are important for both receptor molecular recognition and stimulation. To test this hypothesis, we generated thirteen chimeric peptide ligands based upon the melanocortin agonist peptides NDP-MSH (Ac-Ser-Tyr-Ser-Nle4-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2) and MTII (Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2). In these chimeric ligands, the agonist DPhe-Arg-Trp amino acids were replaced by the AGRP Arg-Phe-Phe residues, and resulted in agonist activity at the mouse melanocortin receptors (mMC1R and mMC3-5Rs), supporting the hypothesis that the AGRP antagonist ligand Arg-Phe-Phe residues mimic the agonist Phe-Arg-Trp amino acids. Interestingly, the Ac-Ser-Tyr-Ser-Nle4-Glu-His-Arg-DPhe-Phe-Gly-Lys-Pro-Val-NH2 peptide possessed 7 nM mMC1R agonist potency, and is 850-fold selective for the mMC1R versus the mMC3R, 2300-fold selective for the mMC1R versus the mMC4R, and 60-fold selective for the MC1R versus the mMC5R, resulting in the discovery of a new peptide template for the design of melanocortin receptor selective ligands.

Glu-Phe from onion (Allium Cepa L.) attenuates lipogenesis in hepatocytes.

PubMed

Lee, Yu Geon; Cho, Jeong-Yong; Hwang, Eom Ji; Jeon, Tae-Il; Moon, Jae-Hak

2017-07-01

A Glu-Phe (EF) was isolated from onion (Allium cepa L. cv. Sunpower). The chemical structure of EF was determined by nuclear magnetic resonance and electrospray ionization-mass (ESI-MS) spectroscopy. We showed that EF reduced lipid accumulation in mouse hepatocytes by inhibiting the expression of sterol regulatory element-binding protein-1c (SREBP-1c) and its lipogenic target genes. We also found that AMP-activated protein kinase (AMPK) was required for the inhibitory effect of EF on lipid accumulation in mouse hepatocytes. Furthermore, EF was qualified in nine onion cultivars by selective multiple reaction-monitoring detection of liquid chromatography-ESI-MS. These results suggest that EF could contribute to the beneficial effect of onion supplement in maintaining hepatic lipid homeostasis.

A rapid and cost-effective method of producing recombinant proBNP and NT-proBNP variants in Escherichia coli for immunoassay of heart failure.

PubMed

Soleh, Muhammad Tarmizi; Foo, Jared Yong Yang; Bailey, Ulla-Maja; Tan, Nikki Yi; Wan, Yunxia; Cooper-White, Justin; Schulz, Benjamin Luke; Punyadeera, Chamindie

2014-01-01

The measurements of plasma natriuretic peptides (NT-proBNP, proBNP and BNP) are used to diagnose heart failure but these are expensive to produce. We describe a rapid, cheap and facile production of proteins for immunoassays of heart failure. DNA encoding N-terminally His-tagged NT-proBNP and proBNP were cloned into the pJexpress404 vector. ProBNP and NT-proBNP peptides were expressed in Escherichia coli, purified and refolded in vitro. The analytical performance of these peptides were comparable with commercial analytes (NT-proBNP EC50 for the recombinant is 2.6 ng/ml and for the commercial material is 5.3 ng/ml) and the EC50 for recombinant and commercial proBNP, are 3.6 and 5.7 ng/ml respectively). Total yield of purified refolded NT-proBNP peptide was 1.75 mg/l and proBNP was 0.088 mg/l. This approach may also be useful in expressing other protein analytes for immunoassay applications. To develop a cost effective protein expression method in E. coli to obtain high yields of NT-proBNP (1.75 mg/l) and proBNP (0.088 mg/l) peptides for immunoassay use.

The hypertrehalosemic neuropeptides of cicadas are structural isomers-evidence by ion mobility mass spectrometry.

PubMed

König, Simone; Marco, Heather; Gäde, Gerd

2017-11-01

It has been known for more than 20 years that the neurosecretory glands of the cicadas, the corpora cardiaca, synthesize two isobaric peptides with hypertrehalosemic activity. Both decapeptides have exactly the same amino acid sequence (pGlu-Val-Asn-Phe-Ser-Pro-Ser-Trp-Gly-Asn-NH 2 ) and mass but differ in their retention time in reversed-phase liquid chromatography. A synthetic peptide with the same sequence elutes together with the second more hydrophobic peptide peak of the natural cicada extract. It is not clear what modification is causing the described observations. Therefore, in the current study, ion mobility separation in conjunction with high-resolution mass spectrometry was used to investigate this phenomenon as it was sensitive to changes in conformation. It detected different drift times in buffer gas for both the intact peptides and some of their fragment ions. Based on the ion mobility and fragment ion intensity of the corresponding ions, it is concluded that the region Pro 6 -Ser 7 -Trp 8 contains a structural feature differing from the L-amino acids present in the known peptide. Whether the conformer is the result of racemization or other biochemical processes needs to be further investigated.

Changes of serum alkaline phosphatase isoenzymes in fasted rats.

PubMed

Wada, H; Niwa, N; Hayakawa, T; Tsuge, H

1996-10-01

Changes of serum alkaline phosphatase (sALP) isoenzymes under fasting conditions were examined using polyacrylamide gel electrophoresis (PAGE), amino-acids (L-phenylalanine (L-Phe), L-homoarginine (L-HArg)) inhibition and wheat germ agglutinin (WGA) treatment. The sALP of non-fasted rats was separated into three bands (S1, S2, S3) by PAGE. The molecular weight (M.W.) of S1 corresponded to that of an isoenzyme found in the ileum. By the addition of L-Phe, the staining intensity of S1 was weakened, S2 and S3 remained unchanged and the total activity of the isoenzymes extracted from intestine decreased. On the other hand, the activity of isoenzymes extracted from kidney and bone decreased by the addition of L-HArg. Therefore, S1 was judged to be derived from intestine. The activities of total sALP and S1 decreased from 16 h of fasting. Total sALP activity and sALP activity of the supernatant prepared by WGA treatment decreased, whereas the ALP activity of the precipitate (difference between total sALP activity and supernatant sALP activity) did not change. The activity band of the precipitate corresponded to that of S3 by PAGE. Therefore, S3 was judged to be derived from bone. In conclusion, under fasting conditions, the activity of S1 decreased while the activities of S2 and S3 remained unchanged.

Breast-feeding success among infants with phenylketonuria.

PubMed

Banta-Wright, Sandra A; Shelton, Kathleen C; Lowe, Nancy D; Knafl, Kathleen A; Houck, Gail M

2012-08-01

Breast milk is the nutrition of choice for human infants (American Academy of Pediatrics, 2005; American Association of Family Physicians, 2008; Association of Women's Health Obstetric and Neonatal Nurses, 2005; Canadian Paediatric Society, 2005; U.S. Preventive Services Task Force, 2008; World Health Organization, 2009). In comparison to standard commercial formula, human breast milk has a lower concentration of protein and a lower content of the amino acid phenylalanine (Phe). For infants with phenylketonuria (PKU), these attributes of human breast milk make it ideal as a base source of nutrition. The purpose of this study was to compare the incidence and duration of breast-feeding and corresponding Phe levels of breast-fed and formula-fed infants with PKU in the caseload of a pediatric metabolic clinic at an urban tertiary-care medical center. Charts were reviewed for infants diagnosed with PKU beginning with 2005 and ending with 1980, the year no further breast-feeding cases were identified in the PKU population. During the first year of life, most of the infants, whether breast-fed or formula-fed, had similar mean Phe levels. However, the frequency distributions revealed that more breast-fed infants with PKU had Phe levels within the normal range (120-360 μmol/L) and were less likely to have low Phe levels (<120 μmol/L) than formula-fed infants with PKU. Further research is needed to understand how mothers manage breast-feeding in the context of PKU. Copyright © 2012 Elsevier Inc. All rights reserved.

Dietary Phenylalanine Requirements are Similar in Small, Medium, and Large Breed Adult Dogs using the Indicator Amino Acid Oxidation Technique.

PubMed

Mansilla, W D; Gorman, A; Fortener, L; Shoveller, A K

2018-05-26

We have previously determined Phenylalanine (Phe) requirements in mature dogs; however, little information is available on differences of Phe minimum requirements (MR) on different breed sizes. The objective of this study was to determine Phe requirements in adult dogs of 3 different breed sizes using the direct amino acid oxidation (DAAO) technique. In total, 12 adult dogs were used, 4 Miniature Dachshunds (5.3 ± 0.6 Kg BW; 1.8 ± 0.1 years old; mean ± SD), 4 Beagles (8.3 ± 0.7 Kg BW; 6.7 ± 0.2 years old; mean ± SD), and 4 Labrador Retrievers (34.9 ± 2.2 Kg BW; 4.4 ± 1.4 years old; mean ± SD). A basal Phe-deficient diet with excess of Tyrosine (Tyr) was formulated. Dogs were randomly fed the basal diet supplemented with increasing levels of Phe; the Phe content in the final experimental diets was 0.24, 0.29, 0.34, 0.44, 0.54, 0.64, and 0.74%. After 2 d of adaptation to the experimental diets, dogs underwent individual DAAO studies. During the DAAO studies, total daily feed was divided in 13 equal meals; at the sixth meal, dogs were fed a bolus of L-[1-13C]-Phe (9.40 mg/kg BW), and thereafter, L-[1-13C]-Phe (2.4 mg/kg BW) was supplied with every meal. Total production of 13CO2 (13CO2) during isotopic steady state was determined by enrichment of 13CO2 in breath samples and total production of CO2 measured using indirect calorimetry. The mean requirement for Phe and the 95% confidence interval (CI) were determined using a 2-phase linear regression model. To account for differences in feed intake, requirements were expressed in mg.kg BW-1.d-1. The mean requirement for Phe were 41.9, 41.3, and 42.6, and upper 95% CI of Phe requirements were 57.3, 58.4, and 64.8 mg.kg BW-1.d-1 for Miniature Dachshunds, Beagles, and Labrador Retrievers, respectively. The mean requirement and the upper 95% CI for the pooled data (all dogs) was 45.3 and 55.4 mg.kg BW-1.d-1, respectively. In conclusion, the Phe requirements for different breeds were similar among dog breeds studied. However, Phe recommendations proposed in this study are lower than those proposed by NRC and AAFCO (mg.kg BW-1.d-1).

Blood-brain barrier transport of an essential amino acid after cerebral ischemia reperfusion injury.

PubMed

Suzuki, Toyofumi; Miyazaki, Yumiko; Ohmuro, Aya; Watanabe, Masaki; Furuishi, Takayuki; Fukami, Toshiro; Tomono, Kazuo

2013-01-01

Under pathophysiological conditions such as -cerebral ischemia-reperfusion (IR), damage to cerebrovascular endothelial cells causes alterations in the blood-brain barrier (BBB) function that can exacerbate neuronal cell injury and death. Clarifying changes in BBB transport in the early period of IR is important for understanding BBB function during therapy after cerebral ischemia. The present study was aimed at clarifying changes during IR in the BBB transport of L-phenylalanine (Phe) as a substrate of L-type amino acid transporter 1. An IR model was produced in mice by blood recirculation following occlusion of the middle cerebral artery. Permeability of the BBB to [(3)H]Phe was measured after IR injury using the brain perfusion method. Confocal microscopy of the IR injury showed no brain penetration of fluorescent tracer, thus confirming BBB integrity during 45 min of ischemia. Tight junction opening was not observed at 30 min after reperfusion following ischemia for 45 min. At the time of IR, [(3)H]Phe uptake into the brain appeared saturated. The Michaelis constant and maximum transport velocity in the IR group was reduced by 22 % compared with those in controls. These results suggest that the intrinsic transport clearance of Phe is slightly decreased in the early phase of IR.

A potentiometric chiral sensor for L-Phenylalanine based on crosslinked polymethylacrylic acid-polycarbazole hybrid molecularly imprinted polymer.

PubMed

Chen, Yu; Chen, Lei; Bi, Ruilin; Xu, Lan; Liu, Yan

2012-11-19

A novel chiral molecularly imprinted polymer (MIP) sensor for L-Phenylalanine has been developed, which is constructed by electrochemically driven cross-linking a pendant polymer precursor, poly[2-(N-carbazolyl)ethyl methacrylate-co-meth-acrylic acid]s (PCEMMAs). In this MIP sensing material, the recognition sites, the insulating polymethylacrylic acid (PMAA), were covalently bonded to the conducting polycarbazole which could be used as signal transfer interface between recognition layer and electrode. The mole ratio of copolymerizing monomers, 2-(N-carbazolyl) ethyl methacrylate:methylacrylic acid (CE:MAA), and the scanning cycles of electropolymerization were adjusted during the preparation of MIP sensing material. The optimized conditions, CE:MAA=3:2 and 20 scanning cycles, were obtained. And then the properties of MIP films were characterized by atomic force microscope (AFM), X-ray photoelectron spectroscopy (XPS) and water contact angle. Open circuit potential-time technique was used to estimate the enantioselectivity of the MIP sensor. The results indicate that the promising sensor preferentially responses L-Phenylalanine (L-Phe) over D-Phenylalanine (D-Phe) with a selectivity coefficient K(D)(L)=5.75×10(-4) and the limit of detection (LOD) is 1.37μM, which reveals its good enantioselectivity and sensitivity. Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

Simple and rapid analytical method for detection of amino acids in blood using blood spot on filter paper, fast-GC/MS and isotope dilution technique.

PubMed

Kawana, Shuichi; Nakagawa, Katsuhiro; Hasegawa, Yuki; Yamaguchi, Seiji

2010-11-15

A simple and rapid method for quantitative analysis of amino acids, including valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met) and phenylalanine (Phe), in whole blood has been developed using GC/MS. In this method, whole blood was collected using a filter paper technique, and a 1/8 in. blood spot punch was used for sample preparation. Amino acids were extracted from the sample, and the extracts were purified using cation-exchange resins. The isotope dilution method using ²H₈-Val, ²H₃-Leu, ²H₃-Met and ²H₅-Phe as internal standards was applied. Following propyl chloroformate derivatization, the derivatives were analyzed using fast-GC/MS. The extraction recoveries using these techniques ranged from 69.8% to 87.9%, and analysis time for each sample was approximately 26 min. Calibration curves at concentrations from 0.0 to 1666.7 μmol/l for Val, Leu, Ile and Phe and from 0.0 to 333.3 μmol/l for Met showed good linearity with regression coefficients=1. The method detection limits for Val, Leu, Ile, Met and Phe were 24.2, 16.7, 8.7, 1.5 and 12.9 μmol/l, respectively. This method was applied to blood spot samples obtained from patients with phenylketonuria (PKU), maple syrup urine disease (MSUD), hypermethionine and neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD), and the analysis results showed that the concentrations of amino acids that characterize these diseases were increased. These results indicate that this method provides a simple and rapid procedure for precise determination of amino acids in whole blood. Copyright © 2010 Elsevier B.V. All rights reserved.

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).

PubMed

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.

Bladder pain induced by prolonged peripheral alpha 1A adrenoceptor stimulation involves the enhancement of transient receptor potential vanilloid 1 activity and an increase of urothelial adenosine triphosphate release.

PubMed

Matos, R; Cordeiro, J M; Coelho, A; Ferreira, S; Silva, C; Igawa, Y; Cruz, F; Charrua, A

2016-12-01

Pathophysiological mechanisms of chronic visceral pain (CVP) are unknown. This study explores the association between the sympathetic system and bladder nociceptors activity by testing the effect of a prolonged adrenergic stimulation on transient receptor potential vanilloid 1 (TRPV1) activity and on urothelial adenosine triphosphate (ATP) release. Female Wistar rats received saline, phenylephrine (PHE), PHE + silodosin, PHE + naftopidil or PHE + prazosin. TRPV1 knockout and wild-type mice received saline or PHE. Visceral pain behaviour tests were performed before and after treatment. Cystometry was performed, during saline and capsaicin infusion. Fos immunoreactivity was assessed in L6 spinal cord segment. Human urothelial ATP release induced by mechanical and thermal stimulation was evaluated. Subcutaneous, but not intrathecal, PHE administration induced pain, which was reversed by silodosin, a selective alpha 1A adrenoceptor antagonist, but not by naftopidil, a relatively selective antagonist for alpha 1D adrenoceptor. Silodosin also reversed PHE-induced bladder hyperactivity and L6 spinal cord Fos expression. Thus, in subsequent experiments, only silodosin was used. Wild-type, but not TRPV1 knockout, mice exhibited phenylephrine-induced pain. Capsaicin induced a greater increase in voiding contractions in PHE-treated rats than in control animals, and silodosin reversed this effect. When treated with PHE, ATP release from human urothelial cells was enhanced either by mechanical stimulation or by lowering the thermal threshold of urothelial TRPV1, which becomes abnormally responsive at body temperature. This study suggests that the activation of peripheral alpha 1A adrenoceptors induces CVP, probably through its interaction with TRPV1 and ATP release. © 2016 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

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

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

Lee, Y.H.

Several phenylalanine derivatives have been found to inhibit the gelation of deoxygenated sickle hemoglobin (deoxy HbS). Proton and /sup 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 ..beta..93 position with N-(2,2,2-trifluoroethyl) iodoacetamide (IA-F/sub 3/), and by monitoring the relaxation rates of the C2 and C4 histidine protons. The results show that the /sup 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 interactionmore » 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 ..beta..2, 4b143 and ..beta..146 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.« less

Ribbon structure stabilized by C10 and C12 turns in αγ hybrid peptide.

PubMed

Wani, Naiem Ahmad; Kant, Rajni; Gupta, Vivek Kumar; Aravinda, Subrayashastry; Rai, Rajkishor

2016-04-01

The present study describes the synthesis and crystallographic analysis of αγ hybrid peptides, Boc-Gpn-L-Pro-NHMe (1), Boc-Aib-Gpn-L-Pro-NHMe (2), and Boc-L-Pro-Aib-Gpn-L-Pro-NHMe (3). Peptides 1 and 2 adopt expanded 12-membered (C12 ) helical turn over γα segment. Peptide 3 promotes the ribbon structure stabilized by type II β-turn (C10 ) followed by the expanded C12 helical γα turn. Both right-handed and left-handed helical conformations for Aib residue are observed in peptides 2 and 3, respectively. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

The Multi-Biomarker Approach for Heart Failure in Patients with Hypertension

PubMed Central

Bielecka-Dabrowa, Agata; Gluba-Brzózka, Anna; Michalska-Kasiczak, Marta; Misztal, Małgorzata; Rysz, Jacek; Banach, Maciej

2015-01-01

We assessed the predictive ability of selected biomarkers using N-terminal pro-brain natriuretic peptide (NT-proBNP) as the benchmark and tried to establish a multi-biomarker approach to heart failure (HF) in hypertensive patients. In 120 hypertensive patients with or without overt heart failure, the incremental predictive value of the following biomarkers was investigated: Collagen III N-terminal propeptide (PIIINP), cystatin C (CysC), lipocalin-2/NGAL, syndecan-4, tumor necrosis factor-α (TNF-α), interleukin 1 receptor type I (IL1R1), galectin-3, cardiotrophin-1 (CT-1), transforming growth factor β (TGF-β) and N-terminal pro-brain natriuretic peptide (NT-proBNP). The highest discriminative value for HF was observed for NT-proBNP (area under the receiver operating characteristic curve (AUC) = 0.873) and TGF-β (AUC = 0.878). On the basis of ROC curve analysis we found that CT-1 > 152 pg/mL, TGF-β < 7.7 ng/mL, syndecan > 2.3 ng/mL, NT-proBNP > 332.5 pg/mL, CysC > 1 mg/L and NGAL > 39.9 ng/mL were significant predictors of overt HF. There was only a small improvement in predictive ability of the multi-biomarker panel including the four biomarkers with the best performance in the detection of HF—NT-proBNP, TGF-β, CT-1, CysC—compared to the panel with NT-proBNP, TGF-β and CT-1 only. Biomarkers with different pathophysiological backgrounds (NT-proBNP, TGF-β, CT-1, CysC) give additive prognostic value for incident HF in hypertensive patients compared to NT-proBNP alone. PMID:25984599

Functional characterization of the modified melanocortin peptides responsible for ligand selectivity at the human melanocortin receptors.

PubMed

Chen, Min; Georgeson, Keith E; Harmon, Carroll M; Haskell-Luevano, Carrie; Yang, Yingkui

2006-11-01

The melanocortin system plays an important role in energy homeostasis as well as skin pigmentation, steroidogenesis and exocrine gland function. In this study, we examined eight Ac-His-Phe-Arg-Trp-NH(2) tetrapeptides that were modified at the Phe position and pharmacologically characterized their activities at the human MCR wild-types and their mutants. Our results indicate that at the hMC1R, all D stereochemical modified residues at the Phe position of peptides increase cAMP production in a dose-dependent manner. At the hMC3R, the DPhe peptide dose dependently increases cAMP production but all other three tetrapeptides were not. At the hMC4R, both the DPhe and DNal(1') peptides induce cAMP production. However, both DTyr and DNal(2') were not able to induce cAMP production. Further studies indicated that at the hMC1R M128L mutant receptor, the all D-configured tetrapeptides reduce their potencies as compared to that of hMC1R wild-type. However, at the hMC3R and hMC4R L165M and L133M mutant receptors, the DNal(2') and DTyr tetrapeptides possess agonist activity. These findings indicate that DPhe in tetrapeptide plays an important role in ligand selectivity and specific residue TM3 of the melanocortin receptors is crucial for ligand selectivity.

Effects of calcitonin gene-related peptide on canine cerebral artery strips and the in-vivo vertebral blood flow in dogs.

PubMed

Ikegaki, I; Suzuki, Y; Satoh, S; Asano, T; Shibuya, M; Sugita, K

1989-10-01

The effects of calcitonin gene-related peptide (CGRP) on canine cerebral arteries and on vertebral blood flow were investigated in-vivo and in-vitro and the findings compared with the effects of vasoactive intestinal peptide (VIP) and substance P. Administration of CGRP into the vertebral artery caused a dose-dependent and long-lasting increase in blood flow. The in-vivo vasodilatory effects of substance P and VIP were short-lasting. CGRP (0.1 to 100 nmol/l) elicited a concentration-dependent relaxation of the isolated middle cerebral and basilar arteries when the tissues were precontracted by exposure to prostaglandin F2 alpha (PGF2 alpha). This effect was not antagonized by propranolol, atropine, tetrodotoxin, (N-Ac-Tyr1, D-Phe2)-growth hormone-releasing factor(1-29)-NH2 or (D-Pro2, D-Trp7,9) substance P. CGRP also reduced concentration-dependently the contraction of cerebral arteries induced by KCl or 9,11-epithio-11,12-metano-thromboxane A2 (STXA2). Mechanical removal of the endothelium did not abolish the vasodilatory response to CGRP. In PGF2 alpha-contracted canine cerebral arteries, VIP (0.1 to 100 nmol/l) was less potent a vasodilator than CGRP. At low concentrations (0.01 to 1 nmol/l) substance P elicited a rapid and short-lasting relaxation, and in the absence of endothelium this relaxation disappeared. These findings are clear evidence that CGRP modulates vascular tone.

Ultrasonic speed, densities and viscosities of xylitol in water and in aqueous tyrosine and phenylalanine solutions at different temperatures

NASA Astrophysics Data System (ADS)

Ali, A.; Bidhuri, P.; Uzair, S.

2014-07-01

Ultrasonic speed u, densities ρ and viscosities η of xylitol in water and in 0.001 m aqueous l-tyrosine (Tyr) and l-phenylalanine (Phe) have been measured at different temperatures. From the density and ultrasonic speed measurements apparent molar isentropic compression κ_{φ}, apparent molar isentropic compressions at infinite dilution κ_{{S,φ}}0 , experimental slope S K , hydration number n H , transfer partial molar isentropic compressibility Δ_{tr} κ_{{S,φ}}0 of xylitol from water to aqueous Tyr and Phe have been obtained. From the viscosity data, B-coefficient and B-coefficient of transfer Δ tr B of xylitol from water to aqueous Phe and Tyr at different temperatures have also been estimated. Gibbs free energies of activation of viscous flow per mole of solvent Δ μ 1 0# and per mole of solute Δ μ 2 0# have been calculated by using Feakins transition state theory for the studied systems. The calculated parameters have been interpreted in terms of solute-solute and solute-solvent interactions and hydration behavior of xylitol.

Diagnostic value of pleural fluid N-terminal pro-brain natriuretic peptide levels in patients with cardiovascular diseases.

PubMed

Liao, Huai; Na, Moon Jun; Dikensoy, Oner; Lane, Kirk B; Randal, Barnette; Light, Richard W

2008-01-01

The diagnosis of the cause of pleural effusions caused by cardiovascular diseases such as congestive heart failure (CHF) and acute pulmonary embolism is sometimes difficult. The purpose of the present study was to evaluate the utility of pleural fluid levels of N-terminal pro-brain natriuretic peptide (NT-proBNP) in differentiating pleural effusions due to CHF, pulmonary embolism and post-coronary artery bypass graft (CABG) surgery. The levels of pleural fluid NT-proBNP were measured by ELISA in a total of 40 patients: 10 with CHF, 10 with pulmonary embolism, 10 post-CABG and 10 with carcinoma. The median level of NT-proBNP in the pleural fluid of patients with CHF was 5390 pg/mL (25th to 75th percentiles, 4566 to 8158 pg/mL), which was significantly higher than that in patients with post-CABG effusions (424 pg/mL, 352 to 873), with pulmonary embolism (311 pg/mL, 212 to 1159), or with carcinoma (302 pg/mL, 208 to 626) (P < 0.001, CHF group vs all other groups). In receiver-operating curve analysis, an NT-proBNP level of >or=2220 pg/mL demonstrated a sensitivity of 100% and a specificity of 96.7% for the identification of CHF. Measurement of the NT-proBNP level in pleural fluid is accurate in diagnosing the etiology of the effusion as CHF. Pleural fluid levels above 2220 pg/mL are essentially diagnostic that the pleural effusion is due to CHF.

Role of aromatic interactions in amyloid formation by islet amyloid polypeptide.

PubMed

Tu, Ling-Hsien; Raleigh, Daniel P

2013-01-15

Aromatic-aromatic and aromatic-hydrophobic interactions have been proposed to play a role in amyloid formation by a range of polypeptides, including islet amyloid polypeptide (IAPP or amylin). IAPP is responsible for amyloid formation in patients with type 2 diabetes. The polypeptide is 37 residues long and contains three aromatic residues, Phe-15, Phe-23, and Tyr-37. The ability of all single aromatic to leucine mutants, all double aromatic to leucine mutants, and the triple leucine mutant to form amyloid were examined. Amyloid formation was almost twice as rapid for the F15L mutant as for the wild type but was almost 3-fold slower for the Y37L mutant and almost 2-fold slower for the F23L mutant. Amyloid fibrils formed from each of the single mutants were effective at seeding amyloid formation by wild-type IAPP, implying that the fibril structures are similar. The F15L/F23L double mutant has a larger effect than the F15L/Y37L double mutant on the rate of amyloid formation, even though a Y37L substitution has more drastic consequences in the wild-type background than does the F23L mutation, suggesting nonadditive effects between the different sites. The triple leucine mutant and the F23L/Y37L double mutant are the slowest to form amyloid. F15 has been proposed to make important contacts early in the aggregation pathway, but the data for the F15L mutant indicate that they are not optimal. A set of variants containing natural and unnatural amino acids at position 15, which were designed to conserve hydrophobicity, but alter α-helix and β-sheet propensity, were analyzed to determine the properties of this position that control the rate of amyloid formation. There is no correlation between β-sheet propensity at this position and the rate of amyloid formation, but there is a correlation with α-helical propensity.

Design of a new peptidomimetic agonist for the melanocortin receptors based on the solution structure of the peptide ligand, Ac-Nle-cyclo[Asp-Pro-DPhe-Arg-Trp-Lys]-NH(2).

PubMed

Fotsch, Christopher; Smith, Duncan M; Adams, Jeffrey A; Cheetham, Janet; Croghan, Michael; Doherty, Elizabeth M; Hale, Clarence; Jarosinski, Mark A; Kelly, Michael G; Norman, Mark H; Tamayo, Nuria A; Xi, Ning; Baumgartner, James W

2003-07-21

The solution structure of a potent melanocortin receptor agonist, Ac-Nle-cyclo[Asp-Pro-DPhe-Arg-Trp-Lys]-NH(2) (1) was calculated using distance restraints determined from 1H NMR spectroscopy. Eight of the lowest energy conformations from this study were used to identify non-peptide cores that mimic the spatial arrangement of the critical tripeptide region, DPhe-Arg-Trp, found in 1. From these studies, compound 2a, containing the cis-cyclohexyl core, was identified as a functional agonist of the melanocortin-4 receptor (MC4R) with an IC(50) and EC(50) below 10 nM. Compound 2a also showed 36- and 7-fold selectivity over MC3R and MC1R, respectively, in the binding assays. Subtle changes in cyclohexane stereochemistry and removal of functional groups led to analogues with lower affinity for the MC receptors.

The influence of water-ethanol mixture on the thermodynamics of complex formation between 18-crown-6 ether and L-phenylalanine

NASA Astrophysics Data System (ADS)

Usacheva, T. R.; Sharnin, V. A.; Chernov, I. V.; Matteoli, E.; Terekhova, I. V.; Kumeev, R. S.

2012-08-01

The influence of water-ethanol mixture composition on the complex formation between 18-crown-6 ether and L-phenylalanine was studied by titration calorimetry at Т = 298.15 K. The standard thermodynamic parameters (ΔrGо, ΔrHо, ТΔrSо) of formation of [Phe18C6] molecular complex were calculated from data obtained by means of the microcalorimetric system TAM III (TA Instruments, USA) at X(EtOH) = 0.0/0.6 mol fraction. The stability of [Phe18C6] and the mechanism of complexation in water were investigated using the 1Н and 13С NMR spectroscopy. The increase of EtOH concentration results in an increase of the complex stability and of the exothermicity of complexation.

Complete mitochondrial genome of the desert toad-headed agama, Phrynocephalus przewalskii (Reptilia, Squamata, Agamidae), a novel gene organization in vertebrate mtDNA.

PubMed

Li, Donghai; Song, Sen; Chen, Tao; Zhang, Caihong; Chang, Cheng

2015-01-01

The mitogenome of the desert toad-headed agama, Phrynocephalus przewalskii, was amplified using polymerase chain reaction (PCR), long-and-accurate PCR and directly sequenced by primer walking. The complete mitogenome was 16,892 bp in size and contained 13 protein-coding, 23 tRNA, and 2 rRNA genes, and 1 control region. The mitogenome of the P. przewalskii was similar to those of other Phrynocephalus sand lizards in gene arrangement and composition, except that tRNA-Phe and tRNA-Pro were exchanged and tRNA-Phe had two copies. The control region comprised three parts, one between tRNA-Thr and tRNA-Phe, a second between the two tRNA-Phe copies, and a third between tRNA-Pro and 12S RNA. The overall nucleotide composition of the H-strand was 36.3% A, 26.7% T, 12.5% G, 24.6% C. The complete mitogenome of P. przewalskii will contribute to understanding the evolution of the genus Phrynocephalus and the family Agamidae.

Cascade Bioassay Evidence for the Existence of Urothelium-Derived Inhibitory Factor in Guinea Pig Urinary Bladder

PubMed Central

Guan, Na N.; Thor, Anna; Hallén, Katarina; Wiklund, N. Peter; Gustafsson, Lars E.

2014-01-01

Our aim was to investigate whether guinea pig urothelium-derived bioactivities compatible with the existence of urothelium-derived inhibitory factor could be demonstrated by in vitro serial bioassay and whether purinergic P1 receptor agonists, nitric oxide, nitrite or prostaglandins might explain observed activities. In a cascade superfusion system, urothelium-denuded guinea pig ureters were used as bioassay tissues, recording their spontaneous rhythmic contractions in presence of scopolamine. Urothelium-intact or -denuded guinea pig urinary bladders were used as donor tissues, stimulated by intermittent application of carbachol before or during the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME), the adenosine/P1 nucleoside receptor antagonist 8-(p-sulfophenyl)theophylline (8-PST) or the cyclo-oxygenase inhibitor diclofenac infused to bath donor and bioassay tissues. The spontaneous contractions of bioassay ureters were unaltered by application of carbachol 1–5 µM in the presence of scopolamine 5–30 µM. When carbachol was applied over the urothelium-denuded bladder, the assay ureter contraction rate was unaltered. Introducing carbachol over the everted urothelium-intact bladder significantly inhibited the contraction frequency of the assay ureter, suggesting the transfer of an inhibitory activity from the bladder to the assay ureter. The transmissible inhibitory activity was not markedly antagonized by L-NAME, 8-PST or diclofenac, while L-NAME nearly abolished nitrite release from the urothelium-intact bladder preparations. We suggest that urothelium-derived inhibitory factor is a transmissible entity over a significant distance as demonstrated in this novel cascade superfusion assay and seems less likely to be nitric oxide, nitrite, an adenosine receptor agonist or subject to inhibition by administration of a cyclo-oxygenase inhibitor. PMID:25084114

Cascade bioassay evidence for the existence of urothelium-derived inhibitory factor in Guinea pig urinary bladder.

PubMed

Guan, Na N; Thor, Anna; Hallén, Katarina; Wiklund, N Peter; Gustafsson, Lars E

2014-01-01

Our aim was to investigate whether guinea pig urothelium-derived bioactivities compatible with the existence of urothelium-derived inhibitory factor could be demonstrated by in vitro serial bioassay and whether purinergic P1 receptor agonists, nitric oxide, nitrite or prostaglandins might explain observed activities. In a cascade superfusion system, urothelium-denuded guinea pig ureters were used as bioassay tissues, recording their spontaneous rhythmic contractions in presence of scopolamine. Urothelium-intact or -denuded guinea pig urinary bladders were used as donor tissues, stimulated by intermittent application of carbachol before or during the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME), the adenosine/P1 nucleoside receptor antagonist 8-(p-sulfophenyl)theophylline (8-PST) or the cyclo-oxygenase inhibitor diclofenac infused to bath donor and bioassay tissues. The spontaneous contractions of bioassay ureters were unaltered by application of carbachol 1-5 µM in the presence of scopolamine 5-30 µM. When carbachol was applied over the urothelium-denuded bladder, the assay ureter contraction rate was unaltered. Introducing carbachol over the everted urothelium-intact bladder significantly inhibited the contraction frequency of the assay ureter, suggesting the transfer of an inhibitory activity from the bladder to the assay ureter. The transmissible inhibitory activity was not markedly antagonized by L-NAME, 8-PST or diclofenac, while L-NAME nearly abolished nitrite release from the urothelium-intact bladder preparations. We suggest that urothelium-derived inhibitory factor is a transmissible entity over a significant distance as demonstrated in this novel cascade superfusion assay and seems less likely to be nitric oxide, nitrite, an adenosine receptor agonist or subject to inhibition by administration of a cyclo-oxygenase inhibitor.

Accidental intoxication with Veratrum album.

PubMed

Grobosch, T; Binscheck, T; Martens, F; Lampe, D

2008-01-01

A 49-year-old man consumed two glasses (approximately 2 x 20 mL) of a beverage containing yellow gentian (Gentiana lutea). Shortly after ingestion, he developed nausea, vomiting, and oral paraesthesia. On admission to the hospital he suffered from severe bradycardia (35 beats/min) and hypotension (50/30 mm Hg), and he was treated with activated charcoal, antiemetics (metoclopramide, ondansetron), atropine, and intravenous electrolytic solution. The initial suspicion of Veratrum poisoning could be confirmed by identifying protoveratrines A (ProA) and protoveratrine B (ProB) in a sample from the beverage as well as in the patients serum by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS). The yellow-colored beverage contained 25% ethanol (by headspace gas chromatography), 20.4 mg/L ProA, and 13.7 mg/L ProB. The serum concentration of ProA was 1162 ng/L and ProB was 402 ng/L. Veratridine, cevadine, and jervine were not detected, neither in the beverage nor in the serum sample. The lower limits of quantitation for all compounds is 10 microg/L (S/N > 10, beverage) and 100 ng/L (S/N > 10, serum). After treatment, the patient completely recovered from the symptoms within 24 h and was discharged from the hospital. The analytical method described was developed for the simultaneous identification and quantitation of five Veratrum alkaloids. The method is based on a liquid-liquid extraction followed by LC-MS-MS analysis. The time needed for analysis was 6 min.

The delta-selective opioid peptide dermenkephalin and the mu-selective hybrid peptide dermenkephalin-[1-4]-dermophin-[5-7] display strikingly different conformations despite identical tetrapeptide N-termini. A quantitative 2-D NMR and molecular modeling analysis.

PubMed

Riand, J; Baron, D; Nicolas, P; Benajiba, A; Teng, Y; Naim, M

1999-12-01

The selective recognition of the aminoterminal binding pharmacophore Tyr-D-Xaa-Phe of the opioid heptapeptide dermorphin, Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2 (DRM)1, and of dermenkephalin, Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2 (DREK), by the mu-opioid receptor and delta-opioid receptor, respectively, depends upon the constitution / conformation of the C-terminal tripeptide. The hybrid peptide DREK-[1-4]-DRM-[5-7] is very potent at, and exquisitely selective for the mu-opioid receptor, and differs only from dermenkephalin by its C-terminal tripeptide. Comparison of the structural features of DREK-[1-4]-DRM-[5-7] and dermenkephalin by nmr analysis and molecular modeling revealed striking differences, as well in the trans (Tyr5 - Pro6) isomer (population 75%) than in the cis isomer.. Whereas the folded C-terminal tail of dermenkephalin influenced the tertiary structure of the N-terminal tetrapeptide and placed the Tyr1 and Phe3 aromatic rings in definite orientations that are best suited for the delta-receptor, there were only weak contacts, as shown by NOE data, between the aminoterminal and carboxyterminal parts of the hybrid peptide. This promoted increased flexibility of the whole backbone and relaxed orientations for the side-chains of Tyr1 and Phe3 that are compatible with the mu-receptor but unsuitable for the delta-receptor. The steric hindrance introduced by Pro6 in DREK-[1-4]-DRM-[5-7], plus the absence of large hydrophobic side-chains in positions 5 and 6 may prevent close contacts between the N-terminal and C-terminal domains and reorientation of the main pharmacophoric elements Tyr1 and Phe3.

Synthesis and antibacterial activity of new peptides from Alfalfa RuBisCO protein hydrolysates and mode of action via a membrane damage mechanism against Listeria innocua.

PubMed

Kobbi, Sabrine; Nedjar, Naima; Chihib, Nourdine; Balti, Rafik; Chevalier, Mickael; Silvain, Amandine; Chaabouni, Semia; Dhulster, Pascal; Bougatef, Ali

2018-02-01

In this work we evaluated the mode of action of six new synthesized peptides (Met-Asp-Asn; Glu-leu-Ala-Ala-Ala-Cys; Leu-Arg-Asp-Asp-Phe; Gly-Asn-Ala-Pro-Gly-Ala-Val-Ala; Ala-Leu-Arg-Met-Ser-Gly and Arg-Asp-Arg-Phe-Leu), previously identified, from the most active peptide fractions of RuBisCO peptic hydrolysate against Listeria innocua via a membrane damage mechanism. Antibacterial effect and the minimum inhibitory concentrations (MIC) of these peptides were evaluated against six strains and their hemolytic activities towards bovine erythrocytes were determined. Prediction of the secondary structure of peptides indicated that these new antibacterial peptides are characterized by a short peptide chains (3-8 amino acid) and a random coli structure. Moreover, it was observed that one key characteristic of antibacterial peptides is the presence of specific amino acids such as cysteine, glycine, arginine and aspartic acid. In addition the determination of the extracellular potassium concentration revealed that treatment with pure RuBisCO peptides could cause morphological changes of L. innocua and destruction of the cell integrity via irreversible membrane damage. The results could provide information for investigating the antibacterial model of antibacterial peptides derived from RuBisCO protein hydrolysates. Copyright © 2017 Elsevier Ltd. All rights reserved.

Electrophysiological effects of protopine in cardiac myocytes: inhibition of multiple cation channel currents.

PubMed

Song, L S; Ren, G J; Chen, Z L; Chen, Z H; Zhou, Z N; Cheng, H

2000-03-01

Protopine (Pro) from Corydalis tubers has been shown to have multiple actions on cardiovascular system, including anti-arrhythmic, anti-hypertensive and negative inotropic effects. Although it was thought that Pro exerts its actions through blocking Ca(2+) currents, the electrophysiological profile of Pro is unclear. The aim of this study is to elucidate the ionic mechanisms of Pro effects in the heart. In single isolated ventricular myocytes from guinea-pig, extracellular application of Pro markedly and reversibly abbreviates action potential duration, and decreases the rate of upstroke (dV/dt)(max), amplitude and overshoot of action potential in a dose-dependent manner. Additionally, it produces a slight, but significant hyperpolarization of the resting membrane potential. Pro at 25, 50 and 100 microM reduces L-type Ca(2+) current (I(Ca,L)) amplitude to 89.1, 61.9 and 45.8% of control, respectively, and significantly slows the decay kinetics of I(Ca,L) at higher concentration. The steady state inactivation of I(Ca,L) is shifted negatively by 5.9 - 7.0 mV (at 50 - 100 microM Pro), whereas the voltage-dependent activation of I(Ca,L) remains unchanged. In contrast, Pro at 100 microM has no evident effects on T-type Ca(2+) current (I(Ca,T)). In the presence of Pro, both the inward rectifier (I(K1)) and delayed rectifier (I(K)) potassium currents are variably inhibited, depending on Pro concentrations. Sodium current (I(Na)), recorded in low [Na(+)](o) (40 mM) solution, is more potently suppressed by Pro. At 25 microM, Pro significantly attenuated I(Na) at most of the test voltages (-60 approximately +40 mV, with a 53% reduction at -30 mV. Thus, Pro is not a selective Ca(2+) channel antagonist. Rather, it acts as a promiscuous inhibitor of cation channel currents including I(Ca,L), I(K), I(K1) as well as I(Na). These findings may provide some mechanistic explanations for the therapeutic actions of Pro in the heart.

Electrophysiological effects of protopine in cardiac myocytes: inhibition of multiple cation channel currents

PubMed Central

Song, Long-Sheng; Ren, Guo-Jun; Chen, Zhao-Luan; Chen, Zhi-He; Zhou, Zhao-Nian; Cheng, Heping

2000-01-01

Protopine (Pro) from Corydalis tubers has been shown to have multiple actions on cardiovascular system, including anti-arrhythmic, anti-hypertensive and negative inotropic effects. Although it was thought that Pro exerts its actions through blocking Ca2+ currents, the electrophysiological profile of Pro is unclear. The aim of this study is to elucidate the ionic mechanisms of Pro effects in the heart. In single isolated ventricular myocytes from guinea-pig, extracellular application of Pro markedly and reversibly abbreviates action potential duration, and decreases the rate of upstroke (dV/dt)max, amplitude and overshoot of action potential in a dose-dependent manner. Additionally, it produces a slight, but significant hyperpolarization of the resting membrane potential. Pro at 25, 50 and 100 μM reduces L-type Ca2+ current (ICa,L) amplitude to 89.1, 61.9 and 45.8% of control, respectively, and significantly slows the decay kinetics of ICa,L at higher concentration. The steady state inactivation of ICa,L is shifted negatively by 5.9–7.0 mV (at 50–100 μM Pro), whereas the voltage-dependent activation of ICa,L remains unchanged. In contrast, Pro at 100 μM has no evident effects on T-type Ca2+ current (ICa,T). In the presence of Pro, both the inward rectifier (IK1) and delayed rectifier (IK) potassium currents are variably inhibited, depending on Pro concentrations. Sodium current (INa), recorded in low [Na+]o (40 mM) solution, is more potently suppressed by Pro. At 25 μM, Pro significantly attenuated INa at most of the test voltages (−60∼+40 mV, with a 53% reduction at −30 mV. Thus, Pro is not a selective Ca2+ channel antagonist. Rather, it acts as a promiscuous inhibitor of cation channel currents including ICa,L, IK, IK1 as well as INa. These findings may provide some mechanistic explanations for the therapeutic actions of Pro in the heart. PMID:10696087

A fragment of the alarmin prothymosin α as a novel biomarker in murine models of bacteria-induced sepsis.

PubMed

Samara, Pinelopi; Miriagou, Vivi; Zachariadis, Michael; Mavrofrydi, Olga; Promponas, Vasilis J; Dedos, Skarlatos G; Papazafiri, Panagiota; Kalbacher, Hubert; Voelter, Wolfgang; Tsitsilonis, Ourania

2017-07-25

Sepsis is a life-threatening condition that requires urgent care. Thus, the identification of specific and sensitive biomarkers for its early diagnosis and management are of clinical importance. The alarmin prothymosin alpha (proTα) and its decapeptide proTα(100-109) are immunostimulatory peptides related to cell death. In this study, we generated bacterial models of sepsis in mice using two Klebsiella pneumoniae strains (L-78 and ATCC 43816) and monitored sepsis progression using proTα(100-109) as a biomarker. Serum concentration of proTα(100-109) gradually increased as sepsis progressed in mice infected with L-78, a strain which, unlike ATCC 43816, was phagocytosed by monocytes/macrophages. Analysis of splenocytes from L-78-infected animals revealed that post-infection spleen monocytes/macrophages were gradually driven to caspase-3-mediated apoptosis. These results were verified in vitro in L-78-infected human monocytes/macrophages. Efficient phagocytosis of L-78 by monocytes stimulated their apoptosis and the concentration of proTα(100-109) in culture supernatants increased. Human macrophages strongly phagocytosed L-78, but resisted cell death. This is the first report suggesting that high levels of proTα(100-109) correlate, both in vitro and in vivo, with increased percentages of cell apoptosis. Moreover, we showed that low levels of proTα(100-109) early post-infection likely correlate with sepsis resolution and thus, the decapeptide could eventually serve as an early surrogate biomarker for predicting bacteria-induced sepsis outcome.

Conformational dynamics of L-lysine, L-arginine, L-ornithine binding protein reveals ligand-dependent plasticity.

PubMed

Silva, Daniel-Adriano; Domínguez-Ramírez, Lenin; Rojo-Domínguez, Arturo; Sosa-Peinado, Alejandro

2011-07-01

The molecular basis of multiple ligand binding affinity for amino acids in periplasmic binding proteins (PBPs) and in the homologous domain for class C G-protein coupled receptors is an unsolved question. Here, using unrestrained molecular dynamic simulations, we studied the ligand binding mechanism present in the L-lysine, L-arginine, L-ornithine binding protein. We developed an analysis based on dihedral angles for the description of the conformational changes upon ligand binding. This analysis has an excellent correlation with each of the two main movements described by principal component analysis (PCA) and it's more convenient than RMSD measurements to describe the differences in the conformational ensembles observed. Furthermore, an analysis of hydrogen bonds showed specific interactions for each ligand studied as well as the ligand interaction with the aromatic residues Tyr-14 and Phe-52. Using uncharged histidine tautomers, these interactions are not observed. On the basis of these results, we propose a model in which hydrogen bond interactions place the ligand in the correct orientation to induce a cation-π interaction with Tyr-14 and Phe-52 thereby stabilizing the closed state. Our results also show that this protein adopts slightly different closed conformations to make available specific hydrogen bond interactions for each ligand thus, allowing a single mechanism to attain multiple ligand specificity. These results shed light on the experimental evidence for ligand-dependent conformational plasticity not explained by the previous crystallographic data. Copyright © 2011 Wiley-Liss, Inc.

Anticancer Activity of a Hexapeptide from Skate (Raja porosa) Cartilage Protein Hydrolysate in HeLa Cells

PubMed Central

Pan, Xin; Zhao, Yu-Qin; Hu, Fa-Yuan; Chi, Chang-Feng; Wang, Bin

2016-01-01

In this study, the hexapeptide Phe-Ile-Met-Gly-Pro-Tyr (FIMGPY), which has a molecular weight of 726.9 Da, was separated from skate (Raja porosa) cartilage protein hydrolysate using ultrafiltration and chromatographic methods, and its anticancer activity was evaluated in HeLa cells. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay indicated that FIMGPY exhibited high, dose-dependent anti-proliferation activities in HeLa cells with an IC50 of 4.81 mg/mL. Acridine orange/ethidium bromide (AO/EB) fluorescence staining and flow cytometry methods confirmed that FIMGPY could inhibit HeLa cell proliferation by inducing apoptosis. Western blot assay revealed that the Bax/Bcl-2 ratio and relative intensity of caspase-3 in HeLa cells treated with 7-mg/mL FIMGPY were 2.63 and 1.83, respectively, significantly higher than those of the blank control (p < 0.01). Thus, FIMGPY could induce apoptosis by upregulating the Bax/Bcl-2 ratio and caspase-3 activation. Using a DNA ladder method further confirmed that the anti-proliferation activity of FIMGPY was attributable to its role in inducing apoptosis. These results suggest that FIMGPY from skate cartilage protein hydrolysate may have applications as functional foods and nutraceuticals for the treatment and prevention of cancer. PMID:27537897

Anticancer Activity of a Hexapeptide from Skate (Raja porosa) Cartilage Protein Hydrolysate in HeLa Cells.

PubMed

Pan, Xin; Zhao, Yu-Qin; Hu, Fa-Yuan; Chi, Chang-Feng; Wang, Bin

2016-08-16

In this study, the hexapeptide Phe-Ile-Met-Gly-Pro-Tyr (FIMGPY), which has a molecular weight of 726.9 Da, was separated from skate (Raja porosa) cartilage protein hydrolysate using ultrafiltration and chromatographic methods, and its anticancer activity was evaluated in HeLa cells. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay indicated that FIMGPY exhibited high, dose-dependent anti-proliferation activities in HeLa cells with an IC50 of 4.81 mg/mL. Acridine orange/ethidium bromide (AO/EB) fluorescence staining and flow cytometry methods confirmed that FIMGPY could inhibit HeLa cell proliferation by inducing apoptosis. Western blot assay revealed that the Bax/Bcl-2 ratio and relative intensity of caspase-3 in HeLa cells treated with 7-mg/mL FIMGPY were 2.63 and 1.83, respectively, significantly higher than those of the blank control (p < 0.01). Thus, FIMGPY could induce apoptosis by upregulating the Bax/Bcl-2 ratio and caspase-3 activation. Using a DNA ladder method further confirmed that the anti-proliferation activity of FIMGPY was attributable to its role in inducing apoptosis. These results suggest that FIMGPY from skate cartilage protein hydrolysate may have applications as functional foods and nutraceuticals for the treatment and prevention of cancer.

Structural Basis for Reversible and Irreversible Inhibition of Human Cathepsin L by their Respective dipeptidyl glyoxal and diazomethylketone Inhibitors

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

R Shenoy; J Sivaraman

Cathepsin L plays a key role in many pathophysiological conditions including rheumatoid arthritis, tumor invasion and metastasis, bone resorption and remodeling. Here we report the crystal structures of two analogous dipeptidyl inhibitor complexes which inhibit human cathepsin L in reversible and irreversible modes, respectively. To-date, there are no crystal structure reports of complexes of proteases with their glyoxal inhibitors or complexes of cathepsin L and their diazomethylketone inhibitors. These two inhibitors - inhibitor 1, an {alpha}-keto-{beta}-aldehyde and inhibitor 2, a diazomethylketone, have different groups in the S1 subsite. Inhibitor 1 [Z-Phe-Tyr (OBut)-COCHO], with a Ki of 0.6 nM, is themore » most potent, reversible, synthetic peptidyl inhibitor of cathepsin L reported to-date. The structure of the inhibitor 1 complex was refined up to 2.2 {angstrom} resolution. The structure of the complex of the inhibitor 2 [Z-Phe-Tyr (t-Bu)-diazomethylketone], an irreversible inhibitor that can inactivate cathepsin L at {micro}M concentrations, was refined up to 1.76 {angstrom} resolution. These two inhibitors have substrate-like interactions with the active site cysteine (Cys25). Inhibitor 1 forms a tetrahedral hemithioacetal adduct, whereas the inhibitor 2 forms a thioester with Cys25. The inhibitor 1 {beta}-aldehyde group is shown to make a hydrogen bond with catalytic His163, whereas the ketone carbonyl oxygen of the inhibitor 2 interacts with the oxyanion hole. tert-Butyl groups of both inhibitors are found to make several non-polar contacts with S' subsite residues of cathepsin L. These studies, combined with other complex structures of cathepsin L, reveal the structural basis for their potency and selectivity.« less

Theoretical prediction and experimental verification on enantioselectivity of haloacid dehalogenase L-DEX YL with chloropropionate

NASA Astrophysics Data System (ADS)

Kondo, Hirotaka; Fujimoto, Kazuhiro J.; Tanaka, Shigenori; Deki, Hiroyuki; Nakamura, Takashi

2015-03-01

L-2-Haloacid dehalogenase (L-DEX YL) is a member of a family of enzymes that decontaminate a variety of environmental pollutants such as L-2-chloropropionate (L-2-CPA). This enzyme specifically catalyzes the hydrolytic dehalogenation of L-2-haloacid to produce D-2-hydroxy acid, and does not catalyze that of D-2-haloacid. Here, using the quantum-mechanical/molecular-mechanical and the fragment molecular orbital calculations, the enzymatic reaction of L-DEX YL to D-2-CPA was compared with that to L-2-CPA. As a result, Tyr12, Leu45 and Phe60 were predicted to affect the enantioselectivity. We then performed the site-directed-mutagenesis experiments and the activity measurement of these mutants, thus finding that the F60Y mutant had the enzymatic activity with D-2-CPA.

Comparison of the post-mortem interval on the effect of vascular responses to the activation of ionotropic and metabotropic receptors.

PubMed

Bloch-Bogusławska, Elżbieta; Grześk, Elżbieta; Grześk, Grzegorz

2015-03-01

The contractibility of blood vessels depends on their normal structure and the availability of calcium ions; it changes under the influence of numerous contracting and relaxing factors, which control the activities of various pathways of intracellular and intercellular signaling. The main aim of the study was to investigate, by means of perfusion pressure in rat tail arteries, the role of Ca 2+ in vascular response to α-1 adrenoceptor activation by phenylephrine (PHE) and Bay K8644 agonist of the L-type calcium channel and caffeine before and after a post-mortem interval (PMI) of 2, 4, 6 and 8 h. A phasic increase of perfusion pressure in rat tail arteries, as induced by PHE or caffeine, in Ca 2+ -free solutions was used as an indicator of intracellular Ca 2+ release through the inositol 1,4,5-triphosphate and ryanodine receptor pathways, respectively. In Ca 2+ -free-ethylene glycol tetraacetic acid (EGTA)-poly(sodium styrenesulfonate) (PSS) and in Ca 2+ -EGTA-PSS, the PHE induced elevation of perfusion pressure significantly decreased. Vascular responses to caffeine (20 mmol/1) in Ca 2+ -free-EGTA-PSS, with an increase of PMI from 2-8 h, did not change significantly. A similar effect was observed with vascular responses to KCl 40 mmol/1 in Ca 2+ -EGTA-PSS. To confirm whether the inhibitory effect of 2, 4, 6 and 8 h PMI was mediated through the formation of NO, nitro-L-arginine (L-NNA), a potent NO synthase inhibitor, was used. Exposure to L-NNA (10 -5 M) blocked the inhibition induced by an increase of PMI. The blocked effects of L-NNA were reversed by L-arginine (10 -4 M). In conclusion, these patterns of change in artery responses provide insight into the post-mortem change in the receptor-mediated signaling components in epithelial and smooth muscle cells, and support the further study of post-mortem vascular responses triggered by G protein-coupled receptors (metabotropic) and channel-linked receptors (ionotropic) as potential markers for estimating short and long-term PMIs, respectively.

[Relaxant effects of protopine on smooth muscles].

PubMed

Huang, Y H; Zhang, Z Z; Jiang, J X

1991-01-01

The relaxant effects of protopine (Pro) on smooth muscles were studied by recording isotonic contraction and radioimmunoassay. Pro relaxed the contraction of rabbit thoracic aorta, mesenteric artery, portal vein and guinea pig ileum and taenia colon induced by high K+ (70 mmol.L-1). Pro also inhibited the contraction of rabbit thoracic aorta, mesenteric artery, portal vein induced by NE (0.3 mumol.L-1) and guinea pig taenia colon induced by BaCl2 (1 mmol.L-1). Pro inhibited the intracellular Ca2+ release, but did not inhibit Ca2+ influx induced by NE. These results suggested that the smooth muscle relaxant mechanism of action of Pro may be the inhibition of intracellular Ca2+ release.

Prohormone convertase 1 (PC1) when expressed with pro cholecystokinin (pro CCK) in L cells performs three endoproteolytic cleavages which are observed in rat brain and in CCK-expressing endocrine cells in culture, including the production of glycine and arginine extended CCK8.

PubMed

Wang, W; Birch, N P; Beinfeld, M C

1998-07-30

Pro CCK was expressed in an L cell line engineered to express PC1 and the products secreted into the media were characterized by a combination of RIA, gel filtration and HPLC. PC1 released from L cells, cleaved pro CCK generating the amino terminal pro peptide. PC1 also generated a peptide which after carboxypeptidase B treatment, was detected with an antiserum specific for CCK Gly. Neither of these peptides was found in media from L cells expressing pro CCK alone. This CCK Gly immunoreactive peptide was similar in size to CCK 8, and after treatment with arylsulfatase and carboxypeptidase B, it co-eluted on HPLC with unsulfated CCK 8 Gly. These results agree with previous studies which support a role for PC1 in generation of CCK 8. This is the first demonstration that PC1 acting alone is able to cleave pro CCK liberating the amino terminal pro peptide and a glycine and arginine extended CCK 8 which is the immediate precursor of CCK 8 amide.

Synthesis and Sensory Characteristics of Kokumi γ-[Glu]n-Phe in the Presence of Glutamine and Phenylalanine: Glutaminase from Bacillus amyloliquefaciens or Aspergillus oryzae as the Catalyst.

PubMed

Yang, Juan; Sun-Waterhouse, Dongxiao; Cui, Chun; Dong, Keming; Wang, Wei

2017-10-04

The transpeptidase activity of glutaminase from Bacillus amyloliquefaciens (GBA) and Aspergillus oryzae (GAO) to yield γ-[Glu] n -Phe peptides were verified for the first time. In the presence of Gln and Phe, γ-Glu-Phe and γ-Glu-γ-Glu-Phe were synthesized by GAO, and γ-Glu-Phe, γ-Glu-γ-Glu-Phe, γ-Glu-γ-Glu-γ-Glu-Phe, γ-Glu-γ-Glu-γ-Glu-γ-Glu-Phe, and γ-Glu-γ-Glu-γ-Glu-γ-Glu-γ-Glu-Phe were synthesized by GBA. The K m values for the transpeptidation catalyzed by GBA and GAO were 47.88 and 153.92 mM (Phe as the acceptor), 84.89 and 236.47 mM (γ-Glu-Phe as the acceptor), indicating that GBA had a greater affinity than GAO for Phe and γ-Glu-Phe in the transpeptidation reaction. The K m values for the transpeptidation catalyzed by GBA against acceptors, Phe and γ-[Glu] (1≤n<5) -Phe (47.88-206.47 mM), increased with an elevated number of γ-glutamyl residue within the acceptor. The optimal conditions for γ-[Glu] n -Phe synthesis were pH 10 and 37 °C for 3 h, 300 mM Gln, 100 mM Phe, 0.05 U/mL GBA. All the γ-[Glu] (1≤n≤5) -Phe exhibited astringency in water and imparted a kokumi taste to commercial soy sauce and model chicken broth. The astringent threshold values (2.5-3.92 mM) were approximately 3-fold of the kokumi threshold concentrations (0.78-1.53 mM). γ-[Glu] n -Phe or the post-enzymatic reaction mixture enhanced the umami intensity of commercial soy sauce and model chicken broth.

Clinical implications of B-type natriuretic peptide and N-terminal pro--B-type natriuretic peptide in the care of the vascular surgery patient.

PubMed

Wayne Causey, Marlin; Singh, Niten

2014-12-01

B-type natriuretic peptide (also known as brain natriuretic peptide or BNP) is a physiologic marker that is often used to assess a patient's global cardiovascular health. BNP is secreted from the ventricular cardiac myocytes in response to stretch that occurs due to increased intravascular volume. PreproBNP is cleaved into BNP and N-terminal proBNP (NT proBNP) to cause diuresis, natriuresis, and vasodilation, and can be measured with a blood laboratory assay test or point-of-care testing. BNP/NT proBNP has been most extensively studied in the diagnosis and management of heart failure, but within the past 5 years, interest has carried over to vascular surgery patients. Studies have demonstrated that elevated levels of BNP/NT-proBNP (typically >100 pg/mL/>300 pg/mL) are associated with major adverse cardiac events at 30 and 180 days. Additional analysis of BNP/NT-proBNP has demonstrated that patients can be classified as very low risk (<19 pg/mL), low risk (<100 pg/mL), intermediate risk (100 to 400 pg/mL), or high risk (>400 pg/mL). BNP/NT-proBNP in the low- and very-low-risk groups suggests patients are unlikely to have a major adverse cardiac event. An elevated BNP/NT-proBNP, excluding those with reasons for abnormal values, suggests the need for additional risk stratification and medical risk factor optimization. A preoperative measure of BNP or NT-proBNP affords an easy and rapid opportunity to individually and objectively quantify perioperative cardiovascular risk. Recent studies have also identified other biomarkers, none superior to BNP or NT-proBNP, but that, when used concomitantly, aid in further stratifying perioperative risk and will likely be the focus of future investigations. Published by Elsevier Inc.

Evaluation of extraction methods for the identification of proteins from date palm (Phoenix dactylifera L.) seed and flesh.

PubMed

Lee, Hooi Xian; Ahmad, Fisal; Saad, Bahruddin; Ismail, Mohd Nazri

2017-11-26

Date fruits are well known to be very nutritious. Nevertheless, the protein contents of the fruit, particularly the seed and flesh, are still understudied, largely due to their difficult physical characteristics. This study was conducted to compare three different protein extraction methods which were the trichloroacetic acid (TCA)-acetone (TCA-A), phenol (Phe), and TCA-acetone-phenol (TCA-A-Phe), and to perform proteomic analysis on date palm seed and flesh. Phe extraction method showed the highest protein yields for both seed (8.26 mg/g) and flesh (1.57 mg/g). Through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Phe, and TCA-A-Phe extraction methods were shown to be efficient in removing interfering compounds and gave well-resolved bands over a wide range of molecular weights. Following liquid chromatography-tandem mass spectrometry analysis, about 50-64% of extracted proteins were identified with known functions including those involved in glycolysis, Krebs cycle, defense, and storage. Phe protein extraction method was proven to be the optimal method for date flesh and seed.

Azidoethoxyphenylalanine as a Vibrational Reporter and Click Chemistry Partner in Proteins

PubMed Central

Tookmanian, Elise M.; Phillips-Piro, Christine M.; Fenlon, Edward E.; Brewer, Scott H.

2016-01-01

An unnatural amino acid, 4-(2-azidoethoxy)-l-phenylalanine (AePhe, 1), was designed and synthesized in three steps from known compounds in 54% overall yield. The sensitivity of the IR absorption of the azide of AePhe was established by comparison of the frequency of the asymmetric stretch vibration in water and dimethyl sulfoxide. AePhe was successfully incorporated into superfolder green fluorescent protein (sfGFP) at the 133 and 149 sites using the amber codon suppression method. The IR spectra of these sfGFP constructs indicated that the azide group at the 149 site was not fully solvated despite the location in sfGFP and the three-atom linker between the azido group and the aromatic ring of AePhe. An X-ray crystal structure of sfGFP-149-AePhe was solved at 1.45 Å resolution and provides an explanation for the IR data as the flexible linker adopts a conformation which partially buries the azide on the protein surface. Both sfGFP-AePhe constructs efficiently undergo a bioorthogonal strain-promoted click cycloaddition with a dibenzocyclooctyne derivative. PMID:26608683

Construction of hybrid peptide synthetases by module and domain fusions

PubMed Central

Mootz, Henning D.; Schwarzer, Dirk; Marahiel, Mohamed A.

2000-01-01

Nonribosomal peptide synthetases are modular enzymes that assemble peptides of diverse structures and important biological activities. Their modular organization provides a great potential for the rational design of novel compounds by recombination of the biosynthetic genes. Here we describe the extension of a dimodular system to trimodular ones based on whole-module fusion. The recombinant hybrid enzymes were purified to monitor product assembly in vitro. We started from the first two modules of tyrocidine synthetase, which catalyze the formation of the dipeptide dPhe-Pro, to construct such hybrid systems. Fusion of the second, proline-specific module with the ninth and tenth modules of the tyrocidine synthetases, specific for ornithine and leucine, respectively, resulted in dimodular hybrid enzymes exhibiting the combined substrate specificities. The thioesterase domain was fused to the terminal module. Upon incubation of these dimodular enzymes with the first tyrocidine module, TycA, incorporating dPhe, the predicted tripeptides dPhe-Pro-Orn and dPhe-Pro-Leu were obtained at rates of 0.15 min-1 and 2.1 min-1. The internal thioesterase domain was necessary and sufficient to release the products from the hybrid enzymes and thereby facilitate a catalytic turnover. Our approach of whole-module fusion is based on an improved definition of the fusion sites and overcomes the recently discovered editing function of the intrinsic condensation domains. The stepwise construction of hybrid peptide synthetases from catalytic subunits reinforces the inherent potential for the synthesis of novel, designed peptides. PMID:10811885

Construction of hybrid peptide synthetases by module and domain fusions.

PubMed

Mootz, H D; Schwarzer, D; Marahiel, M A

2000-05-23

Nonribosomal peptide synthetases are modular enzymes that assemble peptides of diverse structures and important biological activities. Their modular organization provides a great potential for the rational design of novel compounds by recombination of the biosynthetic genes. Here we describe the extension of a dimodular system to trimodular ones based on whole-module fusion. The recombinant hybrid enzymes were purified to monitor product assembly in vitro. We started from the first two modules of tyrocidine synthetase, which catalyze the formation of the dipeptide dPhe-Pro, to construct such hybrid systems. Fusion of the second, proline-specific module with the ninth and tenth modules of the tyrocidine synthetases, specific for ornithine and leucine, respectively, resulted in dimodular hybrid enzymes exhibiting the combined substrate specificities. The thioesterase domain was fused to the terminal module. Upon incubation of these dimodular enzymes with the first tyrocidine module, TycA, incorporating dPhe, the predicted tripeptides dPhe-Pro-Orn and dPhe-Pro-Leu were obtained at rates of 0.15 min(-1) and 2.1 min(-1). The internal thioesterase domain was necessary and sufficient to release the products from the hybrid enzymes and thereby facilitate a catalytic turnover. Our approach of whole-module fusion is based on an improved definition of the fusion sites and overcomes the recently discovered editing function of the intrinsic condensation domains. The stepwise construction of hybrid peptide synthetases from catalytic subunits reinforces the inherent potential for the synthesis of novel, designed peptides.

Prognostic Value of N-Terminal Pro-B-Type Natriuretic Peptide Levels in Heart Failure Patients With and Without Atrial Fibrillation.

PubMed

Kristensen, Søren Lund; Jhund, Pardeep S; Mogensen, Ulrik M; Rørth, Rasmus; Abraham, William T; Desai, Akshay; Dickstein, Kenneth; Rouleau, Jean L; Zile, Michael R; Swedberg, Karl; Packer, Milton; Solomon, Scott D; Køber, Lars; McMurray, John J V

2017-10-01

Patients with heart failure (HF) and atrial fibrillation (AF) have higher circulating levels of NT-proBNP (N-terminal pro-B-type natriuretic peptide) than HF patients without AF. There is uncertainty about the prognostic importance of a given concentration of NT-proBNP in HF patients with and without AF. We investigated this question in a large cohort of patients with HF and reduced ejection fraction. We studied 14 737 patients with HF and reduced ejection fraction and a measurement of NT-proBNP at time of screening, enrolled in either the PARADIGM-HF trial (Prospective Comparison of ARNI With ACEI to Determine Impact on Global Mortality and Morbidity in Heart Failure) or the ATMOSPHERE trial (Aliskiren Trial to Minimize Outcomes in Patients With Heart Failure), of whom 3575 (24%) had AF on their baseline ECG. Median (Q1, Q3) levels of NT-proBNP were 1817 pg/mL (1095-3266 pg/mL) in those with AF and 1271 pg/mL (703-2569 pg/mL) in those without ( P <0.0001). Patients with AF were older (67 versus 62 years), had worse New York Heart Association class (III/IV; 36% versus 24%), and experienced fewer previous HF hospitalizations (52% versus 61%) or myocardial infarction (30% versus 46%); all P <0.001. We categorized patients with and without AF into 5 NT-proBNP bands: <400, 400 to 999 (reference), 1000 to 1999, 2000 to 2999, and ≥3000 pg/mL. For the primary composite outcome of cardiovascular death or HF hospitalization, event rates differed for patients with and without AF in the lowest band (<400 pg/mL; 8.2 versus 5.0 per 100 patient-years), but not for the higher bands (400-999 pg/mL, 7.4 versus 7.7 per 100 patient-years; 1000-1999 pg/mL, 9.8 versus 11.4 per 100 patient-year; 2000-2999 pg/mL, 13.5 versus 13.4 per 100 patient-years; ≥3000 pg/mL, 22.7 versus 23.0 per 100 patient-years). These findings were consistent whether NT-proBNP was examined as a categorical or continuous variable and before and after adjustment for other prognostic variables. We found similar results for the components of the composite outcome and all-cause mortality. HF and reduced ejection fraction patients with AF had higher NT-proBNP than those without AF. However, above a concentration of 400 pg/mL (representing most patients in each group), NT-proBNP had similar predictive value for adverse cardiovascular outcomes, irrespective of AF status. URL: https://www.clinicaltrials.gov. Unique identifier NCT00853658 (ATMOSPHERE) and NCT01035255 (PARADIGM-HF). © 2017 American Heart Association, Inc.

The asparagine residue in the FRNK box of potyviral helper-component protease is critical for its small RNA binding and subcellular localization.

PubMed

Sahana, Nandita; Kaur, Harpreet; Jain, R K; Palukaitis, Peter; Canto, Tomas; Praveen, Shelly

2014-05-01

The multifunctional potyviral helper-component protease (HcPro) contains variable regions with some functionally conserved domains, such as the FRNK box. Natural variants occur at the FRNK box, a conserved central domain, known for its role in RNA binding and RNAi suppression activities, although no dominant natural variants for the N(182) residue are known to occur. Here, a mutant at HcPro(N182L) was developed to investigate its role in natural populations. Using in vitro studies, we found an increase in the small RNA (sRNA) binding potential of HcPro(N182L) without affecting its protein-protein interaction properties, suggesting that the presence of N(182) is critical to maintain threshold levels of sRNAs, but does not interfere in the self-interaction of HcPro. Furthermore, we found that expression of HcPro(N182L) in Nicotiana benthamiana affected plant growth. Transient expression of HcPro(N182L) induced reporter gene expression in 16c GFP transgenic plants more than HcPro did, suggesting that replacement of asparagine in the FRNK box favours RNA silencing suppression. HcPro was found to be distributed in the nucleus and cytoplasm, whereas HcPro(N182L) was observed only in cytoplasmic inclusion bodies in N. benthamiana leaves, when fused to a GFP tag and expressed by agro-infiltration, suggesting mutation favours oligomerization of HcPro. These findings suggest that amino acid N(182) of the conserved FRNK box may regulate RNA silencing mechanisms, and is required for maintenance of the subcellular localization of the protein for its multi-functionality. Hence, the N(182) residue of the FRNK box seems to be indispensable for potyvirus infection during evolution.

Purification, crystallization and preliminary X-ray diffraction study of human ribosomal protein L10 core domain

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

Nishimura, Mitsuhiro; Protein Research Group, RIKEN Yokohama Institute, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045; Kaminishi, Tatsuya

2007-11-01

A truncated variant of human ribosomal protien L10 was prepared and crystallized. Diffraction data were collected to 2.5 Å resolution. Eukaryotic ribosomal protein L10 is an essential component of the large ribosomal subunit, which organizes the architecture of the aminoacyl-tRNA binding site. The human L10 protein is also called the QM protein and consists of 214 amino-acid residues. For crystallization, the L10 core domain (L10CD, Phe34–Glu182) was recombinantly expressed in Escherichia coli and purified to homogeneity. A hexagonal crystal of L10CD was obtained by the sitting-drop vapour-diffusion method. The L10CD crystal diffracted to 2.5 Å resolution and belongs to spacemore » group P3{sub 1}21 or P3{sub 2}21.« less

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

PubMed Central

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

2005-01-01

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

Association of N-terminal pro-B-type natriuretic peptide with contrast-induced nephropathy and long-term outcomes in patients with chronic kidney disease and relative preserved left ventricular function.

PubMed

Liu, Yuan-hui; Liu, Yong; Zhou, Ying-ling; Yu, Dan-qing; He, Peng-cheng; Xie, Nian-jin; Li, Hua-long; Wei-Guo; Chen, Ji-yan; Tan, Ning

2015-04-01

The aim of the present article was to evaluate the association of N-terminal pro-B-type natriuretic peptide (NT-pro-BNP) with contrast-induced nephropathy (CIN) and long-term outcomes in patients with chronic kidney disease (CKD) and relative preserved left ventricular function (LVF) undergoing percutaneous coronary intervention (PCI). We prospectively enrolled 1203 consecutive patients with CKD and preserved LVF undergoing elective PCI. The primary end point was the development of CIN, defined as an absolute increase in serum creatinine (SCr) ≥0.5 mg/dL, from baseline within 48 to 72 hours after contrast medium exposure. CIN incidence varied from 2.2% to 5.2%. Univariate logistic analysis showed that lg-NT-pro-BNP was significantly associated with CIN (odds ratio [OR] = 3.93, 95% confidence interval [CI], 2.22-6.97, P < 0.001). Furthermore, lg-NT-pro-BNP remained a significant predictor of CIN (OR = 3.30, 95% CI, 1.57-6.93, P = 0.002), even after adjusting for potential confounding risk factors. These results were confirmed by using other CIN criteria, which were defined as elevations of the SCr by 25% or 0.5 and 0.3 mg/dL from the baseline. The best cutoff value of lg-NT-pro-BNP for detecting CIN was 2.73 pg/mL (537 pg/mL) with 73.1% sensitivity and 70.0% specificity according to the receiver operating characteristic (ROC) analysis (C statistic = 0.754, 95% CI, 0.67-0.84, P < 0.001). In addition, NT-pro-BNP ≥537 pg/mL (2.73 pg/mL, lg-NT-pro-BNP) was associated with an increased risk of all-cause mortality and composite end points during 2.5 years of follow-up. NT-pro-BNP ≥537 pg/mL is independently associated with an increased risk of CIN with different definitions and poor clinical outcomes in patients with CKD and relative preserved LVF undergoing PCI.

Association of N-Terminal Pro-B-Type Natriuretic Peptide With Contrast-Induced Nephropathy and Long-Term Outcomes in Patients With Chronic Kidney Disease and Relative Preserved Left Ventricular Function

PubMed Central

Liu, Yuan-hui; Liu, Yong; Zhou, Ying-ling; Yu, Dan-qing; He, Peng-cheng; Xie, Nian-Jin; Li, Hua-long; Wei-Guo; Chen, Ji-yan; Tan, Ning

2015-01-01

Abstract The aim of the present article was to evaluate the association of N-terminal pro-B-type natriuretic peptide (NT-pro-BNP) with contrast-induced nephropathy (CIN) and long-term outcomes in patients with chronic kidney disease (CKD) and relative preserved left ventricular function (LVF) undergoing percutaneous coronary intervention (PCI). We prospectively enrolled 1203 consecutive patients with CKD and preserved LVF undergoing elective PCI. The primary end point was the development of CIN, defined as an absolute increase in serum creatinine (SCr) ≥0.5 mg/dL, from baseline within 48 to 72 hours after contrast medium exposure. CIN incidence varied from 2.2% to 5.2%. Univariate logistic analysis showed that lg-NT-pro-BNP was significantly associated with CIN (odds ratio [OR] = 3.93, 95% confidence interval [CI], 2.22–6.97, P < 0.001). Furthermore, lg-NT-pro-BNP remained a significant predictor of CIN (OR = 3.30, 95% CI, 1.57–6.93, P = 0.002), even after adjusting for potential confounding risk factors. These results were confirmed by using other CIN criteria, which were defined as elevations of the SCr by 25% or 0.5 and 0.3 mg/dL from the baseline. The best cutoff value of lg-NT-pro-BNP for detecting CIN was 2.73 pg/mL (537 pg/mL) with 73.1% sensitivity and 70.0% specificity according to the receiver operating characteristic (ROC) analysis (C statistic = 0.754, 95% CI, 0.67–0.84, P < 0.001). In addition, NT-pro-BNP ≥537 pg/mL (2.73 pg/mL, lg-NT-pro-BNP) was associated with an increased risk of all-cause mortality and composite end points during 2.5 years of follow-up. NT-pro-BNP ≥537 pg/mL is independently associated with an increased risk of CIN with different definitions and poor clinical outcomes in patients with CKD and relative preserved LVF undergoing PCI. PMID:25837748

Synthesis, structure, and glutathione peroxidase-like activity of amino acid containing ebselen analogues and diaryl diselenides.

PubMed

Selvakumar, Karuthapandi; Shah, Poonam; Singh, Harkesh B; Butcher, Ray J

2011-11-04

The synthesis of some ebselen analogues and diaryl diselenides, which have amino acid functions as an intramolecularly coordinating group (Se···O) has been achieved by the DCC coupling procedure. The reaction of 2,2'-diselanediylbis(5-tert-butylisophthalic acid) or the activated ester tetrakis(2,5-dioxopyrrolidin-1-yl) 2,2'-diselanediylbis(5-tert-butylisophthalate) with different C-protected amino acids (Gly, L-Phe, L-Ala, and L-Trp) afforded the corresponding ebselen analogues. The used precursor diselenides have been found to undergo facile intramolecular cyclization during the amide bond formation reaction. In contrast, the DCC coupling of 2,2'-diselanediyldibenzoic acid with C-protected amino acids (Gly, L/D-Ala and L-Phe) affords the corresponding amide derivatives and not the ebselen analogues. Some of the representative compounds have been structurally characterized by single-crystal X-ray crystallography. The glutathione peroxidase (GPx)-like activities of the ebselen analogues and the diaryl diselenides have been evaluated by using the coupled reductase assay method. Intramolecularly stabilized ebselen analogues show slightly higher maximal velocity (V(max)) than ebselen. However, they do not show any GPx-like activity at low GSH concentrations at which ebselen and related diselenides are active. This could be attributed to the peroxide-mediated intramolecular cyclization of the corresponding selenenyl sulfide and diaryl diselenide intermediates generated during the catalytic cycle. Interestingly, the diaryl diselenides with alanine (L,L or D,D) amide moieties showed excellent catalytic efficiency (k(cat)/K(M)) with low K(M) values in comparison to the other compounds. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Utility of N-terminal pro-brain natriuretic peptide for assessing hemodynamic significance of patent ductus arteriosus in dogs undergoing ductal repair.

PubMed

Hariu, Crystal D; Saunders, Ashley B; Gordon, Sonya G; Norby, Bo; Miller, Matthew W

2013-09-01

Determine if plasma N-terminal pro-brain natriuretic peptide (NT-proBNP) correlates with markers of hemodynamically significant patent ductus arteriosus (PDA) in dogs. Ten dogs with PDA and 30 healthy dogs of similar ages. Prospective case series with control population. Dogs with PDA were initially evaluated with thoracic radiographs, transthoracic echocardiography, pulmonary capillary wedge pressure (PCWP) and NT-proBNP. Following ductal occlusion, NT-proBNP and echocardiography were repeated within 24 h and at day 90. PCWP was repeated at day 90. Correlation between NT-proBNP and hemodynamic measurements was assessed, and accuracy of NT-proBNP for identifying PDA severity was estimated. NT-proBNP was significantly higher (median; absolute range) in dogs with PDA (895; 490-7118 pmol/L) than controls (663; 50-1318 pmol/L) (p = 0.025). NT-proBNP decreased significantly 90 days post-ductal closure (597; 154-1858 pmol/L) (p = 0.013). Left atrial and ventricular size decreased significantly within 24 h and at day 90 as did PCWP (day 90 only). NT-proBNP correlated with vertebral heart size (VHS) and indexed left ventricular systolic diameter (iLVIDs); concentrations ≥ 1224 pmol/L distinguished dogs with elevated VHS and iLVIDs. NT-proBNP is elevated in dogs with PDA, decreases following PDA closure and correlates with select radiographic and echocardiographic markers of cardiac remodeling. Copyright © 2013 Elsevier B.V. All rights reserved.

Peptide design using alpha,beta-dehydro amino acids: from beta-turns to helical hairpins.

PubMed

Mathur, Puniti; Ramakumar, S; Chauhan, V S

2004-01-01

Incorporation of alpha,beta-dehydrophenylalanine (DeltaPhe) residue in peptides induces folded conformations: beta-turns in short peptides and 3(10)-helices in larger ones. A few exceptions-namely, alpha-helix or flat beta-bend ribbon structures-have also been reported in a few cases. The most favorable conformation of DeltaPhe residues are (phi,psi) approximately (-60 degrees, -30 degrees ), (-60 degrees, 150 degrees ), (80 degrees, 0 degrees ) or their enantiomers. DeltaPhe is an achiral and planar residue. These features have been exploited in designing DeltaPhe zippers and helix-turn-helix motifs. DeltaPhe can be incorporated in both right and left-handed helices. In fact, consecutive occurrence of three or more DeltaPhe amino acids induce left-handed screw sense in peptides containing L-amino acids. Weak interactions involving the DeltaPhe residue play an important role in molecular association. The C--H.O==C hydrogen bond between the DeltaPhe side-chain and backbone carboxyl moiety, pi-pi stacking interactions between DeltaPhe side chains belonging to enantiomeric helices have shown to stabilize folding. The unusual capability of a DeltaPhe ring to form the hub of multicentered interactions namely, a donor in aromatic C--H.pi and C--H.O==C and an acceptor in a CH(3).pi interaction suggests its exploitation in introducing long-range interactions in the folding of supersecondary structures. Copyright 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2004

Production of Cinnamic and p-Hydroxycinnamic Acids in Engineered Microbes.

PubMed

Vargas-Tah, Alejandra; Gosset, Guillermo

2015-01-01

The aromatic compounds cinnamic and p-hydroxycinnamic acids (pHCAs) are phenylpropanoids having applications as precursors for the synthesis of thermoplastics, flavoring, cosmetic, and health products. These two aromatic acids can be obtained by chemical synthesis or extraction from plant tissues. However, both manufacturing processes have shortcomings, such as the generation of toxic subproducts or a low concentration in plant material. Alternative production methods are being developed to enable the biotechnological production of cinnamic and (pHCAs) by genetically engineering various microbial hosts, including Escherichia coli, Saccharomyces cerevisiae, Pseudomonas putida, and Streptomyces lividans. The natural capacity to synthesize these aromatic acids is not existent in these microbial species. Therefore, genetic modification have been performed that include the heterologous expression of genes encoding phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities, which catalyze the conversion of l-phenylalanine (l-Phe) and l-tyrosine (l-Tyr) to cinnamic acid and (pHCA), respectively. Additional host modifications include the metabolic engineering to increase carbon flow from central metabolism to the l-Phe or l-Tyr biosynthetic pathways. These strategies include the expression of feedback insensitive mutant versions of enzymes from the aromatic pathways, as well as genetic modifications to central carbon metabolism to increase biosynthetic availability of precursors phosphoenolpyruvate and erythrose-4-phosphate. These efforts have been complemented with strain optimization for the utilization of raw material, including various simple carbon sources, as well as sugar polymers and sugar mixtures derived from plant biomass. A systems biology approach to production strains characterization has been limited so far and should yield important data for future strain improvement.

Production of Cinnamic and p-Hydroxycinnamic Acids in Engineered Microbes

PubMed Central

Vargas-Tah, Alejandra; Gosset, Guillermo

2015-01-01

The aromatic compounds cinnamic and p-hydroxycinnamic acids (pHCAs) are phenylpropanoids having applications as precursors for the synthesis of thermoplastics, flavoring, cosmetic, and health products. These two aromatic acids can be obtained by chemical synthesis or extraction from plant tissues. However, both manufacturing processes have shortcomings, such as the generation of toxic subproducts or a low concentration in plant material. Alternative production methods are being developed to enable the biotechnological production of cinnamic and (pHCAs) by genetically engineering various microbial hosts, including Escherichia coli, Saccharomyces cerevisiae, Pseudomonas putida, and Streptomyces lividans. The natural capacity to synthesize these aromatic acids is not existent in these microbial species. Therefore, genetic modification have been performed that include the heterologous expression of genes encoding phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities, which catalyze the conversion of l-phenylalanine (l-Phe) and l-tyrosine (l-Tyr) to cinnamic acid and (pHCA), respectively. Additional host modifications include the metabolic engineering to increase carbon flow from central metabolism to the l-Phe or l-Tyr biosynthetic pathways. These strategies include the expression of feedback insensitive mutant versions of enzymes from the aromatic pathways, as well as genetic modifications to central carbon metabolism to increase biosynthetic availability of precursors phosphoenolpyruvate and erythrose-4-phosphate. These efforts have been complemented with strain optimization for the utilization of raw material, including various simple carbon sources, as well as sugar polymers and sugar mixtures derived from plant biomass. A systems biology approach to production strains characterization has been limited so far and should yield important data for future strain improvement. PMID:26347861

Quality-of-Life Impairments Persist Six Months After Treatment of Graves' Hyperthyroidism and Toxic Nodular Goiter: A Prospective Cohort Study.

PubMed

Cramon, Per; Winther, Kristian Hillert; Watt, Torquil; Bonnema, Steen Joop; Bjorner, Jakob Bue; Ekholm, Ola; Groenvold, Mogens; Hegedüs, Laszlo; Feldt-Rasmussen, Ulla; Rasmussen, Åse Krogh

2016-08-01

The treatment of hyperthyroidism is aimed at improving health-related quality of life (HRQoL) and reducing morbidity and mortality. However, few studies have used validated questionnaires to assess HRQoL prospectively in such patients. The purpose of this study was to assess the impact of hyperthyroidism and its treatment on HRQoL using validated disease-specific and generic questionnaires. This prospective cohort study enrolled 88 patients with Graves' hyperthyroidism and 68 with toxic nodular goiter from endocrine outpatient clinics at two Danish university hospitals. The patients were treated with antithyroid drugs, radioactive iodine, or surgery. Disease-specific and generic HRQoL were assessed using the thyroid-related patient-reported outcome (ThyPRO) and the Medical Outcomes Study 36-item Short Form (SF-36), respectively, evaluated at baseline and six-month follow-up. The scores were compared with those from two general population samples who completed ThyPRO (n = 739) and SF-36 (n = 6638). Baseline scores for patients with Graves' hyperthyroidism and toxic nodular goiter were significantly worse than those for the general population scores on all comparable ThyPRO scales and all SF-36 scales and component summaries. ThyPRO scores improved significantly with treatment on all scales in Graves' hyperthyroidism and four scales in toxic nodular goiter, while SF-36 scores improved on five scales and both component summaries in Graves' hyperthyroidism and only one scale in toxic nodular goiter. In Graves' hyperthyroidism, large treatment effects were observed on three ThyPRO scales (Hyperthyroid Symptoms, Tiredness, Overall HRQoL) and moderate effects on three scales (Anxiety, Emotional Susceptibility, Impaired Daily Life), while moderate effects were seen in two ThyPRO scales in toxic nodular goiter (Anxiety, Overall HRQoL). However, significant disease-specific and generic HRQoL deficits persisted on multiple domains across both patient groups. Graves' hyperthyroidism and toxic nodular goiter cause severe disease-specific and generic HRQoL impairments, and HRQoL deficits persist in both patient groups six months after treatment. These data have the potential to improve communication between physicians and patients by offering realistic estimates of expected HRQoL impairments and treatment effects. Future studies should identify risk factors for persistent HRQoL deficits, compare HRQoL effects of the various therapies, and thereby aid in determining the optimal treatment strategies.

Nutritional status in patients with phenylketonuria using glycomacropeptide as their major protein source.

PubMed

Pinto, A; Almeida, M F; Ramos, P C; Rocha, S; Guimas, A; Ribeiro, R; Martins, E; Bandeira, A; MacDonald, A; Rocha, J C

2017-10-01

Low phenylalanine (PHE), glycomacropeptide-based protein substitute (GMP) is an alternative to traditional L-amino acid supplements (AA) used in the dietary management of phenylketonuria (PKU). In a retrospective, longitudinal study, we report the nutritional status of PKU patients taking AA and GMP. Eleven PKU patients aged 27±10 years (1 HPA, 4 mild and 6 classical PKU) on dietary treatment were evaluated (anthropometry, body composition, blood pressure measurements, biochemical markers including vitamin, mineral, lipids, carbohydrates and protein status/metabolism, and nutritional intake assessment) at two different annual reviews. The mean time taking AA was 13±5 months and GMP 13±7 months. Blood phenylalanine (PHE) and tyrosine (TYR) were analysed before and after GMP introduction. Both GMP and AA protein substitutes provided similar protein equivalent intake (0.85 vs 0.75 g/kg/day, P=0.182). In the GMP group, it contributed 57% (27-100%) of the protein substitute intake (with AA delivering the rest of protein substitute intake), providing an additional 34±12 mg/day PHE. Nutritional intake, anthropometry and body composition measurements were similar in both the groups. Median blood PHE did not change (P=0.594), although values within target range improved (36 vs 46%), but this was not statistically significant. Mean blood TYR increased (52.0±19.2 vs 63.2±25.6 μmol/l, P=0.033), and all biochemical markers remained stable, except for a lower A1C haemoglobin (P=0.011). Partial GMP contribution to total protein substitute intake did not affect nutritional status in patients with PKU. Blood PHE control was not adversely affected. The increased blood TYR after GMP introduction necessitates further study.

Acute resistance exercise reduces blood pressure and vascular reactivity, and increases endothelium-dependent relaxation in spontaneously hypertensive rats.

PubMed

Faria, Thaís de Oliveira; Targueta, Gabriel Pelegrineti; Angeli, Jhuli Keli; Almeida, Edna Aparecida Silveira; Stefanon, Ivanita; Vassallo, Dalton Valentim; Lizardo, Juliana Hott de Fúcio

2010-09-01

The aim of the present study was to assess the effects of acute dynamic resistance exercise on resting blood pressure (BP) and on endothelial function of vascular bed of spontaneously hypertensive rats. Hemodynamic measurements were performed before and after acute dynamic resistance exercise in conscious animals. After exercise, the tail artery was cannulated for mean perfusion pressure with constant flow measurement and for performing concentration-response curves to acetylcholine (ACh) and sodium nitroprusside (SNP) and dose-response curves to phenylephrine (PHE). PHE protocol was also repeated with damaged endothelium and after L-NAME and indomethacin perfusion on the tail. The maximal response (E(max)) and sensitivity (pD(2)) were evaluated to these drugs. Exercise reduced resting systolic and diastolic BP (Delta -79 +/- 1.8; -23 +/- 2.3 mmHg, respectively; P < 0.05). ACh-induced relaxation increased in the exercise group (pD(2) = 9.8 +/- 0.06, P < 0.05) when compared with control rats (pD(2) = 8.7 +/- 0.1). The E(max) to PHE with intact endothelium decreased following exercise condition (439 +/- 18 mmHg, P < 0.05) when compared with control rats (276 +/- 22 mmHg). This response was abolished after L-NAME and indomethacin administration. After damage of the endothelium, PHE responses were not significantly different between the groups; however, E(max) and pD(2) increased when compared with responses obtained with intact endothelium. The results demonstrated that acute dynamic resistance exercise decreased resting BP and reactivity to PHE and increased endothelium-dependent relaxation. Nitric oxide and vasodilators prostanoids appear to be involved in post-exercise endothelial and pressor responses.

Validation of the LupusPRO version 1.8: an update to a disease-specific patient-reported outcome tool for systemic lupus erythematosus.

PubMed

Azizoddin, D R; Weinberg, S; Gandhi, N; Arora, S; Block, J A; Sequeira, W; Jolly, M

2018-04-01

Objectives LupusPRO has shown good measurement properties as a disease-specific patient-reported outcome tool in systemic lupus erythematosus (SLE). For the purpose of clinical trials, the version 1.7 (v1.7) domain of Pain-Vitality was separated into distinct Pain, Vitality and Sleep domains in v1.8, and the psychometric properties examined. Methods A total of 131 consecutive SLE patients were self-administered surveys assessing fatigue (FACIT, SF-36), pain (Pain Inventory, SF-36), insomnia (Insomnia Severity Index), emotional health (PHQ-9, SF-36) and quality of life (SF-36, LupusPRO) at routine care visits. Internal consistency reliability (ICR) for each domain was obtained using Cronbach's alpha. The convergent construct validity of LupusPRO domains with corresponding SF-36 domains or tools were tested using Spearman correlation. Varimax rotations were conducted to assess factor structures of the LupusPRO v1.8. Results Mean (SD) age was 40.04 (14.10) years. Scores from the LupusPRO-Sleep domain strongly correlated with insomnia scores, while LupusPRO-Vitality correlated strongly with fatigue (FACIT) and SF-36 vitality. The LupusPRO-Pain domain correlated strongly with pain (SF36 Bodily-Pain, Pain Inventory) scores. Similarly, the LupusPRO domains of Physical and Emotional Health had significant correlations with corresponding SF-36 domains. The ICR for HRQoL and non-HRQoL were 0.96 and 0.81. LupusPRO (domains HRQoL and QoL) scores correlated with disease activity. Principal component analysis included seven factor loadings presenting for the HRQOL subscales (combined Sleep, Vitality, and Pain), and three factors for the NHRQoL (Combined Coping and Social Support). Conclusions LupusPRO v1.8 (including its Sleep, Vitality, and Pain domains) has acceptable reliability and validity. Use of LupusPRO as an outcome measure in clinical trials would facilitate responsiveness assessment.

Conformational Specific Infrared and Ultraviolet Spectroscopy of Cold YA(D-Pro)AA\\cdotH+ Ions: a Sterochemical "twist" on the Proline Effect

NASA Astrophysics Data System (ADS)

Harrilal, Christopher P.; DeBlase, Andrew F.; Burke, Nicole L.; McLuckey, Scott A.; Zwier, Timothy S.

2016-06-01

The "proline effect" is a well-known fragmentation phenomenon in mass spectrometry, in which y-fragments are produced preferentially over b-fragments during the collision induced dissociation of protonated L-proline containing peptide ions. This specific fragmentation channel is favored because of the high basicity of the secondary amine intermediate and the ring instability in alternative bn+ products [ASMS 2014, 25, 1705]. In contrast, peptides containing the D-Pro stereoisomer have been shown to largely favor the production of b4+ ions over y3+ ions. This strongly suggests that differences in the conformational preferences between the D-Pro and L-Pro diastereomers are likely to be responsible but structural evidence has been lacking to date. Using tandem mass spectrometry and IR-UV double resonant action spectroscopy we are able to compare the 3D structures of cold [YA(D-Pro)AA+H]+ to [YA(L-Pro)AA+H]+ ions. The UV action spectra reveals two major conformers in [YA(D-Pro)AA+H]+ and one major conformer in [YA(L-Pro)AA+H]+. Clear differences in the hydrogen bonding patterns are apparent between the two conformers observed in the D-Pro specie which are both distinct from the L-Pro diastereomer. Furthermore, conformer and diastereomer specific photofragmentation patterns are observed. It is also noted that a ten-fold photofragment enhancement unique to one of the D-Pro conformers is observed upon absorption of a resonant IR photon after UV excitation. Differences in the excited state photophysics between the two D-Pro conformers suggest that vibrational excitation of S1 turns on coupling to the dissociative -Tyr channel in one conformer, while this coupling is already present in the vibronic ground state of the other. Calculated harmonic spectra (M052X/6-31+G*) of conformers obtained from Monte Carlo searches to the experimental spectra.

Interferon-alpha therapy in patients with hepatitis C virus infection increases plasma phenylalanine and the phenylalanine to tyrosine ratio.

PubMed

Zoller, Heinz; Schloegl, Anna; Schroecksnadel, Sebastian; Vogel, Wolfgang; Fuchs, Dietmar

2012-05-01

Higher blood levels of the essential amino acid phenylalanine (Phe) together with impaired conversion of Phe to tyrosine (Tyr) have been observed in patients suffering from inflammatory conditions. Data suggest that inflammatory responses may interfere with Phe metabolism. This study aimed to investigate whether treatment with cytokine interferon-α (IFN-α) influences Phe concentrations and the Phe to Tyr ratios (Phe/Tyr) measured by HPLC. Twenty-five patients (9 females, 16 males, aged mean ± SD: 44.5 ± 11.0 years) with hepatitis C virus (HCV) infection were examined before and after 1 month of effective antiviral therapy with pegylated IFN-α and weight-based ribavirin. Results were compared to HCV-RNA titers and concentrations of neopterin. IFN-α treatment was associated with a drop of HCV load (from median 6.3 to 3.2 log10 copies/μL; P<0.001) and an increase of neopterin concentrations (from median 4.83 to 12.1 nM; P=0.001) which confirms effectiveness of therapy. Before therapy, median Phe concentration were 123.9 μM, Tyr was 98.8 μM, and Phe/Tyr was 1.23 μmol/μmol, and under therapy median Phe concentrations increased to 132.6 μM and Phe/Tyr to 1.33 (both P<0.05; paired rank test), Tyr levels remained unchanged. The increase of Phe concentrations and of Phe/Tyr in HCV infected individuals is caused by IFN-α therapy. Data indicate that activity of enzyme phenylalanine 4-hydroxylase becomes impaired. Future studies should show whether side effects of IFN-α treatment such as mood changes and depression will be associated with the alterations of Phe metabolism.

Purification and characterisation of a novel antistaphylococcal peptide (ASP-1) from Bacillus sp. URID 12.1.

PubMed

Chalasani, Ajay Ghosh; Roy, Utpal; Nema, Sushma

2018-01-01

A strong antistaphylococcal peptide (ASP-1) from Bacillus subtilis URID 12.1 strain that is active against cefoxitin- and methicillin-resistant Staphylococcus aureus clinical isolates was purified to homogeneity by solvent extraction, silica gel-based adsorption chromatography and reversed-phase high-performance liquid chromatography. The peptide sequence of ASP-1 as determined by MALDI-TOF/MS and ESI-FTICR-MS was acetylated Phe-Thr-Ala-Val-Dhb-Phe-Ile/Leu. The peptide was further analysed by alkaline hydrolysis, ESI-Q-TOF-MS and an ion mobility assay, which detected the presence of a lactone ring in the intact peptide and a cyclic nature, subsequently revealing the linearised peptide sequence as acPhe-Leu-Phe-Thr-Val-Ala-Dhb. Based on the molecular mass (804.5 Da), peptide sequence and amino acid composition, ASP-1 was identified as a lactone ring-containing peptide similar to TL-119, a poorly studied cyclic depsipeptide. Circular dichroism spectroscopy revealed its predominantly random structure in aqueous solution and its β-sheet conformation in methanol. Minimum inhibitory concentrations (MICs) of the purified peptide against S. aureus and methicillin-resistant S. aureus (MRSA) ranged from 2 µg/mL to 64 µg/mL. At sub-MICs and 1× MIC, ASP-1 showed a strong antibiofilm characteristic. ASP-1 at a concentration of 128 µg/mL did not show haemolytic activity, and no cytotoxicity was observed against hepatic carcinoma and breast carcinoma cell lines at the same concentration. Peptide ASP-1 with anti-MRSA and antibiofilm abilities and non-haemolytic and non-cytotoxic properties has not been reported previously. These findings suggest that it may serve as a lead molecule for developing alternative topical antibacterial agents. Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

Crystal structure of bis­{μ2-2,2′-[(4,10-dimethyl-1,4,7,10-tetra­aza­cyclo­dodecane-1,7-di­yl)bis(meth­yl­ene)]bis­(4-oxo-4H-pyran-3-olato)}dicobalt­calcium bis­(perchlorate) 1.36-hydrate

PubMed Central

Rossi, Patrizia; Paoli, Paola; Giorgi, Luca; Formica, Mauro; Fusi, Vieri

2017-01-01

The title compound, [CaCo2(C22H30N4O6)2](ClO4)2·1.36H2O or {Ca[Co(H–2 L1)]2}·2ClO4·1.36H2O {where L1 is 4,10-bis­[(3-hy­droxy-4-pyron-2-yl)meth­yl]-1,7-dimethyl-1,4,7,10-tetra­aza­cyclo­dodecane}, is a trinuclear complex whose asymmetric unit comprises a quarter of the {Ca[Co(H–2 L1)]2}2+ trinuclear complex, half of a perchlorate ion and 0.34-water mol­ecules. In the neutral [Co(H–2 L1)] moiety, the cobalt ion is hexa­coordinated in a trigonal–prismatic fashion by the surrounding N4O2 donor set. A Ca2+ cation holds together two neutral [Co(H–2 L1)] moieties and is octa­coordinated in a distorted trigonal–dodeca­hedral fashion by the surrounding O atoms belonging to the deprotonated oxide and carbonyl groups of two [Co(H–2 L1)] units. The coordination of the CoII cation preorganizes L1 and an electron-rich area forms, which is able to host hard metal ions. The comparison between the present structure and the previously published ones suggests a high versatility of this ligand; indeed, hard metal ions with different nature and dimensions lead to complexes having different stoichiometry (mono- and dinuclear monomers and trinuclear dimers) or even a polymeric structure. The heterotrinuclear CoII–CaII–CoII complexes are connected in three dimensions via weak C—H⋯O hydrogen bonds, which are also responsible for the inter­actions with the perchlorate anions and the lattice water mol­ecules. The perchlorate anion is disordered about a twofold rotation axis and was refined giving the two positions a fixed occupancy factor of 0.5. The crystal studied was refined as a two-component inversion twin [BASF parameter = 0.14 (4)]. PMID:29250424

Structural analysis and the effect of cyclo(His-Pro) dipeptide on neurotoxins--a dynamics and density functional theory study.

PubMed

Abiram, Angamuthu; Kolandaivel, Ponmalai

2010-02-01

The switching propensity and maximum probability of occurrence of the side chain imidazole group in the dipeptide cyclo(His-Pro) (CHP) were studied by applying molecular dynamics simulations and density functional theory. The atomistic behaviour of CHP with the neurotoxins glutamate (E) and paraquat (Pq) were also explored; E and Pq engage in hydrogen bond formation with the diketopiperazine (DKP) ring of the dipeptide, with which E shows a profound interaction, as confirmed further by NH and CO stretching vibrational frequencies. The effect of CHP was found to be greater on E than on Pq neurotoxin. A ring puckering study indicated a twist boat conformation for the six-membered DKP ring. Molecular electrostatic potential (MESP) mapping was also used to explore the hydrogen bond interactions prevailing between the neurotoxins and the DKP ring. The results of this study reveal that the DKP ring of the dipeptide CHP can be expected to play a significant role in reducing effects such as oxidative stress and cell death caused by neurotoxins.

SX Phoenecis Stars in the Extremely Metal-Poor Globular Clusters NGC 5053

NASA Astrophysics Data System (ADS)

Nemec, James M.; Mateo, Mario; Burke, Morgan; Olszewski, Edward W.

1995-09-01

The results of a major search for photometrically variable blue straggler stars (BSs) in the extremely metal-poor globular cluster NGC 5053 are presented. The survey is based on photometry of over 200 CCD frames (BVI passbands) taken on 18 nights between 1985 and 1994. Five of the 16 BSs monitored for variability are identified as SX Phe stars and their photometric characteristics derived. These five stars are among the shortest-period (49

Visualization of deep ultraviolet photons based on Förster resonance energy transfer and cascade photon reabsorption in diphenylalanine-carbon nitrides composite film

NASA Astrophysics Data System (ADS)

Gan, Zhixing; Zhou, Weiping; Chen, Zhihui; Wang, Huan; Di, Yunsong; Huang, Shisong

2016-11-01

A diphenylalanine (L-Phe-L-Phe, FF)-carbon nitride composite film is designed and fabricated to visualize the deep ultraviolet (DUV, 245-290 nm) photons. The FF film, composed of diphenylalanine molecules, doped with carbon nitrides shows blue emission under excitation of DUV light, which makes the DUV beam observable. Both Förster resonance energy transfer and cascade photon reabsorption contribute to the conversion of photon energy. First, the FF is excited by the DUV photons. On one hand, the energy transfers to the embedded carbon nitrides through nonradiative dipole-dipole couplings. On the other hand, the 284 nm photons emitted from the FF would further excite the carbon nitrides, which will finally convert to blue fluorescence. Herein, the experimental demonstration of a simple device for the visualization of high DUV fluxes is reported.

Fullerene (C60) nanoparticles exert photocytotoxicity through modulation of reactive oxygen species and p38 mitogen-activated protein kinase activation in the MCF-7 cancer cell line

NASA Astrophysics Data System (ADS)

Li, Zhi; Zhang, Fei-long; Wang, Zhiyuan; Pan, Li-li; Shen, Ying-ying; Zhang, Zhen-zhong

2013-12-01

The photocytotoxicity of water-dispersed 100-300 nm fullerene amino acid derivatives nanoparticles was studied. The nanoparticle solution of fullerene derivatives, l-phenylalanine (C60-phe) and glycine (C60-gly), suppressed the in vitro growth of MCF-7 cells lines, induced cancer cells apoptosis, and caused a perturbation of the cell cycle. These nanoparticle solutions increased intracellular reactive oxygen species after irradiation. C60-phe or C60-gly upregulated the expression of phosphorylated (p)p38 mitogen-activated protein kinase (MAPK). N-Acetyl- l-cysteine significantly depressed the composite-induced activation of p38MAPK, and the kinase inhibitor SB203580 significantly prevented C60 derivative-induced cell apoptosis. This study revealed that p38MAPK is activated by C60 nanoparticles through triggering reactive oxygen species generation, leading to cancer cell injuries.

Prosomatostatin-I is processed to somatostatin-26 and somatostatin-14 in the pancreas of the bowfin, Amia calva.

PubMed

Wang, Y; Youson, J H; Conlon, J M

1993-08-13

With the exception of the Agnatha (lampreys and hagfishes), somatostatin-14 is the predominant molecular form of somatostatin in the pancreas of species from all classes of vertebrates yet studied. The pancreas of the holostean fish, Amia calva (bowfin; order Amiiformes) contained somatostatin-like immunoreactivity that was resolved by reversed phase HPLC in two components. The primary structure of the more abundant peptide (somatostatin-26) was established as: Ser-Ala-Asn-Pro-Ala5-Leu-Ala-Pro-Arg-Glu10-Arg-Lys-Ala-Gly-+ ++Cys15-Lys-Asn-Phe- Phe-Trp20-Lys-Thr-Phe-Thr-Ser25-Cys. This amino acid sequence shows one substitution (Leu for Met at position 6) and two deletions compared with mammalian somatostatin-28. The minor component was identical to somatostatin-14. The data show that the pathway of post-translational processing of prosomatostatin-I in the bowfin pancreas is appreciably different from the corresponding pathway in teleost fish and higher vertebrates.

Binding constants of phenylalanine for the four mononucleotides

NASA Technical Reports Server (NTRS)

Khaled, M. A.; Mullins, D. W., Jr.; Lacey, J. C., Jr.

1984-01-01

Earlier work has shown that several properties of amino acids correlate directly with properties of their anticodonic nucleotides. Furthermore, in precipitation studies with thermal proteinoids and homopolyribonucleotides, an anticodonic preference was displayed between Lys-rich, Pro-rich and Gly-rich thermal proteinoids and their anticodonic polyribonucleotides. However, Phe-rich thermal proteinoid displayed a preference for its codonic nucleotide, poly U. This inconsistency seemed to be explained by a folding in of the hydrophobic residues of Phe causing the proteinoid to appear more hydrophilic. The present work used nuclear magnetic resonance techniques to resolve a limited question: to which of the four nucleotides does Phe bind most strongly? The results show quite clearly that Phe binds most strongly to its anticodonic nucleotide, AMP.

L-Aspartic and l-glutamic acid ester-based ProTides of anticancer nucleosides: Synthesis and antitumoral evaluation.

PubMed

Gao, Ling-Jie; De Jonghe, Steven; Daelemans, Dirk; Herdewijn, Piet

2016-05-01

A series of novel aryloxyphosphoramidate nucleoside prodrugs based on l-aspartic acid and l-glutamic acid as amino acid motif has been synthesized and evaluated for antitumoral activity. Depending on the cancer cell line studied and on the nature of the parent nucleoside compound (gemcitabine, 5-iodo-2'-deoxy-uridine, floxuridine or brivudin), the corresponding ProTides are endowed with an improved or decreased cytotoxic activity. Copyright © 2016 Elsevier Ltd. All rights reserved.

The efficacy of the self-adjusting file and ProTaper for removal of calcium hydroxide from root canals

PubMed Central

FARIA, Gisele; KUGA, Milton Carlos; RUY, Alessandra Camila; ARANDA-GARCIA, Arturo Javier; BONETTI-FILHO, Idomeo; GUERREIRO-TANOMARU, Juliane Maria; LEONARDO, Renato Toledo

2013-01-01

Objective The goal of this study was to evaluate the efficacy of the Self-Adjusting File (SAF) and ProTaper for removing calcium hydroxide [Ca(OH)2] from root canals. Material and Methods Thirty-six human mandibular incisors were instrumented with the ProTaper system up to instrument F2 and filled with a Ca(OH)2-based dressing. After 7 days, specimens were distributed in two groups (n=15) according to the method of Ca(OH)2 removal. Group I (SAF) was irrigated with 5 mL of NaOCl and SAF was used for 30 seconds under constant irrigation with 5 mL of NaOCl using the Vatea irrigation device, followed by irrigation with 3 mL of EDTA and 5 mL of NaOCl. Group II (ProTaper) was irrigated with 5 mL of NaOCl, the F2 instrument was used for 30 seconds, followed by irrigation with 5 mL of NaOCl, 3 mL of EDTA, and 5 mL of NaOCl. In 3 teeth Ca(OH)2 was not removed (positive control) and in 3 teeth canals were not filled with Ca(OH)2 (negative control). Teeth were sectioned and prepared for the scanning electron microscopy. The amounts of residual Ca(OH)2 were evaluated in the middle and apical thirds using a 5-score system. Results None of the techniques completely removed the Ca(OH)2 dressing. No difference was observed between SAF and ProTaper in removing Ca(OH)2 in the middle (P=0.11) and the apical (P=0.23) thirds. Conclusion The SAF system showed similar efficacy to rotary instrument for removal of Ca(OH)2 from mandibular incisor root canals. PMID:24037074

Combined sabal and urtica extract compared with finasteride in men with benign prostatic hyperplasia: analysis of prostate volume and therapeutic outcome.

PubMed

Sökeland, J

2000-09-01

To test the hypothesis that in patients with benign prostatic hyperplasia (BPH), the outcome of drug therapy with finasteride may be predictable from the baseline prostate volume and that positive clinical effects might be expected only in patients with prostate volumes of > 40 mL, using a subgroup analysis of results from a previously reported clinical trial of finasteride and phytotherapy. A subgroup of 431 patients was analysed from a randomized, multicentre, double-blind clinical trial involving 543 patients with the early stages of BPH. Patients received a fixed combination of extracts of saw palmetto fruit (Serenoa repens) and nettle root (Urtica dioica) (PRO 160/120) or the synthetic 5alpha-reductase inhibitor finasteride. The patients assessed had valid ultrasonographic measurements and baseline prostate volumes of either 40 mL. All 516 patients were included in the safety analysis. The results of the original trial showed equivalent efficacy for both treatments. The mean (SD) maximum urinary flow (the main outcome variable) increased (from baseline values) after 24 weeks by 1.9 (5.6) mL/s with PRO 160/120 and by 2.4 (6.3) mL/s with finasteride. There were no statistically significant group differences (P = 0.52). The subgroups with small prostates ( 40 mL were similar, at 2.3 (6.1) and 2. 2 (5.3) mL/s, respectively. There were improvements in the International Prostate Symptom Score in both treatment groups, with no statistically significant differences. The subgroup analysis showed slightly better results for voiding symptoms in the patients with prostates of > 40 mL, but there were also improvements in the subgroup with smaller prostates. The safety analysis showed that more patients in the finasteride group reported adverse events and also there were more adverse events in this group than in patients treated with PRO 160/120. The present analysis showed that the efficacy of both PRO 160/120 and finasteride was equivalent and unrelated to prostate volume. However, PRO 160/120 had better tolerability than finasteride.

[In-vitro evaluation of cinnarizine as a competing agent to beta-cyclodextrin inclusion complexes: effect of cinnarizine on the membrane permeation rate of progesterone from its beta-cyclodextrin inclusion complex].

PubMed

Muraoka, Atsushi; Tokumura, Tadakazu; Machida, Yoshiharu

2008-01-01

The use of competing agents is considered a powerful tool for the development of a drug-delivery system with drug/cyclodextrin inclusion complexes. However, there are very few studies examining this issue. To explain this phenomenon, it was thought that a competing agent with a sufficiently high stability constant had not yet been reported. In this study, cinnarizine (CN), which has a high stability constant with beta-cyclodextrin (beta-CD) and unique solubility characteristics, was selected, and its ability as a competing agent was examined in a membrane permeability study. The permeability study showed that the permeation rates of the drugs flurbiprofen, progesterone, and spironolactone decreased with their stability constants with the addition of beta-CD. In one of the drugs, progesterone (Pro), the decrease was restored by the addition of CN. The amount of CN added was a 1:1 molar ratio to the amount of Pro. However, no similar action was induced with the addition of DL-phenylalanine (Phe) in the permeation study at the 1:5 (Pro:Phe) molar ratio. These finding indicate that CN acts as a competing agent, and its action is much stronger than that of Phe.

Purification and identification of corn peptides that facilitate alcohol metabolism by semi-preparative high-performance liquid chromatography and nano liquid chromatography with electrospray ionization tandem mass spectrometry.

PubMed

Ma, Zhi-Li; Hou, Tao; Shi, Wen; Liu, Wei-Wei; Ibrahim, Salam A; He, Hui

2016-11-01

In this study, peptides that facilitate alcohol metabolism were purified and identified from corn protein hydrolysates. The ultra-filtered fraction with a molecular weight < 3 kDa (F3) potential activity was separated into six fractions (F3-H1-F3-H6) by semi-preparative high-performance liquid chromatography. Among the resultant six fractions, F3-H4 and F3-H5 exhibited the highest ability to eliminate alcohol in vivo. A total of 16 peptides with strong signal values were identified from F3-H4 and F3-H5 fractions by nano liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Several identified peptides were then selected and synthesized to determine their potential to facilitate alcohol metabolism. We found that Leu-Leu and Pro-Phe were the key structure units in Gln-Leu-Leu-Pro-Phe responsible for this peptide's ability to facilitate alcohol metabolism. However, the role of Leu-Leu and Pro-Phe may be affected by peptide chain length and hydrophobic properties. Our results have thus provided some insight into the study of the structure-activity relationships of corn peptides. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Prospective evaluation of biomarkers for prediction of quality of life in community-acquired pneumonia.

PubMed

Nickler, Manuela; Schaffner, Daniela; Christ-Crain, Mirjam; Ottiger, Manuel; Thomann, Robert; Hoess, Claus; Henzen, Christoph; Mueller, Beat; Schuetz, Philipp

2016-11-01

Most clinical research investigated prognostic biomarkers for their ability to predict cardiovascular events or mortality. It is unknown whether biomarkers allow prediction of quality of life (QoL) after survival of the acute event. Herein, we investigated the prognostic potential of well-established inflammatory/cardiovascular blood biomarkers including white blood cells (WBC), C-reactive protein (CRP), procalcitonin (PCT), pro-adrenomedullin (proADM) and pro-atrial natriuretic peptide (proANP) in regard to a decline in QoL in a well-defined cohort of patients with community-acquired pneumonia (CAP). Within this secondary analysis including 753 patients with a final inpatient diagnosis of CAP from a multicenter trial, we investigated associations between admission biomarker levels and decline in QoL assessed by the EQ-5D health questionnaire from admission to day 30 and after 6 years. Admission proADM and proANP levels significantly predicted decline of the weighted EQ-5D index after 30 days (n=753) with adjusted odds ratios (ORs) of 2.0 ([95% CI 1.1-3.8]; p=0.027) and 3.7 ([95% CI 2.2-6.0]; p<0.001). Results for 6-year outcomes (n=349) were similar with ORs of 3.3 ([95% CI 1.3-8.3]; p=0.012) and 6.2 ([95% CI 2.7-14.2]; p<0.001). The markers were associated with most of the different QoL dimensions including mobility, self-care, and usual activities, but not pain/discomfort and to a lesser degree anxiety/depression and the visual analogue scale (VAS). Initial WBC, PCT and CRP values did not well predict QoL at any time point. ProADM and proANP accurately predict short- and long-term decline in QoL across most dimensions in CAP patients. It will be interesting to reveal underlying physiopathology in future studies.

Azidoethoxyphenylalanine as a Vibrational Reporter and Click Chemistry Partner in Proteins.

PubMed

Tookmanian, Elise M; Phillips-Piro, Christine M; Fenlon, Edward E; Brewer, Scott H

2015-12-21

An unnatural amino acid, 4-(2-azidoethoxy)-L-phenylalanine (AePhe, 1), was designed and synthesized in three steps from known compounds in 54% overall yield. The sensitivity of the IR absorption of the azide of AePhe was established by comparison of the frequency of the azide asymmetric stretch vibration in water and dimethyl sulfoxide. AePhe was successfully incorporated into superfolder green fluorescent protein (sfGFP) at the 133 and 149 sites by using the amber codon suppression method. The IR spectra of these sfGFP constructs indicated that the azide group at the 149 site was not fully solvated despite the location in sfGFP and the three-atom linker between the azido group and the aromatic ring of AePhe. An X-ray crystal structure of sfGFP-149-AePhe was solved at 1.45 Å resolution and provides an explanation for the IR data as the flexible linker adopts a conformation which partially buries the azide on the protein surface. Both sfGFP-AePhe constructs efficiently undergo a bioorthogonal strain-promoted click cycloaddition with a dibenzocyclooctyne derivative. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Annexin 1 exerts anti-nociceptive effects after peripheral inflammatory pain through formyl-peptide-receptor-like 1 in rat dorsal root ganglion.

PubMed

Pei, L; Zhang, J; Zhao, F; Su, T; Wei, H; Tian, J; Li, M; Shi, J

2011-12-01

Annexin 1 (ANXA1) has analgesic effects in inflammatory pain. We aimed to investigate the anti-nociceptive role of ANXA1, at the dorsal root ganglion (DRG) level, through an interaction with formyl-peptide-receptor-like 1 (FPR2/ALX). Inflammatory pain was evoked by injecting complete Freund's adjuvant (CFA, 50 μl) into the hindpaw of male Sprague-Dawley rats. The distribution of ANXA1 and FPR2/ALX in L4/5 DRGs was evaluated by immunofluorescence. The expression of ANXA1 was measured by western blot. The involvement of FPR2/ALX in the anti-nociception of ANXA1 was investigated by thermal (irradiant heat) and mechanical (von Frey filament) pain tests with intrathecal (i.t.) ANXA1-derived peptide (Anxa1(2-26)), FPR2/ALX agonist 5(S)-6(R)-7-trihydroxyheptanoic-acid-methyl-ester (BML-111), and antagonist N-t-Boc-Phe-Leu-Phe-Leu-Phe (Boc1). ANXA1 and FPR2/ALX localized in the satellite glial cells and neurones in L4/5 DRGs. CFA treatment (n=20) increased ANXA1 expression in L4/5 DRGs within 7 days (P<0.01). I.T. Anxa1(2-26) (20 and 100 µg µl(-1)) and BML-111 (10 and 100 nmol) reduced CFA-induced thermal and mechanical nociception within 48 h (n=40) (P<0.05). However, i.t. Boc1 10 µg intensified inflammatory pain (P<0.05) and reversed the anti-nociceptive effect of Anxa1(2-26) (n=25) (P<0.05). Moreover, ANXA1 expression increased in L4/5 DRGs after i.t. Anxa1(2-26) (20 µg µl(-1)) (P<0.05) and BML-111 (10 nmol) (P<0.01) but decreased after i.t. Boc1 (10 and 100 µg) alone (P<0.01) or Boc1 (10 µg) co-injection with Anxa1(2-26) (20 µg µl(-1)) (P<0.05). Endogenous ANXA1 expression at the DRG level is involved in CFA-induced inflammatory pain, and i.t. ANXA1 20 µg µl(-1) produces its anti-nociceptive effect through FPR2/ALX.

Second Language Perception and Production of English Regular Past Tense: L1 Influence in Phonology and Morphosyntax

ERIC Educational Resources Information Center

Chen, Wen-Hsin

2016-01-01

The goal of this study is to provide a better understanding of the influence from first language (L1) phonology and morphosyntax on second language (L2) production and perception of English regular past tense morphology. (Abstract shortened by ProQuest.) [The dissertation citations contained here are published with the permission of ProQuest LLC.…

Bioorganometallic chemistry. 5. Molecular recognition of aromatic amino acid guests by Cp{sup *} Rh-nucleobase/nucleoside/nucleotide cyclic trimer hosts in aqueous solution

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

Chen, H.; Maestre, M.F.; Fish, R.H.

We report what we believe is the first example of bioorganometallic hosts, 1-4, [Cp{sup *}Rh(9-methyladenine)]{sub 3}(OTf){sub 3} (1), [Cp{sup *}Rh(Me-5`-AMP)]{sub 3} (4), being able to recognize aromatic amino acid guests L-tryptophan (L-Trp) and L-phenylalanine (L-Phe) in aqueous media at pH 7. Results show that the molecular recognition of aromatic amino acids with bioorganometallic hosts 1-4 in aqueous solution, as studied by {sup 1}H NMR and NOE techniques, occurs predominately via a {pi}-{pi} interaction, and, in the case of L-Trp, additional electronic/hydrophobic interactions with hosts are possible. 7 refs., 1 fig., 1 tab.

Expanding the chemical space for natural products by Aspergillus-Streptomyces co-cultivation and biotransformation

PubMed Central

Wu, Changsheng; Zacchetti, Boris; Ram, Arthur F.J.; van Wezel, Gilles P.; Claessen, Dennis; Hae Choi, Young

2015-01-01

Actinomycetes and filamentous fungi produce a wide range of bioactive compounds, with applications as antimicrobials, anticancer agents or agrochemicals. Their genomes contain a far larger number of gene clusters for natural products than originally anticipated, and novel approaches are required to exploit this potential reservoir of new drugs. Here, we show that co-cultivation of the filamentous model microbes Streptomyces coelicolor and Aspergillus niger has a major impact on their secondary metabolism. NMR-based metabolomics combined with multivariate data analysis revealed several compounds that correlated specifically to co-cultures, including the cyclic dipeptide cyclo(Phe-Phe) and 2-hydroxyphenylacetic acid, both of which were produced by A. niger in response to S. coelicolor. Furthermore, biotransformation studies with o-coumaric acid and caffeic acid resulted in the production of the novel compounds (E)-2-(3-hydroxyprop-1-en-1-yl)-phenol and (2E,4E)-3-(2-carboxy-1-hydroxyethyl)-2,4-hexadienedioxic acid, respectively. This highlights the utility of microbial co-cultivation combined with NMR-based metabolomics as an efficient pipeline for the discovery of novel natural products. PMID:26040782

Pro-Gastrin Releasing Peptide: A New Serum Marker for Endometrioid Adenocarcinoma.

PubMed

Kiseli, Mine; Caglar, Gamze Sinem; Yarci Gursoy, Asli; Tasci, Tolga; Candar, Tuba; Akincioglu, Egemen; Pabuccu, Emre Goksan; Boran, Nurettin; Tulunay, Gokhan; Umudum, Haldun

2018-06-13

Gastrin-releasing peptide (GRP) is thought to play a role in the metastatic process of various malignancies. The more stable precursor of GRP, pro-GRP (ProGRP), has been shown to be secreted by neuroendocrine tumors. This study was designed to assess the validity of ProGRP as a diagnostic marker in endometrioid adenocarcinomas (EAs) of the endometrium. Thirty-seven patients with a diagnosis of EA, 23 patients with endometrial hyperplasia, and 32 age-matched controls with normal endometrial histology were recruited for this study. Serum ProGRP and cancer antigen 125 (CA125) values were compared between groups. Median serum ProGRP levels were significantly higher in the cancer group compared to corresponding levels in both the hyperplasia and control groups (p = 0.008 and p < 0.001 respectively; endometrial cancer: 27.5 pg/mL; hyperplasia: 16.1 pg/mL; controls: 12.9 pg/mL). Age and endometrial thickness were positively correlated with ProGRP levels (r = 0.322, p = 0.006 and r = 0.269, p = 0.023, respectively). Receiver Operating Characteristic curve analyses for EA revealed a threshold of 20.81 pg/mL, with a sensitivity of 60.7% and specificity of 81.4%, positive predictive value of 68% and negative predictive value of 76.1%. Significantly higher ProGRP levels were observed in patients with EA than in controls. Serum ProGRP has good diagnostic sensitivity and specificity for EA. © 2018 S. Karger AG, Basel.

Human heart failure biomarker immunosensor based on excessively tilted fiber gratings.

PubMed

Luo, Binbin; Wu, Shengxi; Zhang, Zhonghao; Zou, Wengen; Shi, Shenghui; Zhao, Mingfu; Zhong, Nianbing; Liu, Yong; Zou, Xue; Wang, Lingling; Chai, Weina; Hu, Chuanmin; Zhang, Lin

2017-01-01

A label-free immunosensor platform based on excessively tilted fiber gratings (Ex-TFGs) was developed for highly specific and fast detection of human N-terminal pro-B-type natriuretic peptide (NT-proBNP), which is considered a powerful biomarker for prognosis and risk stratification of heart failure (HF). High-purity anti-NT-proBNP monoclonal antibodies (MAbs) prepared in our laboratory were immobilized on fiber surface through the staphylococcal protein A (SPA) method for subsequent specific binding of the targeted NT-proBNP. Utilizing fiber optic grating demodulation system (FOGDS), immunoassays were carried out in vitro by monitoring the resonance wavelength shift of Ex-TFG biosensor with immobilized anti-NT-proBNP MAbs. Lowest detectable concentration of ~0.5ng/mL for NT-proBNP was obtained, and average sensitivity for NT-proBNP at a concentration range of 0~1.0 ng/mL was approximately 45.967 pm/(ng/mL). Several human serum samples were assessed by the proposed Ex-TFG biomarker sensor, with high specificity for NT-proBNP, indicating potential application in early diagnosing patients with acute HF symptoms.

l-Proline Accumulation and Freeze Tolerance in Saccharomyces cerevisiae Are Caused by a Mutation in the PRO1 Gene Encoding γ-Glutamyl Kinase

PubMed Central

Morita, Yuko; Nakamori, Shigeru; Takagi, Hiroshi

2003-01-01

We previously isolated a mutant which showed a high tolerance to freezing that correlated with higher levels of intracellular l-proline derived from l-proline analogue-resistant mutants. The mutation responsible for the analogue resistance and l-proline accumulation was a single nuclear dominant mutation. By introducing the mutant-derived genomic library into a non-l-proline-utilizing strain, the mutant was found to carry an allele of the wild-type PRO1 gene encoding γ-glutamyl kinase, which resulted in a single amino acid replacement; Asp (GAC) at position 154 was replaced by Asn (AAC). Interestingly, the allele of PRO1 was shown to enhance the activities of γ-glutamyl kinase and γ-glutamyl phosphate reductase, both of which catalyze the first two steps of l-proline synthesis from l-glutamate and which together may form a complex in vivo. When cultured in liquid minimal medium, yeast cells expressing the mutated γ-glutamyl kinase were found to accumulate intracellular l-proline and showed a prominent increase in cell viability after freezing at −20°C compared to the viability of cells harboring the wild-type PRO1 gene. These results suggest that the altered γ-glutamyl kinase results in stabilization of the complex or has an indirect effect on γ-glutamyl phosphate reductase activity, which leads to an increase in l-proline production in Saccharomyces cerevisiae. The approach described in this paper could be a practical method for breeding novel freeze-tolerant yeast strains. PMID:12513997

Mycoremediation of manganese and phenanthrene by Pleurotus eryngii mycelium enhanced by Tween 80 and saponin.

PubMed

Wu, Minghui; Xu, Yongan; Ding, Wenbo; Li, Yuanyuan; Xu, Heng

2016-08-01

Bioremediation of areas co-contaminated with metals and polycyclic aromatic hydrocarbons (PAHs) by mushrooms has attracted considerable attention in recent years. In this study, Pleurotus eryngii was introduced for the removal of Mn and phenanthrene (Phe) from potato liquid medium (PDL) simultaneously. Effects of Tween 80 and saponin on P. eryngii growth together with Mn uptake as well as Phe removal were investigated. Although pollutants had a negative effect on mycelial morphology and growth, P. eryngii could still tolerate and remove Mn and Phe. Tween 80 increased removal of Mn and Phe through increase of P. eryngii growth, Phe solubility, pollutants bioavailability, and specific surface area of mycelium pellets, moreover, the activities of manganese peroxidase (MnP) and laccase, which played an important role on PAHs biodegradation. The maximal removal of Mn and Phe was achieved (92.17 and 93.85 % after 15 days incubation, respectively) with 0.6 g L(-1) Tween 80. Treatments with saponin markedly inhibited P. eryngii growth (50.17-66.32 % lower relative to control) due to its fungistatic activity. Nevertheless, saponin could slightly enhance Phe removal through increasing solubility of Phe, and Phe removal rate varied from 80.53 to 87.06 % in saponin treatments. Joint stress of Mn and Phe induced a strong antioxidative response, and superoxide dismutase (SOD) activity decreased in surfactants-treated mycelium compared with control. Generally, Tween 80 was more suitable for strengthening mycoremediation by P. eryngii than saponin, and could be a promising alternative for the remediation of heavy metals and PAHs co-contaminated sites by mushrooms.

Increased NT-proANP predicts risk of congestive heart failure in Cavalier King Charles spaniels with mitral regurgitation caused by myxomatous valve disease.

PubMed

Eriksson, Anders S; Häggström, Jens; Pedersen, Henrik Duelund; Hansson, Kerstin; Järvinen, Anna-Kaisa; Haukka, Jari; Kvart, Clarence

2014-09-01

To evaluate the predictive value of plasma N-terminal pro-atrial natriuretic peptide (NT-proANP) and nitric oxide end-products (NOx) as markers for progression of mitral regurgitation caused by myxomatous mitral valve disease. Seventy-eight privately owned Cavalier King Charles spaniels with naturally occurring myxomatous mitral valve disease. Prospective longitudinal study comprising 312 measurements over a 4.5 year period. Clinical values were recorded, NT-proANP concentrations were measured by radioimmunoassay, and NOx were analyzed colorimetrically. To predict congestive heart failure (CHF), Cox proportional hazards models with time-varying covariates were constructed. The hazard ratio for NT-proANP (per 1000 pmol/l increase) to predict future CHF was 6.7 (95% confidence interval, 3.6-12.5; p < 0.001). The median time to CHF for dogs with NT-proANP levels >1000 pmol/l was 11 months (95% confidence interval, 5.6-12.6 months), compared to 54 months (46 - infinity) for dogs with concentrations ≤ 1000 pmol/l (p < 0.001). Due to intra- and inter-individual variability, most corresponding analyses for NOx were insignificant but dogs reaching CHF had a lower mean NOx concentration than dogs not reaching CHF (23 vs. 28 μmol/l, p = 0.016). Risk of CHF increased with increase in heart rate (>130 beats per minute) and grade of murmur (≥ 3/6). The risk of CHF due to mitral regurgitation is increased in dogs with blood NT-proANP concentrations above 1000 pmol/l. Measurement of NT-proANP can be a valuable tool to identify dogs that may develop CHF within months. Copyright © 2014 Elsevier B.V. All rights reserved.

Effects of feeding reduced crude protein diets on growth performance, nitrogen excretion, and plasma uric acid concentration of broiler chicks during the starter period.

PubMed

Kriseldi, R; Tillman, P B; Jiang, Z; Dozier, W A

2018-05-01

An experiment (2 trials) was conducted to determine the effects of feeding reduced crude protein (CP) diets to Ross × Ross 708 male broilers while maintaining adequate essential amino acid (AA) concentrations on growth performance, nitrogen excretion, and plasma uric acid (UA) concentration during the starter period. In trial 1, 11 dietary treatments were fed from 1 to 18 d of age containing 1.20% digestible Lys. Diet 1 (23.2% CP) was formulated with DL-Met, L-Lys, and L-Thr to contain 1.70 total Gly + Ser to digestible Lys ratio whereas diets 2 (23.4% CP) to 11 were formulated with additional Gly to contain 1.90 total Gly + Ser to digestible Lys ratio. Free AA were added sequentially in the order of limitation (L-Val, L-Ile, L-Arg, L-Trp, L-His, L-Phe, and L-Leu) from diets 3 to 10 to decrease CP content from 22.6 to 18.8%, respectively. In diet 11, L-Gln was added to increase the CP content to 23.4%. Feed conversion of broilers fed diet 2 was lower (P < 0.05) than those consuming diets 6 to 11 from 1 to 17 d of age. Nitrogen excretion (mg/b/d) decreased (P < 0.001) by 14.1% when broilers were fed diet 4 compared with birds fed diet 2 from 15 to 16 d of age. Broilers fed diet 4 had lower (P = 0.011) plasma UA concentration than birds fed diet 2 at 18 d of age. In trial 2, 8 dietary treatments containing 1.25% digestible Lys and 1.70 total Gly + Ser to digestible Lys ratio were fed from 1 to 21 d of age. Diet 1 (24.0% CP) was supplemented with DL-Met, L-Lys, and L-Thr. Free AA (L-Val, Gly, L-Ile, L-Arg, L-Trp, L-His, and L-Phe) were sequentially supplemented in the order of limitation to decrease CP content in diets 2 to 8 from 23.8 to 20.3%. Broilers fed diet 1 had higher (P < 0.05) body weight gain and lower (P < 0.05) feed conversion when compared with diet 7 or 8. Plasma UA concentration of broiler provided diets 4 to 8 was lower (P < 0.05) compared with diet 1 at 21 d of age. Placing a minimum on dietary CP percentage may not be necessary when proper AA ratios are implemented in diet formulation.

Enantioselective separation of chiral aromatic amino acids with surface functionalized magnetic nanoparticles.

PubMed

Ghosh, Sudipa; Fang, Tan Hui; Uddin, M S; Hidajat, K

2013-05-01

Chiral resolution aromatic amino acids, DL-tryptophan (DL-Trp), DL-phenylalanine (DL-Phe), DL-tyrosine (DL-Tyr) from phosphate buffer solution was achieved in present study employing the concept of selective adsorption by surface functionalized magnetic nanoparticles (MNPs). Surfaces of magnetic nanoparticles were functionalized with silica and carboxymethyl-β-cyclodextrin (CMCD) to investigate their adsorption resolution characteristics. Resolution of enantiomers from racemic mixture was quantified in terms of enantiomeric excess using chromatographic method. The MNPs selectively adsorbed L-enantiomers of DL-Trp, DL-Phe, and DL-Tyr from racemic mixture and enantiomeric excesses (e.e.) were determined as 94%, 73% and 58%, respectively. FTIR studies demonstrated that hydrophobic portion of enantiomer penetrated into hydrophobic cavity of cyclodextrin molecules to form inclusion complex. Furthermore, adsorption site was explored using XPS and it was revealed that amino group at chiral center of the amino acid molecule formed hydrogen bond with secondary hydroxyl group of CMCD molecule and favorability of hydrogen bond formation resulted in selective adsorption of L-enantiomer. Finally, stability constant (K) and Gibbs free energy change (-ΔG°) for inclusion complexation of CMCD with L-/D-enantiomers of amino acids were determined using spectroflurometry in aqueous buffer solution. Higher binding constants were obtained for inclusion complexation of CMCD with L-enantiomers compared to D-enantiomers which stimulated enantioselective properties of CMCD functionalized magnetite silica nanoparticles. Copyright © 2013 Elsevier B.V. All rights reserved.

Can the Pro-Drop Parameter Account for All the Errors in the Acquisition of Non-Referential "It" in L2 English?

ERIC Educational Resources Information Center

Antonova-Ünlü, Elena

2015-01-01

Numerous studies, examining the acquisition of non-referential it in [-pro-drop] English by learners of [+pro-drop] languages, have revealed that their participants omit non-referential subjects in English if their L1 allows null-subject position. However, due to the specificity of their focus, these studies have not considered other difficulties…

Multi-center validation of the Response Biomedical Corporation RAMP NT-proBNP assay with comparison to the Roche Diagnostics GmbH Elecsys proBNP assay.

PubMed

Lee-Lewandrowski, Elizabeth; Januzzi, James L; Green, Sandy M; Tannous, Bakhos; Wu, Alan H B; Smith, Andrew; Wong, Alicia; Murakami, MaryAnn M; Kaczmarek, Jason; Apple, Fred S; Miller, Wayne L; Hartman, Karen; Jaffe, Allan S

2007-01-01

NT-proBNP measurements aid in the evaluation of patients with suspected heart failure (HF) and may facilitate risk stratification in patients with HF and acute coronary syndrome (ACS). Point-of-care (POC) assays may provide more timely results and potentially improve patient outcomes. We evaluated the analytical performance of the Response Biomedical Corporation whole blood RAMP amino-terminal pro-B type natriuretic peptide (NT-proBNP) POC assay compared to the Roche Elecsys proBNP (NT-proBNP) assay. Intra-day and total imprecision (% CV) ranged from 5.5% to 10.3% at 140, 449 and 1675 ng/L. The lowest concentration that yields a 20% CV was 57 ng/L. The lower limit of detection was 18 ng/L. The upper limit of linearity was validated to 23,428 ng/L with an average recovery of 95%. Correlation by Passing and Bablok regression yielded RAMP=1.01 Elecsys+14.6, r=0.98 (n=540; range of Elecsys values <5 to >35,000). Concordance of RAMP versus Elecsys using cut-offs of 125 ng/L for subjects <75 years and 450 ng/L for subjects > or =75 was 92% (95% CI 89-94%) for a group consisting of 127 apparently healthy individuals and 208 non-healthy subjects without HF, and 99% (95% CI 97-100%) for patients with HF, using the New York Heart Association (NYHA) functional classification. Overall, 80%, 87%, 97% and 100% of the RAMP results and 77%, 85%, 96% and 100% of the Elecsys results were greater than the age appropriate cut-off for NYHA I, II, III or IV groups. For both the RAMP and Elecsys results, the median NT-proBNP value was statistically correlated (increasing) with NYHA I, II, III or IV groups, respectively (p<0.0001), with no significant difference between the two methods. The POC Response Biomedical RAMP NT-proBNP assay provides comparable results that measured on the FDA cleared Roche Elecsys central laboratory platform.

Mutations in MAB21L2 result in ocular Coloboma, microcornea and cataracts.

PubMed

Deml, Brett; Kariminejad, Ariana; Borujerdi, Razieh H R; Muheisen, Sanaa; Reis, Linda M; Semina, Elena V

2015-01-01

Ocular coloboma results from abnormal embryonic development and is often associated with additional ocular and systemic features. Coloboma is a highly heterogeneous disorder with many cases remaining unexplained. Whole exome sequencing from two cousins affected with dominant coloboma with microcornea, cataracts, and skeletal dysplasia identified a novel heterozygous allele in MAB21L2, c.151 C>G, p.(Arg51Gly); the mutation was present in all five family members with the disease and appeared de novo in the first affected generation of the three-generational pedigree. MAB21L2 encodes a protein similar to C. elegans mab-21 cell fate-determining factor; the molecular function of MAB21L2 is largely unknown. To further evaluate the role of MAB21L2, zebrafish mutants carrying a p.(Gln48Serfs*5) frameshift truncation (mab21l2Q48Sfs*5) and a p.(Arg51_Phe52del) in-frame deletion (mab21l2R51_F52del) were developed with TALEN technology. Homozygous zebrafish embryos from both lines developed variable lens and coloboma phenotypes: mab21l2Q48Sfs*5 embryos demonstrated severe lens and retinal defects with complete lethality while mab21l2R51_F52del mutants displayed a milder lens phenotype and severe coloboma with a small number of fish surviving to adulthood. Protein studies showed decreased stability for the human p.(Arg51Gly) and zebrafish p.(Arg51_Phe52del) mutant proteins and predicted a complete loss-of-function for the zebrafish p.(Gln48Serfs*5) frameshift truncation. Additionally, in contrast to wild-type human MAB21L2 transcript, mutant p.(Arg51Gly) mRNA failed to efficiently rescue the ocular phenotype when injected into mab21l2Q48Sfs*5 embryos, suggesting this allele is functionally deficient. Histology, immunohistochemistry, and in situ hybridization experiments identified retinal invagination defects, an increase in cell death, abnormal proliferation patterns, and altered expression of several ocular markers in the mab21l2 mutants. These findings support the identification of MAB21L2 as a novel factor involved in human coloboma and highlight the power of genome editing manipulation in model organisms for analysis of the effects of whole exome variation in humans.

Enzymological Basis for Growth Inhibition by l-Phenylalanine in the Cyanobacterium Synechocystis sp. 29108

PubMed Central

Hall, Geraldine C.; Jensen, Roy A.

1980-01-01

The pattern of allosteric control in the biosynthetic pathway for aromatic amino acids provides a basis to explain vulnerability to growth inhibition by l-phenylalanine (0.2 mM or greater) in the unicellular cyanobacterium Synechocystis sp. 29108. We attribute growth inhibition to the hypersensitivity of 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase to feedback inhibition by l-phenylalanine. Hyperregulation of this initial enzyme of aromatic biosynthesis depletes the supply of precursors needed for biosynthesis of l-tyrosine and l-tryptophan. Consistent with this mechanism is the total reversal of phenylalanine inhibition by a combination of tyrosine and tryptophan. Inhibited cultures also contained decreased levels of phycocyanin pigments, a characteristic previously correlated with amino acid starvation in cyanobacteria. l-Phenylalanine is a potent noncompetitive inhibitor (with both substrates) of 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase, whereas l-tyrosine is a very weak inhibitor. Prephenate dehydratase also displays allosteric sensitivity to phenylalanine (inhibition) and to tyrosine (activation). Both 2-fluoro and 4-fluoro derivatives of phenylalanine were potent analog antimetabolites, and these were used in addition to l-phenylalanine as selective agents for resistant mutants. Mutants were isolated which excreted both phenylalanine and tyrosine, the consequence of an altered 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase no longer sensitive to feedback inhibition. Simultaneous insensitivity to l-tyrosine suggests that l-tyrosine acts as a weak analog mimic of l-phenylalanine at a common binding site. Prephenate dehydratase in the regulatory mutants was unaltered. Surprisingly, in view of the lack of regulation in the tyrosine branchlet of the pathway, such mutants excrete more phenylalanine than tyrosine, indicating that l-tyrosine activation dominates l-phenylalanine inhibition of prephenate dehydratase in vivo. In mutant Phe r19 the loss in allosteric sensitivity of 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase was accompanied by a threefold increase in specific activity. This could suggest that existence of a modest degree of repression control (autogenous) over 3-deoxy-d-arabinoheptulosonate synthase, although other explanations are possible. Specific activities of chorismate mutase, prephenate dehydratase, shikimate/nicotinamide adenine dinucleotide phosphate dehydrogenase, and arogenate/nicotinamide adenine dinucleotide phosphate dehydrogenase in mutant Phe r19 were identical with those of the wild type. PMID:6108316

Prognostic value of N-terminal pro-B-type natriuretic peptide in patients with acute coronary syndromes undergoing left main percutaneous coronary intervention.

PubMed

Jaberg, Laurenz; Toggweiler, Stefan; Puck, Marietta; Frank, Michelle; Rufibach, Kaspar; Lüscher, Thomas F; Corti, Roberto

2011-01-01

Patients undergoing acute left main (LM) coronary artery revascularization have a high mortality and natriuretic peptides such as N-terminal pro-B-type (NT-proBNP) have been shown to have prognostic value in patients with acute coronary syndromes. The present study looked at the prognostic value of NT-proBNP in these patients. We studied all consecutive patients undergoing acute LM coronary artery percutaneous coronary intervention between January 2005 and December 2008 in whom NT-proBNP was measured (n=71). We analyzed the clinical characteristics and the short- and long-term outcomes in relation to NT-proBNP level at admission. Median NT-proBNP was 1,364 ng/L, ranging from 46 to 70,000 ng/L. NT-proBNP was elevated in 63 (89%) patients and was ≥1,000ng/L in 42 (59%). Log NT-proBNP (hazard ratio [HR] 3.51, 95% confidence interval [CI] 1.55-7.97, P=0.003) and left ventricular ejection fraction (HR 0.95, 95%CI 0.91-0.99, P=0.007) were predictors for all-cause mortality. Log NT-proBNP was the only independent significant predictor of cardiovascular mortality. In-hospital mortality was 0% for patients with NT-proBNP <1,000, but 17% for those with NT-proBNP ≥1,000 (P=0.036). NT-proBNP is a strong predictor of outcome in patients undergoing acute LM coronary artery stenting. Mortality in such patients is high, but those with NT-proBNP < 1,000ng/L may have a favorable short- and long-term prognosis. Further research, including a larger patient population, is needed to determine the optimal cut-off value for NT-proBNP in patients undergoing acute LM coronary artery intervention.

Determination of the enthalpy of vaporization and prediction of surface tension for ionic liquid 1-alkyl-3-methylimidazolium propionate [C(n)mim][Pro](n = 4, 5, 6).

PubMed

Tong, Jing; Yang, Hong-Xu; Liu, Ru-Jing; Li, Chi; Xia, Li-Xin; Yang, Jia-Zhen

2014-11-13

With the use of isothermogravimetrical analysis, the enthalpies of vaporization, Δ(g)lH(o)m(T(av)), at the average temperature, T(av) = 445.65 K, for the ionic liquids (ILs) 1-alkyl-3-methylimidazolium propionate [C(n)mim][Pro](n = 4, 5, 6) were determined. Using Verevkin's method, the difference of heat capacities between the vapor phase and the liquid phase, Δ(g)lC(p)(o)m, for [C(n)mim][Pro](n = 2, 3, 4, 5, 6), were calculated based on the statistical thermodynamics. Therefore, with the use of Δ(g)lC(p)(o)m, the values of Δ(g)lH(o)m(T(av)) were transformed into Δ(g)lH(o)m(298), 126.8, 130.3, and 136.5 for [C(n)mim][Pro](n = 4, 5, 6), respectively. In terms of the new scale of polarity for ILs, the order of the polarity of [C(n)mim][Pro](n = 2, 3, 4, 5, 6) was predicted, that is, the polarity decreases with increasing methylene. A new model of the relationship between the surface tension and the enthalpy of vaporization for aprotic ILs was put forward and used to predict the surface tension for [C(n)mim][Pro](n = 2, 3, 4, 5, 6) and others. The predicted surface tension for the ILs is in good agreement with the experimental one.

N-terminal pro-B-type Natriuretic Peptide in three different mechanisms of dysnatremia onset after a child's craniopharyngioma surgery.

PubMed

Spatenkova, Vera; Hradil, Jan; Suchomel, Petr

2017-10-01

Craniopharyngioma, due to its sellar location, can be perioperatively complicated by different types of dysnatremia. We present a rare postoperative onset of a combination of three different mechanisms of dysnatremia with N-terminal pro-B-type Natriuretic Peptide (NT-proBNP) and renal function parameters in a boy with a good outcome after craniopharyngioma surgery: 1/ Central diabetes insipidus (CDI) onset immediately after the operation, hypernatremia with peak serum sodium (SNa) 158 mmol/l) caused by free water polyuria (electrolyte-free water clearance, EWC 0.104 ml/s), NT-proBNP 350 pg/ml; 2/ cerebral salt wasting (CSW) onset on day 7, hyponatremia (SNa 128 mmol/l) with hypoosmolality (measured serum osmolality, SOsm 265 mmol/kg) caused by natriuresis (sodium - daily output 605 mmol/day, fractional excretion 0.035), NT-proBNP 191 pg/ml; 3/ Polydypsia onset on day 11 caused hyponatremia (SNa 132 mmol/l), EWC 0.015, NT-proBNP 68 pg/ml.

L-Proline uptake in Saccharomyces cerevisiae mitochondria can contribute to bioenergetics during nutrient stress as alternative mitochondrial fuel.

PubMed

Pallotta, Maria Luigia

2014-01-01

L-Proline (pyrrolidine-2-carboxylic acid) is a distinctive metabolite both biochemically and biotechnologically and is currently recognized to have a cardinal role in gene expression and cellular signaling pathways in stress response. Proline-fueled mitochondrial metabolism involves the oxidative conversion of L-Proline to L-Glutamate in two enzymatic steps by means of Put1p and Put2p that help Saccharomyces cerevisiae to respond to changes in the nutritional environment by initiating the breakdown of L-Proline as a source for nitrogen, carbon, and energy. Compartmentalization of L-Proline catabolic pathway implies that extensive L-Proline transport must take place between the cytosol where its biogenesis via Pro1p, Pro2p, Pro3p occurs and mitochondria. L-Proline uptake in S. cerevisiae purified and active mitochondria was investigated by swelling experiments, oxygen uptake and fluorimetric measurement of a membrane potential generation (ΔΨ). Our results strongly suggest that L-Proline uptake occurs via a carried-mediated process as demonstrated by saturation kinetics and experiments with N-ethylmaleimide, a pharmacological compound that is a cysteine-modifying reagent in hydrophobic protein domains and that inhibited mitochondrial transport. Plasticity of S. cerevisiae cell biochemistry according to background fluctuations is an important factor of adaptation to stress. Thus L-Proline → Glutamate route feeds Krebs cycle providing energy and anaplerotic carbon for yeast survival.

A nonpeptidyl growth hormone secretagogue.

PubMed

Smith, R G; Cheng, K; Schoen, W R; Pong, S S; Hickey, G; Jacks, T; Butler, B; Chan, W W; Chaung, L Y; Judith, F

1993-06-11

A nonpeptidyl secretagogue for growth hormone of the structure 3-amino-3-methyl-N-(2,3,4,5-tetrahydro-2-oxo-1-([2'-(1H-tetrazol-5 -yl) (1,1'-biphenyl)-4-yl]methyl)-1H-1-benzazepin-3(R)-yl)-butanamid e (L-692,429) has been identified. L-692,429 synergizes with the natural growth hormone secretagogue growth hormone-releasing hormone and acts through an alternative signal transduction pathway. The mechanism of action of L-692,429 and studies with peptidyl and nonpeptidyl antagonists suggest that this molecule is a mimic of the growth hormone-releasing hexapeptide His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (GHRP-6). L-692,429 is an example of a nonpeptidyl specific secretagogue for growth hormone.

Usefulness of Age-Stratified N-Terminal Prohormone of Brain Natriuretic Peptide for Diagnosing Kawasaki Disease

PubMed Central

Lee, Sang Hoon; Yoon, Somy; Hong, Seunghee; Yang, Eun Mi; Eom, Gwang Hyeon

2017-01-01

N-terminal prohormone of brain natriuretic peptide (NT-proBNP) was recently reported as a biomarker for diagnosing Kawasaki disease (KD). The basal NT-proBNP level, however, gradually decreases with age. We investigated the usefulness of an age-stratified cutoff value of NT-proBNP for diagnosing KD. All the patients enrolled in this study visited Chonnam National University Hospital between December 2007 and March 2016. The KD groups consisted of 214 patients with complete KD and 129 patients with incomplete KD. The control group included 62 children with simple febrile illness but without heart disease. Laboratory data including NT-proBNP level were evaluated. Each group was divided into subgroups according to patient age (<6 months, 6–12 months, 12–24 months, and >24 months), and different cutoff values of NT-proBNP were calculated. The cutoff values of NT-proBNP used to diagnose total KD and incomplete KD were 762 and 762 pg/mL (<6 months), 310 and 310 pg/mL (6–12 months), 326 and 326 pg/mL (12–24 months), and 208 and 137 pg/mL (>24 months), respectively. In conclusion, age-stratified NT-proBNP is a useful biomarker for the differential diagnosis of KD in patients with a simple febrile illness. PMID:29358841

Evaluation of CA125 and NT-proBNP values in patients undergoing transcatheter aortic valve implantation

PubMed Central

Ayhan, Hüseyin; Kasapkara, Hacı Ahmet; Durmaz, Tahir; Keleş, Telat; Sarı, Cenk; Baştuğ, Serdal; Erdoğan, Kemal Eşref; Bayram, Nihal Akar; Bilen, Emine; Akçay, Murat; Bozkurt, Engin

2015-01-01

Background Transcatheter aortic valve implantation (TAVI) is a minimally invasive, emerging therapy in surgically high risk, or inoperable patients. Parameters used for risk classification have some deficiencies in the selection of patients. The objective of this study is to evaluate the impact of TAVI on carbohydrate antigen 125 (CA125) and N-Terminal pro Brain-type Natriuretic Peptide (NT-proBNP) as biomarkers that have been used frequently in recent years, and also the relationship of these biomarkers to prognosis. Methods & Results Transcatheter aortic valve implantation was practiced on 31 patients in this study. Then, CA125 and NT-proBNP levels studied in patients prior to and after the TAVI were evaluated. The patients were also grouped in accordance with their left ventricular ejection fraction (LVEF) and CA125 levels (LVEF ≥ 40% and < 40%; CA125 ≤ 35 U/L and > 35 U/L). The TAVI operation was successfully performed in all patients. There was no in-hospital mortality and substantial improvement in functional capacity was detected at follow ups. In addition, a statistically significant decrease was detected in post-TAVI CA125 and NT-proBNP levels of all patients (CA125 83.8 ± 18.1 U/L vs. 64.3 ± 14.2 U/L, P = 0.008; NT-proBNP: 4633.6 ± 627.6 pg/mL vs. 2866.3 ± 536.8 pg/mL, P < 0.001). In groups divided according to the CA125 levels, there was also statistically significant post-TAVI decline in CA125 levels. Within CA125 > 35 U/L and LVEF < 40% groups, the permanent need for a pacemaker was required in one (3.2%) patient and mortality was observed in two (6.4%) patients after TAVI at follow up. Conclusions The results show that TAVI can be performed effectively and reliably in patients with high baseline levels of CA125 and NT-proBNP. These biomarkers are reduced substantially with TAVI, while high biomarker levels are associated with undesired events, and certainly, these biomarkers can be used for risk classifications in patient selection for TAVI. PMID:25870611

Immunohistochemical localization of constitutive and inducible cyclo-oxygenases in rat uterus during the oestrous cycle and pregnancy.

PubMed

Fang, L; Chatterjee, S; Dong, Y L; Gangula, P R; Yallampalli, C

1998-06-01

The uterus is a rich source of eicosanoids synthesized from arachidonic acid metabolism through the cyclo-oxygenase pathway. Two isoforms of cyclo-oxygenase, constitutive (COX-I) and inducible (COX-II) enzyme, have been reported. In the present study, we have immunohistochemically mapped the distribution of both COX-I and COX-II during various physiological states of the rat uterus. Uterine tissue was collected from female rats (a) during different stages of the oestrous cycle, (b) on days 1, 4, 8 and 18 of gestation, (c) after spontaneous delivery and (d) post partum, and fixed in Bouin's fixative. After paraffin wax embedding, 5-microm-thick sections were immunohistochemically stained by the ABC technique. Observation of the stained sections under the light microscope revealed that, in non-pregnant rat uterus, both COX-I and COX-II were abundantly expressed in the endometrium, with minimal staining observed in the myometrium. Staining was more prominent in epithelial cells than in stromal cells. The intensity of staining in epithelial cells was highest at pro-oestrus and oestrus and lowest at dioestrus. In pregnant rats, although the expression of both COX-I and COX-II was localized primarily to the endometrium with very little staining in the myometrium on day 1 of gestation, both of these enzymes were also apparent in myometrial cells by day 4 of gestation. The staining intensity of endometrial and myometrial cells increased further with the progression of gestation, being maximal at the time of spontaneous delivery. During the post-partum period, however, the staining intensity for both of the enzymes in endometrium and myometrium was decreased. Thus, our studies show that the expression of cyclo-oxygenases in various uterine cells vary with the oestrous cycle and with pregnancy. Furthermore, prominent increases in the expression of cyclo-oxygenases in the myometrium during pregnancy and parturition imply that the cyclo-oxygenase system in the myometrium may play a major role in modulating uterine contractility during pregnancy and labour.

Monitoring of cyclosporine concentrations by using dry blood-spot samples.

PubMed

Mee, A V; Wong, P Y; Sun, C; Oei, L; Elliott, S; Naik, N; Joaquin, B; Uchimaru, D

1991-01-01

We modified the Incstar Cyclo Trac SP kit to enable its use with dry blood-spots on filter paper. The recovery ranged from 92 to 106%. Dilution studies have shown excellent linearity and parallelism throughout the range of the assay. Precision is demonstrated by within-assay CV's of 6.6 and 4.3% at 96 and 342 micrograms/L respectively and between-assay CV's of 9.1 and 7.0% at 138 and 506 micrograms/L respectively. A comparison study (n = 209) with whole blood assay gave a correlation coefficient of 0.97, a slope of 1.04, and an intercept of 13.2. Whole blood and dry blood-spot cyclosporine assays on heart, kidney, liver, and lung transplants were also compared.

Production of Bombyx mori silk fibroin incorporated with unnatural amino acids.

PubMed

Teramoto, Hidetoshi; Kojima, Katsura

2014-07-14

Silk fibroin incorporated with unnatural amino acids was produced by in vivo feeding of p-chloro-, p-bromo-, and p-azido-substituted analogues of L-phenylalanine (Phe) to transgenic silkworms (Bombyx mori) that expressed a mutant of phenylalanyl-tRNA synthetase with expanded substrate recognition capabilities in silk glands. Cutting down the content of Phe in the diet was effective for increasing the incorporation of Phe analogues but simultaneously caused a decrease of fibroin production. The azide groups incorporated in fibroin were active as chemical handles for click chemistry in both the solubilized and the solid (fibrous) states. The azides survived degumming in the boiling alkaline solution that is required for complete removal of the sericin layer, demonstrating that AzPhe-incorporated silk fibroin could be a versatile platform to produce "clickable" silk materials in various forms. This study indicates the huge potential of UAA mutagenesis as a novel methodology to alter the characteristics of B. mori silk.

Comparative experimental infection of Listeria monocytogenes and Listeria ivanovii in bovine trophoblasts.

PubMed

Rocha, Cláudia E; Mol, Juliana P S; Garcia, Luize N N; Costa, Luciana F; Santos, Renato L; Paixão, Tatiane A

2017-01-01

Listeria monocytogenes is a Gram-positive, facultative intracellular and invasive bacterium that has tropism to the placenta, and causes fetal morbidity and mortality in several mammalian species. While infection with L. monocytogenes and L. ivanovii are known as important causes of abortion and reproductive failure in cattle, the pathogenesis of maternal-fetal listeriosis in this species is poorly known. This study used the bovine chorioallantoic membrane explant model to investigate the kinetics of L. monocytogenes, L. ivanovii, and L. innocua infections in bovine trophoblastic cells for up to 8 h post infection. L. monocytogenes and L. ivanovii were able to invade and multiply in trophoblastic cells without causing cell death or inducing expression of pro-inflammatory genes. Although L. innocua was unable to multiply in bovine trophoblastic cells, it induced transcription of the pro-inflammatory mediator CXCL6. This study demonstrated for the first time the susceptibility of bovine trophoblastic cells to L. monocytogenes and L. ivanovii infection.

Comparative experimental infection of Listeria monocytogenes and Listeria ivanovii in bovine trophoblasts

PubMed Central

Rocha, Cláudia E.; Mol, Juliana P. S.; Garcia, Luize N. N.; Costa, Luciana F.; Santos, Renato L.

2017-01-01

Listeria monocytogenes is a Gram-positive, facultative intracellular and invasive bacterium that has tropism to the placenta, and causes fetal morbidity and mortality in several mammalian species. While infection with L. monocytogenes and L. ivanovii are known as important causes of abortion and reproductive failure in cattle, the pathogenesis of maternal-fetal listeriosis in this species is poorly known. This study used the bovine chorioallantoic membrane explant model to investigate the kinetics of L. monocytogenes, L. ivanovii, and L. innocua infections in bovine trophoblastic cells for up to 8 h post infection. L. monocytogenes and L. ivanovii were able to invade and multiply in trophoblastic cells without causing cell death or inducing expression of pro-inflammatory genes. Although L. innocua was unable to multiply in bovine trophoblastic cells, it induced transcription of the pro-inflammatory mediator CXCL6. This study demonstrated for the first time the susceptibility of bovine trophoblastic cells to L. monocytogenes and L. ivanovii infection. PMID:28467447

NT-pro-BNP predicts worsening renal function in patients with chronic systolic heart failure.

PubMed

Pfister, R; Müller-Ehmsen, J; Hagemeister, J; Hellmich, M; Erdmann, E; Schneider, C A

2011-06-01

Worsening renal function (WRF) is frequently observed in patients with heart failure and is associated with worse outcome. The aim of this study was to examine the association of the cardiac serum marker N-terminal pro-B-type natriuretic peptide (NT-pro-BNP) and WRF. A total of 125 consecutive patients of a tertiary care outpatient clinic for heart failure prospectively underwent evaluation of renal function every 6 months. The association of baseline NT-pro-BNP with WRF was analysed during a follow up of 18 months. Twenty-eight (22.4%) patients developed WRF (increase in serum creatinine ≥0.3 mg/dL). Patients with WRF (2870 pg/mL, interquartile range (IQR) 1063-4765) had significantly higher baseline NT-pro-BNP values than patients without WRF (547 pg/mL, IQR 173-1454). The risk for WRF increased by 4.0 (95% CI 2.1-7.5) for each standard deviation of log NT-pro-BNP. In multivariable analysis including age, baseline renal function, ejection fraction, New York Heart Association class and diuretic dose, only NT-pro-BNP and diabetes were independent predictors of WRF. At a cut-off level of 696 pg/mL, NT-pro-BNP showed a sensitivity of 92.9% and a negative predictive value of 96.4% for WRF. NT-pro-BNP is a strong independent predictor of WRF within 18 months in patients with systolic heart failure with a high negative predictive value. Further studies are needed to evaluate reno-protective strategies in patients with elevated NT-pro-BNP. © 2011 The Authors. Internal Medicine Journal © 2011 Royal Australasian College of Physicians.

Structure and receptor-binding activity of insulin from a holostean fish, the bowfin (Amia calva).

PubMed

Conlon, J M; Youson, J H; Whittaker, J

1991-05-15

The holostean fishes are the extant representatives of the primitive ray-finned fishes from which the present-day teleosts may have evolved. The primary structure of insulin from a holostean fish, the bowfin (Amia calva), was established as: A-chain: Gly-Ile-Val-Glu-Gln-Cys-Cys-Leu-Lys-Pro-Cys-Thr-Ile-Tyr-Glu-Met-Glu- Lys-Tyr-Cys-Asn B-chain: Ala-Ala-Ser-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Phe-Leu- Val-Cys-Gly-Glu-Ser-Gly-Phe-Phe-Tyr-Asn-Pro-Asn-Lys-Ser This amino acid sequence contains several substitutions (methionine at A16, phenylalanine at B16 and serine at B22) at sites that have been strongly conserved in other vertebrate species and that may be expected to influence biological activity. Consistent with this prediction, bowfin insulin was approx. 14-fold less potent than pig insulin in inhibiting the binding of [125I-Tyr-A14](human insulin) to transfected mouse NIH 3T3 cells expressing the human insulin receptor.

Structure and receptor-binding activity of insulin from a holostean fish, the bowfin (Amia calva).

PubMed Central

Conlon, J M; Youson, J H; Whittaker, J

1991-01-01

The holostean fishes are the extant representatives of the primitive ray-finned fishes from which the present-day teleosts may have evolved. The primary structure of insulin from a holostean fish, the bowfin (Amia calva), was established as: A-chain: Gly-Ile-Val-Glu-Gln-Cys-Cys-Leu-Lys-Pro-Cys-Thr-Ile-Tyr-Glu-Met-Glu- Lys-Tyr-Cys-Asn B-chain: Ala-Ala-Ser-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Phe-Leu- Val-Cys-Gly-Glu-Ser-Gly-Phe-Phe-Tyr-Asn-Pro-Asn-Lys-Ser This amino acid sequence contains several substitutions (methionine at A16, phenylalanine at B16 and serine at B22) at sites that have been strongly conserved in other vertebrate species and that may be expected to influence biological activity. Consistent with this prediction, bowfin insulin was approx. 14-fold less potent than pig insulin in inhibiting the binding of [125I-Tyr-A14](human insulin) to transfected mouse NIH 3T3 cells expressing the human insulin receptor. PMID:2039477

Characterization of Molecular Determinants of the Conformational Stability of Macrophage Migration Inhibitory Factor: Leucine 46 Hydrophobic Pocket

PubMed Central

El-Turk, Farah; Fauvet, Bruno; Ashrafi, Amer; Ouertatani-Sakouhi, Hajer; Cho, Min-Kyu; Neri, Marilisa; Cascella, Michele; Rothlisberger, Ursula; Pojer, Florence; Zweckstetter, Markus; Lashuel, Hilal

2012-01-01

Macrophage Migration Inhibitory Factor (MIF) is a key mediator of inflammatory responses and innate immunity and has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. The oligomerization of MIF, more specifically trimer formation, is essential for its keto-enol tautomerase activity and probably mediates several of its interactions and biological activities, including its binding to its receptor CD74 and activation of certain signaling pathways. Therefore, understanding the molecular factors governing the oligomerization of MIF and the role of quaternary structure in modulating its structural stability and multifunctional properties is crucial for understanding the function of MIF in health and disease. Herein, we describe highly conserved intersubunit interactions involving the hydrophobic packing of the side chain of Leu46 onto the β-strand β3 of one monomer within a hydrophobic pocket from the adjacent monomer constituted by residues Arg11, Val14, Phe18, Leu19, Val39, His40, Val41, Val42, and Pro43. To elucidate the structural significance of these intersubunit interactions and their relative contribution to MIF’s trimerization, structural stability and catalytic activity, we generated three point mutations where Leu46 was replaced by glycine (L46G), alanine (L46A) and phenylalanine (L46F), and their structural properties, stability, oligomerization state, and catalytic activity were characterized using a battery of biophysical methods and X-ray crystallography. Our findings provide new insights into the role of the Leu46 hydrophobic pocket in stabilizing the conformational state of MIF in solution. Disrupting the Leu46 hydrophobic interaction perturbs the secondary and tertiary structure of the protein but has no effect on its oligomerization state. PMID:23028743

Role of VIP and substance P in NANC innervation in the longitudinal smooth muscle of the rat jejunum - influence of extrinsic denervation.

PubMed

Kasparek, Michael S; Fatima, Javairiah; Iqbal, Corey W; Duenes, Judith A; Sarr, Michael G

2007-07-01

This study was designed to determine changes in nonadrenergic, noncholinergic (NANC) neurotransmission mediated by Vasoactive Intestinal Polypeptide (VIP) and Substance P after small bowel transplantation (SBT). Six groups of rats (n > or = 6 per group) were studied: naïve controls (NC); 1 wk after anesthesia/sham celiotomy (SC-1); 1 or 8 wk after jejunal and ileal transection/reanastomosis (TA-1, TA-8), or syngeneic, orthotopic SBT (SBT-1, SBT-8). Jejunal longitudinal muscle strips were studied under NANC-conditions for spontaneous contractile activity, response to exogenous VIP and Substance P, and electrical field stimulation (EFS). Spontaneous activity did not differ between the six groups. VIP inhibited contractile activity in all groups 1 wk postoperatively (P < 0.05), which was prevented by the NO synthase inhibitor L-N(G)-nitro arginine (L-NNA). In contrast, VIP had no effect in the other groups. Precontraction with Substance P exposed an inhibitory effect of VIP in all groups (P < 0.05 each). Substance P increased contractile activity in all groups, but to a lesser extent in SBT-8 compared with NC, TA-8, and SBT-1 (P < 0.05). The inhibitory effect of EFS at 6 Hz was prevented by L-NNA in NC and TA-8; addition of the VIP antagonist ([D-p-Cl-Phe(6), Leu(17)]-VIP) increased contractile activity in NC, but not in TA-8 and SBT-8. The Substance P antagonist ([D-Pro(2), D-Trp(7,9)]-Substance P) decreased contractile activity during EFS at 50 Hz in NC and SBT-8. SBT decreased response to exogenous Substance P, although release of endogenous Substance P (EFS) is preserved. Changes in VIP signaling are acute and reversible and not caused by effects of SBT.

Characterization of molecular determinants of the conformational stability of macrophage migration inhibitory factor: leucine 46 hydrophobic pocket.

PubMed

El-Turk, Farah; Fauvet, Bruno; Ashrafi, Amer; Ouertatani-Sakouhi, Hajer; Cho, Min-Kyu; Neri, Marilisa; Cascella, Michele; Rothlisberger, Ursula; Pojer, Florence; Zweckstetter, Markus; Lashuel, Hilal

2012-01-01

Macrophage Migration Inhibitory Factor (MIF) is a key mediator of inflammatory responses and innate immunity and has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. The oligomerization of MIF, more specifically trimer formation, is essential for its keto-enol tautomerase activity and probably mediates several of its interactions and biological activities, including its binding to its receptor CD74 and activation of certain signaling pathways. Therefore, understanding the molecular factors governing the oligomerization of MIF and the role of quaternary structure in modulating its structural stability and multifunctional properties is crucial for understanding the function of MIF in health and disease. Herein, we describe highly conserved intersubunit interactions involving the hydrophobic packing of the side chain of Leu46 onto the β-strand β3 of one monomer within a hydrophobic pocket from the adjacent monomer constituted by residues Arg11, Val14, Phe18, Leu19, Val39, His40, Val41, Val42, and Pro43. To elucidate the structural significance of these intersubunit interactions and their relative contribution to MIF's trimerization, structural stability and catalytic activity, we generated three point mutations where Leu46 was replaced by glycine (L46G), alanine (L46A) and phenylalanine (L46F), and their structural properties, stability, oligomerization state, and catalytic activity were characterized using a battery of biophysical methods and X-ray crystallography. Our findings provide new insights into the role of the Leu46 hydrophobic pocket in stabilizing the conformational state of MIF in solution. Disrupting the Leu46 hydrophobic interaction perturbs the secondary and tertiary structure of the protein but has no effect on its oligomerization state.

Microfluidic assay of platelet deposition on collagen by perfusion of whole blood from healthy individuals taking aspirin.

PubMed

Li, Ruizhi; Fries, Susanne; Li, Xuanwen; Grosser, Tilo; Diamond, Scott L

2013-08-01

Microfluidic devices can create hemodynamic conditions for platelet assays. We validated an 8-channel device in a study of interdonor response to acetylsalicylic acid (ASA, aspirin) with whole blood from 28 healthy individuals. Platelet deposition was assessed before treatment or 24 h after ingestion of 325 mg ASA. Whole blood (plus 100 μmol/L H-d-Phe-Pro-Arg-chloromethylketone to inhibit thrombin) was further treated ex vivo with ASA (0-500 μmol/L) and perfused over fibrillar collagen for 300 s at a venous wall shear rate (200 s(-1)). Ex vivo ASA addition to blood drawn before aspirin ingestion caused a reduction in platelet deposition [half-maximal inhibitory concentration (IC50) approximately 10-20 μmol/L], especially between 150 and 300 s of perfusion, when secondary aggregation mediated by thromboxane was expected. Twenty-seven of 28 individuals displayed smaller deposits (45% mean reduction; range 10%-90%; P < 0.001) from blood obtained 24 h after ASA ingestion (no ASA added ex vivo). In replicate tests, an R value to score secondary aggregation [deposition rate from 150 to 300 s normalized by rate from 60 to 150 s] showed R < 1 in only 2 of 28 individuals without ASA ingestion, with R > 1 in only 3 of 28 individuals after 500 μmol/L ASA addition ex vivo. At 24 h after ASA ingestion, 21 of 28 individuals displayed poor secondary aggregation (R < 1) without ex vivo ASA addition, whereas the 7 individuals with residual secondary aggregation (R > 1) displayed insensitivity to ex vivo ASA addition. Platelet deposition was not correlated with platelet count. Ex vivo ASA addition caused similar inhibition at venous and arterial wall shear rates. Microfluidic devices quantified platelet deposition after ingestion or ex vivo addition of aspirin.

Cardiovirus Leader proteins bind exportins: Implications for virus replication and nucleocytoplasmic trafficking inhibition

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

Ciomperlik, Jessica J.; Basta, Holly A.; Palmenberg, Ann C., E-mail: acpalmen@wisc.edu

2016-01-15

Cardiovirus Leader proteins (L{sub X}) inhibit cellular nucleocytoplasmic trafficking by directing host kinases to phosphorylate Phe/Gly-containing nuclear pore proteins (Nups). Resolution of the Mengovirus L{sub M} structure bound to Ran GTPase, suggested this complex would further recruit specific exportins (karyopherins), which in turn mediate kinase selection. Pull-down experiments and recombinant complex reconstitution now confirm that Crm1 and CAS exportins form stable dimeric complexes with encephalomyocarditis virus L{sub E}, and also larger complexes with L{sub E}:Ran. shRNA knockdown studies support this idea. Similar activities could be demonstrated for recombinant L{sub S} and L{sub T} from Theiloviruses. When mutations were introduced tomore » alter the L{sub E} zinc finger domain, acidic domain, or dual phosphorylation sites, there was reduced exportin selection. These regions are not involved in Ran interactions, so the Ran and Crm1 binding sites on L{sub E} must be non-overlapping. The involvement of exportins in this mechanism is important to viral replication and the observation of trafficking inhibition by L{sub E}.« less

Association of cardiac and renal function with extreme N-terminal fragment pro-B-type natriuretic peptide levels in elderly patients.

PubMed

Cui, Hua; Huo, Guoliang; Liu, Lin; Fan, Li; Ye, Ping; Cao, Jian; Bai, Yongyi; Wang, Fan; Hu, Yixin

2012-07-26

The data are inconsistent regarding whether extreme N-terminal fragment pro-B-type natriuretic peptide (NT pro-BNP) levels are associated with impaired renal function. Furthermore, the relationship between extreme NT pro-BNP levels and cardiac and renal function in elderly patients has not been reported. The aim of the present study was to examine a hypothesis that extreme NT pro-BNP levels may be associated with impaired cardiac and renal function in elderly patients. We retrospectively analyzed the data of demographic, clinical, and echocardiographic features on 152 consecutive elderly patients aged more than 80 years old (average age, 83.65 ± 3.58 years) with NT pro-BNP levels ≥ 3000 pg/ml. The participants were divided into two categories according to their NT pro-BNP levels: (1) 3000-10000 pg/mL and (2) >10000 pg /mL. The number of patients with impaired renal function (P = 0.019) and the mortality (P < 0.001) in the period of inpatient was higher in the group with NT pro-BNP > 10000 pg /mL. The levels of serum creatinine and creatine kinase MB (CK-MB) in the group of NT pro-BNP > 10000 pg / mL were higher than those in the group of NT pro-BNP = 3000-10000 pg/mL (P = 0.001 and P = 0.023, respectively). Furthermore, no significant difference in the distribution by NYHA class in different NT pro-BNP levels was observed. Multiple linear regression analyses demonstrated that with NT pro-BNP levels as the dependent variable, NT pro-BNP levels were positively correlated with CK-MB (β = 0.182, P = 0.024) and creatinine levels (β = 0.281, P = 0.001). The area under the receiver-operating characteristic (ROC) curve of NT pro-BNP levels and clinical diagnosis of impaired renal function was 0.596 and reached significant difference (95%CI:0.503-0.688, P = 0.044). These data suggest that the extreme elevation of NT pro-BNP levels (≥3000 pg/ml) is mainly determined by impaired renal function in elderly patients above 80 years. Extreme NT pro-BNP levels may be useful for assessing the severity of impaired renal function.

Effects of phenylalanine, histidine, and leucine on basal and GHRH-stimulated GH secretion and on PRL, insulin, and glucose levels in short children. Comparison with the effects of arginine.

PubMed

Bellone, J; Valetto, M R; Aimaretti, G; Segni, M; Volta, C; Cardimale, G; Baffoni, C; Pasquino, A M; Bernasconi, S; Bartolotta, E; Mucci, M; Ghigo, E

1996-01-01

Of the amino acids arginine is the most potent GH secretagogue in man. It potentiates the GH response to GHRH, exerts a weaker PRL-releasing effect, stimulates insulin and glucagon and induces a biphasic glucose variation. The potency and effects of other amino acids on pituitary and pancreatic hormones need to be clarified. In 43 children with normal short stature (5.3-14.0 yr; 30 M and 13 F) the effects of the infusion of phenylalanine (Phe, 0.08 g/kg), histidine (His, 0.1 g/kg), and leucine (Leu, 0.08 g/kg) on basal and GHRH-stimulated GH secretion and on PRL, insulin and glucose levels were studied and compared with those of arginine at high (hArg, 0.5 g/kg) or low dose (lArg, 0.2 g/kg). Phe increased basal (p < 0.05) but not GHRH-stimulated GH levels, induced PRL and insulin rises (p < 0.03 and p < 0.03), and did not change glycemia. Though a trend toward an increase in basal GH levels was found after His, His and Leu did not significantly modify either basal or GHRH-induced GH secretion nor basal PRL, insulin and glucose levels. Both hArg and lArg increased basal (p < 0.0001 and p < 0.05, respectively) and GHRH-stimulated GH levels (p < 0.006 and p < 0.006). hArg increased both PRL (p < 0.002) and insulin levels (p < 0.005) more (p < 0.0005 and p < 0.004) than lArg (p < 0.005 and p < 0.005), while glucose levels showed a similar increase followed by a similar decrease. We conclude that in childhood: a) Phe significantly increases GH secretion but, differently from Arg, does not potentiate the response to GHRH, suggesting different mechanisms of action of these amino acids; b) differently from His and Leu, Phe is a PRL and insulin secretagogue but is less potent than Arg; c) Arg has the highest stimulatory effect on pituitary and pancreatic hormones.

Plasma Proenkephalin and Poor Long-Term Outcome in Renal Transplant Recipients

PubMed Central

Kieneker, Lyanne M.; Hartmann, Oliver; Struck, Joachim; Bergmann, Andreas; Gansevoort, Ron T.; Joosten, Michel M.; van den Berg, Else; de Boer, Rudolf A.; Bakker, Stephan J.L.

2017-01-01

Background Proenkephalin (pro-ENK), a stable and reliable surrogate marker for unstable enkephalins, was found to be associated with acute kidney injury and chronic renal failure in previous studies. We aimed to investigate whether pro-ENK is linked to chronic kidney injury and poor long-term outcome in renal transplant recipients (RTR). Methods We included 664 stable RTR and 95 healthy kidney donors. Pro-ENK was measured in plasma with a double monoclonal sandwich immunoassay. Graft failure was defined as return to dialysis therapy or retransplantation. Results Median pro-ENK was 110 pmol/L (interquartile range [IQR], 85-148 pmol/L) in RTR and 48 pmol/L (IQR, 42-55 pmol/L) in kidney donors. Pro-ENK was correlated with estimated glomerular filtration rate (GFR) (rs = −0.80, P < 0.001) in RTR and with measured GFR (rs = −0.74, P < 0.001) in kidney donors. During a median follow-up of 3.1 years (IQR, 2.7-3.9 years), 45 RTR developed graft failure and 76 died. Pro-ENK was positively associated with risk (hazard ratio [HR] per standard deviation increment of the logarithm of pro-ENK; 95% confidence interval [CI]) of graft failure (HR, 4.80; 95% CI, 3.55-6.48) and mortality (HR, 1.50; 95% CI, 1.22-1.85). After adjustment of age, sex, and estimated GFR, the association of pro-ENK with graft failure remained significant (HR, 2.36; 95% CI, 1.37-4.06), whereas no significant association of pro-ENK with risk of all-cause mortality was observed (HR, 1.34; 95% CI, 0.90-2.09). Conclusions Plasma pro-ENK is associated with kidney function as reflected by correlations with measured GFR in both RTR and kidney donors. In addition, pro-ENK was independently associated with increased risk of graft failure in RTR. Pro-ENK may aid in identification of RTR at risk for late graft failure. PMID:28795142

Apple cider vinegar boosted immunomodulatory and health promoting effects of Lactobacillus casei in common carp (Cyprinus carpio).

PubMed

Safari, Roghieh; Hoseinifar, Seyed Hossein; Nejadmoghadam, Shabnam; Khalili, Mohsen

2017-08-01

The present study was performed to investigate the immunomodulatory and health promoting effects of combined or singular administration of apple cider vinegar (ACV) and Lactobacillus casei in common carp (Cyprinus carpio) diet. An 8-week feeding trial was designed with following treatments: Control (basal diet), Pro (contains 10 7  CFU g -1 L. casei), LACV (contains 1% ACV), HACV (contains 2% ACV), Pro + LACV (contains 10 7  CFU g -1 L. casei plus 1% ACV) and Pro + HACV (contains 10 7  CFU g -1 L. casei plus 2% ACV). Evaluation of skin mucus revealed notable increase of total Ig level and lysozyme activity in Pro + LACV and Pro + HACV treatments compared other groups (P < 0.05). Similarly, serum total Ig and lysozyme activity in HACV, Pro + LACV and Pro + HACV fed carps was remarkably higher than other groups (P < 0.05). However, regarding serum alternative complement (ACH50) activity significant difference was observed just between Pro + HACV and control treatment (P < 0.05). The highest expression of immune related (LYZ, TNF-alpha, IL1b, IL8) and antioxidant enzymes genes (GSR, GST) were observed in carps fed Pro + HACV and Pro + LACV. The expression of GH gene expression in Pro, LACV and HACV treatments was significantly higher than those in control group (P < 0.05). The highest expression level of GH and IGF1 was observed in fish fed combined Pro and ACV (P < 0.05). These results indicated that co-administration of ACV boosted immunomodulatory and health promoting effects of L. casei and can be considered as a promising immunostimulants in early stage of common carp culture. Copyright © 2017 Elsevier Ltd. All rights reserved.

Inhibition of Arabidopsis growth by the allelopathic compound azetidine-2-carboxylate is due to the low amino acid specificity of cytosolic prolyl-tRNA synthetase.

PubMed

Lee, Jiyeon; Joshi, Naveen; Pasini, Rita; Dobson, Renwick C J; Allison, Jane; Leustek, Thomas

2016-10-01

The toxicity of azetidine-2-carboxylic acid (A2C), a structural analogue of L-proline, results from its incorporation into proteins due to misrecognition by prolyl-tRNA synthetase (ProRS). The growth of Arabidopsis thaliana seedling roots is more sensitive to inhibition by A2C than is cotyledon growth. Arabidopsis contains two ProRS isozymes. AtProRS-Org (At5g52520) is localized in chloroplasts/mitochondria, and AtProRS-Cyt (At3g62120) is cytosolic. AtProRS-Cyt mRNA is more highly expressed in roots than in cotyledons. Arabidopsis ProRS isoforms were expressed as His-tagged recombinant proteins in Escherichia coli. Both enzymes were functionally active in ATP-PPi exchange and aminoacylation assays, and showed similar K m for L-proline. A major difference was observed in the substrate specificity of the two enzymes. AtProRS-Cyt showed nearly identical substrate specificity for L-proline and A2C, but for AtProRS-Org the specificity constant was 77.6 times higher for L-proline than A2C, suggesting that A2C-sensitivity may result from lower amino acid specificity of AtProRS-Cyt. Molecular modelling and simulation results indicate that this specificity difference between the AtProRS isoforms may result from altered modes of substrate binding. Similar kinetic results were obtained with the ProRSs from Zea mays, suggesting that the difference in substrate specificity is a conserved feature of ProRS isoforms from plants that do not accumulate A2C and are sensitive to A2C toxicity. The discovery of the mode of action of A2C toxicity could lead to development of biorational weed management strategies. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Importance of tetrahedral intermediate formation in the catalytic mechanism of the serine proteases chymotrypsin and subtilisin.

PubMed

Petrillo, Teodolinda; O'Donohoe, Catrina A; Howe, Nicole; Malthouse, J Paul G

2012-08-07

Two new inhibitors in which the terminal α-carboxyl groups of Z-Ala-Ala-Phe-COOH and Z-Ala-Pro-Phe-COOH have been replaced with a proton to give Z-Ala-Ala-Phe-H and Z-Ala-Pro-Phe-H, respectively, have been synthesized. Using these inhibitors, we estimate that for α-chymotrypsin and subtilisin Carlsberg the terminal carboxylate group decreases the level of inhibitor binding 3-4-fold while a glyoxal group increases the level of binding by 500-2000-fold. We show that at pH 7.2 the effective molarities of the catalytic hydroxyl group of the active site serine are 41000-229000 and 101000-159000 for α-chymotrypsin and subtilisin Carlsberg, respectively. It is estimated that oxyanion stabilization and the increased effective molarity of the catalytic serine hydroxyl group can account for the catalytic efficiency of the reaction. We argue that substrate binding induces the formation of a strong hydrogen bond or low-barrier hydrogen bond between histidine-57 and aspartate-102 that increases the pK(a) of the active site histidine, allowing it to be an effective general base catalyst for the formation of the tetrahedral intermediate and increasing the effective molarity of the catalytic hydroxyl group of serine-195. A catalytic mechanism for acyl intermediate formation in the serine proteases is proposed.

Blood N-terminal Pro-brain Natriuretic Peptide and Interleukin-17 for Distinguishing Incomplete Kawasaki Disease from Infectious Diseases.

PubMed

Wu, Ling; Chen, Yuanling; Zhong, Shiling; Li, Yunyan; Dai, Xiahua; Di, Yazhen

2015-06-01

To explore the diagnostic value of blood N-terminal pro-brain natriuretic peptide (NT-proBNP) and interleukin-17(IL-17) for incomplete Kawasaki disease. Patients with Kawasaki disease, Incomplete Kawasaki disease and unclear infectious fever were included in this retrospective study. Their clinical features, and laboratory test results of blood NT-proBNP and IL-17 were collected and compared. 766 patients with complete clinical information were recruited, consisting of 291 cases of Kawasaki disease, 74 cases of incomplete Kawasaki disease, and 401 cases of unclear infectious diseases. When the consistency with indicator 2 and 3 in Kawasaki disease diagnosis criteria was assessed with blood IL-17 ?11.55 pg/mL and blood NT-proBNP ? 225.5 pg/dL as the criteria, the sensitivity and specificity for distinguishing incomplete Kawasaki disease and infectious diseases reached 86.5% and 94.8%, respectively. When we chose the consistency with indicator 1 and 2 in Kawasaki disease diagnosis criteria, the appearance of decrustation and/or the BCG erythema, blood IL-17 ?11.55 pg/mL and blood NT-Pro BNP ?225.5 pg/dL as the criteria, the sensitivity and specificity for distinguishing incomplete Kawasaki disease and infectious diseases was 43.2% and 100%, respectively. Blood NT-proBNP and IL-17 are useful laboratory indicators for distinguishing incomplete Kawasaki disease and infectious diseases at the early stage.

Total plasma proANP increases with atrial dilatation in horses.

PubMed

Van Der Vekens, N; Hunter, I; Timm, A; Decloedt, A; De Clercq, D; Deprez, P; Goetze, J P; van Loon, G

2015-12-19

Equine atrial natriuretic peptide (ANP) plasma concentrations are correlated with left atrial size. However, species-specific assays are lacking and the results from human assays are poorly reproducible. A new methodology called processing independent analysis (PIA) that measures the total proANP product in plasma has proven to be successful in human medicine, but has never been used in horses. The aims were to establish an equine proANP reference interval by measurement of the total proANP product using PIA and to examine the proANP concentrations in horses with atrial dilatation. Sample stability was studied by comparison of storage at -80°C and -20°C. Plasma samples were obtained from 23 healthy horses, 12 horses with moderate or severe valvular regurgitation without atrial dilatation and 42 horses with valvular regurgitation and atrial dilatation. The proANP concentration was significantly (P<0.001) higher in horses with atrial dilatation (761.4 (442.1-1859.1) pmol/l) than in healthy horses (491.6 (429.5-765.9) pmol/l; P<0.001) or horses with cardiac disease but without atrial dilatation (544.4 (457.0-677.6) pmol/l). A cut-off value (573.8 pmol/l) for detection of atrial dilatation was calculated. Sample storage at -80°C did not differ from sample storage at -20°C. The measurement of total proANP in plasma detects atrial dilatation in horses and may be useful for clinical evaluation in equine medicine. British Veterinary Association.

Synthesis and structure analysis of ferrocene-containing pseudopeptides.

PubMed

Angelici, Gaetano; Górecki, Marcin; Pescitelli, Gennaro; Zanna, Nicola; Monari, Magda; Tomasini, Claudia

2017-10-23

Ferrocene with its aromaticity and facile redox properties is an attractive moiety to be incorporated into functional moieties. Medicinal applications of ferrocene are well known and ferrocene itself shows cytotoxic and antianemic properties. In this article, we will describe the synthesis and the structure analysis of two pseudopeptides containing a ferrocene moiety as N-terminal group. After purification, Fc-l-Phe-d-Oxd-OBn [l-Phel-phenylalanine; d-Oxd(4R,5S)-4-Methyl-5-carboxy-oxazolidin-2-one] appears as bright brown solid that spontaneously forms brown needles. The X-ray diffraction of the crystals shows the presence of strong π interactions between the ferrocenyl moiety and the phenyl rings, while no NH•••OC hydrogen bonds are formed. This result is confirmed by FT-IR and 1 H NMR analysis. In contrast, both FT-IR and 1 H NMR analysis suggest that Fc-(l-Phe-d-Oxd) 2 -OBn forms a turn conformation stabilized by intramolecular NH•••OC hydrogen bonds in solution. Chiroptical spectroscopies (ECD and VCD) substantially confirmed the absence of a well-defined folded structure. The presence of the Fc moiety is responsible for specific ECD signals, one of which displayed pronounced temperature dependence and is directly related with the helicity assumed by the Fc core. Solid-state ECD spectra were recorded and rationalized on the basis of the X-ray geometry and quantum-mechanical calculations. © 2017 Wiley Periodicals, Inc.

Analysis of crystal structure of Arabidopsis MPK6 and generation of its mutants with higher activity

PubMed Central

Wang, Bo; Qin, Xinghua; Wu, Juan; Deng, Hongying; Li, Yuan; Yang, Hailian; Chen, Zhongzhou; Liu, Guoqin; Ren, Dongtao

2016-01-01

Mitogen-activated protein kinase (MAPK) cascades, which are the highly conserved signalling modules in eukaryotic organisms, have been shown to play important roles in regulating growth, development, and stress responses. The structures of various MAPKs from yeast and animal have been solved, and structure-based mutants were generated for their function analyses, however, the structures of plant MAPKs remain unsolved. Here, we report the crystal structure of Arabidopsis MPK6 at a 3.0 Å resolution. Although MPK6 is topologically similar to ERK2 and p38, the structures of the glycine-rich loop, MAPK insert, substrate binding sites, and L16 loop in MPK6 show notable differences from those of ERK2 and p38. Based on the structural comparison, we constructed MPK6 mutants and analyzed their kinase activity both in vitro and in planta. MPK6F364L and MPK6F368L mutants, in which Phe364 and Phe368 in the L16 loop were changed to Leu, respectively, acquired higher intrinsic kinase activity and retained the normal MAPKK activation property. The expression of MPK6 mutants with basal activity is sufficient to induce camalexin biosynthesis; however, to induce ethylene and leaf senescence, the expression of MPK6 mutants with higher activity is required. The results suggest that these mutants can be used to analyze the specific biological functions of MPK6. PMID:27160427

Purification and characterization of two isoenzymes of lipoxygenase from soybeans.

PubMed

Diel, E; Stan, H J

1978-01-01

A chromatographic procedure for the purification of two lipoxygenase isoenzymes (linoleate: O2 oxidoreductase, EC 1.13.11.12.) from soybean is described. The procedure for the purification of isoenzyme L-1 includes optimalized extraction, ammonium sulfate fractionation, heat treatment and gradient elution from a CM-Sephadex C-50 column. The purification of L-2 includes ammonium sulfate fractionation, gelfiltration on Sephadex G-150 and gradient elution from a DEAE-cellulose column. Both isoenzymes L-1 and L-2 appear homogeneous after Disc-PAGE. The isoelectric points are 5.6 for L-1 and 5.8 for L-2. Molecular weights are estimated as 100,000 for L-1 as well as L-2 applying three different methods. Both isoenzymes contain 0.9 mol iron per mol protien. The estimated turn over numbers are 8,200 mol linoleate per mol enzyme and min for L-1 and 3,100 for L-2. Amino acid compositions determined after acid hydrolysis show marked differences between L-1 and L-2, particularly with respect to the amino acids Lys, Phe, Ser, Gly and Leu. L-1 posesses a total of 9 cysteine molecules, 6 of which are present as disulfide bonds. L-2 posesses a total of 8 cysteine molecules with only one disulfide bond.

ER-associated degradation is required for vasopressin prohormone processing and systemic water homeostasis

PubMed Central

Somlo, Diane R.M.; Kim, Geun Hyang; Prescianotto-Baschong, Cristina; Sun, Shengyi; Beuret, Nicole; Long, Qiaoming; Rutishauser, Jonas

2017-01-01

Peptide hormones are crucial regulators of many aspects of human physiology. Mutations that alter these signaling peptides are associated with physiological imbalances that underlie diseases. However, the conformational maturation of peptide hormone precursors (prohormones) in the ER remains largely unexplored. Here, we report that conformational maturation of proAVP, the precursor for the antidiuretic hormone arginine-vasopressin, within the ER requires the ER-associated degradation (ERAD) activity of the Sel1L-Hrd1 protein complex. Serum hyperosmolality induces expression of both ERAD components and proAVP in AVP-producing neurons. Mice with global or AVP neuron–specific ablation of Se1L-Hrd1 ERAD progressively developed polyuria and polydipsia, characteristics of diabetes insipidus. Mechanistically, we found that ERAD deficiency causes marked ER retention and aggregation of a large proportion of all proAVP protein. Further, we show that proAVP is an endogenous substrate of Sel1L-Hrd1 ERAD. The inability to clear misfolded proAVP with highly reactive cysteine thiols in the absence of Sel1L-Hrd1 ERAD causes proAVP to accumulate and participate in inappropriate intermolecular disulfide–bonded aggregates, promoted by the enzymatic activity of protein disulfide isomerase (PDI). This study highlights a pathway linking ERAD to prohormone conformational maturation in neuroendocrine cells, expanding the role of ERAD in providing a conducive ER environment for nascent proteins to reach proper conformation. PMID:28920920

Location of alkali metal binding sites in endothelin A selective receptor antagonists, cyclo(D-Trp-D-Asp-Pro-D-Val-Leu) and cyclo(D-Trp-D-Asp-Pro-D-Ile-Leu), from multistep collisionally activated decompositions.

PubMed

Ngoka, L C; Gross, M L

2000-02-01

We previously showed by using mass spectrometry that endothelin A selective receptor antagonists BQ123 and JKC301 form novel coordination compounds with sodium ions. This property may underlie the ability of an ET(A) antagonist to induce net tubular sodium reabsorption in the proximal tubule cells and reverse acute renal failure induced by severe ischemia. We have now defined the metal binding sites on BQ123 and JKC301 by subjecting the metal-containing peptides to multiple stages of collisionally activated decomposition (CAD) in an ion trap mass spectrometer. When submitted to low-energy CAD, the ring opens at the Asp-Pro amide bond. The metal ion, which bonds, inter alia, to the carbonyl oxygen of the proline residue, acts as a fixed charge site, and directs a charge-remote, sequence-specific fragmentation of the ring-opened peptide. Amino acid residues are sequentially cleaved from the C-terminal end, and the terminal aziridinone structure moves one step toward the N-terminus with each C-terminal amino acid residue removed. These observations are the basis of a new method to sequence cyclic peptides. Amino acid residues are observed as sets of three ions, a*(n)PD, b*(n)PD and c*(n)PD where n is the number of amino acid residues in the peptide. Copyright 2000 John Wiley & Sons, Ltd.

AKT activation drives the nuclear localization of CSE1L and a pro-oncogenic transcriptional activation in ovarian cancer cells

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

Lorenzato, Annalisa; Biolatti, Marta; Institute for Cancer Research at Candiolo, Candiolo, Torino

The human homolog of the yeast cse1 gene (CSE1L) is over-expressed in ovarian cancer. CSE1L forms complex with Ran and importin-α and has roles in nucleocytoplasmic traffic and gene expression. CSE1L accumulated in the nucleus of ovarian cancer cell lines, while it was localized also in the cytoplasm of other cancer cell lines. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells, as the CSE1L protein translocated to the cytoplasm when AKT was inactivated. Moreover, the expression of a constitutively active AKT forced the translocation of CSE1L from the cytoplasm to the nucleus in other cancermore » cells. Nuclear accrual of CSE1L was associated to the nuclear accumulation of the phosphorylated Ran Binding protein 3 (RanBP3), which depended on AKT as well. Also in samples of human ovarian cancer, AKT activation was associated to nuclear accumulation of CSE1L and phosphorylation of RanBP3. Expression profiling of ovarian cancer cells after CSE1L silencing showed that CSE1L was required for the expression of genes promoting invasion and metastasis. In agreement, CSE1L silencing impaired motility and invasiveness of ovarian cancer cells. Altogether these data show that in ovarian cancer cells activated AKT by affecting RanBP3 phosphorylation determines the nuclear accumulation of CSE1L and likely the nuclear concentration of transcription factors conveying pro-oncogenic signals. - highlights: • CSE1L is a key player in nucleocytoplasmic traffic by forming complex with Ran. • AKT phosphorylates RanBP3 that regulates the nucleocytoplasmic gradient of Ran. • The activated oncogenic AKT drives the nuclear accumulation of CSE1L. • CSE1L in the nucleus up-regulates genes conveying pro-oncogenic signals. • CSE1L might contribute to tumor progression driven by the activated oncogenic AKT.« less

Purification, biochemical, and structural characterization of a novel fibrinolytic enzyme from Mucor subtilissimus UCP 1262.

PubMed

Nascimento, Thiago Pajeú; Sales, Amanda Emmanuelle; Porto, Tatiana Souza; Costa, Romero Marcos Pedrosa Brandão; Breydo, Leonid; Uversky, Vladimir N; Porto, Ana Lúcia Figueiredo; Converti, Attilio

2017-08-01

Fibrinolytic proteases are enzymes that degrade fibrin. They provide a promising alternative to existing drugs for thrombolytic therapy. A protease isolated from the filamentous fungus Mucor subtilissimus UCP 1262 was purified in three steps by ammonium sulfate fractionation, ion exchange, and molecular exclusion chromatographies, and characterized biochemically and structurally. The purified protease exhibited a molecular mass of 20 kDa, an apparent isoelectric point of 4.94 and a secondary structure composed mainly of α-helices. Selectivity for N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as substrate suggests that this enzyme is a chymotrypsin-like serine protease, whose activity was enhanced by the addition of Cu 2+ , Mg 2+ , and Fe 2+ . The enzyme showed a fibrinolytic activity of 22.53 U/mL at 40 °C and its contact with polyethylene glycol did not lead to any significant alteration of its secondary structure. This protein represents an important example of a novel fibrinolytic enzyme with potential use in the treatment of thromboembolic disorders such as strokes, pulmonary emboli, and deep vein thrombosis.

Facile and selective covalent grafting of an RGD-peptide to electrospun scaffolds improves HUVEC adhesion.

PubMed

Dettin, Monica; Zamuner, Annj; Roso, Martina; Iucci, Giovanna; Samouillan, Valerie; Danesin, Roberta; Modesti, Michele; Conconi, Maria Teresa

2015-10-01

The development of a biomimetic surface able to promote endothelialization is fundamental in the search for blood vessel substitutes that prevent the formation of thrombi or hyperplasia. This study aims at investigating the effect of functionalization of poly-ε-caprolactone or poly(L-lactic acid-co-ɛ-caprolactone) electrospun scaffolds with a photoreactive adhesive peptide. The designed peptide sequence contains four Gly-Arg-Gly-Asp-Ser-Pro motifs per chain and a p-azido-Phe residue at each terminus. Different peptide densities on the scaffold surface were obtained by simply modifying the peptide concentration used in pretreatment of the scaffold before UV irradiation. Scaffolds of poly-ε-caprolactone embedded with adhesive peptides were produced to assess the importance of peptide covalent grafting. Our results show that the scaffolds functionalized with photoreactive peptides enhance adhesion at 24 h with a dose-dependent effect and control the proliferation of human umbilical vein endothelial cells, whereas the inclusion of adhesive peptide in the electrospun matrices by embedding does not give satisfactory results. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

Effect of dietary organic selenium on muscle proteolytic activity and water-holding capacity in pork.

PubMed

Calvo, L; Toldrá, F; Aristoy, M C; López-Bote, C J; Rey, A I

2016-11-01

This study evaluates the effect of dietary selenium (Se) supplementation source (organic, Se-enriched yeast; SY vs. inorganic, sodium selenite; SS), dose (0.2: L vs. 0.4: H mg/kg) and the combination of Se and vitamin E (VITE+SS) for 26days on drip loss, TBARS, colour changes, myofibrillar protein pattern and proteolysis in pork. The lowest water losses were observed in the SY-H group when compared to the others. SY-H and VITE+SS groups presented lower myofibrillar protein hydrolysis/oxidation. VITE+SS supplementation also resulted in higher PRO, TRP and PHE content at days 2 and 7, whereas the SY group showed increased GLY and CAR and tended to have higher TAU and ANS at day 2. The myofibrillar fragmentation index was not modified by the dietary treatment; however, at day 8, it tended to be higher in groups supplemented with SeY and VITE+SS. The results of the present study might indicate a possible relation between muscle proteolysis and water loss. Copyright © 2016 Elsevier Ltd. All rights reserved.

Smad phosphoisoform signals in acute and chronic liver injury: similarities and differences between epithelial and mesenchymal cells.

PubMed

Matsuzaki, Koichi

2012-01-01

Hepatocellular carcinoma (HCC) usually arises from hepatic fibrosis caused by chronic inflammation. In chronic liver damage, hepatic stellate cells undergo progressive activation to myofibroblasts (MFB), which are important extracellular-matrix-producing mesenchymal cells. Concomitantly, perturbation of transforming growth factor (TGF)-β signaling by pro-inflammatory cytokines in the epithelial cells of the liver (hepatocytes) promotes both fibrogenesis and carcinogenesis (fibro-carcinogenesis). Insights into fibro-carcinogenic effects on chronically damaged hepatocytes have come from recent detailed analyses of the TGF-β signaling process. Smad proteins, which convey signals from TGF-β receptors to the nucleus, have intermediate linker regions between conserved Mad homology (MH) 1 and MH2 domains. TGF-β type I receptor and pro-inflammatory cytokine-activated kinases differentially phosphorylate Smad2 and Smad3 to create phosphoisoforms phosphorylated at the COOH-terminal, linker, or both (L/C) regions. After acute liver injury, TGF-β-mediated pSmad3C signaling terminates hepatocytic proliferation induced by the pro-inflammatory cytokine-mediated mitogenic pSmad3L pathway; TGF-β and pro-inflammatory cytokines synergistically enhance collagen synthesis by activated hepatic stellate cells via pSmad2L/C and pSmad3L/C pathways. During chronic liver disease progression, pre-neoplastic hepatocytes persistently affected by TGF-β together with pro-inflammatory cytokines come to exhibit the same carcinogenic (mitogenic) pSmad3L and fibrogenic pSmad2L/C signaling as do MFB, thereby accelerating liver fibrosis while increasing risk of HCC. This review of Smad phosphoisoform-mediated signals examines similarities and differences between epithelial and mesenchymal cells in acute and chronic liver injuries and considers Smad linker phosphorylation as a potential target for the chemoprevention of fibro-carcinogenesis.

A generic HTS assay for kinase screening: Validation for the isolation of an engineered malate kinase

PubMed Central

Irague, Romain; Topham, Christopher M.; Martineau, Nelly; Baylac, Audrey; Auriol, Clément; Walther, Thomas; François, Jean-Marie; Remaud-Siméon, Magali

2018-01-01

An end-point ADP/NAD+ acid/alkali assay procedure, directly applicable to library screening of any type of ATP-utilising/ADP producing enzyme activity, was implemented. Typically, ADP production is coupled to NAD+ co-enzyme formation by the conventional addition of pyruvate kinase and lactate dehydrogenase. Transformation of enzymatically generated NAD+ into a photometrically active alkali derivative product is then achieved through the successive application of acidic/alkali treatment steps. The assay was successfully miniaturized to search for malate kinase activity in a structurally-guided library of LysC aspartate kinase variants comprising 6,700 clones. The screening procedure enabled the isolation of nine positive variants showing novel kinase activity on (L)-malate, the best mutant, LysC V115A:E119S:E434V exhibited strong substrate selectivity for (L)-malate compared to (L)-aspartate with a (kcat/Km)malate/(kcat/Km)aspartate ratio of 86. Double mutants V115A:E119S, V115A:E119C and E119S:E434V were constructed to further probe the origins of stabilising substrate binding energy gains for (L)-malate due to mutation. The introduction of less sterically hindering side-chains in engineered enzymes carrying E119S and V115A mutations increases the effective volume available for substrate binding in the catalytic pocket. Improved binding of the (L)-malate substrate may be assisted by less hindered movement of the Phe184 aromatic side-chain. Additional favourable long-range electostatic effects on binding arising from the E434V surface mutation are conditionally dependent upon the presence of the V115A mutation close to Phe184 in the active-site. PMID:29462203

The associations between metabolic variables and NT-proBNP are blunted at pathological ranges: the Multi-Ethnic Study of Atherosclerosis

PubMed Central

Sanchez, Otto A.; Duprez, Daniel A.; Bahrami, Hossein; Daniels, Lori B.; Folsom, Aaron R.; Lima, Joao A.; Maisel, Alan; Peralta, Carmen A.; Jacobs, David R.

2014-01-01

Objective Under physiological conditions brain natriuretic peptide (BNP) is inversely associated with metabolic risk factors, but under pathological conditions these associations may tend to plateau. Material end methods 5597 individuals in the Multi-Ethnic Study of Atherosclerosis (MESA), 45–84 years of age, free of overt cardiovascular disease in 2000–02 and then again in 2003–05 participated in this study. Associations between NT-proBNP and BMI, blood lipids, homeostasis model of insulin resistance (HOMA-IR) using linear regression models were adjusted for age, race, sex, BMI, % of energy from saturated fats, intentional exercise, statin use, antihypertensive medication use, diabetes and glomerular filtration rate. The inflection points (IP) at which these associations became nonlinear were determined using linear splines with knots at different levels of NT-proBNP. Results Participants with NT-proBNP ≥100 pg/mL (29%) tended to be older, on statins and anti-hypertensive medications vs. those with NT-proBNP <100 pg/mL. The IP point varies among variables and ranged from 50–120 pg/mL. NT-proBNP

Cardiac and Carotid Markers Link With Accelerated Brain Atrophy: The AGES-Reykjavik Study (Age, Gene/Environment Susceptibility-Reykjavik).

PubMed

Sabayan, Behnam; van Buchem, Mark A; Sigurdsson, Sigurdur; Zhang, Qian; Meirelles, Osorio; Harris, Tamara B; Gudnason, Vilmundur; Arai, Andrew E; Launer, Lenore J

2016-11-01

Pathologies in the heart-brain axis might, independently or in combination, accelerate the process of brain parenchymal loss. We aimed to investigate the association of serum N-terminal brain natriuretic peptide (NT-proBNP), as a marker of cardiac dysfunction, and carotid intima media thickness (CIMT), as a marker of carotid atherosclerosis burden, with structural brain changes. In the longitudinal population-based AGES-Reykjavik study (Age, Gene/Environment Susceptibility-Reykjavik), we included 2430 subjects (mean age, 74.6 years; 41.4% men) with baseline data on NT-proBNP and CITM (assessed by ultrasound imaging). Participants underwent a high-resolution brain magnetic resonance imaging at baseline and 5 years later to assess total brain (TBV), gray matter, and white matter volumes. Each unit higher log-transformed NT-proBNP was associated with 3.6 mL (95% confidence interval [CI], -6.0 to -1.1) decline in TBV and 3.5 mL (95% CI, -5.7 to -1.3) decline in gray matter volume. Likewise, each millimeter higher CIMT was associated with 10.8 mL (95% CI, -17.3 to -4.2) decline in TBV and 8.6 mL (95% CI, -14.4 to -2.8) decline in gray matter volume. There was no association between NT-proBNP and CIMT and changes in white matter volume. Compared with participants with low NT-proBNP and CIMT, participants with both high NT-proBNP and CIMT had 3.8 mL (95% CI, -6.0 to -1.6) greater decline in their TBV and 4 mL (95% CI, -6.0 to -2.0) greater decline in GMW. These associations were independent of sociodemographic and cardiovascular factors. Older subjects with both cardiac dysfunction and carotid atherosclerosis are at an increased risk for brain parenchymal loss. Accumulated pathologies in the heart-brain axis might accelerate brain atrophy. © 2016 American Heart Association, Inc.

Determinants of BH3 binding specificity for Mcl-1 vs. Bcl-xL

PubMed Central

Dutta, Sanjib; Gullá, Stefano; Chen, T. Scott; Fire, Emiko; Grant, Robert A.; Keating, Amy E.

2010-01-01

Interactions among Bcl-2 family proteins are important for regulating apoptosis. Pro-survival members of the family interact with pro-apoptotic BH3-only members, inhibiting execution of cell death through the mitochondrial pathway. Structurally, this interaction is mediated by binding of the alpha-helical BH3 region of the pro-apoptotic proteins to a conserved hydrophobic groove on the pro-survival proteins. Native BH3-only proteins exhibit selectivity in binding pro-survival members, as do small molecules that block these interactions. Understanding the sequence and structural basis of interaction specificity in this family is important, as it may allow the prediction of new Bcl-2 family associations and/or the design of new classes of selective inhibitors to serve as reagents or therapeutics. In this work we used two complementary techniques, yeast surface display screening from combinatorial peptide libraries and SPOT peptide array analysis, to elucidate specificity determinants for binding to Bcl-xL vs. Mcl-1, two prominent pro-survival proteins. We screened a randomized library and identified BH3 peptides that bound to either Mcl-1 or Bcl-xL selectively, or to both with high affinity. The peptides competed with native ligands for binding into the conserved hydrophobic groove, as illustrated in detail by a crystal structure of a specific peptide bound to Mcl-1. Mcl-1 selective peptides from the screen were highly specific for binding Mcl-1 in preference to Bcl-xL, Bcl-2, Bcl-w and Bfl-1, whereas Bcl-xL selective peptides showed some cross-interaction with related proteins Bcl-2 and Bcl-w. Mutational analyses using SPOT arrays revealed the effects of 170 point mutations made in the background of a peptide derived from the BH3 region of Bim, and a simple predictive model constructed using these data explained much of the specificity observed in our Mcl-1 vs. Bcl-xL binders. PMID:20363230

Inhibition of Fibrillar Assemblies of l-Phenylalanine by Crown Ethers: A Potential Approach toward Phenylketonuria.

PubMed

Banik, Debasis; Dutta, Rupam; Banerjee, Pavel; Kundu, Sangita; Sarkar, Nilmoni

2016-08-11

In this article, our aim is to investigate the interaction of l-phenylalanine (l-Phe) fibrils with crown ethers (CEs). For this purpose, two different CEs (15-Crown-5 (15C5) and 18-Crown-6 (18C6)) were used. Interestingly, we have observed that both CEs have the ability to arrest fibril formation. However, 18C6 was found to be a better candidate compared to 15C5. Field emission scanning electron microscopy and fluorescence lifetime imaging microscopy were used to monitor the fibril-arresting kinetics of CEs. The arresting process was further confirmed by fluorescence correlation spectroscopy and nuclear magnetic resonance studies.

Ir gene controlled carrier effects in the induction and elicitation of hapten-specific delayed-type hypersensitivity responses.

PubMed

Weinberger, J Z; Benacerraf, B; Dorf, M E

1979-11-01

The genetic requirements of carrier recognition were examined in the priming and elicitation of hapten specific, T-cell mediated, delayed-type hypersensitivity (DTH) responses. It was shown that nitrophenyl acetyl-poly-(L-glu56-L-lys35-L-phe9) (NP-GLO) could prime for NP responses only in strains of mice which are Ir gene responders to GLO. In contrast to this requirement, NO-GLO could elicit an NP-specific response in NP-bovine gamma globulin primed mice, even in GLO nonresponder strains. Furthermore, the nonimmunogenic molecule, NP-GL, could elicit an NP-specific DTH response in animals primed with NP on an immunogenic carrier.

Interaction of hemoglobin Grey Lynn (Vientiane) with a non-deletional α(+)-thalassemia in an adult Thai proband.

PubMed

Singha, Kritsada; Fucharoen, Goonnapa; Fucharoen, Supan

2014-01-01

Hemoglobin (Hb) Grey Lynn is a Hb variant caused by a substitution of Phe for Leu at position 91 of α1-globin chain, originally described in individual of unknown ethnic background. This article addresses the interaction of Hb Grey Lynn with a non-deletional α(+)-thalassemia found in Thailand, a hitherto un-described condition. The proband was adult Thai woman referred for investigation of mild anemia with Hb 90 g/L. Hb analyses using low pressure liquid chromatography raised a suspicion of abnormal Hb presence, which was failed to demonstrate by cellulose acetate electrophoresis and capillary electrophoresis. DNA sequencing identified a CTT (Leu) to TTT (Phe) mutation at codon 91 corresponding to the Hb Grey Lynn (Vientiane) [α91(FG3)Leu>Phe (α1) on α1-globin gene and a C deletion between codons 36 and 37 on α2-globin gene causing α(+)-thalassemia. As compared to those observed in a compound heterozygote for Hb Grey Lynn / α(0)-thalassemia reported previously, higher MCV (81.7 fL) and MCH (26.3 pg) values with a lower level of Hb Grey Lynn (19.7%) were observed in the proband. The normochromic normocytic anemia observed could be due to the interaction of Hb Grey Lynn with α(+)-thalassemia. The two mutations could be identified using PCR-RFLP and allele-specific PCR assays developed.

High terbinafine resistance in Trichophyton interdigitale isolates in Delhi, India harbouring mutations in the squalene epoxidase gene.

PubMed

Singh, Ashutosh; Masih, Aradhana; Khurana, Ananta; Singh, Pradeep Kumar; Gupta, Meenakshi; Hagen, Ferry; Meis, Jacques F; Chowdhary, Anuradha

2018-03-25

In the last few years, infections caused by dermatophytes along with a concomitant increase in the number of difficult to treat cases have increasingly been recognised, indicating that dermatophytosis remains a challenging public health problem. The majority of infections are caused by Trichophyton rubrum and Trichophyton mentagrophytes complex. Terbinafine, an allylamine antifungal used orally and topically is considered to be a first-line drug in the therapy of dermatophyte infections. Terbinafine resistance has been predominately attributed to point mutations in the squalene epoxidase (SQLE) target gene a key enzyme in the ergosterol biosynthetic pathway leading to single amino acid substitutions. Here, we report the largest series of 20 terbinafine-resistant Trichophyton interdigitale isolates obtained predominately from cases of tinea corporis/cruris in three hospitals in Delhi, India exhibiting elevated MICs (4 to ≥32 μg/mL) to terbinafine and all harbouring single-point mutations Leu393Phe or Phe397Leu in the SQLE gene. In 12 (60%) T. interdigitale isolates, the Phe397Leu substitution was observed, whereas in the remaining 8 (40%) isolates the substitution Leu393Phe was reported for the first time in T. interdigitale. Furthermore, 10 susceptible T. interdigitale isolates (0.125-2 μg/mL) had a wild-type genotype. Remarkably, considerably high terbinafine resistance rate of 32% was observed among 63 T. interdigitale isolates identified by sequencing of the internal transcribed spacer region. This high level of terbinafine resistance of Indian dermatophyte isolates is worrisome warranting antifungal susceptibility testing and mutation analysis for monitoring this emerging resistance. © 2018 Blackwell Verlag GmbH.

Analyses of insulin-potentiating fragments of human growth hormone by computative simulation; essential unit for insulin-involved biological responses.

PubMed

Ohkura, K; Hori, H

2000-07-01

We analyzed the structural features of insulin-potentiating fragments of human growth hormone by computative simulations. The peptides were designated from the N-terminus sequences of the hormone positions at 1-15 (hGH(1-15); H2N-Phe1-Pro2-Thr3-Ile4-Pro5-Leu6-Ser7-Arg8-L eu9-Phe10-Asp11-Asn12-Ala13-Met14-Leu15 -COOH), 6-13 (hGH(6-13)), 7-13 (hGH(7-13)) and 8-13 (hGH(8-13)), which enhanced insulin-producing hypoglycemia. In these peptide molecules, ionic bonds were predicted to form between 8th-arginyl residue and 11th-aspartic residue, and this intramolecular interaction caused the formation of a macrocyclic structure containing a tetrapeptide Arg8-Leu9-Phe10-Asp11. The peptide positions at 6-10 (hGH(6-10)), 9-13 (hGH(9-13)) and 10-13 (hGH(10-13)) did not lead to a macrocyclic formation in the molecules, and had no effect on the insulin action. Although beta-Ala13hGH(1-15), in which the 13th-alanine was replaced by a beta-alanyl residue, had no effect on insulin-producing hypoglycemia, the macrocyclic region (Arg8-Leu9-Phe10-Asp11) was observed by the computative simulation. An isothermal vibration analysis of both of beta-Ala13hGH(1-15) and hGH(1-15) peptide suggested that beta-Ala13hGH(1-15) is molecule was more flexible than hGH(1-15); C-terminal carboxyl group of Leu15 easily accessed to Arg8 and inhibited the ionic bond formation between Arg8 and Asp11 in beta-Ala13hGH(1-15). The peptide of hGH(8-13) dose-dependently enhanced the insulin-involved fatty acid synthesis in rat white adipocytes, and stabilized the C6-NBD-PC (1-acyl-2-[6-[(7-nitro-2,1,3benzoxadiazol-4-yl)amino]-caproyl]-sn- glycero-3-phosphatidylcholine) model membranes. In contrast, hGH(9-13) had no effect both on the fatty acid synthesis and the membrane stability. In the same culture conditions as the fatty acid synthesis assay, hGH(8-13) had no effect on the transcript levels of glucose transporter isoforms (GLUT 1, 4) and hexokinase isozymes (HK I, II) in rat white adipocytes. Judging from these results we considered that the macrocyclic structure in human growth hormonal peptides is regarded with the modification of insulin action, and hGH(8-13) is an essential sequence for the modification of insulin action. This hGH(8-13) peptide modifies the insulin action via stabilizing the cell membrane, and does not directly act on the insulin-involved glucose metabolism.

RNAi suppression of Arogenate Dehydratase1 reveals that phenylalanine is synthesized predominantly via the arogenate pathway in petunia petals.

PubMed

Maeda, Hiroshi; Shasany, Ajit K; Schnepp, Jennifer; Orlova, Irina; Taguchi, Goro; Cooper, Bruce R; Rhodes, David; Pichersky, Eran; Dudareva, Natalia

2010-03-01

l-Phe, a protein building block and precursor of numerous phenolic compounds, is synthesized from prephenate via an arogenate and/or phenylpyruvate route in which arogenate dehydratase (ADT) or prephenate dehydratase, respectively, plays a key role. Here, we used Petunia hybrida flowers, which are rich in Phe-derived volatiles, to determine the biosynthetic routes involved in Phe formation in planta. Of the three identified petunia ADTs, expression of ADT1 was the highest in petunia petals and positively correlated with endogenous Phe levels throughout flower development. ADT1 showed strict substrate specificity toward arogenate, although with the lowest catalytic efficiency among the three ADTs. ADT1 suppression via RNA interference in petunia petals significantly reduced ADT activity, levels of Phe, and downstream phenylpropanoid/benzenoid volatiles. Unexpectedly, arogenate levels were unaltered, while shikimate and Trp levels were decreased in transgenic petals. Stable isotope labeling experiments showed that ADT1 suppression led to downregulation of carbon flux toward shikimic acid. However, an exogenous supply of shikimate bypassed this negative regulation and resulted in elevated arogenate accumulation. Feeding with shikimate also led to prephenate and phenylpyruvate accumulation and a partial recovery of the reduced Phe level in transgenic petals, suggesting that the phenylpyruvate route can also operate in planta. These results provide genetic evidence that Phe is synthesized predominantly via arogenate in petunia petals and uncover a novel posttranscriptional regulation of the shikimate pathway.

Plasma N-terminal pro-brain natriuretic peptide concentrations before and after pericardiocentesis in dogs with cardiac tamponade secondary to spontaneous pericardial effusion.

PubMed

Baumwart, R D; Hanzlicek, A S; Lyon, S D; Lee, P M

2017-10-01

To determine if concentrations of plasma N-terminal pro-brain natriuretic peptide (NT-proBNP) are increased in dogs with cardiac tamponade and if there is a significant increase in plasma NT-proBNP after pericardiocentesis. Ten client-owned dogs with spontaneous cardiac tamponade. Prospective clinical study. Cardiac tamponade was suspected from physical examination and confirmed with echocardiography. Blood was collected and plasma NT-proBNP concentrations were measured before and 30-60 min following pericardiocentesis and resolution of cardiac tamponade. Within-subject changes in plasma NT-proBNP were compared by the Wilcoxon signed-rank test. The plasma NT-proBNP concentrations measured within the reference interval in seven of 10 dogs before pericardiocentesis and in six of 10 dogs following pericardiocentesis. Following pericardiocentesis, there was a statistically significant increase in median NT-proBNP concentration (733 pmol/L, range 250-3,297) compared with the values measured before (643 pmol/L, range 250-3,210, P = 0.004). The NT-proBNP concentration increased in 90% of the dogs following pericardiocentesis. An upper reference limit of 900 pmol/L for plasma NT-proBNP is insensitive for the diagnosis of pericardial effusion and cardiac tamponade in dogs. Plasma NT-proBNP concentration commonly increases following pericardiocentesis, perhaps related to improved ventricular filling and stretch. Published by Elsevier B.V.

Salmonella typhimurium gyrA mutations associated with fluoroquinolone resistance.

PubMed Central

Reyna, F; Huesca, M; González, V; Fuchs, L Y

1995-01-01

Spontaneous quinolone-resistant mutants obtained from Salmonella typhimurium Su694 were screened for mutations by direct DNA sequencing of an amplified PCR gyrA fragment. Substitutions Ser-83-->Phe (Ser83Phe), Ser83Tyr, Asp87Tyr, and Asp87Asn and double mutation Ala67Pro-Gly81Ser, which resulted in decreased sensitivities to ciprofloxacin, enoxacin, pefloxacin, norfloxacin, ofloxacin, and nalidixic acid, were found. The levels of resistance to quinolones for each mutant were determined. PMID:7492118

Assessments of pain, functional impairment, anxiety, and depression in US adults with hemophilia across patient-reported outcome instruments in the Pain, Functional Impairment, and Quality of Life (P-FiQ) study.

PubMed

Buckner, Tyler W; Batt, Katharine; Quon, Doris; Witkop, Michelle; Recht, Michael; Kessler, Craig; Baumann, Kimberly; Hernandez, Grace; Wang, Michael; Cooper, David L; Kempton, Christine L

2018-04-01

Pain, functional impairment, anxiety, and depression associated with joint disease may affect health-related quality of life (HRQoL) in people with hemophilia. To report detailed patient-reported outcomes (PRO) assessments related to HRQoL in participants in the Pain, Functional Impairment, and Quality of Life (P-FiQ) study. Pain and HRQoL were assessed via PRO instruments in US adult males with hemophilia A or B and a history of joint pain or bleeding. PRO instruments included EQ-5D-5L with visual analog scale, Brief Pain Inventory v2 Short Form, SF-36v2, and Hemophilia Activities List. Instrument domain and item responses were described. Responses were collected from 381 adult males with a median age of 34 years. Pain was observed across instruments and affected daily activities and quality of life. Respondents reported functional impairment that limited the kind of work and activities they participated in, with activities involving the lower extremities being most affected. A high prevalence of mental health disorders was identified across instruments. Pain and HRQoL were evaluated using multiple PRO instruments, which vary in timescales of assessment and levels of detail. More consistent clinical assessments and patient dialog regarding pain and aspects of HRQoL may help drive improved outcomes. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

A C69-family cysteine dipeptidase from Lactobacillus farciminis JCM1097 possesses strong Gly-Pro hydrolytic activity.

PubMed

Sakamoto, Takuma; Otokawa, Takuya; Kono, Ryosuke; Shigeri, Yasushi; Watanabe, Kunihiko

2013-11-01

Dipeptide Gly-Pro, a hard-to-degrade and collagenous peptide, is thought to be hydrolysed by prolidases that can work on various X-Pro dipeptides. Here, we found an entirely different type of dipeptidase from Lactobacillus farciminis JCM1097 that cleaves Gly-Pro far more efficiently and with higher specificity than prolidases, and then investigated its properties by use of a recombinant enzyme. Although L. farciminis dipeptidase was expressed in the form of an inclusion body in Escherichia coli at 37 °C, it was smoothly over-expressed in a soluble form at a lower temperature. The maximal Gly-Pro hydrolytic activity was attained in E. coli at 30 °C. In contrast to prolidases that are metallopeptidases showing the modest or marginal activity toward Gly-Pro, this L. farciminis dipeptidase belongs to the cysteine peptidase family C69. Lactobacillus farciminis dipeptidase occurs in cytoplasm and utilizes the side chain of an amino-terminal cysteine residue to perform the nucleophilic attack on the target amide bond between Gly-Pro after processing eight amino acid residues at the N-terminus. Furthermore, L. farciminis dipeptidase is potent enough to synthesize Gly-Pro from Gly and Pro by a reverse reaction. These novel properties could be revealed by virtue of the success in preparing recombinant enzymes in higher yield and in a stable form.

Losartan prevents the imbalance between renal dopaminergic and renin angiotensin systems induced by fructose overload. L-dopa/dopamine index as new potential biomarker of renal dysfunction.

PubMed

Mikusic, Natalia Lucía Rukavina; Kouyoumdzian, Nicolás Martín; Uceda, Ana; Del Mauro, Julieta Sofía; Pandolfo, Marcela; Gironacci, Mariela Mercedes; Puyó, Ana María; Toblli, Jorge Eduardo; Fernández, Belisario Enrique; Choi, Marcelo Roberto

2018-05-01

The renin angiotensin system (RAS) and the renal dopaminergic system (RDS) act as autocrine and paracrine systems to regulate renal sodium management and inflammation and their alterations have been associated to hypertension and renal damage. Nearly 30-50% of hypertensive patients have insulin resistance (IR), with a strong correlation between hyperinsulinemia and microalbuminuria. The aim of this study was to demonstrate the existence of an imbalance between RAS and RDS associated to IR, hypertension and kidney damage induced by fructose overload (FO), as well as to establish their prevention, by pharmacological inhibition of RAS with losartan. Ninety-six male Sprague-Dawley rats were randomly divided into four groups and studied at 4, 8 and 12 weeks: control group (C4, C8 and C12; tap water to drink); fructose-overloaded group (F4, F8 and F12; 10% w/v fructose solution to drink); losartan-treated control (L) group (L4, L8 and L12; losartan 30 mg/kg/day, in drinking water); and fructose-overloaded plus losartan group (F + L4, F + L8 and F + L12, in fructose solution). FO induced metabolic and hemodynamic alterations as well as an imbalance between RAS and RDS, characterized by increased renal angiotensin II levels and AT 1 R overexpression, reduced urinary excretion of dopamine, increased excretion of L-dopa (increased L-dopa/dopamine index) and down-regulation of D 1 R and tubular dopamine transporters OCT-2, OCT-N1 and total OCTNs. This imbalance was accompanied by an overexpression of renal tubular Na + , K + -ATPase, pro-inflammatory (NF-kB, TNF-α, IL-6) and pro-fibrotic (TGF-β1 and collagen) markers and by renal damage (microalbuminuria and reduced nephrin expression). Losartan prevented the metabolic and hemodynamic alterations induced by FO from week 4. Increased urinary L-dopa/dopamine index and decreased D 1 R renal expression associated to FO were also prevented by losartan since week 4. The same pattern was observed for renal expression of OCTs/OCTNs, Na + , K + -ATPase, pro-inflammatory and pro-fibrotic markers from week 8. The appearance of microalbuminuria and reduced nephrin expression was prevented by losartan at week 12. The results of this study provide new insight regarding the mechanisms by which a pro-hypertensive and pro-inflammatory system, such as RAS, downregulates another anti-hypertensive and anti-inflammatory system such as RDS. Additionally, we propose the use of L-dopa/dopamine index as a biochemical marker of renal dysfunction in conditions characterized by sodium retention, IR and/or hypertension, and as a predictor of response to treatment and follow-up of these processes. Copyright © 2018. Published by Elsevier Inc.

The acute response of the amygdalar TRH system to psychogenic stressors varies dependent on the paradigm and circadian condition.

PubMed

Gutiérrez-Mariscal, Mariana; Sánchez, Edith; Rebolledo-Solleiro, Daniela; García-Vázquez, Arlene Iskra; Cote-Vélez, Antonieta; Acasuso-Rivero, Cristina; Charli, Jean-Louis; Joseph-Bravo, Patricia

2012-05-03

Central administration of thyrotropin releasing hormone (TRH) reduces anxiety; amygdalar TRH expression is inversely proportional to the anxious behavior displayed in the elevated plus maze performed during the dark phase (EPM-D). To better understand the role of TRH in amygdala function, we evaluated the expression of TRH and the elements involved in its transmission in various stressful paradigms and how they associated with behavior. Wistar male rats were exposed to restraint (RES), EPM, or the open field test (OFT) and sacrificed 0-60 min afterwards; OFT, RES and EPM were performed during the light (L), and OFT during the dark phase. Restraint increased amygdalar levels of proCRH mRNA, without change in proTRH. All paradigms augmented corticosterone release, highest after OFT-L that also enhanced proCRH mRNA levels and decreased those of proTRH. OFT-D activated the TRH system. Levels of anxiety or locomotion were similar in animals tested in light or dark phases but their association with biochemical parameters differed. ProTRH expression and TRH release correlated positively with decreased anxiety in EPM-L and in OFT-D. No association with anxiety was detected in OFT-L where proCRH and proTRH expression correlated with locomotion supporting their involvement in arousal. The responses of TRH amygdalar systems appeared modulated by the extent of the stress response and by the circadian conditions. Increased proTRH expression of animals exposed to OFT-D was specifically observed in the cortical nucleus of the amygdala, area involved in processing fear stimuli; these TRH neurons may thus be part of a circuit with anxiolytic properties. Copyright © 2012 Elsevier B.V. All rights reserved.

N-terminal pro-brain natriuretic peptide is a strong predictor of mortality in systemic sclerosis.

PubMed

Allanore, Yannick; Komocsi, Andras; Vettori, Serena; Hachulla, Eric; Hunzelmann, Nicolas; Distler, Jörg; Avouac, Jérôme; Gobeaux, Camille; Launay, David; Czirjak, Laszlo; Kahan, André; Meune, Christophe

2016-11-15

Cardiovascular involvement is a major contributor to mortality in systemic sclerosis (SSc). We examined whether N-terminal pro-brain natriuretic peptide (NT-proBNP) is a reliable predictor of mortality in SSc. This multicentre prospective cohort study included 523 patients presenting with SSc, whose mean age was 54±13years, mean disease duration 8±9years, and diffuse cutaneous form in 168. Plasma NT-proBNP was measured at baseline and the patients were followed yearly. Overall mortality was measured at 3years. At baseline, cardiovascular involvement was present in 37 patients, including 17 with pulmonary artery hypertension (PAH) and 20 with a left ventricular ejection fraction (LVEF) <55%. At 3years, 32 (7%) patients had died. The median [25th-75th percentile] NT-proBNP concentration was 203ng/l [129-514] in patients who died within 3years, versus 88ng/l [47-167] in survivors (P<0.001). NT-proBNP was an independent predictor of 3-years mortality in multivariate analysis (P=0.046). The optimal cut-off derived from the ROC curve was 129ng/l; sensitivity and specificity to predict 3y mortality were 78.1 and 66.7%. Using the previously recommended 125-ng/l concentration as threshold value, NT-proBNP reliably and independently predicted 3year mortality, with a sensitivity of 78.1 and a negative predictive value of 97.6%, respectively (P=0.006). The consideration of SSc patients without PAH or LVEF<55% at baseline yielded similar results. NT-proBNP appears as a reliable and independent predictor of mortality in patients with SSc. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Plasma NT pro-BNP, hs-CRP and big-ET levels at admission as prognostic markers of survival in hospitalized patients with dilated cardiomyopathy: a single-center cohort study.

PubMed

Li, Xiaoping; Chen, Chengzhi; Gan, Feng; Wang, Yang; Ding, Ligang; Hua, Wei

2014-05-11

Circulating N-terminal pro-B-type natriuretic peptide (NT pro-BNP), high- sensitivity C-reactive protein (hs-CRP) and big endothelin (big-ET) have been shown to be increased in heart failure and to contribute to both hemodynamic deterioration and cardiovascular remodeling. Here, we examined the prognostic value of the three neurohormones at admission in a population of hospitalized patients with dilated cardiomyopathy (DCM). This cohort study was undertaken in 622 hospitalized patients with DCM in Fuwai Hospital from January 2005 to September 2011 (female 26.5%, 51.4 ± 14.6 years old). Standard demographics, echocardiography and routine blood samples were obtained shortly after admission. NT pro-BNP, hs-CRP and big-ET were measured, and their concentrations in relation to all-cause mortality were assessed through a mean follow-up of 2.6 ± 1.6 years. Kaplan-Meier curves showed that the all-cause mortality rates were higher in patients with NT pro-BNP > 2247 pmol/L compared to patients with NT pro-BNP < 2247 pmol/L (11.9% vs 34.8%, log-rank χ2 = 35.588, P < 0.001), in patients with hs-CRP > 3.90 mg/L compared to patients with hs-CRP < 3.90 mg/L (12.8% vs 33.6%, log-rank χ2 = 39.662, P < 0.001) and in patients with big-ET > 0.95 pmol/L compared to patients with big-ET <0.95 pmol/L (12.5% vs 31.0%, log-rank χ2 = 17.890, P < 0.001). High circulating concentrations of NT pro-BNP (HR 2.217, 95% CI 1.015-4.846, P = 0.046) and hs-CRP (HR 1.922, 95% CI 1.236-2.988, P = 0.004), but not big-ET, in addition to left atrial diameter and fasting blood glucose, were independent predictors of the outcome defined as all-cause mortality. In a large population of patients with DCM, the circulating concentrations of NT pro-BNP and hs-CRP, but not big-ET, were independent markers of all-cause mortality.

Misbalance in type III collagen formation/degradation as a novel serological biomarker for penetrating (Montreal B3) Crohn's disease.

PubMed

van Haaften, W T; Mortensen, J H; Karsdal, M A; Bay-Jensen, A C; Dijkstra, G; Olinga, P

2017-07-01

Misbalances in extracellular matrix turnover are key factors in the development of stricturing (Montreal B2) and penetrating (Montreal B3) Crohn's disease. To determine whether serological markers for collagen formation and degradation could serve as biomarkers for complications of Crohn's disease. Serum biomarkers for type I, III, V and VI collagen formation (P1NP, Pro-C3, Pro-C5, Pro-C6) and matrix metalloproteinase mediated degradation (C1M, C3M, C5M and C6M) were measured in a retrospective, single centre cohort of 112 patients with Crohn's disease in the terminal ileum (nonstricturing/nonpenetrating: n=40, stricturing: n=55, penetrating: n=17) and 24 healthy controls. Active inflammation was defined as CRP >5 mg/L. C3M and Pro-C5 levels were higher in penetrating vs nonpenetrating/nonstricturing and stricturing disease (33.6±5 vs 25.8±2.2 [P=.004] and 27.2±2.3 [P=.018] nmol/L C3M, 1262.7±259.4 vs 902.9±109.9 [P=.005] and 953.0±106.4 [P=.015] nmol/L Pro-C5). C1M (71.2±26.1 vs 46.2±6.2 nmol/L [P<.001]), C3M (31.6±3.9 vs 26.1±1.6 nmol/L [P=.002] and Pro-C5 levels (1171.7±171.5 vs 909.6±80.4 nmol/L [P=.002]) were higher in patients with active inflammation vs without active inflammation. Pro-C3/C3M-ratios were best to differentiate between penetrating vs nonstricturing/nonpenetrating and stricturing disease with area under the curves of 0.815±0.109 (P<.001) and 0.746±0.114 (P=.002) respectively. Serological biomarkers show that penetrating Crohn's disease is characterised by increased matrix metalloproteinase-9 degraded type III collagen and formation of type V collagen. Active inflammation in Crohn's disease is characterised by increased formation of type V collagen and increased matrix metalloproteinase mediated breakdown of type I, III collagen. Pro-C3/C3M ratios are superior in differentiating between penetrating Crohn's disease vs inflammatory and stricturing Crohn's disease. © 2017 The Authors. Alimentary Pharmacology and Therapeutics published by John Wiley & Sons Ltd.

Structure-Activity Relationship Studies on a Macrocyclic Agouti-Related Protein (AGRP) Scaffold Reveal Agouti Signaling Protein (ASP) Residue Substitutions Maintain Melanocortin-4 Receptor Antagonist Potency and Result in Inverse Agonist Pharmacology at the Melanocortin-5 Receptor.

PubMed

Ericson, Mark D; Freeman, Katie T; Schnell, Sathya M; Fleming, Katlyn A; Haskell-Luevano, Carrie

2017-10-12

The melanocortin system consists of five reported receptors, agonists from the proopiomelanocortin gene transcript, and two antagonists, agouti-signaling protein (ASP) and agouti-related protein (AGRP). For both ASP and AGRP, the hypothesized Arg-Phe-Phe pharmacophores are on exposed β-hairpin loops. In this study, the Asn and Ala positions of a reported AGRP macrocyclic scaffold (c[Pro-Arg-Phe-Phe-Asn-Ala-Phe-DPro]) were explored with 14-compound and 8-compound libraries, respectively, to generate more potent, selective melanocortin receptor antagonists. Substituting diaminopropionic acid (Dap), DDap, and His at the Asn position yielded potent MC4R ligands, while replacing Ala with Ser maintained MC4R potency. Since these substitutions correlate to ASP loop residues, an additional Phe to Ala substitution was synthesized and observed to maintain MC4R potency. Seventeen compounds also possessed inverse agonist activity at the MC5R, the first report of this pharmacology. These findings are useful in developing molecular probes to study negative energy balance conditions and unidentified functions of the MC5R.

Amino Acids Inhibitory Effects and Mechanism on 2-Amino-1-Methyl-6-Phenylimidazo [4,5-b]Pyridine (PhIP) Formation in the Maillard Reaction Model Systems.

PubMed

Linghu, Ziyi; Karim, Faris; Smith, J Scott

2017-12-01

This study was to investigate the inhibitory effects of amino acids (AAs) on the formation of 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) and to evaluate the inhibition mechanism of PhIP in Maillard model systems. Different AAs were individually added into model systems heat-treated at 180 °C/1 h. The PhIP, phenylacetaldehyde (PheAce), and pyrazines derivatives were determined using HPLC and GC-MS. AAs significantly reduced (P < 0.05) PhIP levels in a dose-dependent response, ranking as: Trp = Lys > Pro > Leu > Met > Val > Ile > Thr > Phe > Asp, at the highest molar ratio. The PheAce content was gradually reduced with increasing AAs levels, suggesting that AAs may inhibit PhIP formation through scavenging the available PheAce. A correlation between PhIP inhibition and PheAce-scavenging activity of AAs was observed when PheAce and AAs were heated. The variety and quantity of pyrazines formed are highly depending on the type of AAs. © 2017 Institute of Food Technologists®.

Post-Transcriptional Regulation of BCL2 mRNA by the RNA-Binding Protein ZFP36L1 in Malignant B Cells

PubMed Central

Zekavati, Anna; Nasir, Asghar; Alcaraz, Amor; Aldrovandi, Maceler; Marsh, Phil; Norton, John D.; Murphy, John J.

2014-01-01

The human ZFP36 zinc finger protein family consists of ZFP36, ZFP36L1, and ZFP36L2. These proteins regulate various cellular processes, including cell apoptosis, by binding to adenine uridine rich elements in the 3′ untranslated regions of sets of target mRNAs to promote their degradation. The pro-apoptotic and other functions of ZFP36 family members have been implicated in the pathogenesis of lymphoid malignancies. To identify candidate mRNAs that are targeted in the pro-apoptotic response by ZFP36L1, we reverse-engineered a gene regulatory network for all three ZFP36 family members using the ‘maximum information coefficient’ (MIC) for target gene inference on a large microarray gene expression dataset representing cells of diverse histological origin. Of the three inferred ZFP36L1 mRNA targets that were identified, we focussed on experimental validation of mRNA for the pro-survival protein, BCL2, as a target for ZFP36L1. RNA electrophoretic mobility shift assay experiments revealed that ZFP36L1 interacted with the BCL2 adenine uridine rich element. In murine BCL1 leukemia cells stably transduced with a ZFP36L1 ShRNA lentiviral construct, BCL2 mRNA degradation was significantly delayed compared to control lentiviral expressing cells and ZFP36L1 knockdown in different cell types (BCL1, ACHN, Ramos), resulted in increased levels of BCL2 mRNA levels compared to control cells. 3′ untranslated region luciferase reporter assays in HEK293T cells showed that wild type but not zinc finger mutant ZFP36L1 protein was able to downregulate a BCL2 construct containing the BCL2 adenine uridine rich element and removal of the adenine uridine rich core from the BCL2 3′ untranslated region in the reporter construct significantly reduced the ability of ZFP36L1 to mediate this effect. Taken together, our data are consistent with ZFP36L1 interacting with and mediating degradation of BCL2 mRNA as an important target through which ZFP36L1 mediates its pro-apoptotic effects in malignant B-cells. PMID:25014217

N-Terminal Pro-B-type Natriuretic Peptide Is Useful to Predict Cardiac Complications Following Lung Resection Surgery

PubMed Central

Lee, Chang Young; Bae, Mi Kyung; Lee, Jin Gu; Kim, Kwan-Wook; Park, In Kyu

2011-01-01

Background Cardiovascular complications are major causes of morbidity and mortality following non-cardiac thoracic operations. Recent studies have demonstrated that elevation of N-Terminal Pro-B-type natriuretic peptide (NT-proBNP) levels can predict cardiac complications following non-cardiac major surgery as well as cardiac surgery. However, there is little information on the correlation between lung resection surgery and NT-proBNP levels. We evaluated the role of NT-proBNP as a potential marker for the risk stratification of cardiac complications following lung resection surgery. Material and Methods Prospectively collected data of 98 patients, who underwent elective lung resection from August 2007 to February 2008, were analyzed. Postoperative adverse cardiac events were categorized as myocardial injury, ECG evidence of ischemia or arrhythmia, heart failure, or cardiac death. Results Postoperative cardiac complications were documented in 9 patients (9/98, 9.2%): Atrial fibrillation in 3, ECG-evidenced ischemia in 2 and heart failure in 4. Preoperative median NT-proBNP levels was significantly higher in patients who developed postoperative cardiac complications than in the rest (200.2 ng/L versus 45.0 ng/L, p=0.009). NT-proBNP levels predicted adverse cardiac events with an area under the receiver operating characteristic curve of 0.76 [95% confidence interval (CI) 0.545~0.988, p=0.01]. A preoperative NT-proBNP value of 160 ng/L was found to be the best cut-off value for detecting postoperative cardiac complication with a positive predictive value of 0.857 and a negative predictive value of 0.978. Other factors related to cardiac complications by univariate analysis were a higher American Society of Anesthesiologists grade, a higher NYHA functional class and a history of hypertension. In multivariate analysis, however, high preoperative NT-proBNP level (>160 ng/L) only remained significant. Conclusion An elevated preoperative NT-proBNP level is identified as an independent predictor of cardiac complications following lung resection surgery. PMID:22263123

Importance of Thomas single-electron transfer in fast p-He collisions

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

Fischer, D.; Max-Planck-Institut fuer Kernphysik, Saupfercheckweg 1 D-69126; Gudmundsson, M.

We report experimental angular differential cross sections for nonradiative single-electron capture in p-He collisions (p+ He -> H + He{sup +}) with a separate peak at the 0.47 mrad Thomas scattering angle for energies in the 1.3-12.5 MeV range. We find that the intensity of this peak scales with the projectile velocity as v{sub P}{sup -11}. This constitutes the first experimental test of the prediction from 1927 by L. H. Thomas [Proc. R. Soc. 114, 561 (1927)]. At our highest energy, the peak at the Thomas angle contributes with 13.5% to the total integrated nonradiative single-electron capture cross section.

Characterization of endopeptidase activity of tripeptidyl peptidase-I/CLN2 protein which is deficient in classical late infantile neuronal ceroid lipofuscinosis.

PubMed

Ezaki, J; Takeda-Ezaki, M; Oda, K; Kominami, E

2000-02-24

Endopeptidase activities of the CLN2 gene product (Cln2p)/tripeptidyl peptidase I (TPP-I), purified from rat spleen, were studied using the synthetic fluorogenic substrates. We designed and constructed decapeptides, based on the known sequence cleavage specificities of bacterial pepstatin-insensitive carboxyl proteases (BPICP). MOCAc-Gly-Lys-Pro-Ile-Pro-Phe-Phe-Arg-Leu-Lys(Dnp)r-NH(2) is readily hydrolyzed by Cln2p/TPP-I (K(cat)/K(m) = 7.8 s(-1) mM(-1)). The enzyme had a maximal activity at pH 3.0 for an endopeptidase substrate, but at pH 4.5 with respect to tripeptidyl peptidase activity. Both endopeptidase and tripeptidyl peptidase activities were strongly inhibited by Ala-Ala-Phe-CH(2)Cl, but not inhibited by tyrostatin, an inhibitor of bacterial pepstatin-insensitive carboxyl proteases, pepstatin, or inhibitors of serine proteases. Fibroblasts from classical late infantile neuronal ceroid lipofuscinosis patients have less than 5% of the normal tripeptidyl peptidase activity and pepstatin-insensitive endopeptidase activity. Cln2p/TPP-I is a unique enzyme with both tripeptidyl peptidase and endopeptidase activities for certain substrate specificity. Copyright 2000 Academic Press.

Preparation and characterization of injectable Mitoxantrone poly (lactic acid)/fullerene implants for in vivo chemo-photodynamic therapy.

PubMed

Li, Zhi; Zhang, Fei-long; Pan, Li-li; Zhu, Xia-li; Zhang, Zhen-zhong

2015-08-01

Fullerene (C60) L-phenylalanine derivative attached with poly (lactic acid) (C60-phe-PLA) was developed to prepare injectable Mitoxantrone (MTX) multifunctional implants. C60-phe-PLA was self-assembled to form microspheres consisting of a hydrophilic antitumor drug (MTX) and a hydrophobic block (C60) by dispersion-solvent diffusion method. The self-assembled microspheres showed sustained release pattern almost 15days in vitro release experiments. According to the tissue distribution of C57BL mice after intratumoral administration of the microspheres, the MTX mainly distributed in tumors, and rarely in heart, liver, spleen, lung, and kidney. Photodynamic antitumor efficacy of blank microsphere was realized. Microspheres afforded high antitumor efficacy without obvious toxic effects to normal organs, owing to its significantly increased MTX tumor retention time, low MTX levels in normal organs and strong photodynamic activity of PLA-phe-C60. These C60-phe-PLA microspheres may be promising for the efficacy with minimal side effects in future treatment of solid tumors. Copyright © 2015 Elsevier B.V. All rights reserved.

Intracellular delivery and antitumor effects of pH-sensitive liposomes based on zwitterionic oligopeptide lipids.

PubMed

Mo, Ran; Sun, Qiong; Li, Nan; Zhang, Can

2013-04-01

pH-sensitive liposomes (HHG2C(18)-L and PEGHG2C(18)-L) based on zwitterionic oligopeptide lipids as anticancer drug carriers were developed and evaluated for effective intracellular delivery and enhanced antitumor activity. The amino acid-based lipids, 1,5-dioctadecyl-l-glutamyl 2-histidyl-hexahydrobenzoic acid (HHG2C(18)) and 1,5-distearyl N-(N-α-(4-mPEG2000) butanedione)-histidyl-l-glutamate (PEGHG2C(18)), were synthesized, which have the multistage pH-response to tumor microenvironmental pH (pH(e), pH 6.0-7.0) and endosomal/lysosomal pH (pH(i), pH 4.0-6.0) successively. HHG2C(18)-L contains HHG2C(18), while PEGHG2C(18)-L includes HHG2C(18) and PEGHG2C(18). Both of them displayed the capability of charge conversion to the surrounding pH. The zeta potentials of HHG2C(18)-L and PEGHG2C(18)-L were negative at pH 7.4, whereas positive at pH 6.5 and more positive at lower pH. Coumarin 6-loaded HHG2C(18)-L (C6/HHG2C(18)-L) and PEGHG2C(18)-L (C6/PEGHG2C(18)-L) showed higher tumor cellular uptake due to electrostatic absorptive endocytosis at pH(e) (pH 6.5), produced proton sponge effect for endo-lysosomal escape, and accumulated to the mitochondria based on stronger positive charge by the hydrolysis of a pH-sensitive linker at pH(i) (pH 5.5 and pH 4.5). Furthermore, temsirolimus (CCI-779)-loaded HHG2C(18)-L (CCI-779/HHG2C(18)-L) and PEGHG2C(18)-L (CCI-779/PEGHG2C(18)-L) had significantly higher antiproliferative and apoptosis inducing effects toward the human renal carcinoma (A498) cells at pH 6.5 relative to that at pH 7.4. The half maximal inhibitory concentration (IC50) of CCI-779/HHG2C(18)-L and CCI-779/PEGHG2C(18)-L were about 3 μg/mL and 5 μg/mL at pH 6.5, 1.67-fold and 1.60-fold improved relative to that at pH 7.4, respectively. The total apoptotic ratio of CCI-779/HHG2C(18)-L and CCI-779/PEGHG2C(18)-L increased from 9.90% and 7.78% at pH 7.4 to 19.53% and 12.10% at pH 6.5, respectively. In vivo, CCI-779/PEGHG2C(18)-L after intravenous administration presented remarkably higher bioavailability and blood persistence compared with unPEGylated CCI-779/HHG2C(18)-L, and had the strongest antitumor efficacy against xenograft renal cancer (Renca) tumor models. Accordingly, the results provide the feasibility of using pH-sensitive zwitterionic oligopeptide lipids to extend the applications of liposomes to efficient anticancer drug delivery in cancer therapy. Copyright © 2013 Elsevier Ltd. All rights reserved.

NT-proBNP Predicts All-Cause Mortality in a Population of Insurance Applicants, Follow-up Analysis and Further Observations.

PubMed

Fulks, Michael; Kaufman, Valerie; Clark, Michael; Stout, Robert L

2017-01-01

- Further refine the independent value of NT-proBNP, accounting for the impact of other test results, in predicting all-cause mortality for individual life insurance applicants with and without heart disease. - Using the Social Security Death Master File and multivariate analysis, relative mortality was determined for 245,322 life insurance applicants ages 50 to 89 tested for NT-proBNP (almost all based on age and policy amount) along with other laboratory tests and measurement of blood pressure and BMI. - NT-proBNP values ≤75 pg/mL included the majority of applicants denying heart disease and had the lowest risk, while values >500 pg/mL for females and >300 pg/mL for males had very high relative risk. Those admitting to heart disease had a higher mortality risk for each band of NT-proBNP relative to those denying heart disease but had a similar and equally predictive risk curve. - NT-proBNP is a strong independent predictor of all-cause mortality in the absence or presence of known heart disease but the range of values associated with increased risk varies by sex.

Characterization of cyclo-Acetoacetyl-L-Tryptophan Dimethylallyltransferase in Cyclopiazonic Acid Biosynthesis: Substrate Promiscuity and Site Directed Mutagenesis Studies

PubMed Central

Liu, Xinyu; Walsh, Christopher T.

2009-01-01

The fungal neurotoxin α-cyclopiazonic acid (CPA), a nanomolar inhibitor of Ca2+-ATPase with a unique pentacyclic indole tetramic acid scaffold is assembled by a three enzyme pathway CpaS, CpaD and CpaO in Aspergillus sp. We recently characterized the first pathway-specific enzyme CpaS, a hybrid two module polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) that generates cyclo-acetoacetyl-L-tryptophan (cAATrp). Here we report the characterization of the second pathway-specific enzyme CpaD that regiospecifically dimethylallylates cAATrp to form β-cyclopiazonic acid. By exploring the tryptophan and tetramate moieties of cAATrp, we demonstrate that CpaD discriminates against free Trp but accepts tryptophan-containing thiohydantoins, diketopiperazines and linear peptides as substrates for C4-prenylation and also acts as regiospecific O-dimethylallyltransferase (DMAT) on a tyrosine-derived tetramic acid. Comparative evaluation of CpaDs from A. oryzae RIB40 and A. flavus NRRL3357 indicated the importance of the N-terminal region for its activity. Sequence alignment of CpaD with eleven homologous fungal Trp-DMATs revealed five regions of conservation suggesting the presense of critical motifs that could be diagonostic for discovering additional Trp-DMATs. Subsequent site-directed mutagenesis studies identified five polar/charged residues and five tyrosine residues within these motifs that are critical for CpaD activity. This motif characerization will enable a gene probe-based approach to discover additional biosynthetic Trp-DMATs. PMID:19877600

Differential gene expression in porcine SK6 cells infected with wild-type and SAP domain-mutant foot-and-mouth disease virus.

PubMed

Ni, Zixin; Yang, Fan; Cao, Weijun; Zhang, Xiangle; Jin, Ye; Mao, Ruoqing; Du, Xiaoli; Li, Weiwei; Guo, Jianhong; Liu, Xiangtao; Zhu, Zixiang; Zheng, Haixue

2016-06-01

Foot-and-mouth disease virus (FMDV) is the causative agent of a highly contagious disease in livestock. The viral proteinase L(pro) of FMDV is involved in pathogenicity, and mutation of the L(pro) SAP domain reduces FMDV pathogenicity in pigs. To determine the gene expression profiles associated with decreased pathogenicity in porcine cells, we performed transcriptome analysis using next-generation sequencing technology and compared differentially expressed genes in SK6 cells infected with FMDV containing L(pro) with either a wild-type or mutated version of the SAP domain. This analysis yielded 1,853 genes that exhibited a ≥ 2-fold change in expression and was validated by real-time quantitative PCR detection of several differentially expressed genes. Many of the differentially expressed genes correlated with antiviral responses corresponded to genes associated with transcription factors, immune regulation, cytokine production, inflammatory response, and apoptosis. Alterations in gene expression profiles may be responsible for the variations in pathogenicity observed between the two FMDV variants. Our results provided genes of interest for the further study of antiviral pathways and pathogenic mechanisms related to FMDV L(pro).

Improved quality of life in hyperthyroidism patients after surgery.

PubMed

Bukvic, Branka; Zivaljevic, Vladan; Sipetic, Sandra; Diklic, Aleksandar; Tausanovic, Katarina; Stojanovic, Dragos; Stevanovic, Dejan; Paunovic, Ivan

2015-02-01

The most common causes of hyperthyroidism are Graves disease (GD) and toxic nodular goiter (TNG). GD and TNG might influence patients' quality of life (QoL). The aim of our study was to analyze and compare the QoL of patients with GD with that of TNG patients and to evaluate the influence of surgical treatment on their QoL. A prospective case-control study was conducted at the Center for Endocrine surgery in Belgrade, Serbia. The ThyPRO questionnaire was used in the QoL assessment of the GD and TNG patients (31 and 28, respectively) pre- and post-operatively. All patients were receiving antithyroid drugs, and none of the patients were overtly hyperthyroid at the time of completing the preoperative questionnaire. The QoL of the GD patients was worse than that of the TNG patients, with significant differences in eye symptoms, anxiety, and sex life domains (P < 0.001, P = 0.005, and P = 0.004, respectively), preoperatively, and in eye symptoms, anxiety, emotional susceptibility, and overall QoL (P = 0.001, P = 0.027, P = 0.005 and P = 0.013, respectively), postoperatively. The improvement in QoL in the GD patients was significant after surgical treatment in all ThyPRO domains. In the TNG patients, the improvement was significant in all but one ThyPRO domain, sex life (P = 0.066). The QoL of GD patients is worse than those of TNG patients. Surgery may improve QoL in patients with GD and TNG even if they have achieved satisfying thyroid status with medication treatment, preoperatively. Copyright © 2015 Elsevier Inc. All rights reserved.

Resistance training controls arterial blood pressure in rats with L-NAME- induced hypertension.

PubMed

Araujo, Ayslan Jorge Santos de; Santos, Anne Carolline Veríssimo dos; Souza, Karine dos Santos; Aires, Marlúcia Bastos; Santana-Filho, Valter Joviniano; Fioretto, Emerson Ticona; Mota, Marcelo Mendonça; Santos, Márcio Roberto Viana

2013-04-01

Arterial hypertension is a multifactorial chronic condition caused by either congenital or acquired factors. To evaluate the effects of Resistance Training (RT) on arterial pressure, and on vascular reactivity and morphology, of L-NAME-treated hypertensive rats. Male Wistar rats (200 - 250 g) were allocated into Sedentary Normotensive (SN), Sedentary Hypertensive (SH) and Trained Hypertensive (TH) groups. Hypertension was induced by adding L-NAME (40 mg/Kg) to the drinking water for four weeks. Arterial pressure was evaluated before and after RT. RT was performed using 50% of 1RM, 3 sets of 10 repetitions, 3 times per week for four weeks. Vascular reactivity was measured in rat mesenteric artery rings by concentration-response curves to sodium nitroprusside (SNP); phenylephrine (PHE) was also used for histological and stereological analysis. Resistance training inhibited the increase in mean and diastolic arterial pressures. Significant reduction was observed in Rmax (maximal response) and pD2 (potency) of PHE between SH and TH groups. Arteries demonstrated normal intima, media and adventitia layers in all groups. Stereological analysis demonstrated no significant difference in luminal, tunica media, and total areas of arteries in the SH and TH groups when compared to the SN group. Wall-to-lumen ratio of SH arteries was significantly different compared to SN arteries (p<0.05) but there was no difference when compared to TH arteries. RT was able to prevent an increase in blood pressure under the conditions in this study. This appears to involve a vasoconstrictor regulation mechanism and maintenance of luminal diameter in L-NAME induced hypertensive rats.

NprR-NprX Quorum-Sensing System Regulates the Algicidal Activity of Bacillus sp. Strain S51107 against Bloom-Forming Cyanobacterium Microcystis aeruginosa.

PubMed

Wu, Lishuang; Guo, Xingliang; Liu, Xianglong; Yang, Hong

2017-01-01

Harmful cyanobacterial blooms have severely impaired freshwater quality and threatened human health worldwide. Here, a Gram-positive bacterium, Bacillus sp. strain S51107, which exhibits strong algicidal activity against Microcystis aeruginosa , was isolated from Lake Taihu. We found that the algicidal activity of strain S51107 was regulated primarily by NprR-NprX quorum sensing (QS), in which the mature form of the signaling peptide NprX was identified as the SKPDIVG heptapeptide. Disruption of the nprR-nprX cassette markedly decreased the algicidal activity, and complemented strains showed significantly recovered algicidal activity. Strain S51107 produced low-molecular-weight algicidal compounds [indole-3-carboxaldehyde and cyclo(Pro-Phe)] and high-molecular-weight algicidal substance(s) (>3 kDa). Moreover, the production of high-molecular-weight algicidal substance(s) was regulated by NprR-NprX QS, but the production of low-molecular-weight algicidal compounds was not. High-molecular-weight algicidal substance(s) played a more important role than low-molecular-weight algicidal compounds in the algicidal activity of strain S51107. The results of this study could increase our knowledge about algicidal characteristics of a potential algicidal bacterium, Bacillus sp. strain S51107, and provide the first evidence that the algicidal activity of Gram-positive algicidal bacteria is regulated by QS, which will greatly enhance our understanding of the interactions between algae and indigenous algicidal bacteria, thereby providing aid in the design and optimization of strategies to control harmful algae blooms.

NprR-NprX Quorum-Sensing System Regulates the Algicidal Activity of Bacillus sp. Strain S51107 against Bloom-Forming Cyanobacterium Microcystis aeruginosa

PubMed Central

Wu, Lishuang; Guo, Xingliang; Liu, Xianglong; Yang, Hong

2017-01-01

Harmful cyanobacterial blooms have severely impaired freshwater quality and threatened human health worldwide. Here, a Gram-positive bacterium, Bacillus sp. strain S51107, which exhibits strong algicidal activity against Microcystis aeruginosa, was isolated from Lake Taihu. We found that the algicidal activity of strain S51107 was regulated primarily by NprR-NprX quorum sensing (QS), in which the mature form of the signaling peptide NprX was identified as the SKPDIVG heptapeptide. Disruption of the nprR-nprX cassette markedly decreased the algicidal activity, and complemented strains showed significantly recovered algicidal activity. Strain S51107 produced low-molecular-weight algicidal compounds [indole-3-carboxaldehyde and cyclo(Pro-Phe)] and high-molecular-weight algicidal substance(s) (>3 kDa). Moreover, the production of high-molecular-weight algicidal substance(s) was regulated by NprR-NprX QS, but the production of low-molecular-weight algicidal compounds was not. High-molecular-weight algicidal substance(s) played a more important role than low-molecular-weight algicidal compounds in the algicidal activity of strain S51107. The results of this study could increase our knowledge about algicidal characteristics of a potential algicidal bacterium, Bacillus sp. strain S51107, and provide the first evidence that the algicidal activity of Gram-positive algicidal bacteria is regulated by QS, which will greatly enhance our understanding of the interactions between algae and indigenous algicidal bacteria, thereby providing aid in the design and optimization of strategies to control harmful algae blooms. PMID:29075240

Proline with or without hydroxyproline influences collagen concentration and regulates prolyl 4-hydroxylase α (I) gene expression in juvenile turbo ( Scophthalmus maximus L.)

NASA Astrophysics Data System (ADS)

Zhang, Kaikai; Mai, Kangsen; Xu, Wei; Zhou, Huihui; Liufu, Zhiguo; Zhang, Yanjiao; Peng, Mo; Ai, Qinghui

2015-06-01

This study was conducted to investigate the effect of dietary proline (Pro), and Pro and hydroxyproline (Hyp) in combination on the growth performance, total Hyp and collagen concentrations of tissues, and prolyl 4-hydroxylase α(I) (P4H α(I)) gene expression in juvenile turbot feeding high plant protein diets. A diet containing 50% crude protein and 12% crude lipid was formulated as the basal and control, on which other two protein and lipid contents identical experimental diets were formulated by supplementing the basal with either 0.75% Pro (Pro-0.75) or 0.75% Pro and 0.75% Hyp (Pro+Hyp). Four groups of fish in indoor seawater recirculating systems, 35 individuals each, were fed twice a day to apparent satiation for 10 weeks. The results showed that dietary Pro and Hyp supplementation had no significant effect on growth performance and feed utilization of juvenile turbot (P > 0.05). Total Hyp and collagen concentrations in muscle were significantly increased when dietary Pro and Hyp increased (P <0.05), and fish fed diet Pro+Hyp showed significantly higher free Hyp content in plasma than those fed other diets (P <0.05). The expression of P4H a(I) gene in liver and muscle was significantly up regulated in fish fed diet Pro-0.75 in comparison with control (P <0.05); however the gene was significantly down regulated in fish fed diet Pro+Hyp in muscle in comparison with fish fed diet Pro-0.75 (P <0.05). It can be concluded that supplement of crystal L-Pro and L-Hyp to high plant protein diets did not show positive effects on growth performance of juvenile turbot, but enhanced total collagen concentrations in muscle.

D- and L-[123I]-2-I-phenylalanine show a long tumour retention compared with D- and L-[123I]-2-I-tyrosine in R1M rhabdomyosarcoma tumour-bearing Wag/Rij rats.

PubMed

Bauwens, Matthias; Lahoutte, Tony; Kersemans, Ken; Caveliers, Vicky; Bossuyt, Axel; Mertens, John

2007-07-01

The aim of this study was the comparison of the tumour uptake and the long-term retention of [(123)I]-2-I-L-phenylalanine and [(123)I]-2-I-D-phenylalanine with those of [(123)I]-2-I-L-tyrosine and [(123)I]-2-I-D-tyrosine in R1M rhabdomyosarcoma tumour-bearing rats. The biodistribution of the radioactivity as a function of time in R1M tumour-bearing rats was measured by planar gamma camera imaging (dynamic and static). If dissection was applied, the activity in the tumours and tissues of interest was measured by gamma counting. [(123)I]-2-iodo-L-phenylalanine, [(123)I]-2-iodo-D-phenylalaine, [(123)I]-2-I-L-tyrosine showed a considerable tumour uptake reaching a maximum between 10 and 30 min. At 30 min p.i. the differential uptake ratio values of this uptake were, respectively, 2.1, 2.3, 2.5 and 1.7. The activity in the tumour was shown to be related to a tumour cell uptake and not to an increased blood pool activity. All the tracers showed a clearance from the blood to the bladder without renal retention. At longer times both L- and D- [(123)I]-2-I-tyrosine were cleared for a large part from the tumours and the body. [(123)I]-2-I-L-Phe and [(123)I]-2-I-D-Phe showed a considerable and equal retention in the tumours: as compared with 0.5 h, 91% at 24 h and 80% at 48 h. This was related to the longer retention of activity in the blood pool noticed for these compounds (81% at 24 h and 65% at 48 h). The tumour-to-background ratio increased with 25% at those longer times. At short times all the tracers were taken up to a considerable extent in the tumours. In the R1M-bearing Wag/Rij rat model only [(123)I]-2-I-L-phenylalanine and [(123)I]-2-I-D-phenylalanine showed an especially high retention at long times without any significant difference between the enantiomers. Copyright 2007 John Wiley & Sons, Ltd.

Extracellular and intracellular cleavages of proBDNF required at two distinct stages of late-phase LTP

NASA Astrophysics Data System (ADS)

Pang, Petti T.; Nagappan, Guhan; Guo, Wei; Lu, Bai

2016-05-01

Although late-phase long-term potentiation (L-LTP) is implicated in long-term memory, its molecular mechanisms are largely unknown. Here we provide evidence that L-LTP can be divided into two stages: an induction stage (I) and a maintenance stage (II). Both stages require mature brain-derived neurotrophic factor (mBDNF), but involve distinct underlying mechanisms. Stage I requires secretion of existing proBDNF followed by extracellular cleavage by tPA/plasmin. Stage II depends on newly synthesized BDNF. Surprisingly, mBDNF at stage II is derived from intracellular cleavage of proBDNF by furin/PC1. Moreover, stage I involves BDNF-TrkB signaling mainly through MAP kinase, whereas all three signaling pathways (phospholipase C-γ, PI3 kinase, and MAP kinase) are required for the maintenance of L-LTP at stage II. These results reveal the molecular basis for two temporally distinct stages in L-LTP, and provide insights on how BDNF modulates this long-lasting synaptic alternation at two critical time windows.

Pro-apoptotic Bid induces membrane perturbation by inserting selected lysolipids into the bilayer.

PubMed

Goonesinghe, Alexander; Mundy, Elizabeth S; Smith, Melanie; Khosravi-Far, Roya; Martinou, Jean-Claude; Esposti, Mauro D

2005-04-01

Bid is a BH3-only member of the Bcl-2 family that regulates cell death at the level of mitochondrial membranes. Bid appears to link the mitochondrial pathway with the death receptor-mediated pathway of cell death. It is generally assumed that the f.l. (full-length) protein becomes activated after proteolytic cleavage, especially by apical caspases like caspase 8. The cleaved protein then relocates to mitochondria and promotes membrane permeabilization, presumably by interaction with mitochondrial lipids and other Bcl-2 proteins that facilitate the release of apoptogenic proteins like cytochrome c. Although the major action may reside in the C-terminus part, tBid (cleaved Bid), un-cleaved Bid also has pro-apoptotic potential when ectopically expressed in cells or in vitro. This pro-apoptotic action of f.l. Bid has remained unexplained, especially at the biochemical level. In the present study, we show that f.l. (full-length) Bid can insert specific lysolipids into the membrane surface, thereby priming mitochondria for the release of apoptogenic factors. This is most effective for lysophosphatidylcholine species that we report to accumulate in mitochondria during apoptosis induction. A Bid mutant that is not pro-apoptotic in vivo is defective in lysophosphatidylcholine-mediated membrane perturbation in vitro. Our results thus provide a biochemical explanation for the pro-apoptotic action of f.l. Bid.

Preferential hydrophobic interactions are responsible for a preference of D-amino acids in the aminoacylation of 5'-AMP with hydrophobic amino acids

NASA Technical Reports Server (NTRS)

Lacey, J. C. Jr; Wickramasinghe, N. S.; Sabatini, R. S.

1992-01-01

We have studied the chemistry of aminoacyl AMP to model reactions at the 3' terminus of aminoacyl tRNA for the purpose of understanding the origin of protein synthesis. The present studies relate to the D, L preference in the esterification of 5'-AMP. All N-acetyl amino acids we studied showed faster reaction of the D-isomer, with a generally decreasing preference for D-isomer as the hydrophobicity of the amino acid decreased. The beta-branched amino acids, Ile and Val, showed an extreme preference for D-isomer. Ac-Leu, the gamma-branched amino acid, showed a slightly low D/L ratio relative to its hydrophobicity. The molecular basis for these preferences for D-isomer is understandable in the light of our previous studies and seems to be due to preferential hydrophobic interaction of the D-isomer with adenine. The preference for hydrophobic D-amino acids can be decreased by addition of an organic solvent to the reaction medium. Conversely, peptidylation with Ac-PhePhe shows a preference for the LL isomer over the DD isomer.

Effect of the Crevice Former on the Corrosion Behavior of 316L Stainless Steel in Chloride-Containing Synthetic Tap Water

NASA Astrophysics Data System (ADS)

Kim, Seon-Hong; Lee, Ji-Hoon; Kim, Jung-Gu; Kim, Woo-Cheol

2018-05-01

To restrain the failure of the plate heat exchanger (PHE) in customer boiler working fluid, the effect of crevice former type on the corrosion behavior of the 316L stainless steel plate was investigated using electrochemical methods and surface analyses in chloride-containing synthetic tap water (60 °C). The localized corrosion under metal-metal crevice condition was initiated more easily than that under the metal-gasket crevice condition due to the restricted mass transport at the gasket crevice mouth. However, the anodic current under the metal-metal crevice condition was lower than that under metal-gasket crevice condition at a higher anodic potential, indicating that that the metal dissolution under EPDM crevice would be higher than that under metal crevice under the accelerated corrosion condition. Because narrow crevice gap that was formed under gasket accelerated the anodic dissolution at the crevice mouth, the perforation tendency under metal-gasket crevice condition is much higher than that under metal-metal crevice condition. As a result, the crevice geometry, especially the crevice gap, mainly affected the corrosion behavior of PHE material.

Effect of the Crevice Former on the Corrosion Behavior of 316L Stainless Steel in Chloride-Containing Synthetic Tap Water

NASA Astrophysics Data System (ADS)

Kim, Seon-Hong; Lee, Ji-Hoon; Kim, Jung-Gu; Kim, Woo-Cheol

2018-03-01

To restrain the failure of the plate heat exchanger (PHE) in customer boiler working fluid, the effect of crevice former type on the corrosion behavior of the 316L stainless steel plate was investigated using electrochemical methods and surface analyses in chloride-containing synthetic tap water (60 °C). The localized corrosion under metal-metal crevice condition was initiated more easily than that under the metal-gasket crevice condition due to the restricted mass transport at the gasket crevice mouth. However, the anodic current under the metal-metal crevice condition was lower than that under metal-gasket crevice condition at a higher anodic potential, indicating that that the metal dissolution under EPDM crevice would be higher than that under metal crevice under the accelerated corrosion condition. Because narrow crevice gap that was formed under gasket accelerated the anodic dissolution at the crevice mouth, the perforation tendency under metal-gasket crevice condition is much higher than that under metal-metal crevice condition. As a result, the crevice geometry, especially the crevice gap, mainly affected the corrosion behavior of PHE material.

Studies on some feed additives and materials giving partial protection against the suppressive effect of ochratoxin A on egg production of laying hens.

PubMed

Stoev, Stoycho D

2010-06-01

The protective effects of various feed supplements against the harmful effect of ochratoxin A on egg production and sexual maturation of two-weeks old Plymouth Rock female chicks designed for laying hens were studied. A significant protective effect of the feed additives or materials: water extract of artichoke (WEA), sesame seed (SS), Roxazyme-G (RG) and l-beta phenylalanine (PHE) against the suppressive effect of ochratoxin A (OTA) on egg production of laying hens was found. A similar protection was also seen on the toxic effect of OTA on various internal organs of the same hens. A significant protection was found against the decrease of the weight or the quantity of eggs as well as against the delay of the beginning of the laying period of chicks, both of which were provoked by ochratoxin A. These protective effects were strongest in chicks treated with SS or WEA, but were slightest in chicks treated with l-beta PHE. Copyright 2009 Elsevier Ltd. All rights reserved.

L-Phenylalanine catabolism and 2-phenylethanol synthesis in Yarrowia lipolytica--mapping molecular identities through whole-proteome quantitative mass spectrometry analysis.

PubMed

Celińska, Ewelina; Olkowicz, Mariola; Grajek, Włodzimierz

2015-08-01

A world-wide effort is now being pursued towards the development of flavors and fragrances (F&F) production independently from traditional sources, as well as autonomously from depleting fossil fuel supplies. Biotechnological production of F&F by microbes has emerged as a vivid solution to the current market limitations. Amongst a wide variety of fragrant chemicals, 2-PE is of significant interest to both scientific and industrial community. Although the general overview of the 2-PE synthesis pathway is commonly known, involvement of particular molecular identities in this pathway has not been elucidated in Yarrowia lipolytica to date. The aim of this study was mapping molecular identities involved in 2-PE synthesis in Y. lipolytica. To acquire a comprehensive landscape of the proteins that are directly and indirectly involved in L-Phe degradation and 2-PE synthesis, we took advantage of comprehensibility and sensitivity of high-throughput LC-MS/MS-quantitative analysis. Amongst a number of proteins involved in amino acid turnover and the central carbon metabolism, enzymes involved in L-Phe conversion to 2-PE have been identified. Results on yeast-to-hyphae transition in relation to the character of the provided nitrogen source have been presented. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Production of a New Plant-Based Milk from Adenanthera pavonina Seed and Evaluation of Its Nutritional and Health Benefits

PubMed Central

Afolabi, Israel Sunmola; Nwachukwu, Irene Chiamaka; Ezeoke, Chinemelum Sandra; Woke, Ruth Chineme; Adegbite, Olawunmi Adebisi; Olawole, Tolulope Dorcas; Martins, Olubukola C.

2018-01-01

A new plant milk was discovered from the seed of Adenanthera pavonina. The physicochemical and nutritional properties of the new pro-milk extract were assessed, and their biochemical effects were compared with those of soy bean extracts. Eleven groups of three albino rats each were used to assess the health benefits of the pro-milk. Groups were separately administered 3.1, 6.1, and 9.2 µl/g animal wt. pro-milk extract from A. pavonina seed, 6.1 µl/g animal wt. milk extract from soybean, and 6.1 µl/g animal wt. normal saline for 7 or 14 days. The “baseline” group consisted of those sacrificed on day 0. Among the physical properties considered, the pro-milk from A. pavonina had significantly higher (P < 0.05) hue color value and significantly lower (P < 0.05) L* than that from soy bean did. The pro-milk from A. pavonina had a significantly higher (P < 0.05) level of protein (36.14 ± 0.12%), Ca (440.99 ± 0.93 mg/l), Mg (96.69 ± 0.03 mg/l), K (190.41 ± 0.11 mg/l), Na (64.24 ± 0.24 mg/l), and Cu (0.55 ± 0.24 mg/l), and a significantly lower (P < 0.05) level of Mn (0.04 ± 0.01 mg/l) and vitamins A (undetectable), C (1.87 ± 0.01 mg/100 g), and E (0.12 ± 0.01 mg/100 g) compared to those of soy milk. The daily consumption of the pro-milk extract from A. pavonina for 14 days significantly reduced (P < 0.05) Ca2+-adenosine triphosphate synthase (Ca2+-ATPase) at low dose (3.1 µl/g animal wt.), but significantly increased (P < 0.05) Mg2+-ATPase at high dose (9.2 µl/g animal wt.). Daily administration of the A. pavonina extract for 14 days caused a significant reduction (P < 0.05) in acetylcholinesterase activity in the liver, intestine, heart, and kidney, suggesting that the pro-milk may facilitate ions transportation across the membrane. The pro-milk offers promising beneficial effects for patients with neurological diseases, as well as supporting general health owing to the high protein and mineral content. Vitamins fortification is recommended during production. PMID:29556498

A Cyclic Altered Peptide Analogue Based on Myelin Basic Protein 87-99 Provides Lasting Prophylactic and Therapeutic Protection Against Acute Experimental Autoimmune Encephalomyelitis.

PubMed

Emmanouil, Mary; Tseveleki, Vivian; Triantafyllakou, Iro; Nteli, Agathi; Tselios, Theodore; Probert, Lesley

2018-01-31

In this report, amide-linked cyclic peptide analogues of the 87-99 myelin basic protein (MBP) epitope, a candidate autoantigen in multiple sclerosis (MS), are tested for therapeutic efficacy in experimental autoimmune encephalomyelitis (EAE). Cyclic altered peptide analogues of MBP 87-99 with substitutions at positions 91 and/or 96 were tested for protective effects when administered using prophylactic or early therapeutic protocols in MBP 72-85 -induced EAE in Lewis rats. The Lys 91 and Pro 96 of MBP 87-99 are crucial T-cell receptor (TCR) anchors and participate in the formation of trimolecular complex between the TCR-antigen (peptide)-MHC (major histocompability complex) for the stimulation of encephalitogenic T cells that are necessary for EAE induction and are implicated in MS. The cyclic peptides were synthesized using Solid Phase Peptide Synthesis (SPPS) applied on the 9-fluorenylmethyloxycarboxyl/tert-butyl Fmoc/tBu methodology and combined with the 2-chlorotrityl chloride resin (CLTR-Cl). Cyclo(91-99)[Ala 96 ]MBP 87-99 , cyclo(87-99)[Ala 91,96 ]MBP 87-99 and cyclo(87-99)[Arg 91 , Ala 96 ]MBP 87-99 , but not wild-type linear MBP 87-99 , strongly inhibited MBP 72-85 -induced EAE in Lewis rats when administered using prophylactic and early therapeutic vaccination protocols. In particular, cyclo(87-99)[Arg 91 , Ala 96 ]MBP 87-99 was highly effective in preventing the onset and development of clinical symptoms and spinal cord pathology and providing lasting protection against EAE induction.

Motility and Adhesiveness in Human Neutrophils

PubMed Central

Smith, C. Wayne; Hollers, James C.; Patrick, Richard A.; Hassett, Clare

1979-01-01

Human peripheral blood neutrophils (PMN) obtained from healthy adults were examined in vitro with techniques adapted to assess the effects of chemotactic factors (CF) on cellular configuration and adhesiveness. The results were compared with those that use certain conventional techniques for assessing chemotaxis and chemokinesis. Exposure of PMN to N-formyl-l-methionyl-l-phenylalanine (f-Met-Phe), zymosan-activated serum, bacterial chemotactic factor, or a low molecular weight chemotactic factor from activated serum (C5a) in the absence of a gradient resulted in a change in cellular shape from a spherical to a polarized configuration in a high percentage of cells. This occurred rapidly in suspension, under conditions designed to exclude a role for cell adhesiveness, and was reversible upon removal of the CF. Restimulation of cells with the CF resulted in reappearance of the polarized configuration to the same extent as on initial stimulation with one exception: f-Met-Phe pretreated cells failed to respond to f-Met-Phe, though they responded fully to the other CF. Each CF caused a significant increase in PMN attachment to protein-coated glass. This enhanced adhesiveness was not reversible upon removal of the CF when the cells were treated under conditions shown to produce chemotactic deactivation. Cells treated under these conditions also exhibited significantly reduced motility on glass and in micropore filters in the absence of a gradient of CF. Bacterial chemotactic factor, even at high concentrations, failed to produce deactivation and did not cause a sustained enhancement of adhesiveness. Images PMID:372238

Efficient Reassignment of a Frequent Serine Codon in Wild-Type Escherichia coli.

PubMed

Ho, Joanne M; Reynolds, Noah M; Rivera, Keith; Connolly, Morgan; Guo, Li-Tao; Ling, Jiqiang; Pappin, Darryl J; Church, George M; Söll, Dieter

2016-02-19

Expansion of the genetic code through engineering the translation machinery has greatly increased the chemical repertoire of the proteome. This has been accomplished mainly by read-through of UAG or UGA stop codons by the noncanonical aminoacyl-tRNA of choice. While stop codon read-through involves competition with the translation release factors, sense codon reassignment entails competition with a large pool of endogenous tRNAs. We used an engineered pyrrolysyl-tRNA synthetase to incorporate 3-iodo-l-phenylalanine (3-I-Phe) at a number of different serine and leucine codons in wild-type Escherichia coli. Quantitative LC-MS/MS measurements of amino acid incorporation yields carried out in a selected reaction monitoring experiment revealed that the 3-I-Phe abundance at the Ser208AGU codon in superfolder GFP was 65 ± 17%. This method also allowed quantification of other amino acids (serine, 33 ± 17%; phenylalanine, 1 ± 1%; threonine, 1 ± 1%) that compete with 3-I-Phe at both the aminoacylation and decoding steps of translation for incorporation at the same codon position. Reassignments of different serine (AGU, AGC, UCG) and leucine (CUG) codons with the matching tRNA(Pyl) anticodon variants were met with varying success, and our findings provide a guideline for the choice of sense codons to be reassigned. Our results indicate that the 3-iodo-l-phenylalanyl-tRNA synthetase (IFRS)/tRNA(Pyl) pair can efficiently outcompete the cellular machinery to reassign select sense codons in wild-type E. coli.

The opiorphin gene (ProL1) and its homologues function in erectile physiology.

PubMed

Tong, Yuehong; Tar, Moses; Melman, Arnold; Davies, Kelvin

2008-09-01

To determine if ProL1, a member of the opiorphin family of genes, can modulate erectile physiology, as it encodes a peptide which acts as a neutral endopeptidase inhibitor, other examples of which (Vcsa1, hSMR3A) modulate erectile physiology. We cloned members of the opiorphin family of genes into the same mammalian expression backbone (pVAX); 100 microg of these plasmids (pVAX-Vcsa1, -hSMR3A, -hSMR3B and -ProL1) were injected intracorporally into retired breeder rats and the affect on erectile physiology assessed visually, by histology and by measuring the intracavernous pressure (ICP) and blood pressure (BP). As a positive control, rats were treated with pVAX-hSlo (expressing the MaxiK potassium channel) and as a negative control the empty backbone plasmid was injected (pVAX). We also compared the level of expression of ProL1 in corporal tissue of patients not reporting erectile dysfunction (ED), ED associated with diabetes and ED not caused by diabetes. Gene transfer of plasmids expressing all members of the opiorphin family had a similar and significant effect on erectile physiology. At the concentration used in these experiments (100 microg) they resulted in higher resting ICP, and histological and visual analysis showed evidence of a priapic-like condition. After electrostimulation of the cavernous nerve, rats had significantly better ICP/BP than the negative control (pVAX). Gene transfer of pVAX-hSlo increased the ICP/BP ratio to a similar extent to the opiorphin homologues, but with no evidence for a priapic-like condition. Corpora cavernosa tissue samples obtained from men with ED, regardless of underlying causes, had significant down-regulation of both hSMR3A and ProL1. All members of the human opiorphin family of genes can potentially modulate erectile physiology. Both hSMR3 and ProL1 are down-regulated in the corpora of men with ED, and therefore both genes can potentially act as markers of ED.

The opiorphin gene (ProL1) and its homologues function in erectile physiology

PubMed Central

Tong, Yuehong; Tar, Moses; Melman, Arnold; Davies, Kelvin

2010-01-01

OBJECTIVE To determine if ProL1, a member of the opiorphin family of genes, can modulate erectile physiology, as it encodes a peptide which acts as a neutral endopeptidase inhibitor, other examples of which (Vcsa1, hSMR3A) modulate erectile physiology. MATERIALS AND METHODS We cloned members of the opiorphin family of genes into the same mammalian expression backbone (pVAX); 100 μg of these plasmids (pVAX-Vcsa1, -hSMR3A, -hSMR3B and -ProL1) were injected intracorporally into retired breeder rats and the affect on erectile physiology assessed visually, by histology and by measuring the intracavernous pressure (ICP) and blood pressure (BP). As a positive control, rats were treated with pVAX-hSlo (expressing the MaxiK potassium channel) and as a negative control the empty backbone plasmid was injected (pVAX). We also compared the level of expression of ProL1 in corporal tissue of patients not reporting erectile dysfunction (ED), ED associated with diabetes and ED not caused by diabetes. RESULTS Gene transfer of plasmids expressing all members of the opiorphin family had a similar and significant effect on erectile physiology. At the concentration used in these experiments (100 μg) they resulted in higher resting ICP, and histological and visual analysis showed evidence of a priapiclike condition. After electrostimulation of the cavernous nerve, rats had significantly better ICP/BP than the negative control (pVAX). Gene transfer of pVAX-hSlo increased the ICP/BP ratio to a similar extent to the opiorphin homologues, but with no evidence for a priapic-like condition. Corpora cavernosa tissue samples obtained from men with ED, regardless of underlying causes, had significant down-regulation of both hSMR3A and ProL1. CONCLUSION All members of the human opiorphin family of genes can potentially modulate erectile physiology. Both hSMR3 and ProL1 are down-regulated in the corpora of men with ED, and therefore both genes can potentially act as markers of ED. PMID:18410445

Association between level of exposure to death and dying and professional quality of life among palliative care workers.

PubMed

Samson, Tali; Shvartzman, Pesach

2017-06-23

Exposure to the death and dying of others is an anxiety-provoking condition that can contribute to psychological stress. However, the results of empirical studies that evaluated work-related outcomes among physicians and nurses with repeated exposure to dying patients are not consistent. Our aim was to evaluate whether a high level of exposure to death and dying (LED) can increase the risk for poor professional quality of life (ProQoL) in most healthcare workers, but it can also improve ProQoL in a subset of healthcare workers with specific characteristics. We employed a cross-sectional survey designed to better understand the role of LED as a predictor of ProQoL among healthcare workers. Comparison of physicians and nurses with high LED (home-based palliative care units) with a matched group of physicians and nurses with low LED (primary care units) and evaluation of possible interaction effects among LED, death anxiety (DA), and engagement as predictors of ProQoL. The final sample included 110 questionnaires from the high-LED group (response rate = 39%) and 131 from the low-LED (response rate = 24%) group. Workers with high LED reported an increased level of compassion satisfaction (CS) and low to moderate levels of burnout (BU) and secondary traumatic stress (STS), with no significant differences with respect to other healthcare providers. Although levels of CS, STS, and BU did not differ between groups, a univariate MANOVA revealed that the interaction effect of LED × Engagement reduced levels of CS and that the interaction effect of LED × DA increased STS among workers with high LED. LED was significantly correlated with ProQoL among healthcare workers with high LED due to the reported interaction effect. These findings imply, for the first time, that there is a possible correlation between engagement and the risk for poor ProQoL among workers with high LED. Further research is essential to gain a better understanding of this issue.

Distinctive proteolytic activity of cell envelope proteinase of Lactobacillus helveticus isolated from airag, a traditional Mongolian fermented mare's milk.

PubMed

Miyamoto, Mari; Ueno, Hiroshi M; Watanabe, Masayuki; Tatsuma, Yumi; Seto, Yasuyuki; Miyamoto, Taku; Nakajima, Hadjime

2015-03-16

Airag is a traditional fermented milk of Mongolia that is usually made from raw mare's milk. Lactobacillus helveticus is one of the lactic acid bacteria most frequently isolated from airag. In this study, we investigated the genetic and physiological characteristics of L. helveticus strains isolated from airag and clarified their significance in airag by comparing them with strains from different sources. Six strains of L. helveticus were isolated from five home-made airag samples collected from different regions of Mongolia. The optimal temperature for acidification in skim milk was 30 to 35°C for all the Mongolian strains, which is lower than those for the reference strains (JCM 1554 and JCM 1120(T)) isolated from European cheeses. All of the strains had a prtH1-like gene encoding a variant type of cell envelope proteinase (CEP). The CEP amino acid sequence in Snow Brand Typeculture (SBT) 11087 isolated from airag shared 71% identity with PrtH of L. helveticus CNRZ32 (AAD50643.1) but 98% identity with PrtH of Lactobacillus kefiranofaciens ZW3 (AEG40278.1) isolated from a traditional fermented milk in Tibet. The proteolytic activities of the CEP from SBT11087 on artificial substrate (N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide) and pure casein were measured using an intact-cell degradation assay. The activity of the CEP from SBT11087 was observed to be weak and exhibited a lower optimal temperature (40°C) than those from the reference strains (45-50°C). The specificity of the SBT11087 CEP for αS1-casein was typical of the CEPs previously reported in L. helveticus, as determined through the degradation profiles obtained through gel electrophoresis and mass spectrometry analyses. In contrast, the degradation profile of β-casein revealed that the CEP of SBT11087 primarily hydrolyzes its C-terminal domain and hydrolyzed nine of the 16 cleavage sites shared among the CEPs of other L. helveticus strains. Thus, the CEP of SBT11087 is distinct from those from previously reported L. helveticus strains in terms of its optimal temperature and its degradation of β-casein. Therefore, the Mongolian L. helveticus strains differ from other strains of the species in different collections and are specifically suited for the natural lactic acid bacterial population in airag. Copyright © 2014 Elsevier B.V. All rights reserved.

Parasite-specific IL-17-type cytokine responses and soluble IL-17 receptor levels in Alveolar Echinococcosis patients.

PubMed

Lechner, Christian J; Grüner, Beate; Huang, Xiangsheng; Hoffmann, Wolfgang H; Kern, Peter; Soboslay, Peter T

2012-01-01

Alveolar Echinococcosis (AE) caused by the cestode Echinococcus multilocularis, is a severe helminth infection of man, where unrestricted parasite growth will ultimately result in organ failure and fatality. The tissue-infiltrative growth of the larval metacestode and the limited efficacy of available drugs complicate successful intervention in AE; patients often need life-long medication, and if possible, surgical resection of affected tissues and organs. Resistance to AE has been reported, but the determinants which confer protection are not known. ln this study, we analyzed in patients at distinct stages of Alveolar Echirococcosis, that is cured, stable and progressive AE, as well as in infection-free controls, the cellular production and plasma levels of pro-inflammatory cytokines lL-17A, lL-17B, lL-17F and their soluble receptors lL-17RA (slL-17RA) and IL-17RB (sIL-17RB). Significantly elevated levels of IL-17B and slL-17RB were observed, whilst lL-17F and slL-17RA were reduced in patients with AE. Similarly, the cellular production of lL-17F and slL-L7RA in response to E. multilocularis antigens was low in AE patients, while levels of slL-17RB were highly enhanced. These observations suggest immune-modulating properties of E. multitocularis on lL-17 cytokine-mediated pro-inflammatory immune responses; this may facilitate the tissue infiltrative growth of the parasite and its persistence in the human host.

L1014F-kdr Mutation in Indian Anopheles subpictus (Diptera: Culicidae) Arising From Two Alternative Transversions in the Voltage-Gated Sodium Channel and a Single PIRA-PCR for Their Detection

PubMed Central

Singh, O. P.; Dykes, C. L.; Sharma, G.; Das, M. K.

2015-01-01

Leucine-to-phenylalanine substitution at residue L1014 in the voltage-gated sodium channel, target site of action for dichlorodiphenyltrichloroethane (DDT) and pyrethroids, is the most common knockdown resistance (kdr) mutation reported in several insects conferring resistance against DDT and pyrethroids. Here, we report presence of two coexisting alternative transversions, A>T and A>C, on the third codon position of L1014 residue in malaria vector Anopheles subpictus Grassi (species A) from Jamshedpur (India), both leading to the same amino acid substitution of Leu-to-Phe with allelic frequencies of 19 and 67%, respectively. A single primer-introduced restriction analysis–polymerase chain reaction (PIRA-PCR) was devised for the identification of L1014F-kdr mutation in An. subpictus resulting from either type of point mutation. Genotyping of samples with PIRA-PCR revealed high frequency (82%) of L1014F-kdr mutation in the study area. PMID:26336276

NMR structure determination of a synthetic analogue of bacillomycin Lc reveals the strategic role of L-Asn1 in the natural iturinic antibiotics

NASA Astrophysics Data System (ADS)

Volpon, Laurent; Tsan, Pascale; Majer, Zsuzsa; Vass, Elemer; Hollósi, Miklós; Noguéra, Valérie; Lancelin, Jean-Marc; Besson, Françoise

2007-08-01

Iturins are a group of antifungal produced by Bacillus subtilis. All are cyclic lipopeptides with seven α-amino acids of configuration LDDLLDL and one β-amino fatty acid. The bacillomycin L is a member of this family and its NMR structure was previously resolved using the sequence Asp-Tyr-Asn-Ser-Gln-Ser-Thr. In this work, we carefully examined the NMR spectra of this compound and detected an error in the sequence. In fact, Asp1 and Gln5 need to be changed into Asn1 and Glu5, which therefore makes it identical to bacillomycin Lc. As a consequence, it now appears that all iturinic peptides with antibiotic activity share the common β-amino fatty acid 8- L-Asn1- D-Tyr2- D-Asn3 sequence. To better understand the conformational influence of the acidic residue L-Asp1, present, for example in the inactive iturin C, the NMR structure of the synthetic analogue SCP [cyclo ( L-Asp1- D-Tyr2- D-Asn3- L-Ser4- L-Gln5- D-Ser6- L-Thr7-β-Ala8)] was determined and compared with bacillomycin Lc recalculated with the corrected sequence. In both cases, the conformers obtained were separated into two families of similar energy which essentially differ in the number and type of turns. A detailed analysis of both cyclopeptide structures is presented here. In addition, CD and FTIR spectra were performed and confirmed the conformational differences observed by NMR between both cyclopeptides.

Unprecedented 22,26-seco physalins from Physalis angulata and their anti-inflammatory potential.

PubMed

Sun, Cheng-Peng; Oppong, Mahmood Brobbey; Zhao, Feng; Chen, Li-Xia; Qiu, Feng

2017-10-25

Two novel physalins, including a 22,26-seco physalin, physalin X (1), and a 11,15-cyclo-9(10),14(17),22(26)-triseco physalin with an unprecedented aromatic ring, aromaphysalin B (2), were isolated from Physalis angulata L. Their structures were determined by IR, UV, HRESIMS, and 2D NMR spectra as well as theoretical calculations. Compounds 1 and 2 exhibited inhibitory activities on NO production with IC 50 values of 68.50 and 29.69 μM, respectively. A plausible biosynthetic pathway for 2 is also discussed.