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.

Effects of a combination of elicitation and precursor feeding on grape amino acid composition through foliar applications to Garnacha vineyard.

PubMed

Gutiérrez-Gamboa, Gastón; Portu, Javier; López, Rosa; Santamaría, Pilar; Garde-Cerdán, Teresa

2018-04-01

Vine foliar applications of phenylalanine (Phe) or methyl jasmonate (MeJ) could improve the synthesis of secondary metabolites. However, there are no reports focusing on the effects of elicitation supported by precursor feeding on must amino acid composition in grapevines. The aim of this research was to study the effect of the elicitation of methyl jasmonate (MeJ) supported by phenylalanine (Phe) as a precursor feeding (MeJ+Phe) and its application individually on must amino acid composition. Results showed that foliar Phe and MeJ treatments decreased the concentration of most of the studied amino acids with respect to the control (p≤0.05). MeJ+Phe treatment did not affect must nitrogen content. Musts obtained from MeJ+Phe showed higher concentration of several amino acids than samples from Phe and MeJ applications. Therefore, other sources of precursor feeding could support elicitation, to improve amino acid composition and be considered as a tool for viticulture. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

The impact of model peptides on structural and dynamic properties of egg yolk lecithin liposomes - experimental and DFT studies.

PubMed

Wałęsa, Roksana; Man, Dariusz; Engel, Grzegorz; Siodłak, Dawid; Kupka, Teobald; Ptak, Tomasz; Broda, Małgorzata A

2015-07-01

Electron spin resonance (ESR), (1) H-NMR, voltage and resistance experiments were performed to explore structural and dynamic changes of Egg Yolk Lecithin (EYL) bilayer upon addition of model peptides. Two of them are phenylalanine (Phe) derivatives, Ac-Phe-NHMe (1) and Ac-Phe-NMe2 (2), and the third one, Ac-(Z)-ΔPhe-NMe2 (3), is a derivative of (Z)-α,β-dehydrophenylalanine. The ESR results revealed that all compounds reduced the fluidity of liposome's membrane, and the highest activity was observed for compound 2 with N-methylated C-terminal amide bond (Ac-Phe-NMe2 ). This compound, being the most hydrophobic, penetrates easily through biological membranes. This was also observed in voltage and resistance studies. (1) H-NMR studies provided a sound evidence on H-bond interactions between the studied diamides and lecithin polar head. The most significant changes in H-atom chemical shifts and spin-lattice relaxation times T1 were observed for compound 1. Our experimental studies were supported by theoretical calculations. Complexes EYLAc-Phe-NMe2 and EYLAc-(Z)-ΔPhe-NMe2 , stabilized by NH⋅⋅⋅O or/and CH⋅⋅⋅O H-bonds were created and optimized at M06-2X/6-31G(d) level of theory in vacuo and in H2 O environment. According to our molecular-modeling studies, the most probable lecithin site of H-bond interaction with studied diamides is the negatively charged O-atom in phosphate group which acts as H-atom acceptor. Moreover, the highest binding energy to hydrocarbon chains were observed in the case of Ac-Phe-NMe2 (2). Copyright © 2015 Verlag Helvetica Chimica Acta AG, Zürich.

Polypharmacotherapy in rheumatology: 1H NMR analysis of binding of phenylbutazone and methotrexate to serum albumin

NASA Astrophysics Data System (ADS)

Maciążek-Jurczyk, M.; Sułkowska, A.; Równicka-Zubik, J.; Bojko, B.; Szkudlarek-Haśnik, A.; Knopik, M.; Sułkowski, W. W.

2011-05-01

The influence of phenylbutazone (Phe) and methotrexate (MTX) on binding of MTX and Phe to human (HSA) and bovine (BSA) serum albumin in the low-affinity binding sites is investigated. The strength and kind of interactions between serum albumin (SA) and drugs used in combination therapy were found using 1H NMR spectroscopy. A stoichiometric molar ratios for Phe-SA and MTX-SA complexes are 36:1 and 31:1, respectively. It appeared these molar ratios are higher for the ternary systems than it were in the binary ones. The presence of the additional drug (MTX or Phe) causes the increase of an affinity of albumin towards Phe and MTX. It was found that the aliphatic groups of MTX are more resistant to the influence of Phe on the MTX-SA complex than the aromatic rings. The results showed the important impact of another drug (MTX or Phe) on the affinity of SA towards Phe and MTX in the low-affinity binding sites. This work is a subsequent part of the spectroscopic study on Phe-MTX-SA interactions (Maciążek-Jurczyk, 2009 [1]).

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.

Aromatic residues located close to the active center are essential for the catalytic reaction of flap endonuclease-1 from hyperthermophilic archaeon Pyrococcus horikoshii.

PubMed

Matsui, Eriko; Abe, Junko; Yokoyama, Hideshi; Matsui, Ikuo

2004-04-16

Flap endonuclease-1 (FEN-1) possessing 5'-flap endonuclease and 5'-->3' exonuclease activity plays important roles in DNA replication and repair. In this study, the kinetic parameters of mutants at highly conserved aromatic residues, Tyr33, Phe35, Phe79, and Phe278-Phe279, in the vicinity of the catalytic centers of FEN-1 were examined. The substitution of these aromatic residues with alanine led to a large reduction in kcat values, although these mutants retained Km values similar to that of the wild-type enzyme. Notably, the kcat of Y33A and F79A decreased 333-fold and 71-fold, respectively, compared with that of the wild-type enzyme. The aromatic residues Tyr33 and Phe79, and the aromatic cluster Phe278-Phe279 mainly contributed to the recognition of the substrates without the 3' projection of the upstream strand (the nick, 5'-recess-end, single-flap, and pseudo-Y substrates) for the both exo- and endo-activities, but played minor roles in recognizing the substrates with the 3' projection (the double flap substrate and the nick substrate with the 3' projection). The replacement of Tyr33, Phe79, and Phe278-Phe279, with non-charged aromatic residues, but not with aliphatic hydrophobic residues, recovered the kcat values almost fully for the substrates without the 3' projection of the upstream strand, suggesting that the aromatic groups of Tyr33, Phe79, and Phe278-Phe279 might be involved in the catalytic reaction, probably via multiple stacking interactions with nucleotide bases. The stacking interactions of Tyr33 and Phe79 might play important roles in fixing the template strand and the downstream strand, respectively, in close proximity to the active center to achieve the productive transient state leading to the hydrolysis.

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 challenges, advantages and future of phenome-wide association studies.

PubMed

Hebbring, Scott J

2014-02-01

Over the last decade, significant technological breakthroughs have revolutionized human genomic research in the form of genome-wide association studies (GWASs). GWASs have identified thousands of statistically significant genetic variants associated with hundreds of human conditions including many with immunological aetiologies (e.g. multiple sclerosis, ankylosing spondylitis and rheumatoid arthritis). Unfortunately, most GWASs fail to identify clinically significant associations. Identifying biologically significant variants by GWAS also presents a challenge. The GWAS is a phenotype-to-genotype approach. As a complementary/alternative approach to the GWAS, investigators have begun to exploit extensive electronic medical record systems to conduct a genotype-to-phenotype approach when studying human disease - specifically, the phenome-wide association study (PheWAS). Although the PheWAS approach is in its infancy, this method has already demonstrated its capacity to rediscover important genetic associations related to immunological diseases/conditions. Furthermore, PheWAS has the advantage of identifying genetic variants with pleiotropic properties. This is particularly relevant for HLA variants. For example, PheWAS results have demonstrated that the HLA-DRB1 variant associated with multiple sclerosis may also be associated with erythematous conditions including rosacea. Likewise, PheWAS has demonstrated that the HLA-B genotype is not only associated with spondylopathies, uveitis, and variability in platelet count, but may also play an important role in other conditions, such as mastoiditis. This review will discuss and compare general PheWAS methodologies, describe both the challenges and advantages of the PheWAS, and provide insight into the potential directions in which PheWAS may lead. © 2013 The Authors. Immunology Published by John Wiley & Sons Ltd.

Short communication: A comparison of biofilm development on stainless steel and modified-surface plate heat exchangers during a 17-h milk pasteurization run.

PubMed

Jindal, Shivali; Anand, Sanjeev; Metzger, Lloyd; Amamcharla, Jayendra

2018-04-01

Flow of milk through the plate heat exchanger (PHE) results in denaturation of proteins, resulting in fouling. This also accelerates bacterial adhesion on the PHE surface, eventually leading to the development of biofilms. During prolonged processing, these biofilms result in shedding of bacteria and cross-contaminate the milk being processed, thereby limiting the duration of production runs. Altering the surface properties of PHE, such as surface energy and hydrophobicity, could be an effective approach to reduce biofouling. This study was conducted to compare the extent of biofouling on native stainless steel (SS) and modified-surface [Ni-P-polytetrafluoroethylene (PTFE)] PHE during the pasteurization of raw milk for an uninterrupted processing run of 17 h. For microbial studies, raw and pasteurized milk samples were aseptically collected from inlets and outlets of both PHE at various time intervals to examine shedding of bacteria in the milk. At the end of the run, 3M quick swabs (3M, St. Paul, MN) and ATP swabs (Charm Sciences Inc., Lawrence, MA) were used to sample plates from different sections of the pasteurizers (regeneration, heating, and cooling) for biofilm screening and to estimate the efficiency of cleaning in place, respectively. The data were tested for ANOVA, and means were compared. Modified PHE experienced lower mesophilic and thermophilic bacterial attachment and biofilm formation (average log 1.0 and 0.99 cfu/cm 2 , respectively) in the regenerative section of the pasteurizer compared with SS PHE (average log 1.49 and 1.47, respectively). Similarly, higher relative light units were observed for SS PHE compared with the modified PHE, illustrating the presence of more organic matter on the surface of SS PHE at the end of the run. In addition, at h 17, milk collected from the outlet of SS PHE showed plate counts of 5.44 cfu/cm 2 , which were significantly higher than those for pasteurized milk collected from modified PHE (4.12 log cfu/cm 2 ). This provided further evidence in favor of the modified PHE achieving better microbial quality of pasteurized milk in long process runs. Moreover, because cleaning SS PHE involves an acid treatment step, whereas an alkali treatment step is sufficient for the modified-surface PHE, use of the latter is both cost and time effective, making it a better surface for thermal processing of milk and other fluid dairy products. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

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.

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.

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.

Bioavailability of phenanthrene in the presence of birnessite-mediated catechol polymers.

PubMed

Russo, Fabio; Rao, Maria A; Gianfreda, Liliana

2005-07-01

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental contaminants and contribute to the pollution of aquatic and terrestrial environments. In soil, their fate may be affected by interactions with the soil biological community and soil colloids. This study was conducted to investigate the fate of phenanthrene (Phe), selected as a representative PAH, in simplified model systems, which simulate processes naturally occurring in soil. Phe was interacted with catechol (Cat), an orthodiphenol, and common intermediate in the microbial degradation of PAHs, and birnessite (Bir), an abiotic strong oxidative catalyst abundant in soil. Two experimental conditions were investigated: Cat (5 mM)+Bir (1 mg ml(-1))+Phe (0.05 mg ml(-1)) mixed at the same time and incubated for 24 h at 25 degrees C (Cat-Bir-Phe) and Cat+Bir incubated for 24 h at 25 degrees C before Phe addition and then incubated for a further 24 h (Cat-Bir+Phe). After incubation, the systems were analysed for residual Cat and Phe, supplied with a selected Phe-degrading mixed bacterial culture, and then the microbial degradation of Phe and the growth of cells were monitored. Complex phenomena simultaneously occurred. Cat was completely removed after a 24-h incubation with Bir, and no interference by Phe in the Bir-mediated transformation of Cat was observed. Elemental analysis and UV-Vis and Fourier transfer infrared spectra showed that Cat transformation by Bir produced soluble and insoluble polymeric aggregates involving Phe. The hydrocarbon also interacted with the surfaces of Bir either previously coated (Cat-Bir+Phe sample) or not by Cat polymers. When a Phe-degrading bacterial culture was added to the systems after Bir-mediated Cat polymerisation, a different behaviour was observed in terms of Phe consumption and bacterial growth, thus suggesting differentiated availability of Phe to the microbial cells. The hydrocarbon was completely transformed in the presence of Bir and/or Bir covered by Cat polymers. By contrast a reduced degradation was measured when the Phe was involved in the polymerisation of Cat and entrapped in the Cat polymers (Cat-Bir-Phe). Although Cat showed a toxic, lethal effect on the bacterial cells, microbial growth was observed in the presence of Cat and Cat polymers, as the only C source. The mechanism leading to the different availability of Phe in the presence of Cat and Bir is still not clear. Further investigations are requested to provide more insight into such a complex phenomenon.

Quenching of p-Cyanophenylalanine Fluorescence by Various Anions.

PubMed

Pazos, Ileana M; Roesch, Rachel M; Gai, Feng

2013-03-20

To expand the spectroscopic utility of the non-natural amino acid p -cyanophenylalanine (Phe CN ), we examine the quenching efficiencies of a series of commonly encountered anions toward its fluorescence. We find that iodide exhibits an unusually large Stern-Volmer quenching constant, making it a convenient choice in Phe CN fluorescence quenching studies. Indeed, using the villin headpiece subdomain as a testbed we demonstrate that iodide quenching of Phe CN fluorescence offers a convenient means to reveal protein conformational heterogeneity. Furthermore, we show that the amino group of Phe CN strongly quenches its fluorescence, suggesting that Phe CN could be used as a local pH sensor.

[Phenylketonuria diagnosed during the neonatal period and breast feeding].

PubMed

Cornejo, Verónica; Manríquez, Viviana; Colombo, Marta; Mabe, Paulina; Jiménez, Mónica; De la Parra, Alicia; Valiente, Alf; Raimann, Erna

2003-11-01

Phenylketonuria (PKU) is due to of a defect in the phenylalanine hydroxylase gene (12q22-24.1) leading to hyperphenylalaninemia. Treatment consists in a low phenylalanine (Phe) diet. To evaluate the evolution of early diagnosed PKU children, receiving direct breast feeding, and a special formula without Phe, during their first six months of life. Nineteen PKU children diagnosed in the neonatal period (19.29 +/- 13.8 days of age), treated with breast feeding and formula without Phe since diagnosis, were studied. Intake of calories, proteins and dietary Phe were quantified. Blood Phe, nutritional status and psychomotor development were also measured. The diet that these children received during the 6 months period of study, had a mean of 127 +/- 19.9 Kcal/kg/day, 1.95 +/- 0.3 g protein/kg/day and 35.3 +/- 9.5 mg Phe/kg/day. Fifteen children maintained the blood level of Phe under 8 mg/dl, considered an excellent metabolic control. Only 4 cases had intermittently high levels, between 10-12 mg/dl. At 6 months of age, 74% of the children maintained breast feeding as the only source of Phe. Sixty three percent had a normal nutritional status, 5.2% were at nutritional risk and 31.6% were overweight. Eighty one percent had a normal mental development. The use of direct breast feeding allows a good metabolic control and improves growth and development of early diagnosed PKU children.

Structural basis for ligand-dependent dimerization of phenylalanine hydroxylase regulatory domain

PubMed Central

Patel, Dipali; Kopec, Jolanta; Fitzpatrick, Fiona; McCorvie, Thomas J.; Yue, Wyatt W.

2016-01-01

The multi-domain enzyme phenylalanine hydroxylase (PAH) catalyzes the hydroxylation of dietary I-phenylalanine (Phe) to I-tyrosine. Inherited mutations that result in PAH enzyme deficiency are the genetic cause of the autosomal recessive disorder phenylketonuria. Phe is the substrate for the PAH active site, but also an allosteric ligand that increases enzyme activity. Phe has been proposed to bind, in addition to the catalytic domain, a site at the PAH N-terminal regulatory domain (PAH-RD), to activate the enzyme via an unclear mechanism. Here we report the crystal structure of human PAH-RD bound with Phe at 1.8 Å resolution, revealing a homodimer of ACT folds with Phe bound at the dimer interface. This work delivers the structural evidence to support previous solution studies that a binding site exists in the RD for Phe, and that Phe binding results in dimerization of PAH-RD. Consistent with our structural observation, a disease-associated PAH mutant impaired in Phe binding disrupts the monomer:dimer equilibrium of PAH-RD. Our data therefore support an emerging model of PAH allosteric regulation, whereby Phe binds to PAH-RD and mediates the dimerization of regulatory modules that would bring about conformational changes to activate the enzyme. PMID:27049649

[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.

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).

Table of Phenylalanine Content of Foods: Comparative Analysis of Data Compiled in Food Composition Tables.

PubMed

Araújo, Ana Claudia Marquim F; Araújo, Wilma M C; Marquez, Ursula M Lanfer; Akutsu, Rita; Nakano, Eduardo Y

2017-01-01

Knowing the phenylalanine (Phe) content of foods is essential for managing the diet of patients with phenylketonuria. Data on the Phe content of foods are scarce and sometimes vary between different Food Composition Tables (FCT). Brazil created its own table of the Phe contents of fruits and vegetables based exclusively on the chemical analysis of protein content, considering that proteins contain 3-4% Phe (TCFA/ANVISA). This study compared the protein and Phe contents of vegetables and fruits provided by the TCFA/ANVISA with those listed in international food composition tables. The Phe content of 71 fruits and vegetables listed in TCFA/ANVISA was classified into four subgroups, and the Wilcoxon nonparametric test compared the Phe and mean protein contents provided by the FCTs. All tests considered the bilateral hypothesis, and the level of significance was set at 5%. The Spearman's correlation coefficient measured the statistical dependence between Phe and protein contents. The mean Phe content was <50 mg Phe/100 g for 15 fruits; >50 mg/100 g for 11 type-A vegetables; <50 mg/100 g for 8 type-B vegetables; ≤50 mg/100 g for 7 type-C vegetables. The percentage of Phe in protein varied from 3.13 ± 1.03% to 3.74 ± 2.55% in fruits; 3.33 ± 1.41 to 4.82 ± 1.17 in type-A vegetables; 3.46 ± 1.25% to 4.83 ± 2.46 in type-B vegetables; and 3.14% ± 1.49 to 4.62% ± 2.26 in type-C vegetables. The Phe and protein contents provided by most FCTs were positively correlated, suggesting that it is possible to estimate the Phe content of fruits by multiplying its protein content by 3%. For type-A, -B, and -C vegetables, 4% may be used.

High phenylalanine levels directly affect mood and sustained attention in adults with phenylketonuria: a randomised, double-blind, placebo-controlled, crossover trial.

PubMed

ten Hoedt, Amber E; de Sonneville, Leo M J; Francois, Baudouin; ter Horst, Nienke M; Janssen, Mirian C H; Rubio-Gozalbo, M Estela; Wijburg, Frits A; Hollak, Carla E M; Bosch, Annet M

2011-02-01

The main debate in the treatment of Phenylketonuria (PKU) is whether adult patients need the strict phenylalanine (Phe)-restricted diet. Physicians and patients lack evidence-based guidelines to help them make well-informed choices. We have carried out the first randomised double-blind placebo-controlled trial into the effects of short-term elevation of Phe levels on neuropsychological functions and mood of adults with PKU. Nine continuously treated adults with PKU underwent two 4-week supplementation periods: one with Phe, mimicking normal dietary intake, and one with placebo in randomly allocated order via a randomisation coding list in a double-blind cross-over design. A set of neuropsychological tests (Amsterdam Neuropsychological Tasks) was administered at the end of each study period. In addition, patients and for each patient a friend or relative, completed weekly Profile of Mood States (POMS) questionnaires, evaluating the patients' mood. Phe levels were measured twice weekly. Mean plasma Phe levels were significantly higher during Phe supplementation compared with placebo (p = 0.008). Neuropsychological tests demonstrated an impairment in sustained attention during Phe supplementation (p = 0.029). Both patients and their friend or relative reported lower scores on the POMS questionnaires during Phe supplementation (p = 0.017 and p = 0.040, respectively). High plasma Phe levels have a direct negative effect on both sustained attention and on mood in adult patients with PKU. A Phe-restricted "diet for life" might be an advisable option for many.

Effectiveness of the population health and environment approach in improving family planning outcomes in the Gurage, Zone South Ethiopia.

PubMed

Sinaga, Makeda; Mohammed, Ahmed; Teklu, Negash; Stelljes, Kristen; Belachew, Tefera

2015-11-13

Family planning is a strategy of balancing population growth with economic development for sustainable use of natural resources. A high population growth induces increased demand for resources and the rate at which these resources are exploited. Population, health and environment are connected inextricably. Population growth unbalanced with economic development leads to food insecurity which exposes households to the consumption of food with reduced quality and quantity leading to increased risk of malnutrition and poor health. Food insecurity again obliges people to encroach into the natural environment leading to a spiraling progress to destitution. A study in the Philippines provided concrete evidence that integrated development programming incorporating population, health, and the environment (PHE) can be more effective in lowering population growth rates and preserving critical coastal ecosystems than single-sector development interventions". Although the PHE approach has been implemented for 5 years (2008-2012) Guraghe Zone of South Ethiopia, its outcomes have not been evaluated. The objective of this study was to evaluate the effectiveness of PHE approach for achieving family planning (FP) outcomes in Gurage Zone. A comparative cross-sectional study was conducted in October, 2012. A total of 962 married women in the reproductive age group were included in the study. Data were collected using an interviewer administered Amharic version questionnaire. Descriptive statistics and multivariable logistic regression analyses were performed to compare the PHE and non-PHE Woredas (district) based on family planning parameters adopted from Measure Evaluation Manual. Comparison of non-new family panning acceptor women showed that PHE Woreda had a significantly high CPR compared to non-PHE (78% vs 52%, P < 0.0001). Among these sub-groups, women in the PHE Woreda were over four times more likely to use family planning methods during the study period (P < 0.0001) compared with women in the non-PHE Woreda. Women whose husbands' supported their use of family planning methods were 17 times as likely to use family panning methods (AOR: 17.2, 95 % CI [11.1, 26.8]), P < 0.0001. This was even increased to 20 times more when we did sub-group analysis only for women who were not new acceptors (AOR: 20.4:95% CI [9.7, 42.7]), P < 0.0001. The qualitative results showed that there was a better integration of FP, health and environmental issues into the grassroots level interventions in the PHE Woreda through using students as a medium for reaching parents on family planning and environmental issues. The findings suggest that overall; PHE has positive outcomes in FP behaviors both among married women and their husbands. Integration of population, health and environmental issues need to be strengthened and scaled up to sustain the positive FP behaviors such as support of FP use. Strategies used in the PHE Woredas such as using schools and students as medium for integrated PHE interventions are commendable approaches that need to be strengthened.

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.

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

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.

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.

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.

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.

Dietary tryptophan depletion in humans using a simplified two amino acid formula - a pilot study.

PubMed

Linden, Maike; Helmbold, Katrin; Kempf, Janina; Sippas, Shabnam; Filss, Christian; Langen, Karl-Josef; Eisert, Albrecht; Zepf, Florian Daniel

2016-01-01

Acute tryptophan depletion (ATD) is a well-established dietary method in translational brain research used to briefly lower central nervous serotonin (5-hydroxytryptamine (5-HT)) synthesis. A simplified two amino acid ATD formula (ATD PHE/LEU ) was developed while reducing the overall amount of amino acids (AAs), with the objective of administration especially in children and adolescents in future studies. This study investigated tryptophan (TRP) influx rates across the blood-brain barrier (BBB) after dietary ATD PHE/LEU administration relative to the ATD Moja-De protocol that has been established for use in children and adolescents. Seventy-two healthy adults (50% females) were randomized into four groups and administered ATD Moja-De, its TRP-balanced control condition (BAL), ATD PHE/LEU , or its respective control mixture (BAL PHE/LEU ) in a counterbalanced, double-blind, between-subjects design. Blood samples were collected at baseline and at hourly intervals for 6 h after AA intake. Questionnaires about mood, taste, and challenge tolerance were completed at fixed time points. Both challenge mixtures significantly reduced central nervous TRP influx as calculated by Michaelis-Menten kinetics relative to baseline and the respective control conditions with only mild and comparable side effects. A greater decline in TRP influx over the BBB after ATD PHE/LEU administration when compared with ATD Moja-De was detected without group effects for taste, challenge tolerance, and mood. There was unintended initial short increase in plasma TRP concentrations observed after ATD PHE/LEU intake, and a possible redistribution between free and protein-bound TRP triggered by protein synthesis stimulated by the ingested AAs may account for this finding. Moreover, a decline in TRP influx after BAL PHE/LEU administration over a 6-h period was observed, and the large amount of PHE in the BAL PHE/LEU mixture may be a possible explanation for this particular phenomenon, which could have led to an unexpected increase in displacement of TRP at the BBB in this control condition. This pilot study provides preliminary evidence for the possibility of lowering TRP influx as calculated by Michaelis-Menten kinetics into the brain by using a simplified ATD protocol in humans. The simplified composition of only two AAs, the lower overall AA amount, and the appropriate tolerance are characteristics of the newly developed ATD PHE/LEU protocol. Future studies focusing on the effects of the ATD PHE/LEU protocol and its respective control condition on CSF 5-HIAA concentrations, as well as neurochemical studies in rodents, are needed to further validate this newly developed AA mixture before definite conclusions about its usability in ATD-related research in humans, its specificity, and additional effects can be made.

Protective effect of kombucha mushroom (KM) tea on phenol-induced cytotoxicity in albino mice.

PubMed

Yapar, Kursad; Cavusoglu, Kultigin; Oruc, Ertan; Yalcin, Emine

2010-09-01

The present study was carried out to evaluate the protective role of kombucha mushroom (KM) tea on cytotoxicity induced by phenol (PHE) in mice. We used weight gain and micronucleus (MN) frequency as indicators of cytotoxicity and supported these parameters with pathological findings. The animals were randomly divided into seven groups: (Group I) only tap water (Group II) 1000 microl kg(-1) b. wt KM-tea, (Group III) 35 mg kg(-1) body wt. PHE (Group IV) 35 mg kg(-1) body wt. PHE + 250 microl kg(-1) b. wt KM-tea (Group V) 35 mg kg(-1) b. wt PHE + 500 microl kg(-1) b. wt KM-tea (Group VI) 35 mg kg(-1) b. wt PHE + 750 microl kg(-1) b. wt KM-tea, (Group VII) 35 mg kg(-1) b. wt PHE + 1000 microl kg(-1) b. wt KM-tea, for 20 consecutive days by oral gavage. The results indicated that all KM-tea supplemented mice showed a lower MN frequency than erythrocytes in only PHE-treated group. There was an observable regression on account of lesions in tissues of mice supplemented with different doses of KM-tea in histopathological observations. In conclusion, the KM-tea supplementation decreases cytotoxicity induced by PHE and its protective role is dose-dependent.

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

Phenanthrene exposure induces cardiac hypertrophy via reducing miR-133a expression by DNA methylation

PubMed Central

Huang, Lixing; Xi, Zhihui; Wang, Chonggang; Zhang, Youyu; Yang, Zhibing; Zhang, Shiqi; Chen, Yixin; Zuo, Zhenghong

2016-01-01

Growing evidence indicates that there is an emerging link between environmental pollution and cardiac hypertrophy, while the mechanism is unclear. The objective of this study was to examine whether phenanthrene (Phe) could cause cardiac hypertrophy, and elucidate the molecular mechanisms involved. We found that: 1) Phe exposure increased the heart weight and cardiomyocyte size of rats; 2) Phe exposure led to enlarged cell size, and increased protein synthesis in H9C2 cells; 3) Phe exposure induced important markers of cardiac hypertrophy, such as atrial natriuretic peptide, B-type natriuretic peptide, and c-Myc in H9C2 cells and rat hearts; 4) Phe exposure perturbed miR-133a, CdC42 and RhoA, which were key regulators of cardiac hypertrophy, in H9C2 cells and rat hearts; 5) Phe exposure induced DNA methyltransferases (DNMTs) in H9C2 cells and rat hearts; 6) Phe exposure led to methylation of CpG sites within the miR-133a locus and reduced miR-133a expression in H9C2 cells; 7) DNMT inhibition and miR-133a overexpression could both alleviate the enlargement of cell size and perturbation of CdC42 and RhoA caused by Phe exposure. These results indicated that Phe could induce cardiomyocyte hypertrophy in the rat and H9C2 cells. The mechanism might involve reducing miR-133a expression by DNA methylation. PMID:26830171

Phenome-Wide Association Study (PheWAS) for Detection of Pleiotropy within the Population Architecture using Genomics and Epidemiology (PAGE) Network

PubMed Central

Pendergrass, Sarah A.; Brown-Gentry, Kristin; Dudek, Scott; Frase, Alex; Torstenson, Eric S.; Goodloe, Robert; Ambite, Jose Luis; Avery, Christy L.; Buyske, Steve; Bůžková, Petra; Deelman, Ewa; Fesinmeyer, Megan D.; Haiman, Christopher A.; Heiss, Gerardo; Hindorff, Lucia A.; Hsu, Chu-Nan; Jackson, Rebecca D.; Kooperberg, Charles; Le Marchand, Loic; Lin, Yi; Matise, Tara C.; Monroe, Kristine R.; Moreland, Larry; Park, Sungshim L.; Reiner, Alex; Wallace, Robert; Wilkens, Lynn R.; Crawford, Dana C.; Ritchie, Marylyn D.

2013-01-01

Using a phenome-wide association study (PheWAS) approach, we comprehensively tested genetic variants for association with phenotypes available for 70,061 study participants in the Population Architecture using Genomics and Epidemiology (PAGE) network. Our aim was to better characterize the genetic architecture of complex traits and identify novel pleiotropic relationships. This PheWAS drew on five population-based studies representing four major racial/ethnic groups (European Americans (EA), African Americans (AA), Hispanics/Mexican-Americans, and Asian/Pacific Islanders) in PAGE, each site with measurements for multiple traits, associated laboratory measures, and intermediate biomarkers. A total of 83 single nucleotide polymorphisms (SNPs) identified by genome-wide association studies (GWAS) were genotyped across two or more PAGE study sites. Comprehensive tests of association, stratified by race/ethnicity, were performed, encompassing 4,706 phenotypes mapped to 105 phenotype-classes, and association results were compared across study sites. A total of 111 PheWAS results had significant associations for two or more PAGE study sites with consistent direction of effect with a significance threshold of p<0.01 for the same racial/ethnic group, SNP, and phenotype-class. Among results identified for SNPs previously associated with phenotypes such as lipid traits, type 2 diabetes, and body mass index, 52 replicated previously published genotype–phenotype associations, 26 represented phenotypes closely related to previously known genotype–phenotype associations, and 33 represented potentially novel genotype–phenotype associations with pleiotropic effects. The majority of the potentially novel results were for single PheWAS phenotype-classes, for example, for CDKN2A/B rs1333049 (previously associated with type 2 diabetes in EA) a PheWAS association was identified for hemoglobin levels in AA. Of note, however, GALNT2 rs2144300 (previously associated with high-density lipoprotein cholesterol levels in EA) had multiple potentially novel PheWAS associations, with hypertension related phenotypes in AA and with serum calcium levels and coronary artery disease phenotypes in EA. PheWAS identifies associations for hypothesis generation and exploration of the genetic architecture of complex traits. PMID:23382687

Introduction of D-phenylalanine enhanced the receptor binding affinities of gonadotropin-releasing hormone peptides.

PubMed

Lu, Jie; Hathaway, Helen J; Royce, Melanie E; Prossnitz, Eric R; Miao, Yubin

2014-02-01

The purpose of this study was to examine whether the introduction of D-Phe could improve the GnRH receptor binding affinities of DOTA-conjugated D-Lys(6)-GnRH peptides. Building upon the construct of DOTA-Ahx-(D-Lys(6)-GnRH1) we previously reported, an aromatic amino acid of D-Phe was inserted either between the DOTA and Ahx or between the Ahx and D-Lys(6) to generate new DOTA-D-Phe-Ahx-(D-Lys(6)-GnRH) or DOTA-Ahx-D-Phe-(D-Lys(6)-GnRH) peptides. Compared to DOTA-Ahx-(D-Lys(6)-GnRH1) (36.1 nM), the introduction of D-Phe improved the GnRH receptor binding affinities of DOTA-D-Phe-Ahx-(D-Lys(6)-GnRH) (16.3 nM) and DOTA-Ahx-D-Phe-(D-Lys(6)-GnRH) (7.6 nM). The tumor targeting and pharmacokinetic properties of (111)In-DOTA-Ahx-D-Phe-(D-Lys(6)-GnRH) was determined in MDA-MB-231 human breast cancer-xenografted nude mice. Compared to (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1), (111)In-DOTA-Ahx-D-Phe-(D-Lys(6)-GnRH) exhibited comparable tumor uptake with faster renal and liver clearance. The MDA-MB-231 human breast cancer-xenografted tumors were clearly visualized by single photon emission computed tomography (SPECT) using (111)In-DOTA-Ahx-D-Phe-(D-Lys(6)-GnRH) as an imaging probe, providing a new insight into the design of new GnRH peptides in the future. Copyright © 2014 Elsevier Ltd. All rights reserved.

Electrokinetic-Enhanced Remediation of Phenanthrene-Contaminated Soil Combined with Sphingomonas sp. GY2B and Biosurfactant.

PubMed

Lin, Weijia; Guo, Chuling; Zhang, Hui; Liang, Xujun; Wei, Yanfu; Lu, Guining; Dang, Zhi

2016-04-01

Electrokinetic-microbial remediation (EMR) has emerged as a promising option for the removal of polycyclic aromatic hydrocarbons (PAHs) from contaminated soils. The aim of this study was to enhance degradation of phenanthrene (Phe)-contaminated soils using EMR combined with biosurfactants. The electrokinetic (EK) remediation, combined with Phe-degrading Sphingomonas sp. GY2B, and biosurfactant obtained by fermentation of Pseudomonas sp. MZ01, degraded Phe in the soil with an efficiency of up to 65.1 % at the anode, 49.9 % at the cathode after 5 days of the treatment. The presence of biosurfactants, electricity, and a neutral electrolyte stimulated the growth of the degrading bacteria as shown by a rapid increase in microbial biomass with time. The electrical conductivity and pH changed little during the course of the treatment, which benefitted the growth of microorganisms and the remediation of Phe-contaminated soil. The EMR system with the addition of biosurfactant had the highest Phe removal, demonstrating the biosurfactant may enhance the bioavailability of Phe and the interaction with the microorganism. This study suggests that the EMR combined with biosurfactants can be used to enhance in situ bioremediation of PAH-contaminated soils.

Purification and characterization of enantioselective N-acetyl-β-Phe acylases from Burkholderia sp. AJ110349.

PubMed

Imabayashi, Yuki; Suzuki, Shun'ichi; Kawasaki, Hisashi; Nakamatsu, Tsuyoshi

2016-01-01

For the production of enantiopure β-amino acids, enantioselective resolution of N-acyl β-amino acids using acylases, especially those recognizing N-acetyl-β-amino acids, is one of the most attractive methods. Burkholderia sp. AJ110349 had been reported to exhibit either (R)- or (S)-enantiomer selective N-acetyl-β-Phe amidohydrolyzing activity, and in this study, both (R)- and (S)-enantioselective N-acetyl-β-Phe acylases were purified to be electrophoretically pure and determined the sequences, respectively. They were quite different in terms of enantioselectivities and in their amino acids sequences and molecular weights. Although both the purified acylases were confirmed to catalyze N-acetyl hydrolyzing activities, neither of them show sequence similarities to the N-acetyl-α-amino acid acylases reported thus far. Both (R)- and (S)-enantioselective N-acetyl-β-Phe acylase were expressed in Escherichia coli. Using these recombinant strains, enantiomerically pure (R)-β-Phe (>99% ee) and (S)-β-Phe (>99% ee) were obtained from the racemic substrate.

A newly synthetic chromium complex-chromium (D-phenylalanine)3 activates AMP-activated protein kinase and stimulates glucose transport.

PubMed

Zhao, Peng; Wang, Jingying; Ma, Heng; Xiao, Yao; He, Leilei; Tong, Chao; Wang, Zhenhua; Zheng, Qiusheng; Dolence, E Kurt; Nair, Sreejayan; Ren, Jun; Li, Ji

2009-03-15

We synthesized the chromium (phenylalanine)(3) [Cr(D-phe)(3)] by chelating chromium(III) with D-phenylalanine ligand in aqueous solution to improve the bioavailability of chromium, and reported that Cr(D-phe)(3) improved insulin sensitivity. AMP-activated protein kinase (AMPK) is a key mediator for glucose uptake and insulin sensitivity. To address the molecular mechanisms by which Cr(d-phe)(3) increases insulin sensitivity, we investigated whether Cr(D-phe)(3) stimulates glucose uptake via activation of AMPK signaling pathway. H9c2 myoblasts and isolated cardiomyocytes were treated with Cr(D-phe)(3) (25microM). Western blotting was used for signaling determination. The glucose uptake was determined by 2-deoxy-D-glucose-(3)H accumulation. HPLC measured concentrations of AMP. The mitochondrial membrane potential (Deltapsi) was detected by JC-1 fluorescence assay. Cr(D-phe)(3) stimulated the phosphorylation of alpha catalytic subunit of AMPK at Thr(172), as well the downstream targets of AMPK, acetyl-CoA carboxylase (ACC, Ser(212)) and eNOS (Ser(1177)). Moreover, Cr(D-phe)(3) significantly stimulated glucose uptake in both H9c2 cells and cardiomyocytes. AMPK inhibitor compound C (10microM) dramatically inhibited the glucose uptake stimulated by Cr(D-phe)(3), while it did not affect insulin stimulation of glucose uptake. Furthermore, in vivo studies showed that Cr(D-phe)(3) also activated cardiac AMPK signaling pathway. The increase of cardiac AMP concentration and the decrease of mitochondrial membrane potential (Deltapsi) may contribute to the activation of AMPK induced by Cr(D-phe)(3). Cr(D-phe)(3) is a novel compound that activates AMPK signaling pathway, which contributes to the regulation of glucose transport during stress conditions that may be associated the role of AMPK in increasing insulin sensitivity.

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.

Multifaceted plant responses to circumvent Phe hyperaccumulation by downregulation of flux through the shikimate pathway and by vacuolar Phe sequestration.

PubMed

Lynch, Joseph H; Orlova, Irina; Zhao, Chengsong; Guo, Longyun; Jaini, Rohit; Maeda, Hiroshi; Akhtar, Tariq; Cruz-Lebron, Junellie; Rhodes, David; Morgan, John; Pilot, Guillaume; Pichersky, Eran; Dudareva, Natalia

2017-12-01

Detrimental effects of hyperaccumulation of the aromatic amino acid phenylalanine (Phe) in animals, known as phenylketonuria, are mitigated by excretion of Phe derivatives; however, how plants endure Phe accumulating conditions in the absence of an excretion system is currently unknown. To achieve Phe hyperaccumulation in a plant system, we simultaneously decreased in petunia flowers expression of all three Phe ammonia lyase (PAL) isoforms that catalyze the non-oxidative deamination of Phe to trans-cinnamic acid, the committed step for the major pathway of Phe metabolism. A total decrease in PAL activity by 81-94% led to an 18-fold expansion of the internal Phe pool. Phe accumulation had multifaceted intercompartmental effects on aromatic amino acid metabolism. It resulted in a decrease in the overall flux through the shikimate pathway, and a redirection of carbon flux toward the shikimate-derived aromatic amino acids tyrosine and tryptophan. Accumulation of Phe did not lead to an increase in flux toward phenylacetaldehyde, for which Phe is a direct precursor. Metabolic flux analysis revealed this to be due to the presence of a distinct metabolically inactive pool of Phe, likely localized in the vacuole. We have identified a vacuolar cationic amino acid transporter (PhCAT2) that contributes to sequestering excess of Phe in the vacuole. In vitro assays confirmed PhCAT2 can transport Phe, and decreased PhCAT2 expression in PAL-RNAi transgenic plants resulted in 1.6-fold increase in phenylacetaldehyde emission. These results demonstrate mechanisms by which plants maintain intercompartmental aromatic amino acid homeostasis, and provide critical insight for future phenylpropanoid metabolic engineering strategies. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Directional cell migration in an extracellular pH gradient: a model study with an engineered cell line and primary microvascular endothelial cells.

PubMed

Paradise, Ranjani K; Whitfield, Matthew J; Lauffenburger, Douglas A; Van Vliet, Krystyn J

2013-02-15

Extracellular pH (pH(e)) gradients are characteristic of tumor and wound environments. Cell migration in these environments is critical to tumor progression and wound healing. While it has been shown previously that cell migration can be modulated in conditions of spatially invariant acidic pH(e) due to acid-induced activation of cell surface integrin receptors, the effects of pH(e) gradients on cell migration remain unknown. Here, we investigate cell migration in an extracellular pH(e) gradient, using both model α(v)β(3) CHO-B2 cells and primary microvascular endothelial cells. For both cell types, we find that the mean cell position shifts toward the acidic end of the gradient over time, and that cells preferentially polarize toward the acidic end of the gradient during migration. We further demonstrate that cell membrane protrusion stability and actin-integrin adhesion complex formation are increased in acidic pH(e), which could contribute to the preferential polarization toward acidic pH(e) that we observed for cells in pH(e) gradients. These results provide the first demonstration of preferential cell migration toward acid in a pH(e) gradient, with intriguing implications for directed cell migration in the tumor and wound healing environments. Copyright © 2012 Elsevier Inc. All rights reserved.

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.

PheProb: probabilistic phenotyping using diagnosis codes to improve power for genetic association studies.

PubMed

Sinnott, Jennifer A; Cai, Fiona; Yu, Sheng; Hejblum, Boris P; Hong, Chuan; Kohane, Isaac S; Liao, Katherine P

2018-05-17

Standard approaches for large scale phenotypic screens using electronic health record (EHR) data apply thresholds, such as ≥2 diagnosis codes, to define subjects as having a phenotype. However, the variation in the accuracy of diagnosis codes can impair the power of such screens. Our objective was to develop and evaluate an approach which converts diagnosis codes into a probability of a phenotype (PheProb). We hypothesized that this alternate approach for defining phenotypes would improve power for genetic association studies. The PheProb approach employs unsupervised clustering to separate patients into 2 groups based on diagnosis codes. Subjects are assigned a probability of having the phenotype based on the number of diagnosis codes. This approach was developed using simulated EHR data and tested in a real world EHR cohort. In the latter, we tested the association between low density lipoprotein cholesterol (LDL-C) genetic risk alleles known for association with hyperlipidemia and hyperlipidemia codes (ICD-9 272.x). PheProb and thresholding approaches were compared. Among n = 1462 subjects in the real world EHR cohort, the threshold-based p-values for association between the genetic risk score (GRS) and hyperlipidemia were 0.126 (≥1 code), 0.123 (≥2 codes), and 0.142 (≥3 codes). The PheProb approach produced the expected significant association between the GRS and hyperlipidemia: p = .001. PheProb improves statistical power for association studies relative to standard thresholding approaches by leveraging information about the phenotype in the billing code counts. The PheProb approach has direct applications where efficient approaches are required, such as in Phenome-Wide Association Studies.

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.

A small-molecule modulator interacts directly with deltaPhe508-CFTR to modify its ATPase activity and conformational stability.

PubMed

Wellhauser, Leigh; Kim Chiaw, Patrick; Pasyk, Stan; Li, Canhui; Ramjeesingh, Mohabir; Bear, Christine E

2009-06-01

The deletion of Phe-508 (DeltaPhe508) constitutes the most prevalent of a number of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) that cause cystic fibrosis (CF). This mutation leads to CFTR misfolding and retention in the endoplasmic reticulum, as well as impaired channel activity. The biosynthetic defect can be partially overcome by small-molecule "correctors"; once at the cell surface, small-molecule "potentiators" enhance the channel activity of DeltaPhe508-CFTR. Certain compounds, such as VRT-532, exhibit both corrector and potentiator functions. In the current studies, we confirmed that the inherent chloride channel activity of DeltaPhe508-CFTR (after biosynthetic rescue) is potentiated in studies of intact cells and membrane vesicles. It is noteworthy that we showed that the ATPase activity of the purified and reconstituted mutant protein is directly modulated by binding of VRT-532 [4-methyl-2-(5-phenyl-1H-pyrazol-3-yl)-phenol] ATP turnover by reconstituted DeltaPhe508-CFTR is decreased by VRT-532 treatment, an effect that may account for the increase in channel open time induced by this compound. To determine whether the modification of DeltaPhe508-CFTR function caused by direct VRT-532 binding is associated with structural changes, we evaluated the effect of VRT-532 binding on the protease susceptibility of the major mutant. We found that binding of VRT-532 to DeltaPhe508-CFTR led to a minor but significant decrease in the trypsin susceptibility of the full-length mutant protein and a fragment encompassing the second half of the protein. These findings suggest that direct binding of this small molecule induces and/or stabilizes a structure that promotes the channel open state and may underlie its efficacy as a corrector of DeltaPhe508-CFTR.

An analysis of hospital preparedness capacity for public health emergency in four regions of China: Beijing, Shandong, Guangxi, and Hainan.

PubMed

Li, Xingming; Huang, Jianshi; Zhang, Hui

2008-09-20

Hospital preparedness is critical for the early detection and management of public health emergency (PHE). Understanding the current status of PHE preparedness is the first step in planning to enhance hospitals' capacities for emergency response. The objective of this study is to understand the current status of hospital PHE preparedness in China. Four hundred hospitals in four city and provinces of China were surveyed using a standardized questionnaire. Data related to hospital demographic data; PHE preparation; response to PHE in community; stockpiles of drugs and materials; detection and identification of PHE; procedures for medical treatment; laboratory diagnosis and management; staff training; and risk communication were collected and analyzed. Valid responses were received from 318 (79.5%) of the 400 hospitals surveyed. Of the valid responses, 264 (85.2%) hospitals had emergency plans; 93.3% had command centres and personnel for PHE; 22.9% included community organisations during the training for PHE; 97.4% could transport needed medical staff to a PHE; 53.1% had evaluated stockpiles of drugs; 61.5% had evaluated their supply systems; 55.5% had developed surveillance systems; and 74.6% could monitor the abnormity(See in appendix). Physicians in 80.2% of the analyzed hospitals reported up-to-date knowledge of their institution's PHE protocol. Of the 318 respondents, 97.4% followed strict laboratory regulations, however, only about 33.5% had protocols for suspected samples. Furthermore, only 59.0% could isolate and identify salmonella and staphylococcus and less than 5% could isolate and identify human H5N1 avian flu and SARS. Staff training or drill programs were reported in 94.5% of the institutions; 50.3% periodically assessed the efficacy of staff training; 45% had experts to provide psychological counselling; 12.1% had provided training for their medical staff to assess PHE-related stress. All of the above capacities related to the demographic characteristics of hospitals and will be discussed in-depth in this paper. Our survey suggested that, at the time of the survey, hospital preparedness for PHE in China was at an early stage of development. Comprehensive measures should be taken to enhance hospital capacity in the prevention and management of PHE.

Characterization of Cu(II) and Cd(II) resistance mechanisms in Sphingobium sp. PHE-SPH and Ochrobactrum sp. PHE-OCH and their potential application in the bioremediation of heavy metal-phenanthrene co-contaminated sites.

PubMed

Chen, Chen; Lei, Wenrui; Lu, Min; Zhang, Jianan; Zhang, Zhou; Luo, Chunling; Chen, Yahua; Hong, Qing; Shen, Zhenguo

2016-04-01

Soil that is co-contaminated with heavy metals (HMs) and polycyclic aromatic hydrocarbons (PAHs) is difficult to bioremediate due to the ability of toxic metals to inhibit PAH degradation by bacteria. We demonstrated the resistance mechanisms to Cu(II) and Cd(II) of two newly isolated strains of Sphingobium sp. PHE-SPH and Ochrobactrum sp. PHE-OCH and further tested their potential application in the bioremediation of HM-phenanthrene (PhA) co-contaminated sites. The PHE-SPH and PHE-OCH strains tolerated 4.63 and 4.34 mM Cu(II) and also showed tolerance to 0.48 and 1.52 mM Cd(II), respectively. Diverse resistance patterns were detected between the two strains. In PHE-OCH cells, the maximum accumulation of Cu(II) occurred in the cell wall, while the maximum accumulation was in the cytoplasm of PHE-SPH cells. This resulted in a sudden suppression of growth in PHE-OCH and a gradual inhibition in PHE-SPH as the concentration of Cu(II) increased. Organic acid production was markedly higher in PHE-OCH than in PHE-SPH, which may also have a role in the resistance mechanisms, and contributes to the higher Cd(II) tolerance of PHE-OCH. The factors involved in the absorption of Cu(II) or Cd(II) in PHE-SPH and PHE-OCH were identified as proteins and carbohydrates by Fourier transform infrared (FT-IR) spectroscopy. Furthermore, both strains showed the ability to efficiently degrade PhA and maintained this high degradation efficiency under HM stress. The high tolerance to HMs and the PhA degradation capacity make Sphingobium sp. PHE-SPH and Ochrobactrum sp. PHE-OCH excellent candidate organisms for the bioremediation of HM-PhA co-contaminated sites.

A urinary metabolite of phenanthrene as a biomarker of polycyclic aromatic hydrocarbon metabolic activation in workers exposed to residual oil fly ash.

PubMed

Kim, Jee Young; Hecht, Stephen S; Mukherjee, Sutapa; Carmella, Steven G; Rodrigues, Ema G; Christiani, David C

2005-03-01

Residual oil fly ash is a chemically complex combustion product containing a significant component of potentially carcinogenic transition metals and polycyclic aromatic hydrocarbons (PAH). Various biomarkers of PAH exposure have been investigated previously, most notably 1-hydroxypyrene (1-OHP), in urine. In this study, we assessed the utility of r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene (trans, anti-PheT), a metabolite of phenanthrene, to detect occupational PAH exposure. Urine samples collected across the workweek were analyzed for 1-OHP and trans, anti-PheT in boilermakers (n = 20) exposed to residual oil fly ash. Median baseline urinary trans, anti-PheT concentrations were 0.50 microg/g creatinine in current tobacco smokers and 0.39 microg/g creatinine in nonsmokers. Median baseline urinary 1-OHP concentrations in smokers and nonsmokers were 0.31 and 0.13 microg/g creatinine, respectively. To study further the effect of smoking exposure on the urinary PAH markers, urinary cotinine was used. Although urinary trans, anti-PheT and 1-OHP concentrations were correlated (Spearman r = 0.63; P < 0.001) for all subjects, the regression coefficient between log-transformed trans, anti-PheT and log 1-OHP was statistically significant only for subjects with low levels of urinary cotinine or for nonsmokers. Each 1-unit increase in log 1-OHP was associated with a 0.77-unit increase (95% confidence interval, 0.45-1.09) in log trans, anti-PheT in subjects with low levels of urinary cotinine (P < 0.001). In these subjects, dichotomized occupational exposure status was a significant predictor of log trans, anti-PheT (P = 0.02) but not of log 1-OHP (P = 0.2). In conclusion, we found that urinary trans, anti-PheT was detected in levels comparable with 1-OHP in occupationally exposed workers, particularly nonsmokers. This study shows that urinary trans, anti-PheT may be an effective biomarker of uptake and metabolic activation of PAHs.

Social-cognitive functioning and social skills in patients with early treated phenylketonuria: a PKU-COBESO study.

PubMed

Jahja, Rianne; van Spronsen, Francjan J; de Sonneville, Leo M J; van der Meere, Jaap J; Bosch, Annet M; Hollak, Carla E M; Rubio-Gozalbo, M Estela; Brouwers, Martijn C G J; Hofstede, Floris C; de Vries, Maaike C; Janssen, Mirian C H; van der Ploeg, Ans T; Langendonk, Janneke G; Huijbregts, Stephan C J

2016-05-01

Early treatment of phenylketonuria (ET-PKU) prevents mental retardation, but many patients still show cognitive and mood problems. In this study, it was investigated whether ET-PKU-patients have specific phenylalanine (Phe-)related problems with respect to social-cognitive functioning and social skills. Ninety five PKU-patients (mean age 21.6 ± 10.2 years) and 95 healthy controls (mean age 19.6 ± 8.7 years) were compared on performance of computerized and paper-and-pencil tasks measuring social-cognitive abilities and on parent- and self-reported social skills, using multivariate analyses of variance, and controlling for general cognitive ability (IQ-estimate). Further comparisons were made between patients using tetrahydrobiopterin (BH4, N = 30) and patients not using BH4. Associations with Phe-levels on the day of testing, during childhood, during adolescence and throughout life were examined. PKU-patients showed poorer social-cognitive functioning and reportedly had poorer social skills than controls (regardless of general cognitive abilities). Quality of social-cognitive functioning was negatively related to recent Phe-levels and Phe-levels between 8 and 12 years for adolescents with PKU. Quality of social skills was negatively related to lifetime phenylalanine levels in adult patients, and specifically to Phe-levels between 0 and 7, and between 8 and 12 years. There were no differences with respect to social outcome measures between the BH4 and non-BH4 groups. PKU-patients have Phe-related difficulties with social-cognitive functioning and social skills. Problems seem to be more evident among adolescents and adults with PKU. High Phe-levels during childhood and early adolescence seem to be of greater influence than current and recent Phe-levels for these patients.

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.

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.

Identification of putative agouti-related protein(87-132)-melanocortin-4 receptor interactions by homology molecular modeling and validation using chimeric peptide ligands.

PubMed

Wilczynski, Andrzej; Wang, Xiang S; Joseph, Christine G; Xiang, Zhimin; Bauzo, Rayna M; Scott, Joseph W; Sorensen, Nicholas B; Shaw, Amanda M; Millard, William J; Richards, Nigel G; Haskell-Luevano, Carrie

2004-04-22

Agouti-related protein (AGRP) is one of only two naturally known antagonists of G-protein-coupled receptors (GPCRs) identified to date. Specifically, AGRP antagonizes the brain melanocortin-3 and -4 receptors involved in energy homeostasis. Alpha-melanocyte stimulating hormone (alpha-MSH) is one of the known endogenous agonists for these melanocortin receptors. Insight into putative interactions between the antagonist AGRP amino acids with the melanocortin-4 receptor (MC4R) may be important for the design of unique ligands for the treatment of obesity related diseases and is currently lacking in the literature. A three-dimensional homology molecular model of the mouse MC4 receptor complex with the hAGRP(87-132) ligand docked into the receptor has been developed to identify putative antagonist ligand-receptor interactions. Key putative AGRP-MC4R interactions include the Arg111 of hAGRP(87-132) interacting in a negatively charged pocket located in a cavity formed by transmembrane spanning (TM) helices 1, 2, 3, and 7, capped by the acidic first extracellular loop (EL1) and specifically with the conserved melanocortin receptor residues mMC4R Glu92 (TM2), mMC4R Asp114 (TM3), and mMC4R Asp118 (TM3). Additionally, Phe112 and Phe113 of hAGRP(87-132) putatively interact with an aromatic hydrophobic pocket formed by the mMC4 receptor residues Phe176 (TM4), Phe193 (TM5), Phe253 (TM6), and Phe254 (TM6). To validate the AGRP-mMC4R model complex presented herein from a ligand perspective, we generated nine chimeric peptide ligands based on a modified antagonist template of the hAGRP(109-118) (Tyr-c[Asp-Arg-Phe-Phe-Asn-Ala-Phe-Dpr]-Tyr-NH(2)). In these chimeric ligands, the antagonist AGRP Arg-Phe-Phe residues were replaced by the melanocortin agonist His/D-Phe-Arg-Trp amino acids. These peptides resulted in agonist activity at the mouse melanocortin receptors (mMC1R and mMC3-5Rs). The most notable results include the identification of a novel subnanomolar melanocortin peptide template Tyr-c[Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH(2) that is equipotent to alpha-MSH at the mMC1, mMC3, and mMC5 receptors but is 30-fold more potent than alpha-MSH at the mMC4R. Additionally, these studies identified a new and novel >200-fold MC4R versus MC3R selective peptide Tyr-c[Asp-D-Phe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH(2) template. Furthermore, when the His-DPhe-Arg-Trp sequence is used to replace the hAGRP Arg-Phe-Phe residues in the "mini"-AGRP (hAGRP87-120, C105A) template, a potent nanomolar agonist resulted at the mMC1R and MC3-5Rs.

Acceptability and tolerance of a low tyrosine and phenylalanine diet in patients with advanced cancer -- a pilot study.

PubMed

Harvie, Michelle N; Campbell, I T; Howell, A; Thatcher, N

2002-06-01

Low phenylalanine (phe) and tyrosine (tyr) diets limit tumour growth in animal models and may offer a novel cancer therapy. We studied the efficacy and acceptability of a low phe and tyr diet in patients with advanced cancer. Patients with advanced metastatic melanoma (n=22) and metastatic breast cancer (n=15) were invited to follow a low phe and tyr diet (10 mg kg-1 phe and tyr per day) for 1 month. In those individuals who followed the diet for 1 month, we attempted to establish the effects on nutritional status (body weight, fat free mass, percentage body fat, serum albumin), immune cell function (white cell count, lymphocytes and neutrophils), plasma levels of phe-tyr and tryptophan and quality of life (Hospital Anxiety and Depression score). Only three of the 22 patients with metastatic melanoma and three of the 15 patients with metastatic breast cancer agreed to start the diet. All patients experienced problems and side-effects and increases in anxiety and depression. There were declines in weight, with loss of fat and fat free mass but slight increases in white cell counts and neutrophils. Low phe and tyr diets do not appear to be a viable treatment option for patients with advanced cancer.

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.

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.

Synergistic cytogenetic and antineoplastic effects by the combined action of esteric steroidal derivatives of nitrogen mustards.

PubMed

Mourelatos, Constantinos; Nikolaropoulos, Sotiris; Fousteris, Manolis; Pairas, Georgios; Argyraki, Maria; Kareli, Dimitra; Dafa, Evaggelia; Mourelatos, Dionisios; Lialiaris, Theodore

2012-06-01

We studied the effect of five newly synthesized steroidal derivatives of nitrogen mustards. These derivatives have as alkylators either P-N, N-bis(2-chloroethyl)aminophenyl-butyrate (CHL) or P-N, N-bis(2-chloroethyl)aminophenyl-acetate (PHE) groups esterified with different modified steroidal nuclei. We examined them alone or in combination, on sister chromatid exchange rates and on human lymphocyte proliferation kinetics. The antitumor activity of these compounds, alone or in combination, was also tested on Leukemia P388-bearing mice. A pronounced cytogenetic and antineoplastic action was demonstrated by the compounds that contain either PHE or CHL as alkylators and are esterified with a steroidal nucleus having added a cholestene group in the 17 position of the D-ring. The exocyclical insertion of an -NHCO- group in the D-ring of the steroidal nucleus esterified with PHE (amide ester of PHE) yielded a compound demonstrating a distinct cytogenetic and antineoplastic effect. In contrast, the ketone group in the D-ring being inserted endocyclically in the steroidal nucleus (androstene) esterified with either CHL or with PHE gave negative cytogenetic and antineoplastic effects. However, the combined action of cholestene esterified with either CHL or with PHE in combination with either the androstene ester of PHE or with the androstene ester of CHL, respectively, gave synergistic cytogenetic and antineoplastic effects. Also the amide ester of PHE in combination with the androstene ester of CHL gave distinct cytogenetic and antineoplastic effects in a synergistic manner.

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.

Extracellular polymeric substances facilitate the biosorption of phenanthrene on cyanobacteria Microcystis aeruginosa.

PubMed

Bai, Leilei; Xu, Huacheng; Wang, Changhui; Deng, Jiancai; Jiang, Helong

2016-11-01

Phytoplankton-derived extracellular polymeric substances (EPS) are of vital importance for the biogeochemical cycles of hydrophobic organic pollutants in lake ecosystems. In this study, roles of loosely-bound EPS (LB-EPS) and tightly bound EPS (TB-EPS) in biosorption of phenanthrene (PHE) on a typical cyanobacteria Microcystis aeruginosa were investigated. The results showed that the biosorption of PHE on M. aeruginosa cell varied lasted 24 h, while the binding of PHE to LB-EPS and TB-EPS reached equilibrium within less than 2 h. The equilibrium biosorption capacities of M. aeruginosa cell, LB-EPS and TB-EPS were 6.78, 12.31, and 9.47 μg mg(-1), respectively, indicating that the binding of PHE to EPS was a considerable process involved in biosorption. Fluorescence quenching titration revealed that increasing temperature induced more binding sites in EPS for PHE and the binding process was driven by electrostatic force and hydrophobic interactions. Interestingly, dynamic and static quenching processes occurred simultaneously for the binding of PHE to protein-like substances in EPS, whereas the binding of PHE to humic-like substances belonged to static quenching. The relatively higher contents of proteins in LB-EPS produced a stronger binding capacity of PHE. Overall, the interactions between hydrophobic organic pollutants and cyanobacterial EPS are favorable to the bioaccumulation of hydrophobic organic pollutants in cyanobacteria and facilitate the regulatory function of cyanobacterial biomass as a biological pump. Copyright © 2016 Elsevier Ltd. All rights reserved.

Reversible and irreversible conformational transitions in myoglobin: role of hydrated amino acid ionic liquid.

PubMed

Sankaranarayanan, Kamatchi; Sathyaraj, Gopal; Nair, B U; Dhathathreyan, A

2012-04-12

Hydrated phenylalanine ionic liquid (Phe-IL) has been used to solubilize myoglobin (Mb). Structural stability of Mb in Phe-IL analyzed using fluorescence and circular dichroism spectroscopy shows that for low levels of hydration of Phe-IL there is a large red shift in the fluorescence emission wavelength and the protein transforms to complete β sheet from its native helical conformation. Rehydration or dilution reverses the β sheet to an α helix which on aging organizes to micrometer-sized fibrils. At concentrations higher than 200 μM, the protein changes from β to a more random coiled structure. Organization of the protein in Phe-IL in a Langmuir film at the air/water interface has been investigated using the surface pressure-molecular area isotherm and shows nearly the same surface tension for both pure Mb and Mb in Phe-IL. Scanning electron microscopy of the films of Mb in Phe-IL transferred using the Langmuir-Blodgett film technique show layered morphology. This study shows that the conformation of Mb is completely reversible going from β → helix → β sheet up to 200 μM of Phe-IL. Similar surface tension values for Mb in water and in Phe-IL suggests that direct ion binding interactions with the protein coupled with the change in local viscosity from the IL seems to not only alter the secondary structure of individual proteins but also drives the self-assembly of the protein molecules leading finally to fibril formation.

Inhibitory assay for degradation of collagen IV by cathepsin B with a surface plasmon resonance sensor.

PubMed

Shoji, Atsushi; Suenaga, Yumiko; Hosaka, Atsushi; Ishida, Yuuki; Yanagida, Akio; Sugawara, Masao

2017-10-25

We describe a simple method for evaluating the inhibition of collagen IV degradation by cathepsin B with a surface plasmon resonance (SPR) biosensor. The change in the SPR signal decreased with an increase in the concentration of cathepsin B inhibitors. The order of the inhibitory constant (Ki) obtained by the SPR method was CA074Me≈Z-Phe-Phe-FMK < leupeptin. This order was different from that obtained by benzyloxycarbonyl-Phe-Phe-Fluoromethylketone (Z-Phe-Phe-FMK) as a peptide substrate. The comparison of Ki suggested that CA074 and Z-Phe-Phe-FMK inhibited exopeptidase activity, and leupeptin inhibited the endopeptidase activity of cathepsin B more strongly. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

Phenotypic stability of B16-BL6 melanoma exposed to low levels of tyrosine and phenylalanine.

PubMed

Elstad, C A; Meadows, G G

1990-01-01

We previously demonstrated that tyrosine (Tyr) and phenylalanine (Phe) restriction suppresses metastatic heterogeneity of B16-BL6 (BL6) melanoma and selects for tumor variants with decreased metastatic potential. In this study, we investigate stability of this Tyr- and Phe-modulated tumor phenotype by sequentially transplanting BL6 in vivo into mice fed Low Tyr and Phe Diet. Metastatic potential of BL6 is suppressed after one subcutaneous passage. Suppression is unlikely to result from inhibition of tumor growth, since growth in vitro is significantly increased. The metastatic potential of the Tyr- and Phe-modulated tumor is unstable after in vivo passage, and lung colonizing ability is regenerated after ten in vivo passages. Conversely, the antimetastatic effect of Tyr and Phe restriction is stable after prolonged in vitro passage. The metastatic potential of tumors from mice fed Normal Diet is unstable after long-term in vitro culture. Sensitivity to adriamycin of BL6 from mice fed Low Tyr and Phe Diet is increased and is not altered by change in metastatic potential.

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+ .

Effect of heating strategies on whey protein denaturation--Revisited by liquid chromatography quadrupole time-of-flight mass spectrometry.

PubMed

Akkerman, M; Rauh, V M; Christensen, M; Johansen, L B; Hammershøj, M; Larsen, L B

2016-01-01

Previous standards in the area of effect of heat treatment processes on milk protein denaturation were based primarily on laboratory-scale analysis and determination of denaturation degrees by, for example, electrophoresis. In this study, whey protein denaturation was revisited by pilot-scale heating strategies and liquid chromatography quadrupole time-of-flight mass spectrometer (LC/MC Q-TOF) analysis. Skim milk was heat treated by the use of 3 heating strategies, namely plate heat exchanger (PHE), tubular heat exchanger (THE), and direct steam injection (DSI), under various heating temperatures (T) and holding times. The effect of heating strategy on the degree of denaturation of β-lactoglobulin and α-lactalbumin was determined using LC/MC Q-TOF of pH 4.5-soluble whey proteins. Furthermore, effect of heating strategy on the rennet-induced coagulation properties was studied by oscillatory rheometry. In addition, rennet-induced coagulation of heat-treated micellar casein concentrate subjected to PHE was studied. For skim milk, the whey protein denaturation increased significantly as T and holding time increased, regardless of heating method. High denaturation degrees were obtained for T >100°C using PHE and THE, whereas DSI resulted in significantly lower denaturation degrees, compared with PHE and THE. Rennet coagulation properties were impaired by increased T and holding time regardless of heating method, although DSI resulted in less impairment compared with PHE and THE. No significant difference was found between THE and PHE for effect on rennet coagulation time, whereas the curd firming rate was significantly larger for THE compared with PHE. Micellar casein concentrate possessed improved rennet coagulation properties compared with skim milk receiving equal heat treatment. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

X-ray diffraction studies of enkephalins. Crystal structure of [(4'-bromo) Phe4,Leu5]enkephalin.

PubMed Central

Ishida, T; Kenmotsu, M; Mino, Y; Inoue, M; Fujiwara, T; Tomita, K; Kimura, T; Sakakibara, S

1984-01-01

In order to investigate the structure-activity relationship of [Leu5]- and [Met5]enkephalins, [(4'-bromo)Phe4, Leu5]-, [(4'-bromo)Phe4, Met5]- and [Met5] enkephalins were synthesized and crystallized. The crystal structure of [(4'-bromo) Phe4, Leu5]- enkephalin was determined by X-ray diffraction method using the heavy atom method and refined to R = 0.092 by the least-squares method. The molecule in this crystal took essentially the same type I' beta-turn conformation found in [Leu5]enkephalin [Smith & Griffin (1978) Science 199, 1214-1216). On the other hand, the preliminary three-dimensional Patterson analyses showed that the most probable conformations of [(4'-bromo)Phe4,Met5]- and [Met5]enkephalins are both the dimeric extended forms. Based on these insights, the biologically active conformation of enkephalin was discussed in relation to the mu- and delta-receptors. PMID:6721829

Stimulatory effect of calcium on metabolism and its sensitivity to pH in kidney mitochondria.

PubMed

Drewnowska, K; Schoolwerth, A C

1994-07-01

The relationship between mitochondrial matrix free Ca2+ concentration ([Ca2+]m) and pH was evaluated by incubating isolated rat kidney mitochondria with different extramitochondrial Ca2+ concentrations ([Ca2+]e) at medium pH (pHe) 7.0 and 7.4. [Ca2+]m was monitored using the fluorescent signal from mitochondria loaded with the Ca2+ indicator fura 2. The changes in [Ca2+]m were compared with alpha-ketoglutarate dehydrogenase (alpha-KGDH) flux, measured as O2 consumption (nmol.min-1.mg protein-1) from 185 microM alpha-ketoglutarate (alpha-KG). The apparent dissociation constant of the matrix fluorescent probe for Ca2+ was determined in each experiment and was 323 +/- 45 nM (n = 14). When mitochondria were exposed to [Ca2+]e below 160 nM, [Ca2+]m was greater at pHe 7.0 than at pHe 7.4. However, above 160 nM [Ca2+]e, [Ca2+]m plateaued at pHe 7.0 but rose progressively at pHe 7.4. Increasing [Ca2+]m by consecutive additions of Ca2+ to the medium had a significantly more pronounced acceleratory effect on alpha-KG oxidation at pHe 7.0 than at pHe 7.4. Kinetic analysis of alpha-KGDH revealed a 45% decrease in the Michaelis constant (Km) for alpha-KG at pHe 7.0, but the Km was unchanged at pHe 7.4 with elevation of [Ca2+]m from 32 to 751 nM. Maximal velocity (Vmax) increased significantly at both pHe values. Half-maximal alpha-KG oxidation occurred at [Ca2+]m of 76 +/- 11 nM and 105 +/- 31 nM at pHe 7.0 and 7.4, respectively. These studies demonstrate a direct, pH-sensitive correlation between [Ca2+]e and [Ca2+]m; [Ca2+]m changed over a range that may regulate alpha-KGDH flux in intact kidney mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)

Phenylalanine as a nitrogen source induces root growth and nitrogen-use efficiency in Populus × canescens.

PubMed

Jiao, Yu; Chen, Yinghao; Ma, Chaofeng; Qin, Jingjing; Nguyen, Thi Hong Nhung; Liu, Di; Gan, Honghao; Ding, Shen; Luo, Zhi-Bin

2018-01-01

To investigate the physiological responses of poplars to amino acids as sole nitrogen (N) sources, Populus × canescens (Ait.) Smith plants were supplied with one of three nitrogen fertilizers (NH4NO3, phenylalanine (Phe) or the mixture of NH4NO3 and Phe) in sand culture. A larger root system, and decreased leaf size and CO2 assimilation rate was observed in Phe- versus NH4NO3-treated poplars. Consistently, a greater root biomass and a decreased shoot growth were detected in Phe-supplied poplars. Decreased enzymatic activities of nitrate reductase (NR), glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH) and elevated activities of nitrite reductase (NiR), phenylalanine ammonia lyase (PAL), glutamine synthetase (GS) and asparagine synthase (AS) were found in Phe-treated roots. Accordingly, reduced concentrations of NH4+, NO3- and total N, and enhanced N-use efficiencies (NUEs) were detected in Phe-supplied poplars. Moreover, the transcript levels of putative Phe transporters ANT1 and ANT3 were upregulated, and the mRNA levels of NR, glutamine synthetase 2 (GS2), NADH-dependent glutamate synthase (NADH-GOGAT), GDH and asparagine synthetase 2 (ASN2) were downexpressed in Phe-treated roots and/or leaves. The 15N-labeled Phe was mainly allocated in the roots and only a small amount of 15N-Phe was translocated to poplar aerial parts. These results indicate that poplar roots can acquire Phe as an N source to support plant growth and that Phe-induced NUEs in the poplars are probably associated with NH4+ re-utilization after Phe deamination and the carbon bonus simultaneously obtained during Phe uptake. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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

PubMed

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

2014-06-01

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

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

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.

Ameliorative effect of chromium-d-phenylalanine complex on indomethacin-induced inflammatory bowel disease in rats.

PubMed

Nagarjun, S; Dhadde, Shivsharan B; Veerapur, Veeresh P; Thippeswamy, B S; Chandakavathe, Baburao N

2017-05-01

Present study was designed to evaluate the effect of chromium-d-phenylalanine complex (Cr (d-phe) 3 ) on indomethacin-induced inflammatory bowel disease (IBD) in rats. Adult Wistar rats were pretreated with vehicle/Cr (d-phe) 3 (30, 60 and 90μg/kg, p.o.) for 11days. On day 8 and 9, after one h of the above mentioned treatment, indomethacin (7.5mg/kg/day,s.c.) was administered to induce IBD. On day 12, blood samples were collected from animals for lactate dehydrogenase (LDH) estimation and ileum was isolated for macroscopic scoring, biochemical estimation (lipid peroxidation, reduced glutathione and myeloperoxidase activity) and histopathological study. Administration of indomethacin significantly altered the serum LDH, macroscopic and microscopic appearance and biochemical parameters in ileum tissue. Cr (d-phe) 3 , at all the tested doses, caused a significant reversal of changes induced by indomethacin. Present study demonstrates the protective effect of Cr (d-phe) 3 against indomethacin-induced IBD in rats. The observed protective effect might be attributed to the antioxidant and anti-inflammatory properties of Cr (d-phe) 3 . Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Acute phenylalanine/tyrosine depletion of phasic dopamine in the rat brain.

PubMed

Shnitko, Tatiana A; Taylor, Sarah C; Stringfield, Sierra J; Zandy, Shannon L; Cofresí, Roberto U; Doherty, James M; Lynch, William B; Boettiger, Charlotte A; Gonzales, Rueben A; Robinson, Donita L

2016-06-01

Dopamine plays a critical role in striatal and cortical function, and depletion of the dopamine precursors phenylalanine and tyrosine is used in humans to temporarily reduce dopamine and probe the role of dopamine in behavior. This method has been shown to alter addiction-related behaviors and cognitive functioning presumably by reducing dopamine transmission, but it is unclear what specific aspects of dopamine transmission are altered. We performed this study to confirm that administration of an amino acid mixture omitting phenylalanine and tyrosine (Phe/Tyr[-]) reduces tyrosine tissue content in the prefrontal cortex (PFC) and nucleus accumbens (NAc), and to test the hypothesis that Phe/Tyr[-] administration reduces phasic dopamine release in the NAc. Rats were injected with a Phe/Tyr[-] amino acid mixture, a control amino acid mixture, or saline. High-performance liquid chromatography was used to determine the concentration of tyrosine, dopamine, or norepinephrine in tissue punches from the PFC and ventral striatum. In a separate group of rats, phasic dopamine release was measured with fast-scan cyclic voltammetry in the NAc core after injection with either the Phe/Tyr[-] mixture or the control amino acid solution. Phe/Tyr[-] reduced tyrosine content in the PFC and NAc, but dopamine and norepinephrine tissue content were not reduced. Moreover, Phe/Tyr[-] decreased the frequency of dopamine transients, but not their amplitude, in freely moving rats. These results indicate that depletion of tyrosine via Phe/Tyr[-] decreases phasic dopamine transmission, providing insight into the mechanism by which this method modifies dopamine-dependent behaviors in human imaging studies.

In vivo EPR extracellular pH-metry in tumors using a triphosphonated trityl radical.

PubMed

Marchand, Valérie; Levêque, Philippe; Driesschaert, Benoit; Marchand-Brynaert, Jacqueline; Gallez, Bernard

2017-06-01

The ability to assess the extracellular pH (pHe) is an important issue in oncology, because extracellular acidification is associated with tumor aggressiveness and resistance to cytotoxic therapies. In this study, a stable triphosphonated triarylmethyl (TPTAM) radical was qualified as a pHe electron paramagnetic resonance (EPR) molecular reporter. Calibration of hyperfine splitting as a function of pH was performed using a 1.2-GHz EPR spectrometer. Gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) was used as an extracellular paramagnetic broadening agent to assess the localization of TPTAM when incubated with cells. In vivo EPR pH-metry was performed in MDA, SiHa, and TLT tumor models and in muscle. Bicarbonate therapy was used to modulate the tumor pHe. EPR measurements were compared with microelectrode readouts. The hyperfine splitting of TPTAM was strongly pH-dependent around the pKa of the probe (pKa = 6.99). Experiments with Gd-DTPA demonstrated that TPTAM remained in the extracellular compartment. pHe was found to be more acidic in the MDA, SiHa, and TLT tumor models compared with muscle. Treatment of animals by bicarbonate induced an increase in pHe in tumors: similar variations in pHe were found when using in vivo EPR or invasive microelectrodes measurements. This study demonstrates the potential usefulness of TPTAM for monitoring pHe in tumors. Magn Reson Med 77:2438-2443, 2017. © 2016 International Society for Magnetic Resonance in Medicine. © 2016 International Society for Magnetic Resonance in Medicine.

Synthesis and Pharmacology of α/β(3)-Peptides Based on the Melanocortin Agonist Ac-His-dPhe-Arg-Trp-NH2 Sequence.

PubMed

Singh, Anamika; Tala, Srinivasa R; Flores, Viktor; Freeman, Katie; Haskell-Luevano, Carrie

2015-05-14

The melanocortin-3 and -4 receptors are expressed in the brain and play key roles in regulating feeding behavior, metabolism, and energy homeostasis. In the present study, incorporation of β(3)-amino acids into a melanocortin tetrapeptide template was investigated. Four linear α/β(3)-hybrid tetrapeptides were designed with the modifications at the Phe, Arg, and Trp residues in the agonist sequence Ac-His-dPhe-Arg-Trp-NH2. The most potent mouse melanocortin-4 receptor (mMC4R) agonist, Ac-His-dPhe-Arg-β(3)hTrp-NH2 (8) showed 35-fold selectivity versus the mMC3R. The study presented here has identified a new template with heterogeneous backbone for designing potent and selective melanocortin receptor ligands.

Synthesis and Pharmacology of α/β3-Peptides Based on the Melanocortin Agonist Ac-His-dPhe-Arg-Trp-NH2 Sequence

PubMed Central

2015-01-01

The melanocortin-3 and -4 receptors are expressed in the brain and play key roles in regulating feeding behavior, metabolism, and energy homeostasis. In the present study, incorporation of β3-amino acids into a melanocortin tetrapeptide template was investigated. Four linear α/β3-hybrid tetrapeptides were designed with the modifications at the Phe, Arg, and Trp residues in the agonist sequence Ac-His-dPhe-Arg-Trp-NH2. The most potent mouse melanocortin-4 receptor (mMC4R) agonist, Ac-His-dPhe-Arg-β3hTrp-NH2 (8) showed 35-fold selectivity versus the mMC3R. The study presented here has identified a new template with heterogeneous backbone for designing potent and selective melanocortin receptor ligands. PMID:26005535

Unravelling the pathogenic role and genotype-phenotype correlation of the USH2A p.(Cys759Phe) variant among Spanish families.

PubMed

Pérez-Carro, Raquel; Blanco-Kelly, Fiona; Galbis-Martínez, Lilián; García-García, Gema; Aller, Elena; García-Sandoval, Blanca; Mínguez, Pablo; Corton, Marta; Mahíllo-Fernández, Ignacio; Martín-Mérida, Inmaculada; Avila-Fernández, Almudena; Millán, José M; Ayuso, Carmen

2018-01-01

Mutations in USH2A cause both isolated Retinitis Pigmentosa (RP) and Usher syndrome (that implies RP and hearing impairment). One of the most frequent variants identified in this gene and among these patients is the p.(Cys759Phe) change. However, the pathogenic role of this allele has been questioned since it was found in homozygosity in two healthy siblings of a Spanish family. To assess the causative role of USH2A p.(Cys759Phe) in autosomal recessive RP (ARRP) and Usher syndrome type II (USH2) and to establish possible genotype-phenotype correlations associated with p.(Cys759Phe), we performed a comprehensive genetic and clinical study in patients suffering from any of the two above-mentioned diseases and carrying at least one p.(Cys759Phe) allele. Diagnosis was set according to previously reported protocols. Genetic analyses were performed by using classical molecular and Next-Generation Sequencing approaches. Probands of 57 unrelated families were molecularly studied and 63 patients belonging to these families were phenotypically evaluated. Molecular analysis characterized 100% of the cases, identifying: 11 homozygous patients for USH2A p.(Cys759Phe), 42 compound heterozygous patients (12 of them with another missense USH2A pathogenic variant and 30 with a truncating USH2A variant), and 4 patients carrying the p.(Cys759Phe) allele and a pathogenic variant in another RP gene (PROM1, CNGB1 or RP1). No additional causative variants were identified in symptomatic homozygous patients. Statistical analysis of clinical differences between zygosity states yielded differences (p≤0.05) in age at diagnosis of RP and hypoacusis, and progression of visual field loss. Homozygosity of p.(Cys759Phe) and compound heterozygosity with another USH2A missense variant is associated with ARRP or ARRP plus late onset hypoacusis (OR = 20.62, CI = 95%, p = 0.041). The present study supports the role of USH2A p.(Cys759Phe) in ARRP and USH2 pathogenesis, and demonstrates the clinical differences between different zygosity states. Phenotype-genotype correlations may guide the genetic characterization based upon specific clinical signs and may advise on the clinical management and prognosis based upon a specific genotype.

Improved measurement of protein synthesis in human subjects using 2H-phenylalanine isotopomers and gas chromatography/mass spectrometry.

PubMed

Preston, Tom; Small, Alexandra C

2010-03-15

Sensitive methods to measure protein synthetic rate in vivo are required to assess changes in protein expression, especially when comparing healthy with infirm subjects. We have previously applied a 'flooding dose' procedure using (2)H(5)-phenylalanine ((2)H(5)-phe) and (2)H(8)-phe isotopomers as tracers, which has proven successful in measuring albumin and fibrinogen synthesis in response to feeding in cancer patients. Using tert-butyldimethylsilyl derivatives, we have observed that (2)H(7)-phe is formed with time in vivo from (2)H(8)-phe, probably during transamination. This increases errors when estimating the fractional synthetic rate (FSR) using the (2)H(8)-phe isotopomer compared with the (2)H(5)-phe isotopomer. We sought to improve this situation by use of an alternative derivative that overcomes this problem whilst also streamlining sample preparation. When using N-ethoxycarbonyltrifluoroethyl (ECTFE) amino acid esters, (2)H(8)-phe is effectively converted into (2)H(7)-phe through fragmentation under electron ionisation (EI), allowing both (2)H(8)-phe and (2)H(7)-phe isotopomers to be measured as a single intense C(7)(2)H(7)(+) fragment at 98 Th. To illustrate the improved situation, the mean RMS residual was calculated for all albumin data, for each isotopomer and for each derivative. Albumin-bound Phe was analysed as ECTFE-phe with improved precision, independent of the isotopomer used, confirming that the new derivative is superior. Copyright 2010 John Wiley & Sons, Ltd.

Biological findings from the PheWAS catalog: focus on connective tissue-related disorders (pelvic floor dysfunction, abdominal hernia, varicose veins and hemorrhoids).

PubMed

Salnikova, Lyubov E; Khadzhieva, Maryam B; Kolobkov, Dmitry S

2016-07-01

Pelvic floor dysfunction, specifically genital prolapse (GP) and stress urinary inconsistency (SUI) presumably co-occur with other connective tissue disorders such as hernia, hemorrhoids, and varicose veins. Observations on non-random coexistence of these disorders have never been summarized in a meta-analysis. The performed meta-analysis demonstrated that varicose veins and hernia are associated with GP. Disease connections on the molecular level may be partially based on shared genetic susceptibility. A unique opportunity to estimate shared genetic susceptibility to disorders is provided by a PheWAS (phenome-wide association study) designed to utilize GWAS data concurrently to many phenotypes. We searched the PheWAS Catalog, which includes the results of the PheWAS study with P value < 0.05, for genes associated with GP, SUI, abdominal hernia, varicose veins and hemorrhoids. We found pronounced signals for the associations of the SLC2A9 gene with SUI (P = 6.0e-05) and the MYH9 gene with varicose veins of lower extremity (P = 0.0001) and hemorrhoids (P = 0.0007). The comparison of the PheWAS Catalog and the NHGRI Catalog data revealed enrichment of genes associated with bone mineral density in GP and with activated partial thromboplastin time in varicose veins of lower extremity. In cross-phenotype associations, genes responsible for peripheral nerve functions seem to predominate. This study not only established novel biologically plausible associations that may warrant further studies but also exemplified an effective use of the PheWAS Catalog data.

Effect of prenatal polycyclic aromatic hydrocarbons exposure on birth outcomes: the Polish mother and child cohort study.

PubMed

Polanska, Kinga; Dettbarn, Gerhard; Jurewicz, Joanna; Sobala, Wojciech; Magnus, Per; Seidel, Albrecht; Hanke, Wojciech

2014-01-01

The aim of this study was to assess the impact of PAH exposure on various anthropometric measures of birth outcomes. The study population consisted of 210 nonsmoking pregnant women. Urine samples collected between 20th and 24th week of pregnancy were used for analysis of the following PAH metabolites: 1-, 2-, 3-, 4-, and 9-hydroxyphenanthrene (1-, 2-, 3-, 4-, and 9-OH-PHE), 1-hydroxypyrene (1-OH-PYR), 1,6 + 1,8-dihydroxypyrene (DI-OH-PYR), phenanthrene trans-1,2-dihydrodiol (PHE-1,2-diol), and phenanthrene trans-9,10-dihydrodiol (PHE-9,10-diol) by gas chromatography-mass spectrometry. Environmental tobacco smoke exposure (ETS) was assessed by cotinine level in saliva using a stable isotope dilution LC-ESI-MS/MS method. The mean PAH metabolite concentrations were in the range of 0.15 µg/g creatinine for 9-OH-PHE to 5.9 µg/g creatinine for PHE-9,10-diol. It was shown that none of the individual PAH exposure markers demonstrate a statistically significant influence on birth outcomes. Interestingly a statistically significant association was found between the sum of OH-PHE along with cotinine level and the cephalization index after adjusting for potential confounders (P = 0.04). This study provides evidence that combined exposure of pregnant women to common environmental pollutants such as PAH and ETS might adversely affect fetal development. Thus, reduction of human exposure to these mixtures of hazardous compounds would in particular result in substantial health benefits for newborns.

Stabilizing and destabilizing effects of phenylalanine --> F5-phenylalanine mutations on the folding of a small protein.

PubMed

Woll, Matthew G; Hadley, Erik B; Mecozzi, Sandro; Gellman, Samuel H

2006-12-20

We report a systematic evaluation of phenylalanine-to-pentafluorophenylalanine (Phe --> F5-Phe) mutants for the 35-residue chicken villin headpiece subdomain (c-VHP), the hydrophobic core of which features a cluster of three Phe side chains (residues 6, 10, and 17). Phe --> F5-Phe mutations are interesting because aryl-perfluoroaryl interactions of optimal geometry are intrinsically more favorable than aryl-aryl interactions and because perfluoroaryl units are more hydrophobic than are analogous aryl units. One mutant, Phe-10 --> F5-Phe, provides enhanced tertiary structural stability relative to the native sequence. The other six mutants analyzed caused a decrease in stability.

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.

Biodegradation, Biosorption of Phenanthrene and Its Trans-Membrane Transport by Massilia sp. WF1 and Phanerochaete chrysosporium

PubMed Central

Gu, Haiping; Lou, Jun; Wang, Haizhen; Yang, Yu; Wu, Laosheng; Wu, Jianjun; Xu, Jianming

2016-01-01

Reducing phenanthrene (PHE) in the environment is critical to ecosystem and human health. Biodegradation, biosorption, and the trans-membrane transport mechanism of PHE by a novel strain, Massilia sp. WF1, and an extensively researched model fungus, Phanerochaete chrysosporium were investigated in aqueous solutions. Results showed that the PHE residual concentration decreased with incubation time and the data fitted well to a first-order kinetic equation, and the t1/2 of PHE degradation by WF1, spores, and mycelial pellets of P. chrysosporium were about 2 h, 87 days, and 87 days, respectively. The biosorbed PHE was higher in P. Chrysosporium than that in WF1, and it increased after microorganisms were inactivated and inhibited, especially in mycelial pellets. The detected intracellular auto-fluorescence of PHE by two-photon excitation microscopy also proved that PHE indeed entered into the cells. Based on regression, the intracellular (Kdin) and extracellular (Kdout) dissipation rate constants of PHE by WF1 were higher than those by spores and mycelial pellets. In addition, the transport rate constant of PHE from outside solution into cells (KinS/Vout) for WF1 were higher than the efflux rate constant of PHE from cells to outside solution (KoutS/Vin), while the opposite phenomena were observed for spores and mycelial pellets. The amount of PHE that transported from outside solution into cells was attributed to the rapid degradation and active PHE efflux in the cells of WF1 and P. Chrysosporium, respectively. Besides, the results under the inhibition treatments of 4°C, and the presence of sodium azide, colchicine, and cytochalasin B demonstrated that a passive trans-membrane transport mechanism was involved in PHE entering into the cells of WF1 and P. Chrysosporium. PMID:26858710

Redox potentials of primary electron acceptor quinone molecule (QA)- and conserved energetics of photosystem II in cyanobacteria with chlorophyll a and chlorophyll d.

PubMed

Allakhverdiev, Suleyman I; Tsuchiya, Tohru; Watabe, Kazuyuki; Kojima, Akane; Los, Dmitry A; Tomo, Tatsuya; Klimov, Vyacheslav V; Mimuro, Mamoru

2011-05-10

In a previous study, we measured the redox potential of the primary electron acceptor pheophytin (Phe) a of photosystem (PS) II in the chlorophyll d-dominated cyanobacterium Acaryochloris marina and a chlorophyll a-containing cyanobacterium, Synechocystis. We obtained the midpoint redox potential (E(m)) values of -478 mV for A. marina and -536 mV for Synechocystis. In this study, we measured the redox potentials of the primary electron acceptor quinone molecule (Q(A)), i.e., E(m)(Q(A)/Q(A)(-)), of PS II and the energy difference between [P680·Phe a(-)·Q(A)] and [P680·Phe a·Q(A)(-)], i.e., ΔG(PhQ). The E(m)(Q(A)/Q(A)(-)) of A. marina was determined to be +64 mV without the Mn cluster and was estimated to be -66 to -86 mV with a Mn-depletion shift (130-150 mV), as observed with other organisms. The E(m)(Phe a/Phe a(-)) in Synechocystis was measured to be -525 mV with the Mn cluster, which is consistent with our previous report. The Mn-depleted downshift of the potential was measured to be approximately -77 mV in Synechocystis, and this value was applied to A. marina (-478 mV); the E(m)(Phe a/Phe a(-)) was estimated to be approximately -401 mV. These values gave rise to a ΔG(PhQ) of -325 mV for A. marina and -383 mV for Synechocystis. In the two cyanobacteria, the energetics in PS II were conserved, even though the potentials of Q(A)(-) and Phe a(-) were relatively shifted depending on the special pair, indicating a common strategy for electron transfer in oxygenic photosynthetic organisms.

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.

Effects of predation and dispersal on bacterial abundance and contaminant biodegradation.

PubMed

Otto, Sally; Harms, Hauke; Wick, Lukas Y

2017-02-01

Research into the biodegradation of soil contaminants has rarely addressed the consequences of predator-prey interactions. Here, we investigated the joint effect of predation and dispersal networks on contaminant degradation by linking spatial abundances of degrader (Pseudomonas fluorescens LP6a) and predator (Bdellovibrio bacteriovorus) bacteria to the degradation of the major soil contaminant phenanthrene (PHE). We used a laboratory microcosm with a PHE passive dosing system and a glass fiber network to facilitate bacterial dispersal. Different predator-to-prey ratios and spatial arrangements of prey and predator inoculation were used to study predation pressure effects on PHE degradation. We observed that predation resulted in (i) enhanced PHE-degradation at low predator counts (PC) compared to controls lacking predation, (ii) reduced PHE-degradation at elevated PC relative to low PC, and (iii) significant effects of the spatial arrangement of prey and predator inoculation on PHE degradation. Our data suggest that predation facilitated by dispersal networks (such as fungal mycelia) may support the build-up of an effective bacterial biomass and, hence, contaminant biodegradation in heterogeneous systems such as soil. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Spanish adaptation of the Patient Health Engagement scale (S.PHE-s)in patients with chronic diseases.

PubMed

Magallares, Alejandro; Graffigna, Guendalina; Barello, Serena; Bonanomi, Andrea; Lozza, Edoardo

2017-08-01

The Patient Health Engagement scale is an instrument based on evidence about the experiences and preferences of patients with chronic diseases regarding their engagement with the treatment they receive. The main goal of this study was to adapt the Patient Health Engagement scale to the Spanish population (S.PHE-s) following the guidelines for cross-cultural adaptations. The sample comprised 413 patients with different chronic diseases. The confirmatory factor analysis showed a one factor model corresponding to the structure proposed by the original authors. The factor structure was invariant by gender. Furthermore, a Rasch Model showed that the S.PHE-s was unidimensional. In addition, every polychoric correlation coefficient was higher than .60. The Ordinal Alpha of the S.PHE-s was .85. Finally, the S.PHE-s was found to be positively related to life satisfaction, positive affect, and treatment adherence and negatively correlated to negative affect, depression, and anxiety. In light of these results, it may be concluded that the S.PHE-s has good psychometric properties and it may be used by the Spanish-speaking scientific community to measure patient engagement.

Stacking and T-shape competition in aromatic-aromatic amino acid interactions.

PubMed

Chelli, Riccardo; Gervasio, Francesco Luigi; Procacci, Piero; Schettino, Vincenzo

2002-05-29

The potential of mean force of interacting aromatic amino acids is calculated using molecular dynamics simulations. The free energy surface is determined in order to study stacking and T-shape competition for phenylalanine-phenylalanine (Phe-Phe), phenylalanine-tyrosine (Phe-Tyr), and tyrosine-tyrosine (Tyr-Tyr) complexes in vacuo, water, carbon tetrachloride, and methanol. Stacked structures are favored in all solvents with the exception of the Tyr-Tyr complex in carbon tetrachloride, where T-shaped structures are also important. The effect of anchoring the two alpha-carbons (C(alpha)) at selected distances is investigated. We find that short and large C(alpha)-C(alpha) distances favor stacked and T-shaped structures, respectively. We analyze a set of 2396 protein structures resolved experimentally. Comparison of theoretical free energies for the complexes to the experimental analogue shows that Tyr-Tyr interaction occurs mainly at the protein surface, while Phe-Tyr and Phe-Phe interactions are more frequent in the hydrophobic protein core. This is confirmed by the Voronoi polyhedron analysis on the database protein structures. As found from the free energy calculation, analysis of the protein database has shown that proximal and distal interacting aromatic residues are predominantly stacked and T-shaped, respectively.

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®.

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.

Application of the desulfurization of phenothiazines for a sensitive detection method by high-performance liquid chromatography.

PubMed

Shimada, K; Mino, T; Nakajima, M; Wakabayashi, H; Yamato, S

1994-11-04

A simple and sensitive high-performance liquid chromatographic (HPLC) method for the determination of phenothiazine (PHE) is described. PHE is converted to diphenylamine (DIP) by desulfurization with Raney nickel catalyst. DIP is highly sensitive to electrochemical detection. The calibration graph for PHE quantification after desulfurization was linear between 0.1 and 2.0 ng per injection. The detection limit (signal-to-noise ratio = 3) of PHE after desulfurization was 10 pg, which is twenty times higher than that of the parent compound PHE. The proposed desulfurization technique was applied to other PHE-related compounds. The structural confirmation of the desulfurized product of PHE was carried out by LC-MS using atmospheric pressure chemical ionization.

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.

Isolation and characterization of biochar-derived organic matter fractions and their phenanthrene sorption.

PubMed

Jin, Jie; Sun, Ke; Liu, Wei; Li, Shiwei; Peng, Xianqiang; Yang, Yan; Han, Lanfang; Du, Ziwen; Wang, Xiangke

2018-05-01

Chemical composition and pollutant sorption of biochar-derived organic matter fractions (BDOMs) are critical for understanding the long-term environmental significance of biochar. Phenanthrene (PHE) sorption by the humic acid-like (HAL) fractions isolated from plant straw- (PLABs) and animal manure-based (ANIBs) biochars, and the residue materials (RES) after HAL extraction was investigated. The HAL fraction comprised approximately 50% of organic carbon (OC) of the original biochars. Results of XPS and 13 C NMR demonstrated that the biochar-derived HAL fractions mainly consisted of aromatic clusters substituted by carboxylic groups. The CO 2 cumulative surface area of BDOMs excluding PLAB-derived RES fractions was obviously lower than that of corresponding biochars. The sorption nonlinearity of PHE by the fresh biochars was significantly stronger than that of the BDOM fractions, implying that the BDOM fractions were more chemically homogeneous. The BDOMs generally exhibited comparable or higher OC-normalized distribution coefficients (K oc ) of PHE than the original biochars. The PHE logK oc values of the fresh biochars correlated negatively with the micropore volumes due to steric hindrance effect. In contrast, a positive relationship between the sorption coefficients (K d ) of BDOMs and the micropore volumes was observed in this study, suggesting that pore filling could dominate PHE sorption by the BDOMs. The positive correlation between the PHE logK oc values of the HAL fractions and the aromatic C contents indicates that PHE sorption by the HAL fractions was regulated by aromatic domains. The findings of this study improve our knowledge of the evolution of biochar properties after application and its potential environmental impacts. Copyright © 2018 Elsevier Ltd. All rights reserved.

A cation-pi interaction in the binding site of the glycine receptor is mediated by a phenylalanine residue.

PubMed

Pless, Stephan A; Millen, Kat S; Hanek, Ariele P; Lynch, Joseph W; Lester, Henry A; Lummis, Sarah C R; Dougherty, Dennis A

2008-10-22

Cys-loop receptor binding sites characteristically contain many aromatic amino acids. In nicotinic ACh and 5-HT3 receptors, a Trp residue forms a cation-pi interaction with the agonist, whereas in GABA(A) receptors, a Tyr performs this role. The glycine receptor binding site, however, contains predominantly Phe residues. Homology models suggest that two of these Phe side chains, Phe159 and Phe207, and possibly a third, Phe63, are positioned such that they could contribute to a cation-pi interaction with the primary amine of glycine. Here, we test this hypothesis by incorporation of a series of fluorinated Phe derivatives using unnatural amino acid mutagenesis. The data reveal a clear correlation between the glycine EC(50) value and the cation-pi binding ability of the fluorinated Phe derivatives at position 159, but not at positions 207 or 63, indicating a single cation-pi interaction between glycine and Phe159. The data thus provide an anchor point for locating glycine in its binding site, and demonstrate for the first time a cation-pi interaction between Phe and a neurotransmitter.

Kinetic Monte Carlo modeling of chemical reactions coupled with heat transfer.

PubMed

Castonguay, Thomas C; Wang, Feng

2008-03-28

In this paper, we describe two types of effective events for describing heat transfer in a kinetic Monte Carlo (KMC) simulation that may involve stochastic chemical reactions. Simulations employing these events are referred to as KMC-TBT and KMC-PHE. In KMC-TBT, heat transfer is modeled as the stochastic transfer of "thermal bits" between adjacent grid points. In KMC-PHE, heat transfer is modeled by integrating the Poisson heat equation for a short time. Either approach is capable of capturing the time dependent system behavior exactly. Both KMC-PHE and KMC-TBT are validated by simulating pure heat transfer in a rod and a square and modeling a heated desorption problem where exact numerical results are available. KMC-PHE is much faster than KMC-TBT and is used to study the endothermic desorption of a lattice gas. Interesting findings from this study are reported.

Kinetic Monte Carlo modeling of chemical reactions coupled with heat transfer

NASA Astrophysics Data System (ADS)

Castonguay, Thomas C.; Wang, Feng

2008-03-01

In this paper, we describe two types of effective events for describing heat transfer in a kinetic Monte Carlo (KMC) simulation that may involve stochastic chemical reactions. Simulations employing these events are referred to as KMC-TBT and KMC-PHE. In KMC-TBT, heat transfer is modeled as the stochastic transfer of "thermal bits" between adjacent grid points. In KMC-PHE, heat transfer is modeled by integrating the Poisson heat equation for a short time. Either approach is capable of capturing the time dependent system behavior exactly. Both KMC-PHE and KMC-TBT are validated by simulating pure heat transfer in a rod and a square and modeling a heated desorption problem where exact numerical results are available. KMC-PHE is much faster than KMC-TBT and is used to study the endothermic desorption of a lattice gas. Interesting findings from this study are reported.

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.

Hydrolytic properties of phenylalanyl- and N-acetylphenylalanyl adenylate anhydrides

NASA Technical Reports Server (NTRS)

Lacey, J. C., Jr.; Mullins, D. W., Jr.; Senaratne, N.

1984-01-01

The hydrolysis of phenylalynyl- and N-acetylephenylalanyl adenylate anhydrides (AcPhe-AMP) is studied experimentally using a new spectrophotometric method. The hydrolysis process was analyzed at low concentrations (0.0001 M), constant temperature of 25 C, constant buffer concentration (0.05 M), and as a function of pH. It is found that while Phe-AMP is susceptible to attack by OH(-), AcPhe-AMP is susceptible to acid decomposition as well. At a pH of 4 to 8, Phe-AMP hydolyzes faster than AcPhe-AMP, but at pH less than four or greater than eight, the blocked form hydrolyzes faster. Both forms are attacked by H2O at the same rate. The rate laws for the various hydrolytic mechanisms and the activation energies for the hydrolyses at pH 7.1 are given in a table, and the possible relevance of the findings to the origin and evolution of the process of protein synthesis is discussed.

Human beta-endorphin: synthesis and biological activity of analogs with substitutions in positions 2, 9, 18, 27 and 31.

PubMed

Yeung, H W; Yamashiro, D; Tseng, L F; Chang, W C; Li, C H

1981-02-01

Four analogs of the opioid peptide human beta-endorphin (Bh-EP) have been synthesized: [D-Lys9, Phe27, Gly31]-beta h-EP, [D-PHe18,Phe27,Gly31]-beta h-EP, [D-Thr2,D-Lys9,Phe27,Gly31]-beta h-EP, and [D-Thr2,D-Phe18,Phe27,Gly31]-beta h-EP. All are practically indistinguishable from beta h-EP in the guinea pig ileum assay. All show diminished analgesic potency in the mouse tail-flick assay.

Intercalation of amino acids and oligopeptides into Zn Al layered double hydroxide by coprecipitation reaction

NASA Astrophysics Data System (ADS)

Aisawa, Sumio; Sasaki, Shuji; Takahashi, Satoshi; Hirahara, Hidetoshi; Nakayama, Hirokazu; Narita, Eiichi

2006-05-01

The coprecipitation of amino acids and oligopeptides with the Zn Al LDH was investigated using phenylalanine (Phe), phenylalanyl-phenylalanine (Phe-Phe), glycyl-phenylalanine (Gly Phe), glycine (Gly), glycyl-glycine (Gly Gly), glycyl-glycyl-glycine (Gly Gly Gly) and N-(N-γ-glutamyl-cysteinyl)-glycine (GSH) as guest species. The coprecipitation behavior of amino acids and oligopeptides was found to be influenced by the solution pH and the kind of their side chain groups, and reached the maximum at pH 8 or 9. The basal spacing, d003, of the Phe, Phe-Phe and GSH/LDH was 1.81, 2.41 and 1.64 nm, supporting that guests were arranged vertical to the LDH basal layer. Acceding to the basal spacing of the Gly, Gly Gly and Gly Gly Gly/LDH (d003=0.84 0.88 nm), these guests were oriented horizontal to the LDH basal layer with the co-intercalated NO3-. Moreover, the amount of Phe-Phe, Gly Gly and Gly Gly Gly intercalated was almost the same as that of Phe and Gly despite increasing the number peptide bond and the molecular size. GSH was intercalated into the LDH interlayer space as GSH oxidized form with bridged LDH layers by their carboxylate groups.

Vibrio cholerae ToxR downregulates virulence factor production in response to cyclo(Phe-Pro).

PubMed

Bina, X Renee; Taylor, Dawn L; Vikram, Amit; Ante, Vanessa M; Bina, James E

2013-08-27

Vibrio cholerae is an aquatic organism that causes the severe acute diarrheal disease cholera. The ability of V. cholerae to cause disease is dependent upon the production of two critical virulence determinants, cholera toxin (CT) and the toxin-coregulated pilus (TCP). The expression of the genes that encode for CT and TCP production is under the control of a hierarchical regulatory system called the ToxR regulon, which functions to activate virulence gene expression in response to in vivo stimuli. Cyclic dipeptides have been found to be produced by numerous bacteria, yet their biological function remains unknown. V. cholerae has been shown to produce cyclo(Phe-Pro). Previous studies in our laboratory demonstrated that cyclo(Phe-Pro) inhibited V. cholerae virulence factor production. For this study, we report on the mechanism by which cyclo(Phe-Pro) inhibited virulence factor production. We have demonstrated that exogenous cyclo(Phe-Pro) activated the expression of leuO, a LysR-family regulator that had not been previously associated with V. cholerae virulence. Increased leuO expression repressed aphA transcription, which resulted in downregulation of the ToxR regulon and attenuated CT and TCP production. The cyclo(Phe-Pro)-dependent induction of leuO expression was found to be dependent upon the virulence regulator ToxR. Cyclo(Phe-Pro) did not affect toxR transcription or ToxR protein levels but appeared to enhance the ToxR-dependent transcription of leuO. These results have identified leuO as a new component of the ToxR regulon and demonstrate for the first time that ToxR is capable of downregulating virulence gene expression in response to an environmental cue. The ToxR regulon has been a focus of cholera research for more than three decades. During this time, a model has emerged wherein ToxR functions to activate the expression of Vibrio cholerae virulence factors upon host entry. V. cholerae and other enteric bacteria produce cyclo(Phe-Pro), a cyclic dipeptide that we identified as an inhibitor of V. cholerae virulence factor production. This finding suggested that cyclo(Phe-Pro) was a negative effector of virulence factor production and represented a molecule that could potentially be exploited for therapeutic development. In this work, we investigated the mechanism by which cyclo(Phe-Pro) inhibited virulence factor production. We found that cyclo(Phe-Pro) signaled through ToxR to activate the expression of leuO, a new virulence regulator that functioned to repress virulence factor production. Our results have identified a new arm of the ToxR regulon and suggest that ToxR may play a broader role in pathogenesis than previously known.

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

Effect of the Abdominal Hollowing and Bracing Maneuvers on Activity Pattern of the Lumbopelvic Muscles During Prone Hip Extension in Subjects With or Without Chronic Low Back Pain: A Preliminary Study.

PubMed

Kahlaee, Amir H; Ghamkhar, Leila; Arab, Amir M

2017-02-01

The purpose of this study was to compare the effect of abdominal hollowing (AH) and abdominal bracing (AB) maneuvers on the activity pattern of lumbopelvic muscles during prone hip extension (PHE) in participants with or without nonspecific chronic low back pain (CLBP). Twenty women with or without CLBP participated in this cross-sectional observational study. The electromyographic activity (amplitude and onset time) of the contralateral erector spinae (CES), ipsilateral erector spinae (IES), gluteus maximus, and biceps femoris muscles was measured during PHE with and without abdominal maneuvers. A 3-way mixed model analysis of variance and post hoc tests were used for statistical analysis. Between-group comparisons showed that the CES onset delay during PHE alone was greater (P = .03) and the activity level of IES, CES, and biceps femoris in all maneuvers (P < .05) was higher in patients with CLBP than in asymptomatic participants. In asymptomatic participants, PHE + AH significantly decreased the signal amplitude (AMP) of IES (P = .01) and CES (P = .02) muscles. In participants with CLBP, IES muscle AMP was lower during PHE + AH compared with PHE + AB and PHE alone. With regard to onset delay, the results also showed no significant difference between maneuvers within either of the 2 groups (P > .05). Performance of the AH maneuver decreased the erector spinae muscle AMP in both groups, and neither maneuver altered the onset delay of any of the muscles in either group. The low back pain group showed higher levels of activity in all muscles (not statistically significant in gluteus maximus during all maneuvers). The groups were similar according to the onset delay of any of the muscles during either maneuver. Copyright © 2016. Published by Elsevier Inc.

Adaptation and Validation of a Chinese Version of Patient Health Engagement Scale for Patients with Chronic Disease.

PubMed

Zhang, Yaying; Graffigna, Guendalina; Bonanomi, Andrea; Choi, Kai-Chow; Barello, Serena; Mao, Pan; Feng, Hui

2017-01-01

The Patient Health Engagement Scale (PHE-s) was designed to assess the emotional and psychological attitudes of patients' engagement along their healthcare management journey. The aim of this study was to validate a culturally adapted Chinese version of the PHE-s (CPHE-s). Three hundred and seventy-seven participants were recruited from eight community health centers in a sample of patients with chronic disease in Hunan Province, China. The original Italian PHE-s was translated into Mandarin Chinese using a standardized forward-backward translation. The Rasch model was utilized and presented uni-dimensionality and good items fitness of the PHE-s. The internal consistency was 0.89 and the weighted Kappa coefficients of the items (test-retest reliability) ranged from 0.52 to 0.79. Both principal component analysis and confirmatory factor analysis supported a single-factor structure of the PHE-s. In testing the external validity, the PHE-s showed a significant moderate correlation with patient activation but not with medicine adherence behavior, which requires further exploration. The result suggested that the PHE-s is a reliable and valid instrument to assess the level of patient engagement in his or her own health management among chronic patients in China. Further analysis of reliability and validity should be assessed among other patient cohorts in China, and future directions for testing changes after patient engagement interventions should be developed by exploring some clinical relevance.

Investigating the effects of peptoid substitutions in self-assembly of Fmoc-diphenylalanine derivatives.

PubMed

Rajbhandary, Annada; Nilsson, Bradley L

2017-03-01

Low molecular weight agents that undergo self-assembly into fibril networks with hydrogel properties are promising biomaterials. Most low molecular weight hydrogelators are discovered empirically or serendipitously due to imperfect understanding of the mechanisms of self-assembly, the packing structure of self-assembled materials, and how the self-assembly process corresponds to emergent hydrogelation. Herein, the mechanisms of self-assembly and hydrogelation of N-fluorenylmethoxycarbonyl diphenylalanine (Fmoc-PhePhe), a well-studied low molecular weight hydrogelator, is probed by systematic comparison with derivatives in which Phe residues are replaced by corresponding N-benzyl glycine peptoid (Nphe) analogs. Peptoids are peptidomimetics that shift display of side chain functionality from the α-carbon to the terminal nitrogen. This alters the hydrogen bonding capacity, the side chain presentation geometry, amide cis/trans isomerization equilibrium, and β-sheet potential of the peptoid relative to the corresponding amino acid in the context of peptidic polymers. It was found that amino acid/peptoid hybrids Fmoc-Phe-Nphe and Fmoc-Nphe-Phe have altered fibril self-assembly propensity and reduced hydrogelation capacity relative to the parent dipeptide, and that fibril self-assembly of the dipeptoid, Fmoc-Nphe-Nphe, is completely curtailed. These findings provide insight into the potential of low molecular weight peptoids and peptide/peptoid hybrids as hydrogelation agents and illuminate the importance of hydrogen bonding and π-π interaction geometry in facilitating self-assembly of Fmoc-Phe-Phe. © 2016 Wiley Periodicals, Inc.

Adaptation and Validation of a Chinese Version of Patient Health Engagement Scale for Patients with Chronic Disease

PubMed Central

Zhang, Yaying; Graffigna, Guendalina; Bonanomi, Andrea; Choi, Kai-chow; Barello, Serena; Mao, Pan; Feng, Hui

2017-01-01

The Patient Health Engagement Scale (PHE-s) was designed to assess the emotional and psychological attitudes of patients' engagement along their healthcare management journey. The aim of this study was to validate a culturally adapted Chinese version of the PHE-s (CPHE-s). Three hundred and seventy-seven participants were recruited from eight community health centers in a sample of patients with chronic disease in Hunan Province, China. The original Italian PHE-s was translated into Mandarin Chinese using a standardized forward–backward translation. The Rasch model was utilized and presented uni-dimensionality and good items fitness of the PHE-s. The internal consistency was 0.89 and the weighted Kappa coefficients of the items (test–retest reliability) ranged from 0.52 to 0.79. Both principal component analysis and confirmatory factor analysis supported a single-factor structure of the PHE-s. In testing the external validity, the PHE-s showed a significant moderate correlation with patient activation but not with medicine adherence behavior, which requires further exploration. The result suggested that the PHE-s is a reliable and valid instrument to assess the level of patient engagement in his or her own health management among chronic patients in China. Further analysis of reliability and validity should be assessed among other patient cohorts in China, and future directions for testing changes after patient engagement interventions should be developed by exploring some clinical relevance. PMID:28220090

Impact of periodic health examination on surgical treatment for uterine fibroids in Beijing: a case-control study.

PubMed

Wu, Hai-Yun; Yang, Ling-Ling; Zhou, Shan

2010-12-07

During the past 2 decades, there has been a rapid proliferation of "health examination center (HEC)" across China. The effects of their services on public's health have not been systemically investigated. This study aimed to assess the impact of periodic health examination (PHE) at HEC on surgical treatment for uterine fibroids in Beijing residents. We identified 224 patients with a primary diagnosis of uterine fibroids who had surgical treatment at four Level-1 general hospitals in Beijing, from June 1, 2009 to October 20, 2009. Controls were women who did not have surgery for uterine fibroids, matched (1:1 ratio) for age (within 2 years). A standard questionnaire was used to inquire about whether participants had PHE at HEC during the previous 2 years. PHE at HEC within 2 years were associated with surgical treatment for uterine fibroids. Odds ratios was 4.05 (95% CI, 2.61-6.29 P < 0.001), after adjustment for marital status, whether have children, annual family income, health insurance, education level and self-rated uterine fibroids-related symptom severity. Our study showed PHE currently provided at HEC in China were associated with significantly increased use of surgical treatment for uterine fibroids in women. Further studies are needed to assess the effects of PHE on clinical as well as on broad societal outcomes in Chinese in contemporary medical settings.

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.

RhoA mediates the expression of acidic extracellular pH-induced matrix metalloproteinase-9 mRNA through phospholipase D1 in mouse metastatic B16-BL6 melanoma cells.

PubMed

Maeda, Toyonobu; Yuzawa, Satoshi; Suzuki, Atsuko; Baba, Yuh; Nishimura, Yukio; Kato, Yasumasa

2016-03-01

Solid tumors are characterized by acidic extracellular pH (pHe). The present study examined the contribution of small GTP-binding proteins to phospholipase D (PLD) activation of acidic pHe-induced matrix metalloproteinase-9 (MMP-9) production. Acidic pHe-induced MMP-9 production was reduced by C3 exoenzyme, which inhibits the Rho family of GTPases; cytochalasin D, which inhibits actin reorganization; and simvastatin, which inhibits geranylgeranylation of Rho. Small interfering RNA (siRNA) against RhoA, but not against Rac1 or Cdc42, significantly inhibited acidic pHe induction of MMP-9. Pull-down assays showed that acidic pHe increased the activated form of RhoA. Forced expression of constitutively active RhoA induced MMP-9 production, even at neutral pHe. RhoA siRNA also reduced acidic pHe induced PLD activity. Specific inhibition of PLD1 and Pld1 gene knockout significantly reduced acidic pHe-induced MMP-9 expression. In contrast, PLD2 inhibition or knockout had no effect on MMP-9 expression. These findings suggested that RhoA-PLD1 signaling is involved in acidic pHe induction of MMP-9.

Resolution Mechanism and Characterization of an Ammonium Chloride-Tolerant, High-Thermostable, and Salt-Tolerant Phenylalanine Dehydrogenase from Bacillus halodurans.

PubMed

Jiang, Wei; Wang, Ya-Li; Fang, Bai-Shan

2018-05-09

As phenylalanine dehydrogenase (PheDH) plays an important role in the synthesis of chiral drug intermediates and detection of phenylketonuria, it is significant to obtain a PheDH with specific and high activity. Here, a PheDH gene, pdh, encoding a novel BhPheDH with 61.0% similarity to the known PheDH from Microbacterium sp., was obtained. The BhPheDH showed optimal activity at 60 °C and pH 7.0, and it showed better stability in hot environment (40-70 °C) than the PheDH from Nocardia sp. And its activity and thermostability could be significantly increased by sodium salt. After incubation for 2 h in 3 M NaCl at 60 °C, the residual activity of the BhPheDH was found to be 1.8-fold higher than that of the control group (without NaCl). The BhPheDH could tolerate high concentration of ammonium chloride and its activity could be also enhanced by the high concentration of ammonium salts. These characteristics indicate that the BhPheDH possesses better thermostability, ammonium chloride tolerance, halophilic mechanism, and high salt activation. The mechanism of thermostability and high salt tolerance of the BhPheDH was analyzed by molecular dynamics simulation. These results provide useful information about the enzyme with high-temperature activity, thermostability, halophilic mechanism, tolerance to high concentration of ammonium chloride, higher salt activation and enantio-selectivity, and the application of molecular dynamics simulation in analyzing the mechanism of these distinctive characteristics.

Structure of human cytosolic phenylalanyl-tRNA synthetase: evidence for kingdom-specific design of the active sites and tRNA binding patterns.

PubMed

Finarov, Igal; Moor, Nina; Kessler, Naama; Klipcan, Liron; Safro, Mark G

2010-03-10

The existence of three types of phenylalanyl-tRNA synthetase (PheRS), bacterial (alphabeta)(2), eukaryotic/archaeal cytosolic (alphabeta)(2), and mitochondrial alpha, is a prominent example of structural diversity within the aaRS family. PheRSs have considerably diverged in primary sequences, domain compositions, and subunit organizations. Loss of the anticodon-binding domain B8 in human cytosolic PheRS (hcPheRS) is indicative of variations in the tRNA(Phe) binding and recognition as compared to bacterial PheRSs. We report herein the crystal structure of hcPheRS in complex with phenylalanine at 3.3 A resolution. A novel structural module has been revealed at the N terminus of the alpha subunit. It stretches out into the solvent of approximately 80 A and is made up of three structural domains (DBDs) possessing DNA-binding fold. The dramatic reduction of aminoacylation activity for truncated N terminus variants coupled with structural data and tRNA-docking model testify that DBDs play crucial role in hcPheRS activity.

Mutation of a Rice Gene Encoding a Phenylalanine Biosynthetic Enzyme Results in Accumulation of Phenylalanine and Tryptophan[W

PubMed Central

Yamada, Tetsuya; Matsuda, Fumio; Kasai, Koji; Fukuoka, Shuichi; Kitamura, Keisuke; Tozawa, Yuzuru; Miyagawa, Hisashi; Wakasa, Kyo

2008-01-01

Two distinct biosynthetic pathways for Phe in plants have been proposed: conversion of prephenate to Phe via phenylpyruvate or arogenate. The reactions catalyzed by prephenate dehydratase (PDT) and arogenate dehydratase (ADT) contribute to these respective pathways. The Mtr1 mutant of rice (Oryza sativa) manifests accumulation of Phe, Trp, and several phenylpropanoids, suggesting a link between the synthesis of Phe and Trp. Here, we show that the Mtr1 mutant gene (mtr1-D) encodes a form of rice PDT with a point mutation in the putative allosteric regulatory region of the protein. Transformed callus lines expressing mtr1-D exhibited all the characteristics of Mtr1 callus tissue. Biochemical analysis revealed that rice PDT possesses both PDT and ADT activities, with a preference for arogenate as substrate, suggesting that it functions primarily as an ADT. The wild-type enzyme is feedback regulated by Phe, whereas the mutant enzyme showed a reduced feedback sensitivity, resulting in Phe accumulation. In addition, these observations indicate that rice PDT is critical for regulating the size of the Phe pool in plant cells. Feeding external Phe to wild-type callus tissue and seedlings resulted in Trp accumulation, demonstrating a connection between Phe accumulation and Trp pool size. PMID:18487352

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

A comparison of exogenous and endogenous CEST MRI methods for evaluating in vivo pH.

PubMed

Lindeman, Leila R; Randtke, Edward A; High, Rachel A; Jones, Kyle M; Howison, Christine M; Pagel, Mark D

2018-05-01

Extracellular pH (pHe) is an important biomarker for cancer cell metabolism. Acido-chemical exchange saturation transfer (CEST) MRI uses the contrast agent iopamidol to create spatial maps of pHe. Measurements of amide proton transfer exchange rates (k ex ) from endogenous CEST MRI were compared to pHe measurements by exogenous acido-CEST MRI to determine whether endogenous k ex could be used as a proxy for pHe measurements. Spatial maps of pHe and k ex were obtained using exogenous acidoCEST MRI and an endogenous CEST MRI analyzed with the omega plot method, respectively, to evaluate mouse kidney, a flank tumor model, and a spontaneous lung tumor model. The pHe and k ex results were evaluated using pixelwise comparisons. The k ex values obtained from endogenous CEST measurements did not correlate with the pHe results from exogenous CEST measurements. The k ex measurements were limited to fewer pixels and had a limited dynamic range relative to pHe measurements. Measurements of k ex with endogenous CEST MRI cannot substitute for pHe measurements with acidoCEST MRI. Whereas endogenous CEST MRI may still have good utility for evaluating some specific pathologies, exogenous acido-CEST MRI is more appropriate when evaluating pathologies based on pHe values. Magn Reson Med 79:2766-2772, 2018. © 2017 International Society for Magnetic Resonance in Medicine. © 2017 International Society for Magnetic Resonance in Medicine.

Novel phenanthridine (PHE-4i) derivative inhibits carrageenan-induced rat hind paw oedema through suppression of hydrogen sulfide.

PubMed

George, Leema; Ramasamy, Tamizhselvi; Manickam, Venkatraman; Iyer, Sathiyanarayanan Kulathu; Radhakrishnan, Vidya

2016-08-01

This study was conducted to assess the anti-inflammatory effect of a novel synthesized phenanthridine alkaloid (PHE-4i) and to examine the possible involvement of hydrogen sulfide (H2S) in anti-inflammatory mechanism. The synthesized phenanthridine derivative PHE-4i (2, 5, and 10 mg/kg) was administered intraperitoneally to rats. One hour following treatment, inflammation was induced by intraplantar injection of carrageenan (1 %), in the hind paw. Paw volume as the index of inflammation was measured before and after carrageenan injection. Neutrophil sequestration into the hind paw was quantified by measuring tissue myeloperoxidase (MPO) activity and was compared for the inhibition of H2S production. Pretreatment with PHE-4i significantly reduced carrageenan-induced hind paw weight, MPO activity, leukocyte infiltration, and H2S production in a dose-dependent manner (p < 0.001). These results indicate that the anti-inflammatory effect of PHE-4i on carrageenan-induced rat paw oedema could be via the inhibition of the gaseous mediator H2S.

Assessing Entrepreneurship in Governmental Public Health

PubMed Central

Wasserman, Jeffrey; Wu, Helen W.; Lauer, Johanna R.

2015-01-01

Objectives. We assessed the feasibility and desirability of public health entrepreneurship (PHE) in governmental public health. Methods. Using a qualitative case study approach with semistructured interview protocols, we conducted interviews between April 2010 and January 2011 at 32 local health departments (LHDs) in 18 states. Respondents included chief health officers and senior LHD staff, representatives from national public health organizations, health authorities, and public health institutes. Results. Respondents identified PHE through 3 overlapping practices: strategic planning, operational efficiency, and revenue generation. Clinical services offer the strongest revenue-generating potential, and traditional public health services offer only limited entrepreneurial opportunities. Barriers include civil service rules, a risk-averse culture, and concerns that PHE would compromise core public health values. Conclusions. Ongoing PHE activity has the potential to reduce LHDs’ reliance on unstable general public revenues. Yet under the best of circumstances, it is difficult to generate revenue from public health services. Although governmental public health contains pockets of entrepreneurial activity, its culture does not sustain significant entrepreneurial activity. The question remains as to whether LHDs’ current public revenue sources are sustainable and, if not, whether PHE is a feasible or desirable alternative. PMID:25689182

Assessing entrepreneurship in governmental public health.

PubMed

Jacobson, Peter D; Wasserman, Jeffrey; Wu, Helen W; Lauer, Johanna R

2015-04-01

We assessed the feasibility and desirability of public health entrepreneurship (PHE) in governmental public health. Using a qualitative case study approach with semistructured interview protocols, we conducted interviews between April 2010 and January 2011 at 32 local health departments (LHDs) in 18 states. Respondents included chief health officers and senior LHD staff, representatives from national public health organizations, health authorities, and public health institutes. Respondents identified PHE through 3 overlapping practices: strategic planning, operational efficiency, and revenue generation. Clinical services offer the strongest revenue-generating potential, and traditional public health services offer only limited entrepreneurial opportunities. Barriers include civil service rules, a risk-averse culture, and concerns that PHE would compromise core public health values. Ongoing PHE activity has the potential to reduce LHDs' reliance on unstable general public revenues. Yet under the best of circumstances, it is difficult to generate revenue from public health services. Although governmental public health contains pockets of entrepreneurial activity, its culture does not sustain significant entrepreneurial activity. The question remains as to whether LHDs' current public revenue sources are sustainable and, if not, whether PHE is a feasible or desirable alternative.

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

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

Complexes of polyadenylic acid and the methyl esters of amino acids

NASA Technical Reports Server (NTRS)

Khaled, M. A.; Mulins, D. W., Jr.; Swindle, M.; Lacey, J. C., Jr.

1983-01-01

A study of amino acid methyl esters binding to polyadenylic acid supports the theory that the genetic code originated through weak but selective affinities between amino acids and nucleotides. NMR, insoluble complex analysis, and ultraviolet spectroscopy are used to illustrate a correlation between the hydrophybicities of A amino acids and their binding constants, which, beginning with the largest, are in the order of Phe (having nominally a hydrophobic AAA anticodon), Ile, Leu, Val and Gly (having a hydrophilic anticodon with no A). In general, the binding constants are twice the values by Reuben and Polk (1980) for monomeric AMP, which suggests that polymer amino acids are interacting with only one base. No real differences are found betwen poly A binding for free Phe, Phe methyl ester or Phe amide, except that the amide value is slightly lower.

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.

Tetrahydrobiopterin responsiveness after extended loading test of 12 Danish PKU patients with the Y414C mutation.

PubMed

Nielsen, Jytte Bieber; Nielsen, Karin E; Güttler, Flemming

2010-02-01

Phenylketonuria (PKU) is an inherited metabolic disease characterized by phenylalanine (Phe) accumulation due to defects in the enzyme phenylalanine hydroxylase (PAH). Phe accumulation can lead to cognitive impairment. Some individuals with PKU respond to tetrahydrobiopterin (BH4) treatment, the natural cofactor of PAH, by a reduction in blood Phe concentrations.We tested 12 patients with PKU, 8-29 years of age, all carrying the common Y414C mutation in the PAH gene. Three were homozygous and nine were compound heterozygous, with the second mutation being a putative null mutation. During the study period, genuine protein was increased to approximately 1 g/kg. The patients were treated with 20, 10, and 5 mg BH4/kg/day for 1 week on each dose, starting with 20 mg/kg. A positive response was defined as a decline in blood Phe>30%. Blood Phe was measured four times a week. Nonresponding children were excluded from the study. Eleven of 12 patients had a positive response with 20 mg/kg, 5/10 responded on 10 mg/kg, and 1/9 on 5 mg/kg. Two were late responders, with a response on 20 mg/kg after >48 h. We could confirm the previously reported inconsistent responsiveness of Y414C in the nine heterozygous patients, whereas the three homozygous patients had early median Phe declines of 73%, 51%, and 27%, respectively, on the three different doses. The varying responses despite uniform trial conditions and genotypes may be due to individual differences in BH4 absorption or metabolism. No side effects were observed.

Inoculating plants with the endophytic bacterium Pseudomonas sp. Ph6-gfp to reduce phenanthrene contamination.

PubMed

Sun, Kai; Liu, Juan; Gao, Yanzheng; Sheng, Yuehui; Kang, Fuxing; Waigi, Michael Gatheru

2015-12-01

Plant organic contamination poses a serious threat to the safety of agricultural products and human health worldwide, and the association of endophytic bacteria with host plants may decrease organic pollutants in planta. In this study, we firstly determined the growth response and biofilm formation of endophytic Pseudomonas sp. Ph6-gfp, and then systematically evaluated the performance of different plant colonization methods (seed soaking (SS), root soaking (RS), leaf painting (LP)) for circumventing the risk of plant phenanthrene (PHE) contamination. After inoculation for 48 h, strain Ph6-gfp grew efficiently with PHE, oxalic acid, or malic acid as the sole sources of carbon and energy. Moreover, strain Ph6-gfp could form robust biofilms in LB medium. In greenhouse hydroponic experiments, strain Ph6-gfp could actively colonize inoculated plants internally, and plants colonized with Ph6-gfp showed a higher capacity for PHE removal. Compared with the Ph6-gfp-free treatment, the accumulations of PHE in Ph6-gfp-colonized plants via SS, RS, and LP were 20.1, 33.1, and 7.1 %, respectively, lower. Our results indicate that inoculating plants with Ph6-gfp could lower the risk of plant PHE contamination. RS was most efficient for improving PHE removal in whole plant bodies by increasing the cell numbers of Ph6-gfp in plant roots. The findings in this study provide an optimized method to strain Ph6-gfp reduce plant PAH residues, which may be applied to agricultural production in PAH-contaminated soil.

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.

Impacts of Pantoea agglomerans strain and cation-modified clay minerals on the adsorption and biodegradation of phenanthrene.

PubMed

Tao, Kelin; Zhao, Song; Gao, Pin; Wang, Lijin; Jia, Hanzhong

2018-06-06

Interactions between microorganisms and minerals have the potential contribution to remove polycyclic aromatic hydrocarbons (PAHs) in model systems. In this study, phenanthrene (PHE) was used as a probe molecule to explore the potential adsorption and biotransformation processes in the presence of microorganisms and various reference clays, such as montmorillonite (M), kaolinite (K), and pyrophyllite (P). Equilibrium adsorption experiments and scanning electron microscopy (SEM) technique were used to investigate the sorption of Pantoea agglomerans strains on clay minerals saturated with cations (Na + and Fe 3+ ). The adsorption isotherms of PHE and Pantoea agglomerans strains on cation-modified clay minerals fitted to Langmuir equation, and their adsorbed amounts both followed the sequence: montmorillonite > kaolinite > pyrophyllite. For six types of cation-modified minerals, the behavior of PHE adsorbed and Pantoea agglomerans adhered onto mentioned minerals was in the order of Na(I)-M > Fe(Ⅲ)-M, Na(I)-K > Fe(Ⅲ)-K and Fe(Ⅲ)-P > Na(I)-P, respectively. The biodegradation results showed that cation-modified clay minerals could enhance the biodegradation of PHE, ascribing to their large specific surface area, and cation exchange capability, as well as the difference in zeta potential between minerals and Pantoea agglomerans strains. Comparison of biodegradation rates displayed that PHE was degraded the highest in the presence of Na-M (93.285%). In addition, the obtained results suggested that the adhesion of bacteria onto cation-exchanged clay minerals was beneficial to the biodegradation of PHE. Anthracen-9-ylmethanol and 3,4-dimethyl-2-(3-methylbutanoyl)benzoic acid were detected as the main intermediate compounds, which can be further biodegraded into small molecules. The overall results obtained in this study are of valuable significance for the understanding of the behavior of PHE in soil and associated environment. Copyright © 2018 Elsevier Inc. All rights reserved.

Structure-activity relationships of the unique and potent agouti-related protein (AGRP)-melanocortin chimeric Tyr-c[beta-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide template.

PubMed

Wilczynski, Andrzej; Wilson, Krista R; Scott, Joseph W; Edison, Arthur S; Haskell-Luevano, Carrie

2005-04-21

The melanocortin receptor system consists of endogenous agonists, antagonists, G-protein coupled receptors, and auxiliary proteins that are involved in the regulation of complex physiological functions such as energy and weight homeostasis, feeding behavior, inflammation, sexual function, pigmentation, and exocrine gland function. Herein, we report the structure-activity relationship (SAR) of a new chimeric hAGRP-melanocortin agonist peptide template Tyr-c[beta-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH(2) that was characterized using amino acids previously reported in other melanocortin agonist templates. Twenty peptides were examined in this study, and six peptides were selected for (1)H NMR and computer-assisted molecular modeling structural analysis. The most notable results include the identification that modification of the chimeric template at the His position with Pro and Phe resulted in ligands that were nM mouse melanocortin-3 receptor (mMC3R) antagonists and nM mouse melanocortin-4 receptor (mMC4R) agonists. The peptides Tyr-c[beta-Asp-His-DPhe-Ala-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH(2) and Tyr-c[beta-Asp-His-DNal(1')-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH(2) resulted in 730- and 560-fold, respectively, mMC4R versus mMC3R selective agonists that also possessed nM agonist potency at the mMC1R and mMC5R. Structural studies identified a reverse turn occurring in the His-DPhe-Arg-Trp domain, with subtle differences observed that may account for the differences in melanocortin receptor pharmacology. Specifically, a gamma-turn secondary structure involving the DPhe(4) in the central position of the Tyr-c[beta-Asp-Phe-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH(2) peptide may differentiate the mixed mMC3R antagonist and mMC4R agonist pharmacology.

A Study of the Long Term Behavior of the SX Phe Star KZ Hya1

NASA Astrophysics Data System (ADS)

Peña, J. H.; Piña, D. S.; Rentería, A.; Villarreal, C.; Soni, A. A.; Guillen, J.; Calderón, J.

2018-04-01

From the newly determined times of maximum light of the SX Phe star KZ Hya and others from the literature, as well as from uvby - β photoelectric photometry, we determined the nature of this star and its physical parameters.

Transport characteristics of endomorphin-2 analogues in brain capillary endothelial cells.

PubMed

Mallareddy, Jayapal Reddy; Tóth, Géza; Fazakas, Csilla; Molnár, Judit; Nagyőszi, Péter; Lipkowski, Andrzej W; Krizbai, István A; Wilhelm, Imola

2012-04-01

Because of their poor metabolic stability and limited blood-brain barrier permeability, endomorphins have a low analgesic efficacy when administered systemically. Therefore, it is of great importance to design analogues with improved peptidase resistance and better delivery to the central nervous system. Recently, novel endomorphin-2 analogues have been synthesized, which proved to bind with high affinity and selectivity to the μ-opioid receptors and showed proteolytic resistance. In this study, we have analysed the transport characteristics of endomorphin-2 and three of its analogues [Dmt-Pro-Phe-Phe-NH(2) , Tyr-(1S,2R)Acpc-Phe-Phe-NH(2) and Tyr-(1S,2R)Achc-Phe-Phe-NH(2) ] using an in vitro blood-brain barrier model. The lipophilicity of the analogues, as assessed by their octanol/water partition coefficients, was higher than that of endomorphin-2. The flux of all four peptides from the apical (blood) side to the basolateral (brain) side was not saturable in the 10nm-1mm concentration range, suggesting that a passive mechanism plays a major role in their transport. The permeability coefficient of the analogues was significantly higher than that of endomorphin-2, suggesting increased blood-brain barrier penetration properties. We conclude that because of their good peptidase resistance and improved transport through brain endothelial cells, these endomorphin-2 analogues will have better analgesic properties in vivo. © 2011 John Wiley & Sons A/S.

Biochar based remediation of water and soil contaminated by phenanthrene and pentachlorophenol.

PubMed

Rao, Maria A; Di Rauso Simeone, Giuseppe; Scelza, Rosalia; Conte, Pellegrino

2017-11-01

Phenanthrene (Phe) and pentachlorophenol (PCP) are classified as persistent organic pollutants and represent serious concern for the environment as they are toxic and ubiquitous. Biochar based remediation is an emerging technology used in water and soil contamination. In this study we used poplar (BP) and conifer (BC) biochars to remediate water and soil contaminated by Phe and PCP. BP and BC were able to remove completely either Phe or PCP from contaminated water within one to three days. When biochar was confined in a porous membrane, BC and BP maintained their sorption efficiency for several remediation cycles. However, in these conditions BC allowed faster Phe removal. In soil remediation experiments, addition of two biochar rates, i.e. 2.5 and 5 mg g -1 , strongly reduced Phe extractability (up to 2.7% of the initially added Phe with the larger BC dose). This was similar to the behavior observed when compost was applied in order to verify the role of soil organic matter in the fate of both contaminants. PCP extractability was reduced only up to 75% (in average) in all samples including those with compost amendment. Only larger amount of biochar (20 and 50 mg g -1 ) allowed reduction of the extractable PCP and nullified phytotoxicity of the contaminant. Copyright © 2017 Elsevier Ltd. All rights reserved.

Redoubling the ring size of an endomorphin-2 analog transforms a centrally acting mu-opioid receptor agonist into a pure peripheral analgesic.

PubMed

Piekielna, Justyna; De Marco, Rossella; Gentilucci, Luca; Cerlesi, Maria Camilla; Calo', Girolamo; Tömböly, Csaba; Artali, Roberto; Janecka, Anna

2016-05-01

The study reports the synthesis and biological evaluation of two opioid analogs, a monomer and a dimer, obtained as products of the solid-phase, side-chain to side-chain cyclization of the pentapeptide Tyr-d-Lys-Phe-Phe-AspNH2 . The binding affinities to the mu, delta, and kappa opioid receptors, as well as results obtained in a calcium mobilization functional assay are reported. Tyr-[d-Lys-Phe-Phe-Asp]2 -NH2 1 was a potent and selective full agonist of mu with sub-nanomolar affinity, while the dimer (Tyr-[d-Lys-Phe-Phe-Asp]2 -NH2 )2 2 showed a significant mixed mu/kappa affinity, acting as an agonist at the mu. Molecular docking computations were utilized to explain the ability of the dimeric cyclopeptide 2 to interact with the receptor. Interestingly, in spite of the increased ring size, the higher flexibility allowed 2 to fold and fit into the mu receptor binding pocket. Both cyclopeptides were shown to elicit strong antinociceptive activity after intraventricular injection but only cyclomonomer 1 was able to cross the blood-brain barrier. However, the cyclodimer 2 displayed a potent peripheral antinociceptive activity in a mouse model of visceral inflammatory pain. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 309-317, 2016. © 2016 Wiley Periodicals, Inc.

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.

Role of brassinosteroids in alleviation of phenanthrene–cadmium co-contamination-induced photosynthetic inhibition and oxidative stress in tomato

PubMed Central

Ahammed, Golam Jalal; Yu, Jingquan

2013-01-01

Heavy metal pollution often occurs together with organic contaminants. Brassinosteroids (BRs) induce plant tolerance to several abiotic stresses, including phenanthrene (PHE) and cadmium (Cd) stress. However, the role of BRs in PHE+Cd co-contamination-induced stress amelioration is unknown. Here, the interactive effects of PHE, Cd, and 24-epibrassinolide (EBR; a biologically active BR) were investigated in tomato plants. The application of Cd (100 µM) alone was more phytotoxic than PHE applied alone (100 µM); however, their combined application resulted in slightly improved photosynthetic activity and pigment content compared with Cd alone after a 40 d exposure. Accumulation of reactive oxygen species and membrane lipid peroxidation were induced by PHE and/or Cd; however, the differences in effect were insignificant between Cd and PHE+Cd. The foliar application of EBR (0.1 µM) to PHE- and/or Cd-stressed plants alleviated photosynthetic inhibition and oxidative stress by causing enhancement of the activity of the enzymes and related transcript levels of the antioxidant system, secondary metabolism, and the xenobiotic detoxification system. Additionally, PHE and/or Cd residues were significantly decreased in both the leaves and roots after application of EBR, more specifically in PHE+Cd-stressed plants when treated with EBR, indicating a possible improvement in detoxification of these pollutants. The findings thus suggest a potential interaction of EBR and PHE for Cd stress alleviation. These results advocate a positive role for EBR in reducing pollutant residues for food safety and also strengthening phytoremediation. PMID:23201830

A mathematical model of tumour and blood pHe regulation: The HCO3-/CO2 buffering system.

PubMed

Martin, Natasha K; Gaffney, Eamonn A; Gatenby, Robert A; Gillies, Robert J; Robey, Ian F; Maini, Philip K

2011-03-01

Malignant tumours are characterised by a low, acidic extracellular pH (pHe) which facilitates invasion and metastasis. Previous research has proposed the potential benefits of manipulating systemic pHe, and recent experiments have highlighted the potential for buffer therapy to raise tumour pHe, prevent metastases, and prolong survival in laboratory mice. To examine the physiological regulation of tumour buffering and investigate how perturbations of the buffering system (via metabolic/respiratory disorders or changes in parameters) can alter tumour and blood pHe, we develop a simple compartmentalised ordinary differential equation model of pHe regulation by the HCO3-/CO2 buffering system. An approximate analytical solution is constructed and used to carry out a sensitivity analysis, where we identify key parameters that regulate tumour pHe in both humans and mice. From this analysis, we suggest promising alternative and combination therapies, and identify specific patient groups which may show an enhanced response to buffer therapy. In addition, numerical simulations are performed, validating the model against well-known metabolic/respiratory disorders and predicting how these disorders could change tumour pHe. Copyright © 2010 Elsevier Inc. 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.

Impact of phenanthrene on the properties of biogeochemical interfaces in soil: A two-layer column study

NASA Astrophysics Data System (ADS)

Reichel, Katharina; Totsche, Kai Uwe

2013-04-01

Biogeochemical interfaces in soils (Totsche et al. 2010) are the "hot spots" of microbial activity and the processing of organic compounds in soils. The production and relocation of mobile organic matter (MOM) and biocolloids like microorganisms are key processes for the formation and depth propagation of biogeochemical interfaces in soils (BGI). Phenanthrene (PHE) has been shown to affect microbial communities in soils (Ding et al. 2012) and may induce shifts in MOM quantity and quality (amount, type and properties of MOM). We hypothesize that the properties of BGI in soil change significantly due to the presence of PHE. The objectives of this study are (i) to evaluate the effect of PHE on soil microbial communities and on MOM quantity and quality under flow conditions with single- and two-layer column experiments and (ii) to assess the role of these processes for the physicochemical, mechanical and sorptive properties of BGI in soils. The soil columns were operated under water-unsaturated conditions. The top layer (source layer, SL, 2 cm) is made of sieved soil material (Luvisol, Scheyern, Germany) spiked with PHE (0.2 mg/g). The bottom layer (reception layer, RL, 10 cm) comprised the same soil without PHE. PHE-free columns were conducted in parallel as reference. Release and transport of MOM in mature soil of a single-layer column experiment was found to depend on the transport regime. The release of larger sized MOM (>0.45 µm) was restricted to an increased residence time during flow interruptions. Steady flow conditions favor the release of smaller MOM (<0.45 µm). Compared to the reference, in the two-layer column experiments higher OC concentrations were detected in the effluent from PHE spiked columns after enhanced flow interruptions (26d, 52d). That indicated the PHE influenced production or mobilization of MOM. Parallel factor analysis of fluorescence excitation and emission matrices revealed the presence of a constant DOM background and two new unknown components in the effluent, probably PHE metabolites. The emergence of new components emphasizes the role of metabolization processes in the release of MOM. The identification of key microbial actors and communities are currently in progress. Totsche, K.U. et al. (2010): Biogeochemical interfaces in soil: The interdisciplinary challenge for soil science. J. Plant Nutr. Soil Sci., 173(1), 88-99 Ding, G.-C., Heuer, H. & Smalla, K. (2012): Dynamics of bacterial communities in two unpolluted soils after spiking with phenanthrene: soil type specific and common responders. Front Microbio 10.3389/fmicb.2012.00290.

Adult phenylketonuria outcome and management.

PubMed

Trefz, F; Maillot, F; Motzfeldt, K; Schwarz, M

2011-01-01

The problem to evaluate treatment outcome in adult PKU (phenylketonuric) patients lies in the heterogeneity of the adult PKU population. This heterogeneity is not only based on the different treatment history of every individual patient but also on the different severity of the underlying defect of the enzyme phenylalanine hydroxylase. Recent, partly double blind studies in adult PKU patients further support recommendation for lifelong treatment. However, it has become evident that dietary treatment is suboptimal and continuation to adulthood often not accepted. Late detected PKU patients (up to 4-6 years of age) benefit from strict dietary treatment and are able to catch up in intellectual performance. Untreated, severely retarded patients with behavioral changes may benefit from introduction of dietary treatment. However, individual decision is necessary and based on the personal situation of the patient. In early and well treated patients a number of studies have demonstrated that cognitive and neurosychologic tests are different from controls. In addition there is evidence that patients with higher blood phenylalanine (phe) levels demonstrate more often psychiatric symptoms like depression and anxiety. Medical problems are more often observed: there are certain risks as impaired growth, decreased bone mineral density and nutrional deficits probably caused by dietary treatment with an artificial protein substitute and/or missing compliance with an unpleasant diet. The long term risk of a strict dietary treatment must be balanced with the risk of higher blood phe (mean blood phenylalanine >600-900 μmol/L) on cognitive and neuropsychological functions and psychiatric symptoms. Further studies should consider the role of blood phe exposure for brain development in childhood and for brain function in all ages. Besides mean blood phe, fluctuation of blood phe over time is important. Fluctuation of blood phe is decreased by sapropterin treatment in responsive patients which would on the long term may have positive effects on cognitive outcome. Further studies also should include adult PKU patients. Copyright © 2011 Elsevier Inc. All rights reserved.

Structural studies of α-melanocyte-stimulating hormone and a novel β-melanocyte-stimulating hormone from the neurointermediate lobe of the pituitary of the dogfish Squalus acanthias

PubMed Central

Bennett, Hugh P. J.; Lowry, Philip J.; McMartin, Colin; Scott, Alexander P.

1974-01-01

A melanocyte-stimulating hormone (MSH) has been isolated from extracts of the neurointermediate lobe of the pituitary of the dogfish Squalus acanthias by gel-filtration and ion-exchange chromatography. It had approximately 1% of the potency of mammalian α-MSH on bioassays in vitro on frog skin and dogfish skin. Sequence analysis revealed it to be a hexadecapeptide with the following primary structure: Asp-Gly-Asp-Asp-Tyr-Lys-Phe-Gly-His-Phe-Arg-Trp-Ser-Val-Pro-Leu. It appears to be related to the β-MSH species of mammalian species but has only the sequence -His-Phe-Arg-Trp- in common with the heptapeptide core -Met-Glu-His-Phe-Arg-Trp-Gly- which is characteristic not only of the MSH peptides but also of the adrenocorticotrophins and lipotrophins studied so far. An α-MSH was also isolated, 50% of which was amidated at the C-terminus group. Sequence data from this study taken in conjunction with those from a previous study (Lowry & Chadwick, 1970b) revealed it to be a tridecapeptide which is identical with the N-terminal sequence of dogfish adrenocorticotrophin. PMID:4375978

Longitudinal Study of [D10]Phenanthrene Metabolism by the Diol Epoxide Pathway in Smokers

PubMed Central

Hecht, Stephen S.; Hochalter, J. Bradley; Carmella, Steven G.; Zhang, Yan; Rauch, Diane M.; Fujioka, Naomi; Jensen, Joni; Hatsukami, Dorothy K.

2013-01-01

The extent of metabolism of [D10]phenanthrene to [D10]r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetradeuterophenanthrene ([D10]PheT) could be a biomarker of human metabolic activation of carcinogenic polycyclic aromatic hydrocarbons, leading to identification of smokers particularly susceptible to lung cancer. The longitudinal stability of [D10]PheT was evaluated in 24 cigarette smokers given 7 – 8 oral doses of [D10]phenanthrene (10 μg) over 5.5 months. [D10]PheT in 6 h urine was quantified after each dose. The overall coefficient of variation for 24 subjects was (mean ± S.D.) 27.4 ± 8.83%. Thus, a single administration of [D10]phenanthrene is likely sufficient to determine a smoker’s ability to metabolize it to [D10]PheT. PMID:23336104

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.

The effect of chronic phenytoin administration on single prolonged stress induced extinction retention deficits and glucocorticoid upregulation in the rat medial prefrontal cortex.

PubMed

George, Sophie A; Rodriguez-Santiago, Mariana; Riley, John; Rodriguez, Elizabeth; Liberzon, Israel

2015-01-01

Post-traumatic stress disorder (PTSD) is a chronic, debilitating disorder. Only two pharmacological agents are approved for PTSD treatment, and they often do not address the full range of symptoms nor are they equally effective in all cases. Animal models of PTSD are critical for understanding the neurobiology involved and for identification of novel therapeutic targets. Using the rodent PTSD model, single prolonged stress (SPS), we have implicated aberrant excitatory neural transmission and glucocorticoid receptor (GR) upregulation in the medial prefrontal cortex (mPFC) and hippocampus (HPC) in fear memory abnormalities associated with PTSD. The objective of this study is to examine the potential protective effect of antiepileptic phenytoin (PHE) administration on SPS-induced extinction retention deficits and GR expression. Forty-eight SPS-treated male Sprague Dawley rats or controls were administered PHE (40, 20 mg/kg, vehicle) for 7 days following SPS stressors; then, fear conditioning, extinction, and extinction retention were tested. Fear conditioning and extinction were unaffected by SPS or PHE, but SPS impaired extinction retention, and both doses of PHE rescued this impairment. Similarly, SPS increased GR expression in the mPFC and dorsal HPC, and PHE prevented SPS-induced GR upregulation in the mPFC. These data demonstrate that PHE administration can prevent the development of extinction retention deficits and upregulation of GR. PHE exerts inhibitory effects on voltage-gated sodium channels and decreases excitatory neural transmission via glutamate antagonism. If glutamate hyperactivity in the days following SPS contributes to SPS-induced deficits, then these data may suggest that the glutamatergic system constitutes a target for secondary prevention.

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.

Substrate and inhibitor studies of thermolysin-like neutral metalloendopeptidase from kidney membrane fractions. Comparison with bacterial thermolysin.

PubMed

Pozsgay, M; Michaud, C; Liebman, M; Orlowski, M

1986-03-25

The inhibitory constants of a series of synthetic N-carboxymethyl peptide inhibitors and the kinetic parameters (Km, kcat, and kcat/Km) of a series of model synthetic substrates were determined for the membrane-bound kidney metalloendopeptidase isolated from rabbit kidney and compared with those of bacterial thermolysin. The two enzymes show striking similarities with respect to structural requirements for substrate binding to the hydrophobic pocket at the S1' subsite of the active site. Both enzymes showed the highest reaction rates with substrates having leucine residues in this position while phenylalanine residues gave the lowest Km. The two enzymes were also inhibited by the same N-carboxymethyl peptide inhibitors. Although the mammalian enzyme was more susceptible to inhibition than its bacterial counterpart, structural variations in the inhibitor molecules affected the inhibitory constants for both enzymes in a similar manner. The two enzymes differed significantly, however, with respect to the effect of structural changes in the P1 and P2' positions of the substrate on the kinetic parameters of the reaction. The mammalian enzyme showed the highest reaction rates and specificity constants with substrates having the sequence -Phe-Gly-Phe- or -Phe-Ala-Phe- in positions P2, P1, and P1', respectively, while the sequence -Ala-Phe-Phe- was the most favored by the bacterial enzyme. The sequence -Gly-Gly-Phe- as found in enkephalins was not favored by either of the enzymes. Of the substrates having an aminobenzoate group in the P2' position, the mammalian enzyme favored those with the carboxyl group in the meta position while the bacterial enzyme favored those with the carboxyl group in the para position.(ABSTRACT TRUNCATED AT 250 WORDS)

Behavioural alterations from exposure to Cu, phenanthrene, and Cu-phenanthrene mixtures: linking behaviour to acute toxic mechanisms in the aquatic amphipod, Hyalella azteca.

PubMed

Gauthier, Patrick T; Norwood, Warren P; Prepas, Ellie E; Pyle, Greg G

2016-01-01

Phenanthrene (PHE) and Cu are two contaminants commonly co-occurring in marine and freshwater environments. Mixtures of PHE and Cu have been reported to induce more-than-additive lethality in the amphipod, Hyalella azteca, a keystone aquatic invertebrate, yet little is understood regarding the interactive toxic mechanisms that mediate more-than-additive toxicity. Understanding the interactions among toxic mechanisms among Cu and PHE will allow for better predictive power in assessing the ecological risks of Cu-PHE mixtures in aquatic environments. Here we use behavioural impairment to help understand the toxic mechanisms of Cu, PHE, and Cu-PHE mixture toxicity in the aquatic amphipod crustacean, Hyalella azteca. Our principal objective was to link alterations in activity and ventilation with respiratory rates, oxidative stress, and neurotoxicity in adult H. azteca. Adult amphipods were used for all toxicity tests. Amphipods were tested at sublethal exposures of 91.8- and 195-μgL(-1) Cu and PHE, respectively, and a Cu-PHE mixture at the same concentrations for 24h. Neurotoxicity was measured as acetylcholinesterase (AChE) activity, where malathion was used as a positive control. Oxidative stress was measured as reactive oxygen species (ROS) production. Phenanthrene-exposed amphipods exhibited severe behavioural impairment, being hyperstimulated to the extent that they were incapable of coordinating muscle movements. In addition, respiration and AChE activity in PHE-exposed amphipods were increased and reduced by 51% and 23% respectively. However, ROS did not increase following exposure to phenanthrene. In contrast, Cu had no effect on amphipod behaviour, respiration or AChE activity, but did lead to an increase in ROS. However, co-exposure to Cu antagonized the PHE-induced reduction in ventilation and negated any increase in respiration. The results suggest that PHE acts like an organophosphate pesticide (e.g., malathion) in H. azteca following 24h sublethal exposures, and that AChE inhibition is the likely mechanism by which PHE alters H. azteca behaviour. However, interactive aspects of neurotoxicity do not account for the previously observed more-than-additive mortality in H. azteca following exposure to Cu-PHE mixtures. Copyright © 2015 Elsevier B.V. All rights reserved.

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

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.

Investigation of PAH Biomarkers in the Urine of Workers Exposed to Hot Asphalt

PubMed Central

Sobus, Jon R.; Mcclean, Michael D.; Herrick, Robert F.; Waidyanatha, Suramya; Onyemauwa, Frank; Kupper, Lawrence L.; Rappaport, Stephen M.

2009-01-01

Airborne emissions from hot asphalt contain mixtures of polycyclic aromatic hydrocarbons (PAHs), including several carcinogens. We investigated urinary biomarkers of three PAHs, namely naphthalene (Nap), phenanthrene (Phe), and pyrene (Pyr) in 20 road-paving workers exposed to hot asphalt and in 6 road milling workers who were not using hot asphalt (reference group). Our analysis included baseline urine samples as well as postshift, bedtime, and morning samples collected over three consecutive days. We measured unmetabolized Nap (U-Nap) and Phe (U-Phe) as well as the monohydroxylated metabolites of Nap (OH-Nap), Phe (OH-Phe), and Pyr (OH-Pyr) in each urine sample. In baseline samples, no significant differences in biomarker levels were observed between pavers and millers, suggesting similar background exposures. In postshift, bedtime, and morning urine samples, the high pairwise correlations observed between levels of all biomarkers suggest common exposure sources. Among pavers, levels of all biomarkers were significantly elevated in postshift samples, indicating rapid uptake and elimination of PAHs following exposure to hot asphalt (biomarker levels were not elevated among millers). Results from linear mixed-effects models of levels of U-Nap, U-Phe, OH-Phe, and OH-Pyr across pavers showed significant effects of work assignments with roller operators having lower biomarker levels than the other workers. However, no work-related effect was observed for levels of OH-Nap, apparently due to the influence of cigarette smoking. Biological half-lives, estimated from regression coefficients for time among pavers, were 8 h for U-Phe, 10 h for U-Nap, 13 h for OH-Phe and OH-Pyr, and 26 h for OH-Nap. These results support the use of U-Nap, U-Phe, OH-Phe, and OH-Pyr, but probably not OH-Nap, as short-term biomarkers of exposure to PAHs emanating from hot asphalt. PMID:19602500

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

Phenome-Wide Association Study of Rheumatoid Arthritis Subgroups Identifies Association between Seronegative Disease and Fibromyalgia

PubMed Central

Doss, Jayanth; Mo, Huan; Carroll, Robert J.; Crofford, Leslie J.; Denny, Joshua C.

2016-01-01

Objective The differences between seronegative and seropositive rheumatoid arthritis (RA) have not been widely reported. We performed electronic health record (EHR)-based phenome-wide association studies (PheWAS) to identify disease associations in seropositive and seronegative RA. Methods A validated algorithm identified RA subjects from the de-identified EHR. Serotypes were determined by values of rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibody (ACPA). We tested EHR-derived phenotypes using PheWAS comparing seropositive RA against seronegative RA, yielding disease associations. PheWAS was also performed on RF-positive versus RF-negative subjects and ACPA-positive versus ACPA-negative subjects. Following PheWAS, select phenotypes were then manually reviewed and fibromyalgia was specifically evaluated using a validated algorithm. Results There were 2199 individuals identified with RA and either RF or ACPA testing. Of these, 1382 (63%) were seropositive. Seronegative RA was associated with “Myalgia and Myositis” (odds ratio [OR] 2.1, P=3.7×10−10) and back pain. A manual record review showed 80% of Myalgia and Myositis codes were used for fibromyalgia, and follow-up with a specific EHR algorithm for fibromyalgia confirmed that seronegative RA was associated with fibromyalgia (OR=1.8, P=4.0×10−6). Seropositive RA was associated with Chronic Airway Obstruction (OR=2.2, P=1.4×10−4) and tobacco use (OR=2.2, P=7.0×10−4). Conclusion This PheWAS in RA patients identifies a strong association between seronegativity and fibromyalgia. It also affirms relationships between seropositivity with chronic airway obstruction and seropositivity with tobacco use. These findings demonstrate the utility of the PheWAS approach to discover novel phenotype associations within different subgroups of a disease. PMID:27589350

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.

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.

A DKP Cyclo(L-Phe-L-Phe) Found in Chicken Essence Is a Dual Inhibitor of the Serotonin Transporter and Acetylcholinesterase

PubMed Central

Tsuruoka, Nobuo; Beppu, Yoshinori; Koda, Hirofumi; Doe, Nobutaka; Watanabe, Hiroshi; Abe, Keiichi

2012-01-01

Diketopiperazines (DKPs) are naturally-occurring cyclic dipeptides with a small structure and are found in many organisms and in large amounts in some foods and beverages. We found that a chicken essence beverage, which is popular among Southeast Asians as a traditional remedy and a rich source of DKPs, inhibited the serotonin transporter (SERT) and suppressed serotonin uptake from rat brain synaptosomes, which prompted us to isolate and identify the active substance(s). We purified a SERT inhibitor from the chicken essence beverage and identified it as the DKP cyclo(L-Phe-L-Phe). Interestingly, it was a naturally occurring dual inhibitor that inhibited both SERT and acetylcholinesterase (AChE) in vitro. The DKP increased extracellular levels of the cerebral monoamines serotonin, norepinephrine, and dopamine in the medial prefrontal cortex and acetylcholine in the ventral hippocampus of freely moving rats when administered orally. Moreover, cyclo(L-Phe-L-Phe) significantly shortened escape latency in the water maze test in depressed mice previously subjected to a repeated open-space swimming task, which induces a depression-like state. Cyclo(L-Phe-L-Phe) also significantly improved accuracy rates in a radial maze test in rats and increased step-through latencies in a passive avoidance test in mice with scopolamine-induced amnesia. These animal test results suggest that cyclo(L-Phe-L-Phe), which is present abundantly in some foods such as chicken essence, may abrogate the onset of depression and, thus, contribute to preventing the development of Alzheimer’s disease and other dementia, because senile depression is a risk factor for dementia. PMID:23209830

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.

Using testate amoeba as potential biointegrators of atmospheric deposition of phenanthrene (polycyclic aromatic hydrocarbon) on "moss/soil interface-testate amoeba community" microecosystems.

PubMed

Meyer, Caroline; Desalme, Dorine; Bernard, Nadine; Binet, Philippe; Toussaint, Marie-Laure; Gilbert, Daniel

2013-03-01

Microecosystem models could allow understanding of the impacts of pollutants such as polycyclic aromatic hydrocarbons on ecosystem functioning. We studied the effects of atmospheric phenanthrene (PHE) deposition on the microecosystem "moss/soil interface-testate amoebae (TA) community" over a 1-month period under controlled conditions. We found that PHE had an impact on the microecosystem. PHE was accumulated by the moss/soil interface and was significantly negatively correlated (0.4 < r(2) < 0.7) with total TA abundance and the abundance of five species of TA (Arcella sp., Centropyxis sp., Nebela lageniformis, Nebela tincta and Phryganella sp.). Among sensitive species, species with a superior trophic level (determined by the test aperture size) were more sensitive than other TA species. This result suggests that links between microbial groups in the microecosystems are disrupted by PHE and that this pollutant had effects both direct (ingestion of the pollutant or direct contact with cell) and/or indirect (decrease of prey) on the TA community. The TA community seems to offer a potential integrative tool to understand mechanisms and processes by which the atmospheric PHE deposition affects the links between microbial communities.

Extracellular pH monitoring for use in closed-loop vagus nerve stimulation

NASA Astrophysics Data System (ADS)

Cork, Simon C.; Eftekhar, Amir; Mirza, Khalid B.; Zuliani, Claudio; Nikolic, Konstantin; Gardiner, James V.; Bloom, Stephen R.; Toumazou, Christofer

2018-02-01

Objective. Vagal nerve stimulation (VNS) has shown potential benefits for obesity treatment; however, current devices lack physiological feedback, which limit their efficacy. Changes in extracellular pH (pHe) have shown to be correlated with neural activity, but have traditionally been measured with glass microelectrodes, which limit their in vivo applicability. Approach. Iridium oxide has previously been shown to be sensitive to fluctuations in pH and is biocompatible. Iridium oxide microelectrodes were inserted into the subdiaphragmatic vagus nerve of anaesthetised rats. Introduction of the gut hormone cholecystokinin (CCK) or distension of the stomach was used to elicit vagal nerve activity. Main results. Iridium oxide microelectrodes have sufficient pH sensitivity to readily detect changes in pHe associated with both CCK and gastric distension. Furthermore, a custom-made Matlab script was able to use these changes in pHe to automatically trigger an implanted VNS device. Significance. This is the first study to show pHe changes in peripheral nerves in vivo. In addition, the demonstration that iridium oxide microelectrodes are sufficiently pH sensitive as to measure changes in pHe associated with physiological stimuli means they have the potential to be integrated into closed-loop neurostimulating devices.

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.

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.

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.

Intra-Sectoral Diversity: A Political Economy of Thai Private Higher Education

ERIC Educational Resources Information Center

Praphamontripong, Prachayani

2010-01-01

Private higher education (PHE) worldwide has been a rapid development in the last several decades. The private sector will continue to grow, diversify and undoubtedly play a significant role in the political economy of higher education. Nevertheless, systematically empirical studies on the trio relationships among PHE, institutional diversity and…

Association of GABA(B)R1 receptor gene polymorphism with obstructive sleep apnea syndrome.

PubMed

Bayazit, Yildirim A; Yilmaz, Metin; Kokturk, Oguz; Erdal, M Emin; Ciftci, Tansu; Gokdogan, Tuba; Kemaloglu, Yusuf; Ileri, Fikret

2007-01-01

GABA(B)R (gamma-amino butyric acid B receptor)-mediated neurotransmission has been implicated in the pathophysiology of a variety of neuropsychiatric disorders. GABA(B)R1 gene variants were identified by single-strand conformation analysis. The nucleotide exchanges cause a substitution of alanine to valine in exon 1a1 (Ala20Val), a substitution of glycine to serine in exon 7 (Gly489Ser) and a silent C to G nucleotide exchange encoding the amino acid phenylalanine in exon 11 (Phe658Phe). The significance of GABA(B)R1a gene polymorphism in obstructive sleep apnea syndrome (OSAS) as well as the association of these polymorphisms with the polysomnography findings in OSAS patients are not known. In this study, we aimed to assess the significance of 3 different GABA(B)R1 gene polymorphisms (Ala20Val, Gly489Ser and Phe658Phe) in OSAS. Seventy-five patients (23 female and 52 male) with OSAS and 99 healthy volunteers (51 female, 48 male) were included in the study to assess Ala20Val, Gly489Ser and Phe658Phe polymorphisms of the GABA(B)R1 gene. For the Ala20Val variants, there was no significant difference between the genotypes and allele frequencies of the patients and controls, nor between both genders (p > 0.05). For Phe658Phe polymorphism, there was no significant difference between genotypes and allele frequencies of the patients and controls (p > 0.05). However, the C/C genotype was overrepresented and the T/C genotype was less frequent in male than female patients (p = 0.03). The C/C genotype was overrepresented and the T/C genotype was less frequent in male patients than male controls (p = 0.01). For GABA(B)R1-Gly489Ser polymorphism, all of the patients and controls had G/G genotype. The apnea arousal index scores of the male patients with C/C genotype were significantly higher than in the patients with C/T genotype (p = 0.01). The percent total sleep time in non-REM 1 scores of the male patients with T/T genotype were significantly higher than in the patients with T/C genotype (p = 0.021). The percent total sleep time in non-REM 2 scores of the female patients with C/C genotype were significantly higher than in the patients with C/T genotype (p = 0.006). The Ala20Val polymorphism of the GABA(B)R1 gene may be associated with OSAS, whereas Gly489Ser polymorphism does not seem to be involved in OSAS. The C/C variant of the Phe658Phe polymorphism GABA(B)R1 gene seems associated with the occurrence of OSAS and is also associated with some sleep related parameters (apnea arousal index and percent total sleep time in non-REM) recorded by polysomnography. Copyright (c) 2007 S. Karger AG, Basel.

Dietary aspartyl-phenylalanine-1-methyl ester delays osteoarthritis and prevents associated bone loss in STR/ORT mice

PubMed Central

Manion, Carl V.; Hochgeschwender, Ute; Edmundson, Allen B.; Hugli, Tony E.

2011-01-01

Objective. STR/ORT mice provide a well-known model for murine idiopathic OA, with histological joint lesions resembling those of human OA. This model was used to investigate protective effects of the dipeptide aspartyl-phenylalanine-1-methyl ester (Asp-Phe-OMe or aspartame) via the oral route vs a regular diet. Methods. STR/ORT mice were housed individually and fed diets with or without Asp-Phe-OMe (4 mg/kg), after weaning at the age of 3 weeks, until 15 months of age (average of 20 animals per group). The study groups were kept blinded to the investigators, who measured food consumption and body weight and performed gait mobility tests. Radiographic scans were also performed at regular time intervals to evaluate differential radiographic anomalies associated with progress of OA in response to oral Asp-Phe-OMe therapy. Results. The Asp-Phe-OMe-fed animals presented a pattern of significantly delayed disease onset. In addition, their muscle and bone mass were highly preserved, even at later time points after OA was established. Moreover, control animals presented a higher variability in gait motility in comparison with the Asp-Phe-OMe-fed animals, suggesting a protective effect from movement limitations associated with advanced OA. Conclusion. Asp-Phe-OMe, given orally, delays OA in the spontaneous STR/ORT model, improves bone cortical density and muscle mass, and may contribute to a better quality of life for these diseased animals. PMID:21372000

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

Phenome-Wide Association Studies as a Tool to Advance Precision Medicine

PubMed Central

Denny, Joshua C.; Bastarache, Lisa; Roden, Dan M.

2017-01-01

Beginning in the early 2000s, the accumulation of biospecimens linked to electronic health records (EHRs) made possible genome-phenome studies (i.e., comparative analyses of genetic variants and phenotypes) using only data collected as a by-product of typical health care. In addition to disease and trait genetics, EHRs proved a valuable resource for analyzing pharmacogenetic traits and developing reverse genetics approaches such as phenome-wide association studies (PheWASs). PheWASs are designed to survey which of many phenotypes may be associated with a given genetic variant. PheWAS methods have been validated through replication of hundreds of known genotype-phenotype associations, and their use has differentiated between true pleiotropy and clinical comorbidity, added context to genetic discoveries, and helped define disease subtypes, and may also help repurpose medications. PheWAS methods have also proven to be useful with research-collected data. Future efforts that integrate broad, robust collection of phenotype data (e.g., EHR data) with purpose-collected research data in combination with a greater understanding of EHR data will create a rich resource for increasingly more efficient and detailed genome-phenome analysis to usher in new discoveries in precision medicine. PMID:27147087

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.

Pegvaliase for the treatment of phenylketonuria: A pivotal, double-blind randomized discontinuation Phase 3 clinical trial.

PubMed

Harding, Cary O; Amato, R Stephen; Stuy, Mary; Longo, Nicola; Burton, Barbara K; Posner, John; Weng, Haoling H; Merilainen, Markus; Gu, Zhonghua; Jiang, Joy; Vockley, Jerry

2018-05-01

Pegvaliase is a recombinant Anabaena variabilis phenylalanine ammonia lyase (PAL) enzyme under investigation for treatment of adult phenylketonuria (PKU). This manuscript describes results of a randomized discontinuation trial (RDT) designed to evaluate the effects of pegvaliase treatment on blood phenylalanine (Phe) and neuropsychiatric outcomes in adults with PKU. PRISM-2 is a 4-part, Phase 3 study that enrolled adults with PKU receiving pegvaliase treatment (initiated in a prior Phase 2 or Phase 3 study). The RDT, Part 2 of PRISM-2, was an 8-week trial that evaluated change in blood Phe concentrations, neuropsychiatric and neurocognitive measures, and safety outcomes in PRISM-2 participants who had achieved at least a 20% blood Phe reduction from pre-treatment baseline with pegvaliase treatment. Participants were randomized 2:1 to either continue pegvaliase (20 mg/day or 40 mg/day) or switch to matching placebo. The pooled pegvaliase group enrolled 66 participants and each placebo group enrolled 14 participants. The primary endpoint of change in blood Phe concentration from RDT entry to RDT Week 8 was met with clinically meaningful and statistically significant differences between the pegvaliase and placebo groups. Mean (SD) blood Phe at the beginning of the RDT when all participants were receiving pegvaliase was 563.9 μM (504.6) in the group assigned to the 20 mg/day placebo group (n = 14), 508.2 μM (363.7) in those assigned to the 40 mg/day placebo group (n = 14), and 503.9 μM (520.3) in those assigned to continue pegvaliase treatment (n = 58). At Week 8 of the RDT, the least squares mean change (95% confidence interval) in blood Phe was 949.8 μM (760.4 to 1139.1) for the 20 mg/day placebo group and 664.8 μM (465.5 to 864.1) for the 40 mg/day placebo group in comparison to 26.5 μM (-68.3 to 121.3) for the pooled (20 mg/day and 40 mg/day) pegvaliase group (P < 0.0001 for pooled pegvaliase group vs each placebo group). Adverse events (AEs) were usually lower in the pooled placebo group when compared to the pooled pegvaliase group. The most common AEs for the pooled pegvaliase and pooled placebo groups were arthralgia (13.6% and 10.3%, respectively), headache (12.1% and 24.1%), anxiety (10.6% and 6.9%), fatigue (10.6% and 10.3%), and upper respiratory tract infection (1.5% and 17.2%). Mean blood Phe reduction was sustained in the pegvaliase group, while placebo groups had mean blood Phe concentration increase toward pre-treatment baseline levels. Results from this study confirmed the efficacy of pegvaliase in maintaining reduced blood Phe concentrations with a manageable safety profile for most participants. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Utilization of [14C]phenylalanine derived from arylphorin or free amino acid in Manduca sexta pharate adults

NASA Technical Reports Server (NTRS)

Wu, M.; Tischler, M. E.

1995-01-01

The role of arylphorin as a storage protein was studied using 14C-arylphorin. 14C-arylphorin was produced optimally by incubating one-half fat body from Manduca sexta fifth instar larvae at 22 degrees C for 24 h, in 1 ml of medium containing amino acids at 25% of their physiological concentration with [U-14C]-phenylalanine (phe) provided initially without nonlabeled phenylalanine. Nonlabeled phe was provided after 1 h at 16% of its physiological concentration. The specific activity of 14C-arylphorin produced in vitro was 30 times greater than that generated in vivo. Injection of 14C-arylphorin into pharate adults was used to study the distribution of 14C-phe derived from this protein into 14CO2 and tissues for comparison with injection of free 14C-phe during the middle (days 6 to 12 pharate adult) and late (days 12 to 17 pharate adult) stages of adult development. Appearance of 14CO2 from 14C-arylphorin as compared to 14C-phenylalanine showed a slower time course during both the middle and late stages of development, in keeping with the time needed for degradation of the protein. In accord with faster phe turnover near the end of adult development, total 14CO2 production was greater and the retention of 14C in hemolymph and fat body was less compared to the middle stage of development regardless of whether 14C-arylphorin or 14C-phe was injected. In the middle stage of development, the appearance of 14C in the cuticle and head parts was greater, whereas incorporation into abdomen and thorax was less than during the late stage of development. Since the pattern of 14C distribution from 14C-arylphorin and 14C-phe was similar, one major function of arylphorin must be as a storage protein replenishing the supply of free amino acids used for synthesis of adult tissues. These results also suggest a limited contribution of M. sexta arylphorin to formation of the cuticle subsequent to day-6 pharate adult.

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.

Complementary π-π interactions induce multicomponent coassembly into functional fibrils.

PubMed

Ryan, Derek M; Doran, Todd M; Nilsson, Bradley L

2011-09-06

Noncovalent self-assembled materials inspired by amyloid architectures are useful for biomedical applications ranging from regenerative medicine to drug delivery. The selective coassembly of complementary monomeric units to provide ordered multicomponent fibrils is a possible strategy for enhancing the sophistication of these noncovalent materials. Herein we report that complementary π-π interactions can be exploited to promote the coassembly of phenylalanine (Phe) derivatives that possess complementary aromatic side-chain functionality. Specifically, equimolar mixtures of Fmoc-Phe and Fmoc-F(5)-Phe, which possess side-chain groups with complementary quadrupole electronics, readily coassemble to form two-component fibrils and hydrogels under conditions where Fmoc-Phe alone fails to self-assemble. In addition, it was found that equimolar mixtures of Fmoc-Phe with monohalogenated (F, Cl, and Br) Fmoc-Phe derivatives also coassembled into two-component fibrils. These results collectively indicate that face-to-face quadrupole stacking between benzyl side-chain groups does not account for the molecular recognition between Phe and halogenated Phe derivatives that promote cofibrillization but that coassembly is mediated by more subtle π-π effects arising from the halogenation of the benzyl side chain. The use of complementary π-π interactions to promote the coassembly of two distinct monomeric units into ordered two-component fibrils dramatically expands the repertoire of noncovalent interactions that can be used in the development of sophisticated noncovalent materials. © 2011 American Chemical Society

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.

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.

Surface-enhanced Raman difference between bombesin and its modified analogues on the colloidal and electrochemically roughen silver surfaces.

PubMed

Podstawka, Edyta; Ozaki, Yukihiro

2008-10-01

In this article, surface-enhanced Raman scattering (SERS) spectra of bombesin (BN) and its six modified analogues ([D-Phe(12)]BN, [Tyr(4)]BN, [Tyr(4),D-Phe(12)]BN, [D-Phe(12),Leu(14)]BN, [Leu(13)-(R)-Leu(14)]BN, and [Lys(3)]BN) on a colloidal silver surface are reported and compared with SERS spectra of these species immobilized onto an ellectrochemically roughen silver electrode. Changes in enhancement and wavenumber of proper bands upon adsorption on different silver surfaces are consistent with BN and its analogues adsorption primarily through Trp(8). Slightly different adsorption states of these molecules are observed depending upon natural amino acids substitution. For example, the indole ring in all the peptides interacts with silver nanoparticles in a edge-on orientation. It is additionally coordinated to the silver through the N(1)--H bond for all the peptides, except [Phe(12)]BN. This is in contrary to the results obtained for the silver roughen electrode that show direct but not strong N(1)--H/Ag interaction for all peptides except [D-Phe(12),Leu(14)]BN and [Leu(13)-(R)-Leu(14)]BN. For BN only C==O is not involved in the chemical coordination with the colloidal surface. [Lys(3)]BN and BN also adsorb with the C--N bond of NH(2) group normal and horizontal, respectively, to the colloidal surface, whereas C--NH(2) in other peptides is tilted to this surface. Also, the Trp(8) --CH(2)-- moiety of only [Tyr(4)]BN, [Lys(3)]BN, and [Tyr(4),D-Phe(12)]BN coordinates to Ag, whereas the Phe(12) ring of [Phe(12)]BN, [Tyr(4),D-Phe(12)]BN, and [D-Phe(12),Leu(14)]BN assists in the peptides binding only on the colloidal silver. (c) 2008 Wiley Periodicals, Inc.

Identification and characterization of a locus which regulates multiple functions in Pseudomonas tolaasii, the cause of brown blotch disease of Agaricus bisporus.

PubMed Central

Grewal, S I; Han, B; Johnstone, K

1995-01-01

Pseudomonas tolaasii, the causal agent of brown blotch disease of Agaricus bisporus, spontaneously gives rise to morphologically distinct stable sectors, referred to as the phenotypic variant form, at the margins of the wild-type colonies. The phenotypic variant form is nonpathogenic and differs from the wild type in a range of biochemical and physiological characteristics. A genomic cosmid clone (pSISG29) from a wild-type P. tolaasii library was shown to be capable of restoring a range of characteristics of the phenotypic variant to those of the wild-type form, when present in trans. Subcloning and saturation mutagenesis analysis with Tn5lacZ localized a 3.0-kb region from pSISG29, designated the pheN locus, required for complementation of the phenotypic variant to the wild-type form. Marker exchange of the Tn5lacZ-mutagenized copy of the pheN locus into the wild-type strain demonstrated that a functional copy of the pheN gene is required to maintain the wild-type pathogenic phenotype and that loss of the pheN gene or its function results in conversion of the wild-type form to the phenotypic variant form. The pheN locus contained a 2,727-bp open reading frame encoding an 83-kDa protein. The predicted amino acid sequence of the PheN protein showed homology to the sensor and regulator domains of the conserved family of two component bacterial sensor regulator proteins. Southern hybridization analysis of pheN genes from the wild type and the phenotypic variant form revealed that DNA rearrangement occurs within the pheN locus during phenotypic variation. Analysis of pheN expression with a pheN::lacZ fusion demonstrated that expression is regulated by environmental factors. These results are related to a model for control for phenotypic variation in P. tolaasii. PMID:7642492

VLITL is a major cross-β-sheet signal for fibrinogen Aα-chain frameshift variants

PubMed Central

Garnier, Cyrille; Briki, Fatma; Le Pogamp, Patrick; Dogan, Ahmet; Rioux-Leclercq, Nathalie; Goude, Renan; Beugnet, Caroline; Martin, Laurent; Delpech, Marc; Bridoux, Frank; Grateau, Gilles; Doucet, Jean

2017-01-01

The first case of hereditary fibrinogen Aα-chain amyloidosis was recognized >20 years ago, but disease mechanisms still remain unknown. Here we report detailed clinical and proteomics studies of a French kindred with a novel amyloidogenic fibrinogen Aα-chain frameshift variant, Phe521Leufs, causing a severe familial form of renal amyloidosis. Next, we focused our investigations to elucidate the molecular basis that render this Aα-chain variant amyloidogenic. We show that a 49-mer peptide derived from the C-terminal part of the Phe521Leufs chain is deposited as fibrils in the patient’s kidneys, establishing that only a small portion of Phe521Leufs directly contributes to amyloid formation in vivo. In silico analysis indicated that this 49-mer Aα-chain peptide contained a motif (VLITL), with a high intrinsic propensity for β-aggregation at residues 44 to 48 of human renal fibrils. To experimentally verify the amyloid propensity of VLITL, we generated synthetic Phe521Leufs-derived peptides and compared their capacity for fibril formation in vitro with that of their VLITL-deleted counterparts. We show that VLITL forms typical amyloid fibrils in vitro and is a major signal for cross-β-sheet self-association of the 49-mer Phe521Leufs peptide identified in vivo, whereas its absence abrogates fibril formation. This study provides compelling evidence that VLITL confers amyloidogenic properties to Aα-chain frameshift variants, yielding a previously unknown molecular basis for the pathogenesis of Aα-chain amyloidosis. PMID:29089309

Preliminary in vivo evaluation of [131I]-2-iodo-D-phenylalanine as a potential radionuclide therapeutic agent in R1M-fluc rhabdomyosarcoma tumor-bearing NuNu mice using bioluminescent imaging.

PubMed

Bauwens, Matthias; Wimana, Lena; Keyaerts, Marleen; Peleman, Cindy; Lahoutte, Tony; Kersemans, Ken; Snykers, Sarah; Vinken, Mathieu; Mertens, John; Bossuyt, Axel

2010-04-01

Carrier-added [(123)I]-2-iodo-D-phenylalanine (CA [(123)I]-2-I-D-Phe) was previously found to have a preferential retention in tumors with a high tumor background contrast in animal models. A previous human dosimetry study demonstrated a favorable biodistribution and radiation burden in human subjects. The aim of this study was to investigate the potential of CA [(131)I]-2-I-D-Phe as an agent for radionuclide therapy. Sixty (60) nude athymic mice were inoculated subcutaneously with firefly luciferase-transduced R1M rhabdomyosarcoma cells. The mice in the therapy group were injected intravenously (i.v.) with 148 MBq [(131)I]-2-I-D-Phe (432 GBq/mmol) in kit solution. Controls were injected with kit solution without radioactivity, with physiological saline, or with 148 MBq [(131)I](-) in physiological saline. Tumor growth was quantified using bioluminescent imaging and caliper measurements. [(131)I]-2-I-D-Phe clearly reduced tumor growth in the treated mice compared with the control groups. A tumor growth-rate reduction of at least 33% was found for mice receiving a therapeutic dose. There were no serious adverse side-effects of the therapy. In conclusion, i.v. injection of CA 148 MBq [(131)I]-2-I-D-Phe specifically reduces tumor growth in athymic nude mice without relevant side-effects on the animals' health.

Modulation of B16-BL6 murine melanoma metastatic phenotype by tyrosine and phenylalanine restriction in the absence of host selection pressures.

PubMed

Elstad, C A; Meadows, G G

1993-01-01

We previously showed that restriction of tyrosine (Tyr) and phenylalanine (Phe) in vivo dramatically suppresses the metastatic phenotype of B16-BL6 (BL6) murine melanoma. Present results indicate a direct effect of Tyr and Phe restriction on the tumor in the absence of host selection pressures. Lung colonizing ability of BL6 is dramatically suppressed after one passage in vitro in media containing low levels of Tyr and Phe. This antimetastatic effect is immediate, stable for at least 5 in vitro passages in Tyr and Phe restricted media, and evident event after levels of Tyr and Phe are restored to normal. Heterogeneity for lung colonizing ability is suppressed, as evidence by fewer tumor colonies formed by clones following i.v. inoculation into mice fed normal diet. This suppression of BL6 metastatic phenotype is not due to differential clearance and retention in the lung or to decreased growth, but is specific for these two amino acids. As the mechanism(s) for the antitumor effects of Tyr and Phe restriction are detailed, the relevance of Tyr and Phe restriction as an early adjuvant to effective cancer treatment can be explored.

Utilization of deep-sea microbial esterase PHE21 to generate chiral sec-butyl acetate through kinetic resolutions.

PubMed

Wang, Yilong; Xu, Yongkai; Zhang, Yun; Sun, Aijun; Hu, Yunfeng

2018-06-08

We previously identified and characterized 1 novel deep-sea microbial esterase PHE21 and used PHE21 as a green biocatalyst to generate chiral ethyl (S)-3-hydroxybutyrate, 1 key chiral chemical, with high enantiomeric excess and yield through kinetic resolution. Herein, we further explored the potential of esterase PHE21 in the enantioselective preparation of secondary butanol, which was hard to be resolved by lipases/esterases. Despite the fact that chiral secondary butanols and their ester derivatives were hard to prepare, esterase PHE21 was used as a green biocatalyst in the generation of (S)-sec-butyl acetate through hydrolytic reactions and the enantiomeric excess, and the conversion of (S)-sec-butyl acetate reached 98% and 52%, respectively, after process optimization. Esterase PHE21 was also used to generate (R)-sec-butyl acetate through asymmetric transesterification reactions, and the enantiomeric excess and conversion of (R)-sec-butyl acetate reached 64% and 43%, respectively, after process optimization. Deep-sea microbial esterase PHE21 was characterized to be a useful biocatalyst in the kinetic resolution of secondary butanol and other valuable chiral secondary alcohols. © 2018 Wiley Periodicals, Inc.

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.

Experimental hyperphenylalaninemia in the pregnant guinea pig: possible phenylalanine teratogenesis and p-chlorophenylalanine embryotoxicity.

PubMed

Kronick, J B; Whelan, D T; McCallion, D J

1987-10-01

Maternal hyperphenylalaninemia (HPH) due to deficient phenylalanine (Phe) hydroxylation is a recognized human teratogen associated with an increased incidence of intrauterine growth retardation, microcephaly, congenital heart disease, and mental retardation. There are no previous reports of experimental HPH during organogenesis. Sustained HPH was produced in pregnant guinea pigs by adding 3.5% Phe and 1.0% parachlorophenylalanine (pCPA), an inhibitor of Phe hydroxylase, to standard guinea pig chow. Animals consumed the supplemented test diets from gestation day 1 until killed on gestation day 17. Examination of day 17 embryos revealed that embryonic mortality was associated only with maternal pCPA administration and was independent of the degree of maternal HPH. Embryonic malformation was associated with maternal HPH as well as maternal pCPA administration. Both maternal HPH and pCPA administration were associated with embryonic growth retardation. There was no association between maternal food intake or plasma tyrosine levels and embryonic abnormality or mortality. Both Phe and tyrosine were found to be concentrated in gestation day 17 yolk sac fluid when compared to maternal plasma Phe and tyrosine. The association of embryonic malformation and maternal HPH is consistent with human data. The embryotoxicity of pCPA requires further study and highlights the necessity of appropriate controls in models of experimental HPH.

Expression of the cystic fibrosis transmembrane conductance regulator gene in the respiratory tract of normal individuals and individuals with cystic fibrosis

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

Trapnell, B.C.; Chinshyan Chu; Paakko, P.K.

1991-08-01

The most common mutation of the cystic fibrosis transmembrane conductance regulator gene, CFTR, associated with the clinical disorder cystic fibrosis (CF) is called {Delta}Phe{sup 508}, a triple-base deletion resulting in loss of phenylalanine at residue 508 of the predicted 1480-amino acid CFTR protein. In the context that the lung is the major site of morbidity and mortality in CF, the authors evaluated airway epithelial cells for CFTR mRNA transcripts in normal individuals, normal-{Delta}Phe{sup 508} heterozygotes, and {Delta}Phe{sup 508} homozygotes to determine if the normal and {Delta}Phe{sup 508} CFTR alleles are expressed in the respiratory epithelium, to what extent they aremore » expressed, and whether there are relative differences in the expression of the normal and abnormal alleles at the mRNA level. Respiratory tract epithelial cells recovered by fiberoptic bronchoscopy with a cytology brush demonstrated CFTR mRNA transcripts with sequences appropriately reflecting the normal and {Delta}Phe{sup 508} CFTR alleles of the various study groups. CFTR gene expression quantified by limited polymerase chain reaction amplification showed that in normal individuals, CFTR mRNA transcripts are expressed in nasal, tracheal, and bronchial epithelial cells.« less

Biosynthesis and Metabolic Fate of Phenylalanine in Conifers

PubMed Central

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

2016-01-01

The amino acid phenylalanine (Phe) is a critical metabolic node that plays an essential role in the interconnection between primary and secondary metabolism in plants. Phe is used as a protein building block but it is also as a precursor for numerous plant compounds that are crucial for plant reproduction, growth, development, and defense against different types of stresses. The metabolism of Phe plays a central role in the channeling of carbon from photosynthesis to the biosynthesis of phenylpropanoids. The study of this metabolic pathway is particularly relevant in trees, which divert large amounts of carbon into the biosynthesis of Phe-derived compounds, particularly lignin, an important constituent of wood. The trunks of trees are metabolic sinks that consume a considerable percentage of carbon and energy from photosynthesis, and carbon is finally immobilized in wood. This paper reviews recent advances in the biosynthesis and metabolic utilization of Phe in conifer trees. Two alternative routes have been identified: the ancient phenylpyruvate pathway that is present in microorganisms, and the arogenate pathway that possibly evolved later during plant evolution. Additionally, an efficient nitrogen recycling mechanism is required to maintain sustained growth during xylem formation. The relevance of phenylalanine metabolic pathways in wood formation, the biotic interactions, and ultraviolet protection is discussed. The genetic manipulation and transcriptional regulation of the pathways are also outlined. PMID:27468292

Biosynthesis and Metabolic Fate of Phenylalanine in Conifers.

PubMed

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

2016-01-01

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

Phe317 Is Essential for Rubber Oxygenase RoxA Activity

PubMed Central

Birke, Jakob; Hambsch, Nadja; Schmitt, Georg; Altenbuchner, Josef

2012-01-01

RoxA is an extracellular c-type diheme cytochrome secreted by Xanthomonas sp. strain 35Y during growth on rubber. RoxA cleaves poly(cis-1,4-isoprene) to 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD). Analysis of the RoxA structure revealed that Phe317 is located in close proximity (≈5 Å) to the N-terminal heme that presumably represents the active site. To find evidence of whether Phe317 is important for catalysis, we changed it to tyrosine, tryptophan, leucine, histidine, or alanine. All five RoxA muteins were expressed after integration of the respective gene into the chromosome of a Xanthomonas sp. ΔroxA strain. Residual clearing zone formation on opaque latex agar was found for Xanthomonas sp. strains expressing the Phe317Leu, Phe317Ala, or Phe317His variant (wild type > Leu > Ala > His). Strains in which Phe317 was changed to tyrosine or tryptophan were inactive. Phe317Ala and Phe312Leu RoxA muteins were purified, and polyisoprene cleavage activities were reduced to ≈3% and 10%, respectively. UV-visible spectroscopy of RoxA muteins confirmed that both heme groups were present in an oxidized form, but spectral responses to the addition of low-molecular-weight (inhibitory) ligand molecules such as imidazole and pyridine were different from those of wild-type RoxA. Our results show that residue 317 is involved in interaction with substrates. This is the first report on structure-function analysis of a polyisoprene-cleaving enzyme and on the identification of an amino acid that is essential for polyisoprene cleavage activity. PMID:22941074

New Class of Bacterial Phenylalanyl-tRNA Synthetase Inhibitors with High Potency and Broad-Spectrum Activity

PubMed Central

Beyer, Dieter; Kroll, Hein-Peter; Endermann, Rainer; Schiffer, Guido; Siegel, Stephan; Bauser, Marcus; Pohlmann, Jens; Brands, Michael; Ziegelbauer, Karl; Haebich, Dieter; Eymann, Christine; Brötz-Oesterhelt, Heike

2004-01-01

Phenylalanyl (Phe)-tRNA synthetase (Phe-RS) is an essential enzyme which catalyzes the transfer of phenylalanine to the Phe-specific transfer RNA (tRNAPhe), a key step in protein biosynthesis. Phenyl-thiazolylurea-sulfonamides were identified as a novel class of potent inhibitors of bacterial Phe-RS by high-throughput screening and chemical variation of the screening hit. The compounds inhibit Phe-RS of Escherichia coli, Haemophilus influenzae, Streptococcus pneumoniae, and Staphylococcus aureus, with 50% inhibitory concentrations in the nanomolar range. Enzyme kinetic measurements demonstrated that the compounds bind competitively with respect to the natural substrate Phe. All derivatives are highly selective for the bacterial Phe-RS versus the corresponding mammalian cytoplasmic and human mitochondrial enzymes. Phenyl-thiazolylurea-sulfonamides displayed good in vitro activity against Staphylococcus, Streptococcus, Haemophilus, and Moraxella strains, reaching MICs below 1 μg/ml. The antibacterial activity was partly antagonized by increasing concentrations of Phe in the culture broth in accordance with the competitive binding mode. Further evidence that inhibition of tRNAPhe charging is the antibacterial principle of this compound class was obtained by proteome analysis of Bacillus subtilis. Here, the phenyl-thiazolylurea-sulfonamides induced a protein pattern indicative of the stringent response. In addition, an E. coli strain carrying a relA mutation and defective in stringent response was more susceptible than its isogenic relA+ parent strain. In vivo efficacy was investigated in a murine S. aureus sepsis model and a S. pneumoniae sepsis model in rats. Treatment with the phenyl-thiazolylurea-sulfonamides reduced the bacterial titer in various organs by up to 3 log units, supporting the potential value of Phe-RS as a target in antibacterial therapy. PMID:14742205

Low uptake of periodic health examinations in the Kingdom of Saudi Arabia, 2013.

PubMed

El Bcheraoui, Charbel; Tuffaha, Marwa; Daoud, Farah; AlMazroa, Mohammad A; Al Saeedi, Mohammad; Memish, Ziad A; Basulaiman, Mohammed; Al Rabeeah, Abdullah A; Mokdad, Ali H

2015-01-01

It is unknown whether Saudis receive health examinations periodically. To inform health authorities on the health-seeking behavior of the Saudi population, we investigated patterns of periodic health examination (PHE) use by Saudis. We conducted a nationally representative multistage survey of individuals aged 15 years or older on sociodemographic characteristics, healthcare utilization, and self-reported chronic conditions. We used a backward elimination multivariate logistic regression model to measure associations between PHE and sociodemographic, behavioral, and health characteristics. Between April and June 2013, a total of 12,000 households were contacted, and 10,735 participants completed the survey (response rate of 89.4%). Among participants, 2542 (22.9%), representing more than 2.7 million Saudis aged 15 years or older, received a PHE during the past 2 years. Moreover, 7463 (73.5%) participants, representing 9.1 million Saudis, visited a healthcare setting in the past 2 years due to illness or injury. The likelihood of receiving a PHE in the past 2 years increased with age, education, being married, consumption of five servings of fruits and vegetables per day, diagnoses of prediabetes, diabetes, or hypercholesterolemia, and a visit to a healthcare setting within the last 2 years due to an illness or an injury. This is the first national study to investigate the use of PHE in Kingdom of Saudi Arabia (KSA) where healthcare is freely available. Few Saudis seek preventive healthcare and most healthcare visits are for injuries or sickness. KSA may reduce its health expenditures by routinizing PHE and detecting chronic conditions at early stages.

Structural determinants for antagonist pharmacology that distinguish the rho1 GABAC receptor from GABAA receptors.

PubMed

Zhang, Jianliang; Xue, Fenqin; Chang, Yongchang

2008-10-01

GABA receptor (GABAR) types C (GABACR) and A (GABAAR) are both GABA-gated chloride channels that are distinguished by their distinct competitive antagonist properties. The structural mechanism underlying these distinct properties is not well understood. In this study, using previously identified binding residues as a guide, we made individual or combined mutations of nine binding residues in the rho1 GABACR subunit to their counterparts in the alpha1beta2gamma2 GABAAR or reverse mutations in alpha1 or beta2 subunits. The mutants were expressed in Xenopus laevis oocytes and tested for sensitivities of GABA-induced currents to the GABAA and GABAC receptor antagonists. The results revealed that bicuculline insensitivity of the rho1 GABACR was mainly determined by Tyr106, Phe138 and Phe240 residues. Gabazine insensitivity of the rho1 GABACR was highly dependent on Tyr102, Tyr106, and Phe138. The sensitivity of the rho1 GABACR to 3-aminopropyl-phosphonic acid and its analog 3-aminopropyl-(methyl)phosphinic acid mainly depended on residues Tyr102, Val140, FYS240-242, and Phe138. Thus, the residues Tyr102, Tyr106, Phe138, and Phe240 in the rho1 GABACR are major determinants for its antagonist properties distinct from those in the GABAAR. In addition, Val140 in the GABACR contributes to 3-APA binding. In conclusion, we have identified the key structural elements underlying distinct antagonist properties for the GABACR. The mechanistic insights were further extended and discussed in the context of antagonists docking to the homology models of GABAA or GABAC receptors.

Chromium (D-phenylalanine)3 supplementation alters glucose disposal, insulin signaling, and glucose transporter-4 membrane translocation in insulin-resistant mice.

PubMed

Dong, Feng; Kandadi, Machender Reddy; Ren, Jun; Sreejayan, Nair

2008-10-01

Chromium has gained popularity as a nutritional supplement for diabetic and insulin-resistant subjects. This study was designed to evaluate the effect of chronic administration of a novel chromium complex of d-phenylalanine [Cr(D-phe)(3)] in insulin-resistant, sucrose-fed mice. Whole-body insulin resistance was generated in FVB mice by 9 wk of sucrose feeding, following which they were randomly assigned to be unsupplemented (S group) or to receive oral Cr(D-phe)(3) in drinking water (SCr group) at a dose of 45 mug.kg(-1).d(-1) ( approximately 3.8 mug of elemental chromium.kg(-1).d(-1)). A control group (C) did not consume sucrose and was not supplemented. Sucrose-fed mice had an elevated serum insulin concentration compared with controls and this was significantly lower in sucrose-fed mice that received Cr(D-phe)(3), which did not differ from controls. Impaired glucose tolerance in sucrose-fed mice, evidenced by the poor glucose disposal rate following an intraperitoneal glucose tolerance test, was significantly improved in mice receiving Cr(D-phe)(3). Chromium supplementation significantly enhanced insulin-stimulated Akt phosphorylation and membrane-associated glucose transporter-4 in skeletal muscles of sucrose-fed mice. In cultured adipocytes rendered insulin resistant by chronic exposure to high concentrations of glucose and insulin, Cr(D-phe)(3) augmented Akt phosphorylation and glucose uptake. These results indicate that dietary supplementation with Cr(D-phe)(3) may have potential beneficial effects in insulin-resistant, prediabetic conditions.

Effects of amino acid supplementations on metabolic and physiological parameters in Atlantic cod (Gadus morhua) under stress.

PubMed

Herrera, Marcelino; Herves, María Antonia; Giráldez, Inmaculada; Skar, Kristin; Mogren, Hanne; Mortensen, Atle; Puvanendran, Velmurugu

2017-04-01

The effects of tryptophan (Trp) and phenylalanine (Phe) diet supplementation on the stress and metabolism of the Atlantic cod have been studied. Fish were fed diet supplemented with Trp or Phe or control diet for 1 week. At the end of the feeding trial, fish were subjected to air exposure or heat shock. Following samples of blood, liver and muscle were taken from the fish and were analyzed for stress and metabolic indicators. After an air exposure, plasma cortisol levels in fish fed with Trp and Phe diets were lower compared to the fish fed the control diet. Diets containing both amino acids increased significantly the liver transaminase activities in juvenile cod. During thermal stress, high Trp contents had significant effects on fructose biphosphatase activity though Phe did not. Overall, activities of glucose 6-phosphate dehydrogenase, pyruvate kinase, and phosphofructokinase increased significantly for both amino acid diets. For the thermal stress, fish had the highest values of those activities for the 3Trp diet. Trp content in the diet had significant effects on the transaminase activity in muscle during air stress compared to fish fed control and Phe diets. Muscle alanine transaminase activity for thermal stress in fish fed any diet was not significantly different from the control. Both Trp and Phe supplementations reduced the stress markers in the cod; hence, they could be used as additives for the stress attenuation. However, they also raised the activity of key enzymes in glycolysis and gluconeogenesis, mainly the Trp diets.

Solvent selection and optimization of α-chymotrypsin-catalyzed synthesis of N-Ac-Phe-Tyr-NH2 using mixture design and response surface methodology.

PubMed

Hu, Shih-Hao; Kuo, Chia-Hung; Chang, Chieh-Ming J; Liu, Yung-Chuan; Chiang, Wen-Dee; Shieh, Chwen-Jen

2012-01-01

A peptide, N-Ac-Phe-Tyr-NH(2) , with angiotensin I-converting enzyme (ACE) inhibitor activity was synthesized by an α-chymotrypsin-catalyzed condensation reaction of N-acetyl phenylalanine ethyl ester (N-Ac-Phe-OEt) and tyrosinamide (Tyr-NH(2) ). Three kinds of solvents: a Tris-HCl buffer (80 mM, pH 9.0), dimethylsulfoxide (DMSO), and acetonitrile were employed in this study. The optimum reaction solvent component was determined by simplex centroid mixture design. The synthesis efficiency was enhanced in an organic-aqueous solvent (Tris-HCl buffer: DMSO: acetonitrile = 2:1:1) in which 73.55% of the yield of N-Ac-Phe-Tyr-NH(2) could be achieved. Furthermore, the effect of reaction parameters on the yield was evaluated by response surface methodology (RSM) using a central composite rotatable design (CCRD). Based on a ridge max analysis, the optimum condition for this peptide synthesis included a reaction time of 7.4 min, a reaction temperature of 28.1°C, an enzyme activity of 98.9 U, and a substrate molar ratio (Phe:Tyr) of 1:2.8. The predicted and the actual (experimental) yields were 87.6 and 85.5%, respectively. The experimental design and RSM performed well in the optimization of synthesis of N-Ac-Phe-Tyr-NH(2) , so it is expected to be an effective method for obtaining a good yield of enzymatic peptide. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

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.

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.

One-Pot Synthesis of D-Phenylalanine-Functionalized Multi-Walled Carbon Nanotubes: a Metal-Free Chiral Material for the Asymmetric Electroreduction of Aromatic Ketones.

PubMed

Yue, Ying-Na; Zeng, Sheng; Wang, Hui; Wang, Shuo; Wang, Huan; Lu, Jia-Xing

2018-06-19

A simple protocol to synthesize D-phenylalanine (D-PHE)-functionalized multi-walled carbon nanotubes (MWCNTs) via one-pot method was established by grafting D-PHE onto MWCNTs to obtain D-PHE-MWCNTs under mild reaction conditions. The resulting D-PHE-MWCNTs were detailedly characterized via spectroscopy and surface analysis. The electroreduction of 2,2,2-trifluoroacetophenone at D-PHE-MWCNTs cathode afforded (S)-α-(trifluoromethyl) benzyl alcohol whose yield was 65% and the enantiomeric excess was 40%. No extra catalysts were required in this electrochemical reaction solution compared with other reactions requiring homogeneous catalysis. The metal-free chiral material also showed acceptable asymmetric electroreduction performance, considerable stability and favorable reusability.

Detection of pleiotropy through a Phenome-wide association study (PheWAS) of epidemiologic data as part of the Environmental Architecture for Genes Linked to Environment (EAGLE) study.

PubMed

Hall, Molly A; Verma, Anurag; Brown-Gentry, Kristin D; Goodloe, Robert; Boston, Jonathan; Wilson, Sarah; McClellan, Bob; Sutcliffe, Cara; Dilks, Holly H; Gillani, Nila B; Jin, Hailing; Mayo, Ping; Allen, Melissa; Schnetz-Boutaud, Nathalie; Crawford, Dana C; Ritchie, Marylyn D; Pendergrass, Sarah A

2014-12-01

We performed a Phenome-wide association study (PheWAS) utilizing diverse genotypic and phenotypic data existing across multiple populations in the National Health and Nutrition Examination Surveys (NHANES), conducted by the Centers for Disease Control and Prevention (CDC), and accessed by the Epidemiological Architecture for Genes Linked to Environment (EAGLE) study. We calculated comprehensive tests of association in Genetic NHANES using 80 SNPs and 1,008 phenotypes (grouped into 184 phenotype classes), stratified by race-ethnicity. Genetic NHANES includes three surveys (NHANES III, 1999-2000, and 2001-2002) and three race-ethnicities: non-Hispanic whites (n = 6,634), non-Hispanic blacks (n = 3,458), and Mexican Americans (n = 3,950). We identified 69 PheWAS associations replicating across surveys for the same SNP, phenotype-class, direction of effect, and race-ethnicity at p<0.01, allele frequency >0.01, and sample size >200. Of these 69 PheWAS associations, 39 replicated previously reported SNP-phenotype associations, 9 were related to previously reported associations, and 21 were novel associations. Fourteen results had the same direction of effect across more than one race-ethnicity: one result was novel, 11 replicated previously reported associations, and two were related to previously reported results. Thirteen SNPs showed evidence of pleiotropy. We further explored results with gene-based biological networks, contrasting the direction of effect for pleiotropic associations across phenotypes. One PheWAS result was ABCG2 missense SNP rs2231142, associated with uric acid levels in both non-Hispanic whites and Mexican Americans, protoporphyrin levels in non-Hispanic whites and Mexican Americans, and blood pressure levels in Mexican Americans. Another example was SNP rs1800588 near LIPC, significantly associated with the novel phenotypes of folate levels (Mexican Americans), vitamin E levels (non-Hispanic whites) and triglyceride levels (non-Hispanic whites), and replication for cholesterol levels. The results of this PheWAS show the utility of this approach for exposing more of the complex genetic architecture underlying multiple traits, through generating novel hypotheses for future research.

Antinociceptive potency of a fluorinated cyclopeptide Dmt-c[D-Lys-Phe-p-CF3-Phe-Asp]NH2.

PubMed

Piekielna-Ciesielska, Justyna; Mollica, Adriano; Pieretti, Stefano; Fichna, Jakub; Szymaszkiewicz, Agata; Zielińska, Marta; Kordek, Radzisław; Janecka, Anna

2018-12-01

Opioid peptides and opiate drugs such as morphine, mediate their analgesic effects, but also undesired side effects, mostly through activation of the mu opioid receptor. However, delta- and kappa-opioid receptors can also contribute to the analgesic effects of opioids. Recent findings showed that simultaneous activation of multiple opioid receptors may result in additional analgesia with fewer side effects. Here, we evaluated the pharmacological profile of our formerly developed mixed mu/kappa-opioid receptor ligands, Dmt-c[D-Lys-Phe-Phe-Asp]NH 2 (C-36) and Dmt-c[D-Lys-Phe-p-CF 3 -Phe-Asp]NH 2 (F-81). The ability of these peptides to cross the blood-brain barrier was tested in the parallel artificial membrane permeability (PAMPA) assay. On the basis of the hot-plate test in mice after central and peripheral administration, analog F-81 was selected for the anti-nociceptive and anti-inflammatory activity assessment after peripheral administration.

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

Functional variants in the sucrase–isomaltase gene associate with increased risk of irritable bowel syndrome

PubMed Central

Henström, Maria; Diekmann, Lena; Bonfiglio, Ferdinando; Hadizadeh, Fatemeh; Kuech, Eva-Maria; von Köckritz-Blickwede, Maren; Thingholm, Louise B; Zheng, Tenghao; Assadi, Ghazaleh; Dierks, Claudia; Heine, Martin; Philipp, Ute; Distl, Ottmar; Money, Mary E; Belheouane, Meriem; Heinsen, Femke-Anouska; Rafter, Joseph; Nardone, Gerardo; Cuomo, Rosario; Usai-Satta, Paolo; Galeazzi, Francesca; Neri, Matteo; Walter, Susanna; Simrén, Magnus; Karling, Pontus; Ohlsson, Bodil; Schmidt, Peter T; Lindberg, Greger; Dlugosz, Aldona; Agreus, Lars; Andreasson, Anna; Mayer, Emeran; Baines, John F; Engstrand, Lars; Portincasa, Piero; Bellini, Massimo; Stanghellini, Vincenzo; Barbara, Giovanni; Chang, Lin; Camilleri, Michael; Franke, Andre; Naim, Hassan Y

2018-01-01

Objective IBS is a common gut disorder of uncertain pathogenesis. Among other factors, genetics and certain foods are proposed to contribute. Congenital sucrase–isomaltase deficiency (CSID) is a rare genetic form of disaccharide malabsorption characterised by diarrhoea, abdominal pain and bloating, which are features common to IBS. We tested sucrase–isomaltase (SI) gene variants for their potential relevance in IBS. Design We sequenced SI exons in seven familial cases, and screened four CSID mutations (p.Val557Gly, p.Gly1073Asp, p.Arg1124Ter and p.Phe1745Cys) and a common SI coding polymorphism (p.Val15Phe) in a multicentre cohort of 1887 cases and controls. We studied the effect of the 15Val to 15Phe substitution on SI function in vitro. We analysed p.Val15Phe genotype in relation to IBS status, stool frequency and faecal microbiota composition in 250 individuals from the general population. Results CSID mutations were more common in patients than asymptomatic controls (p=0.074; OR=1.84) and Exome Aggregation Consortium reference sequenced individuals (p=0.020; OR=1.57). 15Phe was detected in 6/7 sequenced familial cases, and increased IBS risk in case–control and population-based cohorts, with best evidence for diarrhoea phenotypes (combined p=0.00012; OR=1.36). In the population-based sample, 15Phe allele dosage correlated with stool frequency (p=0.026) and Parabacteroides faecal microbiota abundance (p=0.0024). The SI protein with 15Phe exhibited 35% reduced enzymatic activity in vitro compared with 15Val (p<0.05). Conclusions SI gene variants coding for disaccharidases with defective or reduced enzymatic activity predispose to IBS. This may help the identification of individuals at risk, and contribute to personalising treatment options in a subset of patients. PMID:27872184

Functional variants in the sucrase-isomaltase gene associate with increased risk of irritable bowel syndrome.

PubMed

Henström, Maria; Diekmann, Lena; Bonfiglio, Ferdinando; Hadizadeh, Fatemeh; Kuech, Eva-Maria; von Köckritz-Blickwede, Maren; Thingholm, Louise B; Zheng, Tenghao; Assadi, Ghazaleh; Dierks, Claudia; Heine, Martin; Philipp, Ute; Distl, Ottmar; Money, Mary E; Belheouane, Meriem; Heinsen, Femke-Anouska; Rafter, Joseph; Nardone, Gerardo; Cuomo, Rosario; Usai-Satta, Paolo; Galeazzi, Francesca; Neri, Matteo; Walter, Susanna; Simrén, Magnus; Karling, Pontus; Ohlsson, Bodil; Schmidt, Peter T; Lindberg, Greger; Dlugosz, Aldona; Agreus, Lars; Andreasson, Anna; Mayer, Emeran; Baines, John F; Engstrand, Lars; Portincasa, Piero; Bellini, Massimo; Stanghellini, Vincenzo; Barbara, Giovanni; Chang, Lin; Camilleri, Michael; Franke, Andre; Naim, Hassan Y; D'Amato, Mauro

2018-02-01

IBS is a common gut disorder of uncertain pathogenesis. Among other factors, genetics and certain foods are proposed to contribute. Congenital sucrase-isomaltase deficiency (CSID) is a rare genetic form of disaccharide malabsorption characterised by diarrhoea, abdominal pain and bloating, which are features common to IBS. We tested sucrase-isomaltase ( SI ) gene variants for their potential relevance in IBS. We sequenced SI exons in seven familial cases, and screened four CSID mutations (p.Val557Gly, p.Gly1073Asp, p.Arg1124Ter and p.Phe1745Cys) and a common SI coding polymorphism (p.Val15Phe) in a multicentre cohort of 1887 cases and controls. We studied the effect of the 15Val to 15Phe substitution on SI function in vitro. We analysed p.Val15Phe genotype in relation to IBS status, stool frequency and faecal microbiota composition in 250 individuals from the general population. CSID mutations were more common in patients than asymptomatic controls (p=0.074; OR=1.84) and Exome Aggregation Consortium reference sequenced individuals (p=0.020; OR=1.57). 15Phe was detected in 6/7 sequenced familial cases, and increased IBS risk in case-control and population-based cohorts, with best evidence for diarrhoea phenotypes (combined p=0.00012; OR=1.36). In the population-based sample, 15Phe allele dosage correlated with stool frequency (p=0.026) and Parabacteroides faecal microbiota abundance (p=0.0024). The SI protein with 15Phe exhibited 35% reduced enzymatic activity in vitro compared with 15Val (p<0.05). SI gene variants coding for disaccharidases with defective or reduced enzymatic activity predispose to IBS. This may help the identification of individuals at risk, and contribute to personalising treatment options in a subset of patients. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Hydroxylation of D-phenylalanine as a novel approach to detect hydroxyl radicals: application to cardiac pathophysiology.

PubMed

Biondi, Roberto; Ambrosio, Giuseppe; Liebgott, Tibaud; Cardounel, Arturo J; Bettini, Marco; Tritto, Isabella; Zweier, Jay L

2006-07-15

Research in the pathophysiology of ischemia/reperfusion or redox signaling is hindered by lack of simple methodology to measure short-lived oxygen radicals. In the presence of hydroxyl radical ((*)OH), d-phenylalanine (d-Phe) yields para-, meta- and ortho-tyrosine. We have previously demonstrated that d-Phe can accurately detect (*)OH formation in chemical, enzymatic and cellular systems by simple HPLC methodology [Anal Biochem 290:138;2001]. In the present study, we tested whether d-Phe hydroxylation can be used to detect (*)OH formation in intact organs. Rat hearts were perfused with buffer containing 5 mM d-Phe and subjected to 30 min of total global ischemia at 37 degrees C followed by 45 min of reperfusion. Quantitative analysis of the three hydroxytyrosine isomers was achieved by HPLC-based electrochemical detection of cardiac venous effluent, with the analytical cells operating in the oxidative mode. The detection limit of this assay was <10 fmol. Under baseline conditions, hydroxytyrosine release from the heart was very low ( congruent with0.8 nmol/min/g). However, a prominent tyrosine burst occurred immediately upon post-ischemic reflow. In cardiac effluent collected 40 s into reperfusion, the hydroxytyrosine concentration was more than 40 times greater than at baseline; hydroxytyrosine concentration then progressively declined, to return to pre-ischemic values by 5 min of reperfusion. In parallel experiments, formation of hydroxytyrosines was markedly reduced in hearts reperfused in the presence of the (*)OH scavenger mannitol. Inclusion of 5 mm d-Phe in the perfusion medium altered neither basal cardiac function nor coronary vascular tone, but it enhanced recovery of myocardial function during post-ischemic reperfusion, consistent with direct reaction with (*)OH. Our results demonstrate that d-Phe is a sensitive method for detection of (*)OH generation in the heart. Since d-Phe is not a substrate for endogenous enzymes, it can be exploited as a reliable method to measure (*)OH formation under a variety of pathophysiological conditions.

Dietary phenylalanine-improved intestinal barrier health in young grass carp (Ctenopharyngodon idella) is associated with increased immune status and regulated gene expression of cytokines, tight junction proteins, antioxidant enzymes and related signalling molecules.

PubMed

Feng, Lin; Li, Wen; Liu, Yang; Jiang, Wei-Dan; Kuang, Sheng-Yao; Jiang, Jun; Tang, Ling; Wu, Pei; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu

2015-08-01

The present work evaluated the effects of dietary phenylalanine (Phe) on the intestinal immune response, tight junction proteins transcript abundance, and the gene expression of immune- and antioxidant-related signalling molecules in the intestine. In addition, the dietary Phe (and Phe + Tyr) requirement of young grass carp (Ctenopharyngodon idella) was also estimated. Fish were fed fish meal-casein-gelatin based diets (302.3 g crude protein kg(-1)) containing 3.4 (basal diet), 6.1, 9.1, 11.5, 14.0 and 16.8 g Phe kg(-1) with a fixed amount of 10.7 g tyrosine kg(-1) for 8 weeks. The results showed that Phe deficiency or excess Phe reduced the lysozyme and acid phosphatase activities and complement C 3 content in the intestine (P < 0.05). Moreover, zonula occludens-1 (ZO-1), occludin and claudin c mRNA levels were highest in the fish fed the diet containing 11.5 g Phe kg(-1) (P < 0.05). However, claudin 12 and claudin b mRNA levels were not significantly affected by dietary Phe (P > 0.05). Gene expression of interleukin-10 (IL-10), transforming growth factor-β1 (TGF-β1), target of rapamycin (TOR) and inhibitor of nuclear factor κBα (IκBα) in proximal intestine (PI), mid intestine (MI) and distal intestine (DI) increased as dietary Phe increased up to 6.1, 9.1, 11.5 and 14.0 g kg(-1), respectively (P < 0.05). However, interleukin-8 (IL-8), tumour necrosis factor-α (TNF-α) and nuclear factor-κB p65 (NF-κB p65) mRNA levels showed opposite tendencies. In addition, the mRNA level of superoxide dismutase (SOD) was significantly lower in the intestinal tissue of the group fed a diet with Phe levels of 16.8 g kg(-1) than in those of other groups (P < 0.05). The expression of NF-E2-related factor 2 (Nrf2) gene was increased as dietary Phe increased up to 9.1 g kg(-1) (P < 0.05). In conclusion, Phe improved intestinal immune status, and regulated gene expression of cytokines, tight junction proteins, antioxidant enzymes, NF-κB p65, IκBα, TOR, and Nrf2 in the fish intestine. Based on the quadratic regression analysis of lysozyme activity at a 95% maximum, the dietary Phe requirement of young grass carp (256-629 g) was estimated to be 8.31 g kg(-1), corresponding to 2.75 g 100 g(-1) protein. Copyright © 2015 Elsevier Ltd. All rights reserved.

Phenotype Instance Verification and Evaluation Tool (PIVET): A Scaled Phenotype Evidence Generation Framework Using Web-Based Medical Literature.

PubMed

Henderson, Jette; Ke, Junyuan; Ho, Joyce C; Ghosh, Joydeep; Wallace, Byron C

2018-05-04

Researchers are developing methods to automatically extract clinically relevant and useful patient characteristics from raw healthcare datasets. These characteristics, often capturing essential properties of patients with common medical conditions, are called computational phenotypes. Being generated by automated or semiautomated, data-driven methods, such potential phenotypes need to be validated as clinically meaningful (or not) before they are acceptable for use in decision making. The objective of this study was to present Phenotype Instance Verification and Evaluation Tool (PIVET), a framework that uses co-occurrence analysis on an online corpus of publically available medical journal articles to build clinical relevance evidence sets for user-supplied phenotypes. PIVET adopts a conceptual framework similar to the pioneering prototype tool PheKnow-Cloud that was developed for the phenotype validation task. PIVET completely refactors each part of the PheKnow-Cloud pipeline to deliver vast improvements in speed without sacrificing the quality of the insights PheKnow-Cloud achieved. PIVET leverages indexing in NoSQL databases to efficiently generate evidence sets. Specifically, PIVET uses a succinct representation of the phenotypes that corresponds to the index on the corpus database and an optimized co-occurrence algorithm inspired by the Aho-Corasick algorithm. We compare PIVET's phenotype representation with PheKnow-Cloud's by using PheKnow-Cloud's experimental setup. In PIVET's framework, we also introduce a statistical model trained on domain expert-verified phenotypes to automatically classify phenotypes as clinically relevant or not. Additionally, we show how the classification model can be used to examine user-supplied phenotypes in an online, rather than batch, manner. PIVET maintains the discriminative power of PheKnow-Cloud in terms of identifying clinically relevant phenotypes for the same corpus with which PheKnow-Cloud was originally developed, but PIVET's analysis is an order of magnitude faster than that of PheKnow-Cloud. Not only is PIVET much faster, it can be scaled to a larger corpus and still retain speed. We evaluated multiple classification models on top of the PIVET framework and found ridge regression to perform best, realizing an average F1 score of 0.91 when predicting clinically relevant phenotypes. Our study shows that PIVET improves on the most notable existing computational tool for phenotype validation in terms of speed and automation and is comparable in terms of accuracy. ©Jette Henderson, Junyuan Ke, Joyce C Ho, Joydeep Ghosh, Byron C Wallace. Originally published in the Journal of Medical Internet Research (http://www.jmir.org), 04.05.2018.

Phenotype Instance Verification and Evaluation Tool (PIVET): A Scaled Phenotype Evidence Generation Framework Using Web-Based Medical Literature

PubMed Central

Ke, Junyuan; Ho, Joyce C; Ghosh, Joydeep; Wallace, Byron C

2018-01-01

Background Researchers are developing methods to automatically extract clinically relevant and useful patient characteristics from raw healthcare datasets. These characteristics, often capturing essential properties of patients with common medical conditions, are called computational phenotypes. Being generated by automated or semiautomated, data-driven methods, such potential phenotypes need to be validated as clinically meaningful (or not) before they are acceptable for use in decision making. Objective The objective of this study was to present Phenotype Instance Verification and Evaluation Tool (PIVET), a framework that uses co-occurrence analysis on an online corpus of publically available medical journal articles to build clinical relevance evidence sets for user-supplied phenotypes. PIVET adopts a conceptual framework similar to the pioneering prototype tool PheKnow-Cloud that was developed for the phenotype validation task. PIVET completely refactors each part of the PheKnow-Cloud pipeline to deliver vast improvements in speed without sacrificing the quality of the insights PheKnow-Cloud achieved. Methods PIVET leverages indexing in NoSQL databases to efficiently generate evidence sets. Specifically, PIVET uses a succinct representation of the phenotypes that corresponds to the index on the corpus database and an optimized co-occurrence algorithm inspired by the Aho-Corasick algorithm. We compare PIVET’s phenotype representation with PheKnow-Cloud’s by using PheKnow-Cloud’s experimental setup. In PIVET’s framework, we also introduce a statistical model trained on domain expert–verified phenotypes to automatically classify phenotypes as clinically relevant or not. Additionally, we show how the classification model can be used to examine user-supplied phenotypes in an online, rather than batch, manner. Results PIVET maintains the discriminative power of PheKnow-Cloud in terms of identifying clinically relevant phenotypes for the same corpus with which PheKnow-Cloud was originally developed, but PIVET’s analysis is an order of magnitude faster than that of PheKnow-Cloud. Not only is PIVET much faster, it can be scaled to a larger corpus and still retain speed. We evaluated multiple classification models on top of the PIVET framework and found ridge regression to perform best, realizing an average F1 score of 0.91 when predicting clinically relevant phenotypes. Conclusions Our study shows that PIVET improves on the most notable existing computational tool for phenotype validation in terms of speed and automation and is comparable in terms of accuracy. PMID:29728351

Kinetic characteristic of phenanthrene sorption in aged soil amended with biochar

NASA Astrophysics Data System (ADS)

Kim, Chanyang; Kim, Yong-Seong; Hyun, Seunghun

2015-04-01

Biochar has been recently highlighted as an amendment that affects yield of the crops by increasing pH, cation exchange capacity and water retention, and reduces the lability of contaminants by increasing sorption capacity in the soil system. Biochar's physico-chemical properties, high CEC, surfaces containing abundant micropores and macropores, and various types of functional groups, play important roles in enhancing sorption capacity of contaminants. Aging through a natural weathering process might change physico-chemical properties of biochar amended in soils, which can affect the sorption behavior of contaminants. Thus, in this study, the sorption characteristics of phenanthrene (PHE) on biochar-amended soils were studied with various types of chars depending on aging time. To do this, 1) soil was amended with sludge waste char (SWC), wood char (WC), and municipal waste char (MWC) during 0, 6, and 12 month. Chars were applied to soil at 1% and 2.5% (w/w) ratio. 2) Several batch kinetic and equilibrium studies were conducted. One-compartment first order and two-compartment first order model apportioning the fraction of fast and slow sorbing were selected for kinetic models. Where, qt is PHE concentration in biochar-amended soils at each time t, qeis PHE concentration in biochar-amended soils at equilibrium. ff is fastly sorbing fraction and (1-ff) is slowly sorbing fraction. k is sorption rate constant from one-compartment first order model, k1 and k2 are sorption rate constant from two-compartment first order model, t is time (hr). The equilibrium sorption data were fitted with Fruendlich and Langmuir equation. 3) Change in physico-chemical properties of biochar-amended soils was investigated with aging time. Batch equilibrium sorption results suggested that sorbed amount of PHE on WC was greater than SWC and MWC. The more char contents added to soil, the greater sorption capacity of PHE. Sorption equilibrium was reached after 4 hours and equilibrium pH ranged from 6.5 to 8.0. Sorption capacity was reduced with aging time. From kinetic results, two-compartment first order model was more suitable than one-compartment first order model. Fast sorption site of biochar-amended soils dominated total sorption process (i.e., Fraction of fast sorption site ranged from 0.55 to 0.96). Reduced sorption capacity with aging time could be attributed to changes in physico-chemical properties of biochar-amended soils (e.g., reduced pores and increased hydrophilic carboxyl and carbonyl functional groups). Verification is FI-IR and SSA. It is assumed that biochar is a suitable material for PHE contaminated soil in order to reduce the lability of PHE. However, aging effects would lessen biochar benefit for reducing the sorption capacity of PHE by forming hydrophilic functional group and reducing pores.

Altered muscular activation during prone hip extension in women with and without low back pain.

PubMed

Arab, Amir M; Ghamkhar, Leila; Emami, Mahnaz; Nourbakhsh, Mohammad R

2011-08-14

Altered movement pattern has been associated with the development of low back pain (LBP). The purpose of this study was to investigate the activity pattern of the ipsilateral erector spinae (IES) and contralateral erectorspinae (CES), gluteus maximus (GM) and hamstring (HAM) muscles during prone hip extension (PHE) test in women with and without LBP. A cross-sectional non-experimental design was used. Convenience sample of 20 female participated in the study. Subjects were categorized into two groups: with LBP (n = 10) and without LBP (n = 10). The electromyography (EMG) signal amplitude of the tested muscles during PHE (normalized to maximum voluntary electrical activity (MVE)) was measured in the dominant lower extremity in all subjects. Statistical analysis revealed greater normalized EMG signal amplitude in women with LBP compared to non-LBP women. There was significant difference in EMG activity of the IES (P = 0.03) and CES (P = 0.03) between two groups. However, no significant difference was found in EMG signals of the GM (P = 0.11) and HAM (P = 0.14) among two groups. The findings of this study demonstrated altered activation pattern of the lumbo-pelvic muscles during PHE in the women with chronic LBP. This information is important for investigators using PHE as either an evaluation tool or a rehabilitation exercise.

Main regulatory pathways, key genes and microRNAs involved in flower formation and development of moso bamboo (Phyllostachys edulis).

PubMed

Ge, Wei; Zhang, Ying; Cheng, Zhanchao; Hou, Dan; Li, Xueping; Gao, Jian

2017-01-01

Moso bamboo is characterized by infrequent sexual reproduction and erratic flowering habit; however, the molecular biology of flower formation and development is not well studied in this species. We studied the molecular regulation mechanisms of moso bamboo development and flowering by selecting three key regulatory pathways: plant-pathogen interaction, plant hormone signal transduction and protein processing in endoplasmic reticulum at different stages of flowering in moso bamboo. We selected PheDof1, PheMADS14 and six microRNAs involved in the three pathways through KEGG pathway and cluster analysis. Subcellular localization, transcriptional activation, Western blotting, in situ hybridization and qRT-PCR were used to further investigate the expression patterns and regulatory roles of pivotal genes at different flower development stages. Differential expression patterns showed that PheDof1, PheMADS14 and six miRNAs may play vital regulatory roles in flower development and floral transition in moso bamboo. Our research paves way for further studies on metabolic regulatory networks and provides insight into the molecular regulation mechanisms of moso bamboo flowering and senescence. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Phenome-Wide Association Study of Rheumatoid Arthritis Subgroups Identifies Association Between Seronegative Disease and Fibromyalgia.

PubMed

Doss, Jayanth; Mo, Huan; Carroll, Robert J; Crofford, Leslie J; Denny, Joshua C

2017-02-01

The differences between seronegative and seropositive rheumatoid arthritis (RA) have not been widely reported. We performed electronic health record (EHR)-based phenome-wide association studies (PheWAS) to identify disease associations in seropositive and seronegative RA. A validated algorithm identified RA subjects from the de-identified version of the Vanderbilt University Medical Center EHR. Serotypes were determined by rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibody (ACPA) values. We tested EHR-derived phenotypes using PheWAS comparing seropositive RA and seronegative RA, yielding disease associations. PheWAS was also performed in RF-positive versus RF-negative subjects and ACPA-positive versus ACPA-negative subjects. Following PheWAS, select phenotypes were then manually reviewed, and fibromyalgia was specifically evaluated using a validated algorithm. A total of 2,199 RA individuals with either RF or ACPA testing were identified. Of these, 1,382 patients (63%) were classified as seropositive. Seronegative RA was associated with myalgia and myositis (odds ratio [OR] 2.1, P = 3.7 × 10 -10 ) and back pain. A manual review of the health record showed that among subjects coded for Myalgia and Myositis, ∼80% had fibromyalgia. Follow-up with a specific EHR algorithm for fibromyalgia confirmed that seronegative RA was associated with fibromyalgia (OR 1.8, P = 4.0 × 10 -6 ). Seropositive RA was associated with chronic airway obstruction (OR 2.2, P = 1.4 × 10 -4 ) and tobacco use (OR 2.2, P = 7.0 × 10 -4 ). This PheWAS of RA patients identifies a strong association between seronegativity and fibromyalgia. It also affirms relationships between seropositivity and chronic airway obstruction and between seropositivity and tobacco use. These findings demonstrate the utility of the PheWAS approach to discover novel phenotype associations within different subgroups of a disease. © 2016, American College of Rheumatology.

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.

Redesign of negatively charged 111In-DTPA-octreotide derivative to reduce renal radioactivity.

PubMed

Oshima, Nobuhiro; Akizawa, Hiromichi; Kawashima, Hidekazu; Zhao, Songji; Zhao, Yan; Nishijima, Ken-Ichi; Kitamura, Yoji; Arano, Yasushi; Kuge, Yuji; Ohkura, Kazue

2017-05-01

Radiolabeled octreotide derivatives have been studied as diagnostic and therapeutic agents for somatostatin receptor-positive tumors. To prevent unnecessary radiation exposure during their clinical application, the present study aimed to develop radiolabeled peptides which could reduce radioactivity levels in the kidney at both early and late post-injection time points by introducing a negative charge with an acidic amino acid such as L-aspartic acid (Asp) at a suitable position in 111 In-DTPA-conjugated octreotide derivatives. Biodistribution of the radioactivity was evaluated in normal mice after administration of a novel radiolabeled peptide by a counting method. The radiolabeled species remaining in the kidney were identified by comparing their HPLC data with those obtained by alternative synthesis. The designed and synthesized radiolabeled peptide 111 In-DTPA-d-Phe -1 -Asp 0 -d-Phe 1 -octreotide exhibited significantly lower renal radioactivity levels than those of the known 111 In-DTPA-d-Phe 1 -octreotide at 3 and 24h post-injection. The radiolabeled species in the kidney at 24h after the injection of new octreotide derivative represented 111 In-DTPA-d-Phe-OH and 111 In-DTPA-d-Phe-Asp-OH as the metabolites. Their radiometabolites and intact 111 In-DTPA-conjugated octreotide derivative were observed in urine within 24h post-injection. The present study provided a new example of an 111 In-DTPA-conjugated octreotide derivative having the characteristics of both reduced renal uptake and shortened residence time of radioactivity in the kidney. It is considered that this kinetic control was achieved by introducing a negative charge on the octreotide derivative thereby suppressing the reabsorption in the renal tubules and affording the radiometabolites with appropriate lipophilicity. Copyright © 2017 Elsevier Inc. All rights reserved.

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

Evaluating statistical approaches to leverage large clinical datasets for uncovering therapeutic and adverse medication effects.

PubMed

Choi, Leena; Carroll, Robert J; Beck, Cole; Mosley, Jonathan D; Roden, Dan M; Denny, Joshua C; Van Driest, Sara L

2018-04-18

Phenome-wide association studies (PheWAS) have been used to discover many genotype-phenotype relationships and have the potential to identify therapeutic and adverse drug outcomes using longitudinal data within electronic health records (EHRs). However, the statistical methods for PheWAS applied to longitudinal EHR medication data have not been established. In this study, we developed methods to address two challenges faced with reuse of EHR for this purpose: confounding by indication, and low exposure and event rates. We used Monte Carlo simulation to assess propensity score (PS) methods, focusing on two of the most commonly used methods, PS matching and PS adjustment, to address confounding by indication. We also compared two logistic regression approaches (the default of Wald vs. Firth's penalized maximum likelihood, PML) to address complete separation due to sparse data with low exposure and event rates. PS adjustment resulted in greater power than propensity score matching, while controlling Type I error at 0.05. The PML method provided reasonable p-values, even in cases with complete separation, with well controlled Type I error rates. Using PS adjustment and the PML method, we identify novel latent drug effects in pediatric patients exposed to two common antibiotic drugs, ampicillin and gentamicin. R packages PheWAS and EHR are available at https://github.com/PheWAS/PheWAS and at CRAN (https://www.r-project.org/), respectively. The R script for data processing and the main analysis is available at https://github.com/choileena/EHR. leena.choi@vanderbilt.edu. Supplementary data are available at Bioinformatics online.

Biochemical, metabolic, and behavioral characteristics of immature chronic hyperphenylalanemic rats

PubMed Central

Dienel, Gerald A.; Cruz, Nancy F.

2015-01-01

Phenylketonuria and hyperphenylalanemia are inborn errors in metabolism of phenylalanine arising from defects in steps to convert phenylalanine to tyrosine. Phe accumulation causes severe mental retardation that can be prevented by timely identification of affected individuals and their placement on a Phe-restricted diet. In spite of many studies in patients and animal models, the basis for acquisition of mental retardation during the critical period of brain development is not adequately understood. All animal models for human disease have advantages and limitations, and characteristics common to different models are most likely to correspond to the disorder. This study established similar levels of Phe exposure in developing rats between 3 and 16 days of age using three models to produce chronic hyperphenylalanemia, and identified changes in brain amino acid levels common to all models that persist for ~16h of each day. In a representative model, local rates of glucose utilization (CMRglc) were determined at 25–27 days of age, and only selective changes that appeared to depend on Phe exposure were observed. CMRglc was reduced in frontal cortex and thalamus and increased in hippocampus and globus pallidus. Behavioral testing to evaluate neuromuscular competence revealed poor performance in chronically-hyperphenylalanemic rats that persisted for at least three weeks after cessation of Phe injections and did not occur with mild or acute hyperphenylalanemia. Thus, the abnormal amino acid environment, including hyperglycinemia, in developing rat brain is associated with selective regional changes in glucose utilization and behavioral abnormalities that are not readily reversed after they are acquired. PMID:26224289

Mass Spectrometry Identification of N-Chlorinated Dipeptides in Drinking Water.

PubMed

Huang, Guang; Jiang, Ping; Li, Xing-Fang

2017-04-04

We report the identification of N-chlorinated dipeptides as chlorination products in drinking water using complementary high-resolution quadrupole time-of-flight (QTOF) and quadrupole ion-trap mass spectrometry techniques. First, three model dipeptides, tyrosylglycine (Tyr-Gly), tyrosylalanine (Tyr-Ala), and phenylalanylglycine (Phe-Gly), reacted with sodium hypochlorite, and these reaction solutions were analyzed by QTOF. N-Cl-Tyr-Gly, N,N-di-Cl-Tyr-Gly, N-Cl-Phe-Gly, N,N-di-Cl-Phe-Gly, N-Cl-Tyr-Ala, and N,N-di-Cl-Tyr-Ala were identified as the major products based on accurate masses, 35 Cl/ 37 Cl isotopic patterns, and MS/MS spectra. These identified N-chlorinated dipeptides were synthesized and found to be stable in water over 10 days except N,N-di-Cl-Phe-Gly. To enable sensitive detection of N-chlorinated dipeptides in authentic water, we developed a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method with multiple reaction monitoring (MRM) mode. N-Cl-Tyr-Gly, N,N-di-Cl-Tyr-Gly, N-Cl-Phe-Gly, N-Cl-Tyr-Ala, and N,N-di-Cl-Tyr-Ala along with their corresponding dipeptides were detected in authentic tap water samples. The dipeptides were clearly detected in the raw water, but the N-chlorinated dipeptides were at background levels. These results suggest that the N-chlorinated dipeptides are produced by chlorination. This study has identified N-chlorinated dipeptides as new disinfection byproducts in drinking water. The strategy developed in this study can be used to identify chlorination products of other peptides in drinking water.

Effects of cowpea (Vigna unguiculata) root mucilage on microbial community response and capacity for phenanthrene remediation.

PubMed

Sun, Ran; Belcher, Richard W; Liang, Jianqiang; Wang, Li; Thater, Brian; Crowley, David E; Wei, Gehong

2015-07-01

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) is normally limited by their low solubility and poor bioavailability. Prior research suggests that biosurfactants are synthesized as intermediates during the production of mucilage at the root tip. To date the effects of mucilage on PAH degradation and microbial community response have not been directly examined. To address this question, our research compared 3 cowpea breeding lines (Vigna unguiculata) that differed in mucilage production for their effects on phenanthrene (PHE) degradation in soil. The High Performance Liquid Chromatography results indicated that the highest PHE degradation rate was achieved in soils planted with mucilage producing cowpea line C1, inoculated with Bradyrhizobium, leading to 91.6% PHE disappearance in 5 weeks. In root printing tests, strings treated with mucilage and bacteria produced larger clearing zones than those produced on mucilage treated strings with no bacteria or bacteria inoculated strings. Experiments with 14C-PHE and purified mucilage in soil slurry confirmed that the root mucilage significantly enhanced PHE mineralization (82.7%), which is 12% more than the control treatment without mucilage. The profiles of the PHE degraders generated by Denaturing gradient gel electrophoresis suggested that cowpea C1, producing a high amount of root mucilage, selectively enriched the PHE degrading bacteria population in rhizosphere. These findings indicate that root mucilage may play a significant role in enhancing PHE degradation and suggests that differences in mucilage production may be an important criterion for selection of the best plant species for use in phytoremediation of PAH contaminated soils. Copyright © 2015. Published by Elsevier B.V.

A novel missense mutation close to the charge-stabilizing system in a patient with congenital factor VII deficiency.

PubMed

Jiang, Minghua; Wang, Zhaoyue; Yu, Ziqiang; Bai, Xia; Su, Jian; Cao, Lijuan; Zhang, Wei; Ruan, Changgeng

2011-06-01

Congenital factor VII (FVII) deficiency is a rare autosomal recessive bleeding disorder. Its clinical manifestation and mutational spectrum are highly variable. The purpose of this study was to identify and characterize the mutation causing the FVII deficiency in a Chinese patient and his family. The FVII gene was analyzed by genomic DNA sequencing, and the FVII levels in patient's plasma were measured with an enzyme-linked immunoabsorbent assay (ELISA) and one-stage prothrombin time based method. In addition, the FVII-Phe190 mutant identified in the pedigree was expressed in the HEK293 cells, and the subcellular localization experiments in the Chinese hamster ovary (CHO) cells were performed. The patient had a prolonged prothrombin time and low levels of both FVII antigen and activity, and two heterozygous mutations were identified in F7 gene (NG-009262.1): a g.15975 G>A in the splice receptor site of intron 6 and a novel g.16750 C>T in exon 8 resulting in Ser190 to Phe190 replacement. In expression experiments, the reduced antigen and activity levels of FVII-Phe190 in the culture medium were found, whereas an ELISA and Western blotting analysis of FVII revealed that mutant FVII-Phe190 was synthesized in the cells as the wild-type FVII-Ser190. And FVII-Phe190 was found in endoplasmic reticulum and Golgi apparatus. Compound heterozygous mutations in F7 gene should be responsible for the FVII deficiency in this patient. The FVII-Phe190 can normally be synthesized and transported from endoplasmic reticulum to Golgi apparatus, but degraded or inefficiently secreted.

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

Enabling phenotypic big data with PheNorm.

PubMed

Yu, Sheng; Ma, Yumeng; Gronsbell, Jessica; Cai, Tianrun; Ananthakrishnan, Ashwin N; Gainer, Vivian S; Churchill, Susanne E; Szolovits, Peter; Murphy, Shawn N; Kohane, Isaac S; Liao, Katherine P; Cai, Tianxi

2018-01-01

Electronic health record (EHR)-based phenotyping infers whether a patient has a disease based on the information in his or her EHR. A human-annotated training set with gold-standard disease status labels is usually required to build an algorithm for phenotyping based on a set of predictive features. The time intensiveness of annotation and feature curation severely limits the ability to achieve high-throughput phenotyping. While previous studies have successfully automated feature curation, annotation remains a major bottleneck. In this paper, we present PheNorm, a phenotyping algorithm that does not require expert-labeled samples for training. The most predictive features, such as the number of International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) codes or mentions of the target phenotype, are normalized to resemble a normal mixture distribution with high area under the receiver operating curve (AUC) for prediction. The transformed features are then denoised and combined into a score for accurate disease classification. We validated the accuracy of PheNorm with 4 phenotypes: coronary artery disease, rheumatoid arthritis, Crohn's disease, and ulcerative colitis. The AUCs of the PheNorm score reached 0.90, 0.94, 0.95, and 0.94 for the 4 phenotypes, respectively, which were comparable to the accuracy of supervised algorithms trained with sample sizes of 100-300, with no statistically significant difference. The accuracy of the PheNorm algorithms is on par with algorithms trained with annotated samples. PheNorm fully automates the generation of accurate phenotyping algorithms and demonstrates the capacity for EHR-driven annotations to scale to the next level - phenotypic big data. © The Author 2017. Published by Oxford University Press on behalf of the American Medical Informatics Association. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Rationally designed chimeric peptide of met-enkephalin and FMRFa-[D-Ala2,p-Cl-Phe4]YFa induce multiple opioid receptors mediated antinociception and up-regulate their expression.

PubMed

Vats, Ishwar Dutt; Chaudhary, Snehlata; Sharma, Ahuti; Nath, Mahendra; Pasha, Santosh

2010-07-25

The physiological role of NPFF/FMRFa family of peptides appears to be complex and exact mechanism of action of these peptides is not yet completely understood. In same line of scrutiny, another analog of YGGFMKKKFMRFamide (YFa), a chimeric peptide of met-enkephalin and FMRFamide, was rationally designed and synthesized which contain D-alanine and p-Cl-phenylalanine residues at 2nd and 4th positions, respectively i.e., Y-(D-Ala)-G-(p-Cl-Phe)-MKKKFMRFamide ([D-Ala(2), p-Cl-Phe(4)]YFa) in order to achieve improved bioavailability and blood brain barrier penetration. Therefore, present study investigates the possible antinociceptive effect of [D-Ala(2), p-Cl-Phe(4)]YFa on intra-peritoneal (i.p.) administration using tail-flick test in rats followed by its opioid receptor(s) specificity using mu, delta and kappa receptor antagonists. Further, its antinociceptive effect was examined during 6 days of chronic i.p. treatment and assessed effect of this treatment on differential expression of opioid receptors. [D-Ala(2), p-Cl-Phe(4)]YFa in comparison to parent peptide YFa, induce significantly higher dose dependent antinociception in rats which was mediated by all three opioid receptors (mu, delta and kappa). Importantly, it induced comparable antinociception in rats throughout the chronic i.p. treatment and significantly up-regulated the overall expression (mRNA and protein) of mu, delta and kappa opioid receptors. Therefore, pharmacological and molecular behavior of [D-Ala(2), p-Cl-Phe(4)]YFa demonstrate that incorporation of D-alanine and p-Cl-phenylalanine residues at appropriate positions in chimeric peptide leads to altered opioid receptor selectivity and enhanced antinociceptive potency, relative to parent peptide. (c) 2010 Elsevier B.V. All rights reserved.

Cultivation-dependent and cultivation-independent characterization of hydrocarbon-degrading bacteria in Guaymas Basin sediments.

PubMed

Gutierrez, Tony; Biddle, Jennifer F; Teske, Andreas; Aitken, Michael D

2015-01-01

Marine hydrocarbon-degrading bacteria perform a fundamental role in the biodegradation of crude oil and its petrochemical derivatives in coastal and open ocean environments. However, there is a paucity of knowledge on the diversity and function of these organisms in deep-sea sediment. Here we used stable-isotope probing (SIP), a valuable tool to link the phylogeny and function of targeted microbial groups, to investigate polycyclic aromatic hydrocarbon (PAH)-degrading bacteria under aerobic conditions in sediments from Guaymas Basin with uniformly labeled [(13)C]-phenanthrene (PHE). The dominant sequences in clone libraries constructed from (13)C-enriched bacterial DNA (from PHE enrichments) were identified to belong to the genus Cycloclasticus. We used quantitative PCR primers targeting the 16S rRNA gene of the SIP-identified Cycloclasticus to determine their abundance in sediment incubations amended with unlabeled PHE and showed substantial increases in gene abundance during the experiments. We also isolated a strain, BG-2, representing the SIP-identified Cycloclasticus sequence (99.9% 16S rRNA gene sequence identity), and used this strain to provide direct evidence of PHE degradation and mineralization. In addition, we isolated Halomonas, Thalassospira, and Lutibacterium sp. with demonstrable PHE-degrading capacity from Guaymas Basin sediment. This study demonstrates the value of coupling SIP with cultivation methods to identify and expand on the known diversity of PAH-degrading bacteria in the deep-sea.

Cultivation-dependent and cultivation-independent characterization of hydrocarbon-degrading bacteria in Guaymas Basin sediments

PubMed Central

Gutierrez, Tony; Biddle, Jennifer F.; Teske, Andreas; Aitken, Michael D.

2015-01-01

Marine hydrocarbon-degrading bacteria perform a fundamental role in the biodegradation of crude oil and its petrochemical derivatives in coastal and open ocean environments. However, there is a paucity of knowledge on the diversity and function of these organisms in deep-sea sediment. Here we used stable-isotope probing (SIP), a valuable tool to link the phylogeny and function of targeted microbial groups, to investigate polycyclic aromatic hydrocarbon (PAH)-degrading bacteria under aerobic conditions in sediments from Guaymas Basin with uniformly labeled [13C]-phenanthrene (PHE). The dominant sequences in clone libraries constructed from 13C-enriched bacterial DNA (from PHE enrichments) were identified to belong to the genus Cycloclasticus. We used quantitative PCR primers targeting the 16S rRNA gene of the SIP-identified Cycloclasticus to determine their abundance in sediment incubations amended with unlabeled PHE and showed substantial increases in gene abundance during the experiments. We also isolated a strain, BG-2, representing the SIP-identified Cycloclasticus sequence (99.9% 16S rRNA gene sequence identity), and used this strain to provide direct evidence of PHE degradation and mineralization. In addition, we isolated Halomonas, Thalassospira, and Lutibacterium sp. with demonstrable PHE-degrading capacity from Guaymas Basin sediment. This study demonstrates the value of coupling SIP with cultivation methods to identify and expand on the known diversity of PAH-degrading bacteria in the deep-sea. PMID:26217326

The Potential Role of Systemic Buffers in Reducing Intratumoral Extracellular pH and Acid-Mediated Invasion

PubMed Central

Silva, Ariosto S.; Yunes, Jose A.; Gillies, Robert J.; Gatenby, Robert A.

2013-01-01

A number of studies have shown that the extracellular pH (pHe) in cancers is typically lower than that in normal tissue and that an acidic pHe promotes invasive tumor growth in primary and metastatic cancers. Here, we investigate the hypothesis that increased systemic concentrations of pH buffers reduce intratumoral and peritumoral acidosis and, as a result, inhibit malignant growth. Computer simulations are used to quantify the ability of systemic pH buffers to increase the acidic pHe of tumors in vivo and investigate the chemical specifications of an optimal buffer for such purpose. We show that increased serum concentrations of the sodium bicarbonate (NaHCO3) can be achieved by ingesting amounts that have been used in published clinical trials. Furthermore, we find that consequent reduction of tumor acid concentrations significantly reduces tumor growth and invasion without altering the pH of blood or normal tissues. The simulations also show that the critical parameter governing buffer effectiveness is its pKa. This indicates that NaHCO3, with a pKa of 6.1, is not an ideal intratumoral buffer and that greater intratumoral pHe changes could be obtained using a buffer with a pKa of ~7. The simulations support the hypothesis that systemic pH buffers can be used to increase the tumor pHe and inhibit tumor invasion. PMID:19276380

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.

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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

Selection and identification of fungi isolated from sugarcane bagasse and their application for phenanthrene removal from soil.

PubMed

Cortés-Espinosa, D V; Fernández-Perrino, F J; Arana-Cuenca, A; Esparza-García, F; Loera, O; Rodríguez-Vázquez, R

2006-01-01

This work investigated the identification and selection of fungi isolated from sugarcane bagasse and their application for phenanthrene (Phe) removal from soil. Fungi were identified by PCR amplification of ITS regions as Aspergillus terrus, Aspergillus fumigatus and Aspergillus niger, Penicillium glabrum and Cladosporium cladosporioides. A primary selection of fungi was accomplished in plate, considering Phe tolerance of every strain in two different media: potato dextrose agar (PDA) and mineral medium (MM). The radial extension rate (r(r)) in PDA exhibited significant differences (p<0.05) at 200 and 400 ppm of Phe. A secondary selection of A. niger, C. cladosporoides, and P. glabrum sp. was achieved based on their tolerance to 200, 400, 600 and 800 ppm of Phe, in solid culture at a sugarcane bagasse/contaminated soil ratio of 95:5, in Toyamas, Czapeck and Wunder media. Under these conditions, a maximum (70%) Phe removal by A. niger was obtained. In addition C. cladosporioides and A. niger were able to remove high (800 ppm) Phe concentrations.

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.

From Genome-Wide Association Study to Phenome-Wide Association Study: New Paradigms in Obesity Research.

PubMed

Zhang, Y-P; Zhang, Y-Y; Duan, D D

2016-01-01

Obesity is a condition in which excess body fat has accumulated over an extent that increases the risk of many chronic diseases. The current clinical classification of obesity is based on measurement of body mass index (BMI), waist-hip ratio, and body fat percentage. However, these measurements do not account for the wide individual variations in fat distribution, degree of fatness or health risks, and genetic variants identified in the genome-wide association studies (GWAS). In this review, we will address this important issue with the introduction of phenome, phenomics, and phenome-wide association study (PheWAS). We will discuss the new paradigm shift from GWAS to PheWAS in obesity research. In the era of precision medicine, phenomics and PheWAS provide the required approaches to better definition and classification of obesity according to the association of obese phenome with their unique molecular makeup, lifestyle, and environmental impact. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

Synergistic effect with Phe-Gly-Leu-Met-NH2 of the C-terminal of substance P and insulin-like growth factor-1 on epithelial wound healing of rabbit cornea

PubMed Central

Nakamura, Masatsugu; Chikama, Tai-ichiro; Nishida, Teruo

1999-01-01

We previously reported that substance P and insulin-like growth factor-1 (IGF-1) synergistically stimulate corneal epithelial wound healing in vitro and in vivo. We wished to identify which portion of the amino acid sequence of substance P might be responsible for this synergism.Corneal epithelial migration was not affected by the addition of any one of the following factors: substance P; Phe-Gly-Leu-Met-NH2 (C-terminal of substance P); Val-Gly-Leu-Met-NH2 (C-terminal of neurokinin A, neurokinin B, and kassinin); Tyr-Gly-Leu-Met-NH2 (C-terminal of physalaemin); Ile-Gly-Leu-Met-NH2 (C-terminal of eledoisin); or Gly-Leu-Met-NH2 (common C-terminal of tachykinins).In the presence of IGF-1, only substance P and Phe-Gly-Leu-Met-NH2 were synergistic in stimulating corneal epithelial migration in a dose-dependent fashion.The combination of Phe-Gly-Leu-Met-NH2 and IGF-1 did not affect the incorporation of [3H]-thymidine into corneal epithelial cells.Treatment with Phe-Gly-Leu-Met-NH2 and IGF-1, but not with Phe-Gly-Leu-Met-NH2 or IGF-1 alone, increased attachment of corneal epithelial cells to a fibronectin matrix.The levels of α5 and β1 integrin were not affected by Phe-Gly-Leu-Met-NH2 or IGF-1 alone, but they were significantly increased by the combination of Phe-Gly-Leu-Met-NH2 and IGF-1.Topical application of the same combination facilitated corneal epithelial wound closure in vivo.These results demonstrated that Phe-Gly-Leu-Met-NH2, a sequence of 4 amino-acids of the C-terminal of substance P, is the minimum sequence necessary to produce the synergistic effects of substance P and IGF-1 on corneal epithelial wound healing. PMID:10385250

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.

Elicitor and nitrogen applications to Garnacha, Graciano and Tempranillo vines: effect on grape amino acid composition.

PubMed

Gutiérrez-Gamboa, Gastón; Portu, Javier; López, Rosa; Santamaría, Pilar; Garde-Cerdán, Teresa

2018-04-01

Elicitors and nitrogen foliar applications to vineyards could regulate grape nitrogen composition, which has an important effect on grape and wine quality. Thus the aim of this research was to study the effect of foliar elicitor treatments, methyl jasmonate (MeJ) and yeast extract (YE), and foliar nitrogen applications, urea (Ur) and phenylalanine (Phe), to Garnacha, Graciano and Tempranillo vines on grape amino acid composition. The results showed that elicitor and nitrogen foliar applications to Garnacha and Tempranillo grapevines decreased the must amino acid concentration. However, Phe application to these two grapevines increased the must Phe content. The treatments applied to Graciano grapevines barely effected the grape amino acid content. According to the percentage of variance attributable, the variety had a higher impact on the must amino acid composition than the treatments and their interaction, except in certain amino acids such as Phe. The influence of elicitor and nitrogen foliar applications to grapevines on grape amino acid concentration was strongly conditioned by the variety. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Properties of the plant- and manure-derived biochars and their sorption of dibutyl phthalate and phenanthrene

NASA Astrophysics Data System (ADS)

Qiu, Mengyi; Sun, Ke; Jin, Jie; Gao, Bo; Yan, Yu; Han, Lanfang; Wu, Fengchang; Xing, Baoshan

2014-06-01

The properties of plant residue-derived biochars (PLABs) and animal waste-derived biochars (ANIBs) obtained at low and high heating treatment temperatures (300 and 450°C) as well as their sorption of dibutyl phthalate (DBP) and phenanthrene (PHE) were investigated in this study. The higher C content of PLABs could explain that CO2-surface area (CO2-SA) of PLABs was remarkably high relative to ANIBs. OC and aromatic C were two key factors influencing the CO2-SA of the biochars. Much higher surface C content of the ANIBs than bulk C likely explained that the ANIBs exhibited higher sorption of DBP and PHE compared to the PLABs. H-bonding should govern the adsorption of DBP by most of the tested biochars and π-π interaction play an important role in the adsorption of PHE by biochars. High CO2-SA (>200 m2 g-1) demonstrated that abundant nanopores of OC existed within the biochars obtained 450°C (HTBs), which likely result in high and nonlinear sorption of PHE by HTBs.

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.

The Roles of Hemagglutinin Phe-95 in Receptor Binding and Pathogenicity of Influenza B Virus

PubMed Central

Ni, Fengyun; Mbawuike, Innocent Nnadi; Kondrashkina, Elena; Wang, Qinghua

2014-01-01

Diverged ~4,000 years ago, influenza B virus has several important differences from influenza A virus, including lower receptor-binding affinity and highly restricted host range. Based on our prior structural studies, we hypothesized that a single-residue difference in the receptor-binding site of hemagglutinin (HA), Phe-95 in influenza B virus versus Tyr-98 in influenza A/H1~H15, is possibly a key determinant for the low receptor-binding affinity. Here we demonstrate that the mutation Phe95→Tyr in influenza B virus HA restores all three hydrogen bonds made by Tyr-98 in influenza A/H3 HA and has the potential to enhance receptor binding. However, the full realization of this potential is influenced by the local environment into which the mutation is introduced. The binding and replication of the recombinant viruses correlate well with the receptor-binding capabilities of HA. These results are discussed in relation to the roles of Phe-95 in receptor binding and pathogenicity of influenza B virus. PMID:24503069

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.

Acceptable low-phenylalanine foods and beverages can be made with glycomacropeptide from cheese whey for individuals with PKU

PubMed Central

Lim, Kyungwha; van Calcar, Sandra C.; Nelson, Kathryn L.; Gleason, Sally T.; Ney, Denise M.

2007-01-01

Glycomacropeptide (GMP) is a whey protein that contains no aromatic amino acids including phenylalanine (phe). The objective of this study was to make a variety of palatable, low-phe foods and beverages with GMP and to assess their acceptability by conducting consumer sensory studies in individuals with PKU. Results demonstrate acceptability of products made with GMP. GMP supplemented with limiting indispensable amino acids could provide an alternative protein source for individuals with PKU. PMID:17644019

Different partition of polycyclic aromatic hydrocarbon on environmental particulates in freshwater: Microplastics in comparison to natural sediment.

PubMed

Wang, Wenfeng; Wang, Jun

2018-01-01

Microplastics pollution in the aquatic ecosystems has aroused increasing concerns in recent years. Though microplastics are known to sorb organic contaminants from water, the interaction mechanisms between microplastics and organic chemicals are not yet well understood. Here we investigated the partition characteristics of phenanthrene (Phe) in three mass-produced plastic particles, including polyethylene (PE), polystyrene (PS) and polyvinylchloride (PVC)， and one natural sediment, as a comparison. The sorption kinetics of Phe onto microplastics and natural sediment were successfully described by the pseudo-second-order model (R 2 > 0.992), while the equilibrium data were best-fitted to the Langmuir isotherm (R 2 > 0.995). Compared with natural sediment, microplastics exhibited higher capacities for Phe which followed an order of PE > PS > PVC. As the aqueous concentration of pyrene (Pyr) increased, both uptakes and distribution coefficients (K d ) of Phe within the solids decreased, with natural sediment giving the largest decline. Although proportions of Phe desorbed from the contaminated microplastics were low, due to the high Phe uptake, microplastics released larger amounts of the sorbed Phe to water than the natural sediment during the desorption process. Given their minimal abundance relative to natural sediment, microplastics may play a less important role in the transport of organic pollutants in a natural aquatic environment. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

Phenylketonuria and Gut Microbiota: A Controlled Study Based on Next-Generation Sequencing.

PubMed

Pinheiro de Oliveira, Felipe; Mendes, Roberta Hack; Dobbler, Priscila Thiago; Mai, Volker; Pylro, Victor Salter; Waugh, Sheldon G; Vairo, Filippo; Refosco, Lilia Farret; Roesch, Luiz Fernando Würdig; Schwartz, Ida Vanessa Doederlein

2016-01-01

Phenylketonuria (PKU) is an inborn error of metabolism associated with high blood levels of phenylalanine (Phe). A Phe-restricted diet supplemented with L-amino acids is the main treatment strategy for this disease; if started early, most neurological abnormalities can be prevented. The healthy human gut contains trillions of commensal bacteria, often referred to as the gut microbiota. The composition of the gut microbiota is known to be modulated by environmental factors, including diet. In this study, we compared the gut microbiota of 8 PKU patients on Phe-restricted dietary treatment with that of 10 healthy individuals. The microbiota were characterized by 16S rRNA sequencing using the Ion Torrent™ platform. The most dominant phyla detected in both groups were Bacteroidetes and Firmicutes. PKU patients showed reduced abundance of the Clostridiaceae, Erysipelotrichaceae, and Lachnospiraceae families, Clostridiales class, Coprococcus, Dorea, Lachnospira, Odoribacter, Ruminococcus and Veillonella genera, and enrichment of Prevotella, Akkermansia, and Peptostreptococcaceae. Microbial function prediction suggested significant differences in starch/glucose and amino acid metabolism between PKU patients and controls. Together, our results suggest the presence of distinct taxonomic groups within the gut microbiome of PKU patients, which may be modulated by their plasma Phe concentration. Whether our findings represent an effect of the disease itself, or a consequence of the modified diet is unclear.

Phenylketonuria and Gut Microbiota: A Controlled Study Based on Next-Generation Sequencing

PubMed Central

Pinheiro de Oliveira, Felipe; Mendes, Roberta Hack; Dobbler, Priscila Thiago; Mai, Volker; Pylro, Victor Salter; Waugh, Sheldon G; Vairo, Filippo; Refosco, Lilia Farret; Schwartz, Ida Vanessa Doederlein

2016-01-01

Phenylketonuria (PKU) is an inborn error of metabolism associated with high blood levels of phenylalanine (Phe). A Phe-restricted diet supplemented with L-amino acids is the main treatment strategy for this disease; if started early, most neurological abnormalities can be prevented. The healthy human gut contains trillions of commensal bacteria, often referred to as the gut microbiota. The composition of the gut microbiota is known to be modulated by environmental factors, including diet. In this study, we compared the gut microbiota of 8 PKU patients on Phe-restricted dietary treatment with that of 10 healthy individuals. The microbiota were characterized by 16S rRNA sequencing using the Ion Torrent™ platform. The most dominant phyla detected in both groups were Bacteroidetes and Firmicutes. PKU patients showed reduced abundance of the Clostridiaceae, Erysipelotrichaceae, and Lachnospiraceae families, Clostridiales class, Coprococcus, Dorea, Lachnospira, Odoribacter, Ruminococcus and Veillonella genera, and enrichment of Prevotella, Akkermansia, and Peptostreptococcaceae. Microbial function prediction suggested significant differences in starch/glucose and amino acid metabolism between PKU patients and controls. Together, our results suggest the presence of distinct taxonomic groups within the gut microbiome of PKU patients, which may be modulated by their plasma Phe concentration. Whether our findings represent an effect of the disease itself, or a consequence of the modified diet is unclear. PMID:27336782

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.

Food Products Made With Glycomacropeptide, a Low Phenylalanine Whey Protein, Provide a New Alternative to Amino Acid-Based Medical Foods for Nutrition Management of Phenylketonuria

PubMed Central

Van Calcar, Sandra C.; Ney, Denise M.

2012-01-01

Phenylketonuria (PKU), an inborn error in phenylalanine (phe) metabolism, requires lifelong nutrition management with a low-phe diet, which includes a phe-free amino acid-based medical formula to provide the majority of an individual’s protein needs. Compliance with this diet is often difficult for older children, adolescents and adults with PKU. The whey protein glycomacropeptide (GMP) is ideally suited for the PKU diet since it is naturally low in phe. Nutritionally complete, acceptable medical foods and beverages can be made with GMP to increase the variety of protein sources for the PKU diet. As an intact protein, GMP improves protein utilization and increases satiety compared with amino acids. Thus, GMP provides a new, more physiologic source of low-phe dietary protein for those with PKU. PMID:22818728

New cytotoxic cyclic peptides and dianthramide from Dianthus superbus.

PubMed

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

2004-09-01

Four new cyclic peptides, dianthins C-F (1-4), and a new dianthramide, 4-methoxydianthramide B (5), were isolated from the MeOH extract of the traditional Chinese medicinal plant Dianthus superbus. The sequences of cyclic peptides 1-4 were elucidated as cyclo(Gly(1)-Pro(2)-Phe(3)-Tyr(4)-Val(5)-Ile(6)-), cyclo(Gly(1)-Ser(2)-Leu(3)-Pro(4)-Pro(5)-Ile(6)-Phe(7)-), cyclo(Gly(1)-Pro(2)-Ile(3)-Ser(4)-Phe(5)-Val(6)-), and cyclo(Gly(1)-Pro(2)-Phe(3)-Val(4)-Phe(5)-) on the basis of ESI tandem mass fragmentation analysis, chemical evidence, and extensive 2D NMR methods. The conformation of compound 1 was established as an alpha-helix by CD analysis. Furthermore, compounds 3 and 5 showed cytotoxicities toward the Hep G2 cancer cell line with IC(50) values of 2.37 and 4.08, respectively.

Magnetic Activity and Period Variation Studies of the Short-period Eclipsing Binaries. II. V1101 Her, AD Phe, and NSV 455 (J011636.15-394955.7)

NASA Astrophysics Data System (ADS)

Pi, Qing-feng; Zhang, Li-yun; Bi, Shao-lan; Han, Xianming L.; Wang, Dai-mei; Lu, Hong-peng

2017-12-01

In this paper, we present new BVRI light curves of short-period contact eclipsing binaries V1101 Her and AD Phe from our observations carried out from 2014 to 2015 using the SARA KP and SARA CT telescopes. There is an eclipsing binary located at α(2000) = 01h16m36.ˢ15 and δ(2000) = -39°49‧55.″7 in the field of view of AD Phe. We derived an updated ephemeris and found there a cyclic variation overlaying a continuous period increase (V1101 Her) and decrease (AD Phe). This kind of cyclic variation may be attributed to the light time effect via the presence of the third body or magnetic activity cycle. The orbital period increase suggests that V1101 Her is undergoing a mass-transfer from the primary to the secondary component (dM 1/dt = 2.64(±0.11) × 10-6 M ⊙ yr-1) with the third body (P 3 = 13.9(±1.9) years), or 2.81(±0.07) × 10-6 M ⊙ yr-1 for an increase andmagnetic cycle (12.4(±0.5) years). The long-term period decrease suggests that AD Phe is undergoing a mass-transfer from the secondary component to the primary component at a rate of -8.04(±0.09) × 10-8 M ⊙ yr-1 for a period decrease and the third body (P 3 = 56.2(±0.8) years), or -7.11(±0.04) × 10-8 M ⊙ yr-1 for a decrease and magnetic cycle (50.3(±0.5) years). We determined their orbital and geometrical parameters. For AD Phe, we simultaneously analyzed our BVRI light curves and the spectroscopic observations obtained by Duerbeck & Rucinski. The spectral type of V1101 Her was classified as G0 ± 2V by LAMOST stellar spectra survey. The asymmetry of the R-band light curve of AD Phe obtained by McFarlane & Hilditch in 1987 is explained by a cool spot on the primary component.

Identification of Dmt-D-Lys-Phe-Phe-OH as a highly antinociceptive tetrapeptide metabolite of the opioid-neurotensin hybrid peptide PK20.

PubMed

Kleczkowska, Patrycja; Bojnik, Engin; Leśniak, Anna; Kosson, Piotr; Van den Eynde, Isabelle; Ballet, Steven; Benyhe, Sandor; Tourwé, Dirk; Lipkowski, Andrzej W

2013-01-01

Recently, we presented a novel compound (PK20, Dmt-D-Lys-Phe-Phe-Lys-Lys-Pro-Phe-Tle-Leu-OH) that targets single entity opioid and neurotensin pharmacophores. This endomorphin-2-like opioid peptide was introduced as a highly active analgesic because it elicited a strong dose- and time-dependent antinociceptive response when administered centrally and peripherally. Its pain-relieving activity was observed as rapidly as 5 min after drug injection. Such promising results led us to perform further studies, such as determining the resistance to enzymatic degradation, which resulted in obtaining a very stable opioid pharmacore PK20 metabolite. The synthesis of PK20 and its N-terminal tetrapeptide fragment has been accomplished using solid phase peptide chemistry. The biological stability of peptides has been measured in human serum and analyzed by HPLC/MS. Peptides were pharmacologically characterized in in vitro MOP and DOP receptor binding as well as [(35)S]GTPγS receptor binding assays. Antinociceptive properties of compounds were measured by in vivo assays in C57Bl6 mice after intravenous or intrathecal applications. Dmt-D-Lys-Phe-Phe-OH (PK20M), an N-terminal tetrapeptide metabolite of the opioid-neurotensin hybrid peptide PK20, is characterized by a long duration of action, as demonstrated by a preserved, long-lasting analgesic effect even 2 h post-injection (average % MPE = 69.33). In rat brain membranes, PK20M efficiently displaced both the MOP and DOP receptor selective radioprobes [(3)H]DAMGO and [(3)H]DIDI (pKi of 9.52 and 7.86, respectively) and potently stimulated [(35)S]GTPγS binding, proving full agonism at both receptor types. In the [(35)S]GTPγS assay, which measured the agonist-mediated G protein activation, PK20M together with PK20 and Met-enkephalin were potent stimulators of the regulatory G proteins. The relative affinities of PK20M for the μ and δ receptor subtypes revealed μ-receptor selectivity. The novel MOP receptor selective metabolite has been shown to possess opioid subtype receptor selectivity, high potency, and effective analgesic activities as measured in various bioassays.

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.

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.

Predicting the safety and efficacy of buffer therapy to raise tumour pHe: an integrative modelling study.

PubMed

Martin, N K; Robey, I F; Gaffney, E A; Gillies, R J; Gatenby, R A; Maini, P K

2012-03-27

Clinical positron emission tomography imaging has demonstrated the vast majority of human cancers exhibit significantly increased glucose metabolism when compared with adjacent normal tissue, resulting in an acidic tumour microenvironment. Recent studies demonstrated reducing this acidity through systemic buffers significantly inhibits development and growth of metastases in mouse xenografts. We apply and extend a previously developed mathematical model of blood and tumour buffering to examine the impact of oral administration of bicarbonate buffer in mice, and the potential impact in humans. We recapitulate the experimentally observed tumour pHe effect of buffer therapy, testing a model prediction in vivo in mice. We parameterise the model to humans to determine the translational safety and efficacy, and predict patient subgroups who could have enhanced treatment response, and the most promising combination or alternative buffer therapies. The model predicts a previously unseen potentially dangerous elevation in blood pHe resulting from bicarbonate therapy in mice, which is confirmed by our in vivo experiments. Simulations predict limited efficacy of bicarbonate, especially in humans with more aggressive cancers. We predict buffer therapy would be most effectual: in elderly patients or individuals with renal impairments; in combination with proton production inhibitors (such as dichloroacetate), renal glomular filtration rate inhibitors (such as non-steroidal anti-inflammatory drugs and angiotensin-converting enzyme inhibitors), or with an alternative buffer reagent possessing an optimal pK of 7.1-7.2. Our mathematical model confirms bicarbonate acts as an effective agent to raise tumour pHe, but potentially induces metabolic alkalosis at the high doses necessary for tumour pHe normalisation. We predict use in elderly patients or in combination with proton production inhibitors or buffers with a pK of 7.1-7.2 is most promising.

Delivery of Cancer Screening

PubMed Central

Fenton, Joshua J.; Cai, Yong; Weiss, Noel S.; Elmore, Joann G.; Pardee, Roy E.; Reid, Robert J.; Baldwin, Laura-Mae

2012-01-01

Background Patients and physicians strongly endorse the importance of preventive or periodic health examinations (PHEs). However, the extent to which PHEs contribute to the delivery of cancer screening is uncertain. Methods In a retrospective cohort study, we determined the association between receipt of a PHE and cancer testing in a population-based sample of enrollees in a Washington State health plan who were aged 52 to 78 years and eligible for colorectal, breast, or prostate cancer screening in 2002–2003 (N = 64 288). Outcomes included completion of any colorectal cancer testing (fecal occult blood testing, sigmoidoscopy, colonoscopy, or barium enema), screening mammography, and prostate-specific antigen testing. Results More than half (52.4%) of the enrollees received a PHE during the study period. After adjusting for demographics, comorbidity, number of outpatient visits, and historical preventive service use before January 1, 2002, receipt of a PHE was significantly associated with completion of colorectal cancer testing (incidence difference, 40.4% [95% confidence interval (CI), 39.4%–41.3%]; relative incidence, 3.47 [95% CI, 3.34–3.59]), screening mammography [incidence difference, 14.2% [95% CI, 12.7%–15.7%]; relative incidence, 1.23 [95% CI, 1.20–1.25]), and prostate-specific antigen testing (incidence difference, 39.4% [95% CI, 38.3%–40.5%]; relative incidence, 3.06 [95% CI, 2.95–3.18]). Conclusions Among managed care enrollees eligible for cancer screening, PHE receipt is associated with completion of colorectal, breast, and prostate cancer testing. In similar populations, the PHE may serve as a clinically important forum for the promotion of evidence-based colorectal cancer and breast cancer screening and of screening with relatively less empirical support, such as prostate cancer screening. PMID:17389289

How Important Is Private Higher Education in Europe? A Regional Analysis in Global Context

ERIC Educational Resources Information Center

Levy, Daniel C.

2012-01-01

The importance of private higher education (PHE) is increasingly clear globally. But does Europe fit the global generalisation? This question can be assessed with reference to two major considerations: the size of PHE and the degree of private-public difference. The growth of PHE in Europe has been delayed and limited compared to that in most of…

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.

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.

Identification of Genes in the Phenylalanine Metabolic Pathway by Ectopic Expression of a MYB Transcription Factor in Tomato Fruit[W

PubMed Central

Dal Cin, Valeriano; Tieman, Denise M.; Tohge, Takayuki; McQuinn, Ryan; de Vos, Ric C.H.; Osorio, Sonia; Schmelz, Eric A.; Taylor, Mark G.; Smits-Kroon, Miriam T.; Schuurink, Robert C.; Haring, Michel A.; Giovannoni, James; Fernie, Alisdair R.; Klee, Harry J.

2011-01-01

Altering expression of transcription factors can be an effective means to coordinately modulate entire metabolic pathways in plants. It can also provide useful information concerning the identities of genes that constitute metabolic networks. Here, we used ectopic expression of a MYB transcription factor, Petunia hybrida ODORANT1, to alter Phe and phenylpropanoid metabolism in tomato (Solanum lycopersicum) fruits. Despite the importance of Phe and phenylpropanoids to plant and human health, the pathway for Phe synthesis has not been unambiguously determined. Microarray analysis of ripening fruits from transgenic and control plants permitted identification of a suite of coregulated genes involved in synthesis and further metabolism of Phe. The pattern of coregulated gene expression facilitated discovery of the tomato gene encoding prephenate aminotransferase, which converts prephenate to arogenate. The expression and biochemical data establish an arogenate pathway for Phe synthesis in tomato fruits. Metabolic profiling and 13C flux analysis of ripe fruits further revealed large increases in the levels of a specific subset of phenylpropanoid compounds. However, while increased levels of these human nutrition-related phenylpropanoids may be desirable, there were no increases in levels of Phe-derived flavor volatiles. PMID:21750236

Perihematomal Edema Expansion Rates and Patient Outcomes in Deep and Lobar Intracerebral Hemorrhage.

PubMed

Grunwald, Zachary; Beslow, Lauren A; Urday, Sebastian; Vashkevich, Anastasia; Ayres, Alison; Greenberg, Steven M; Goldstein, Joshua N; Leasure, Audrey; Shi, Fu-Dong; Kahle, Kristopher T; Battey, Thomas W K; Simard, J Marc; Rosand, Jonathan; Kimberly, W Taylor; Sheth, Kevin N

2017-04-01

Perihematomal edema (PHE) expansion rate may predict functional outcome following spontaneous intracerebral hemorrhage (ICH). We hypothesized that the effect of PHE expansion rate on outcome is greater for deep versus lobar ICH. Subjects (n = 115) were retrospectively identified from a prospective ICH cohort enrolled from 2000 to 2013. Inclusion criteria were age ≥ 18 years, spontaneous supratentorial ICH, and known onset time. Exclusion criteria were primary intraventricular hemorrhage (IVH), trauma, subsequent surgery, or warfarin-related ICH. ICH and PHE volumes were measured from CT scans and used to calculate expansion rates. Logistic regression assessed the association between PHE expansion rates and 90-day mortality or poor functional outcome (modified Rankin Scale > 2). Odds ratios are per 0.04 mL/h. PHE expansion rate from baseline to 24 h (PHE24) was associated with mortality for deep (p = 0.03, OR 1.13[1.02-1.26]) and lobar ICH (p = 0.02, OR 1.03[1.00-1.06]) in unadjusted regression and in models adjusted for age (deep p = 0.02, OR 1.15[1.02-1.28]; lobar p = 0.03, OR 1.03[1.00-1.06]), Glasgow Coma Scale (deep p = 0.03, OR 1.13[1.01-1.27]; lobar p = 0.02, OR 1.03[1.01-1.06]), or time to baseline CT (deep p = 0.046, OR 1.12[1.00-1.25]; lobar p = 0.047, OR 1.03[1.00-1.06]). PHE expansion rate from baseline to 72 h (PHE72) was associated with mRS > 2 for deep ICH in models that were unadjusted (p = 0.02, OR 4.04[1.25-13.04]) or adjusted for ICH volume (p = 0.02, OR 4.3[1.25-14.98]), age (p = 0.03, OR 5.4[1.21-24.11]), GCS (p = 0.02, OR 4.19[1.2-14.55]), or time to first CT (p = 0.03, OR 4.02[1.19-13.56]). PHE72 was associated with poor functional outcomes after deep ICH, whereas PHE24 was associated with mortality for deep and lobar ICH.

Regulation of neuronal pH by the metabotropic Zn(2+)-sensing Gq-coupled receptor, mZnR/GPR39.

PubMed

Ganay, Thibault; Asraf, Hila; Aizenman, Elias; Bogdanovic, Milos; Sekler, Israel; Hershfinkel, Michal

2015-12-01

Synaptically released Zn(2+) acts as a neurotransmitter, in part, by activating the postsynaptic metabotropic Zn(2+)-sensing Gq protein-coupled receptor (mZnR/GPR39). In previous work using epithelial cells, we described crosstalk between Zn(2+) signaling and changes in intracellular pH and/or extracellular pH (pHe). As pH changes accompany neuronal activity under physiological and pathological conditions, we tested whether Zn(2+) signaling is involved in regulation of neuronal pH. Here, we report that up-regulation of a major H(+) extrusion pathway, the Na(+)/H(+) exchanger (NHE), is induced by mZnR/GPR39 activation in an extracellular-regulated kinase 1/2-dependent manner in hippocampal neurons in vitro. We also observed that changes in pHe can modulate neuronal mZnR/GPR39-dependent signaling, resulting in reduced activity at pHe 8 or 6.5. Similarly, Zn(2+)-dependent extracellular-regulated kinase 1/2 phosphorylation and up-regulation of NHE activity were absent at acidic pHe. Thus, our results suggest that when pHe is maintained within the physiological range, mZnR/GPR39 activation can up-regulate NHE-dependent recovery from intracellular acidification. During acidosis, as pHe drops, mZnR/GPR39-dependent NHE activation is inhibited, thereby attenuating further H(+) extrusion. This mechanism may serve to protect neurons from excessive decreases in pHe. Thus, mZnR/GPR39 signaling provides a homeostatic adaptive process for regulation of intracellular and extracellular pH changes in the brain. We show that the postsynaptic metabotropic Zn(2+)-sensing Gq protein-coupled receptor (mZnR/GPR39) activation induces up-regulation of a major neuronal H(+) extrusion pathway, the Na(+)/H(+) exchanger (NHE), thereby enhancing neuronal recovery from intracellular acidification. Changes in extracellular pH (pHe), however, modulate neuronal mZnR/GPR39-dependent signaling, resulting in reduced activity at pHe 8 or 6.5. This mechanism may serve to protect neurons from excessive decreases in pHe during acidosis. Hence, mZnR/GPR39 signaling provides a homeostatic adaptive process for regulation of intracellular and extracellular pH changes in the brain. © 2015 International Society for Neurochemistry.

Insights into the Catalytic Mechanisms of Phenylalanine and Tryptophan Hydroxylase from Kinetic Isotope Effects on Aromatic Hydroxylation†

PubMed Central

Pavon, Jorge Alex; Fitzpatrick, Paul F.

2006-01-01

Phenylalanine hydroxylase (PheH) and tryptophan hydroxylase (TrpH) catalyze the aromatic hydroxylation of phenylalanine and tryptophan, forming tyrosine and 5-hydroxytryptophan, respectively. The reactions of PheH and TrpH have been investigated with [4-2H]-, [3,5-2H2]-, and 2H5-phenylalanine as substrates. All Dkcat values are normal with Δ117PheH, the catalytic core of rat phenylalanine hydroxylase, ranging from 1.12–1.41. In contrast, for Δ117PheH V379D, a mutant protein in which the stoichiometry between tetrahydropterin oxidation and amino acid hydroxylation is altered, the Dkcat value with [4-2H]-phenylalanine is 0.92 but is normal with [3,5-2H2]-phenylalanine. The ratio of tetrahydropterin oxidation to amino acid hydroxylation for Δ117PheH V379D shows a similar inverse isotope effect with [4-2H]-phenylalanine. Intramolecular isotope effects, determined from the deuterium contents of the tyrosine formed from [4-2H]-and [3,52H2]-phenylalanine, are identical for Δ117PheH and Δ117PheH V379D, suggesting that steps subsequent to oxygen addition are unaffected in the mutant protein. The inverse effects are consistent with the reaction of an activated ferryl-oxo species at the para position of the side chain of the amino acid to form a cationic intermediate. The normal effects on the Dkcat value for the wild-type enzyme are attributed to an isotope effect of 5.1 on the tautomerization of a dienone intermediate to tyrosine with a rate constant 6- to7-fold that for hydroxylation. In addition, there is a slight (∼34%) preference for the loss of the hydrogen originally at C4 of phenylalanine. With 2H5-indole-tryptophan as a substrate for Δ117PheH, the Dkcat value is 0.89, consistent with hydroxylation being rate-limiting in this case. When deuterated phenylalanines are used as substrates for TrpH, the Dkcat values are within error of those for Δ117PheH V379D. Overall, these results are consistent with the aromatic amino acid hydroxylases all sharing the same chemical mechanism, but with the isotope effect for hydroxylation by PheH being masked by tautomerization of an enedione intermediate to tyrosine. PMID:16953590

TRPM5 mediates acidic extracellular pH signaling and TRPM5 inhibition reduces spontaneous metastasis in mouse B16-BL6 melanoma cells

PubMed Central

Maeda, Toyonobu; Suzuki, Atsuko; Koga, Kaori; Miyamoto, Chihiro; Maehata, Yojiro; Ozawa, Shigeyuki; Hata, Ryu-Ichiro; Nagashima, Yoji; Nabeshima, Kazuki; Miyazaki, Kaoru; Kato, Yasumasa

2017-01-01

Extracellular acidity is a hallmark of solid tumors and is associated with metastasis in the tumor microenvironment. Acidic extracellular pH (pHe) has been found to increase intracellular Ca2+ and matrix metalloproteinase-9 (MMP-9) expression by activating NF-κB in the mouse B16 melanoma model. The present study assessed whether TRPM5, an intracellular Ca2+-dependent monovalent cation channel, is associated with acidic pHe signaling and induction of MMP-9 expression in this mouse melanoma model. Treatment of B16 cells with Trpm5 siRNA reduced acidic pHe-induced MMP-9 expression. Enforced expression of Trpm5 increased the rate of acidic pHe-induced MMP-9 expression, as well as increasing experimental lung metastasis. This genetic manipulation did not alter the pHe critical for MMP-9 induction but simply amplified the percentage of inducible MMP-9 at each pHe. Treatment of tumor bearing mice with triphenylphosphine oxide (TPPO), an inhibitor of TRPM5, significantly reduced spontaneous lung metastasis. In silico analysis of clinical samples showed that high TRPM5 mRNA expression correlated with poor overall survival rate in patients with melanoma and gastric cancer but not in patients with cancers of the ovary, lung, breast, and rectum. These results showed that TRPM5 amplifies acidic pHe signaling and may be a promising target for preventing metastasis of some types of tumor. PMID:29108231

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.

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

Structural Mimicry of Receptor Interaction by Antagonistic Interleukin-6 (IL-6) Antibodies*

PubMed Central

Blanchetot, Christophe; De Jonge, Natalie; Desmyter, Aline; Ongenae, Nico; Hofman, Erik; Klarenbeek, Alex; Sadi, Ava; Hultberg, Anna; Kretz-Rommel, Anke; Spinelli, Silvia; Loris, Remy; Cambillau, Christian; de Haard, Hans

2016-01-01

Interleukin 6 plays a key role in mediating inflammatory reactions in autoimmune diseases and cancer, where it is also involved in metastasis and tissue invasion. Neutralizing antibodies against IL-6 and its receptor have been approved for therapeutic intervention or are in advanced stages of clinical development. Here we describe the crystal structures of the complexes of IL-6 with two Fabs derived from conventional camelid antibodies that antagonize the interaction between the cytokine and its receptor. The x-ray structures of these complexes provide insights into the mechanism of neutralization by the two antibodies and explain the very high potency of one of the antibodies. It effectively competes for binding to the cytokine with IL-6 receptor (IL-6R) by using side chains of two CDR residues filling the site I cavities of IL-6, thus mimicking the interactions of Phe229 and Phe279 of IL-6R. In the first antibody, a HCDR3 tryptophan binds similarly to hot spot residue Phe279. Mutation of this HCDR3 Trp residue into any other residue except Tyr or Phe significantly weakens binding of the antibody to IL-6, as was also observed for IL-6R mutants of Phe279. In the second antibody, the side chain of HCDR3 valine ties into site I like IL-6R Phe279, whereas a LCDR1 tyrosine side chain occupies a second cavity within site I and mimics the interactions of IL-6R Phe229. PMID:27129274

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.

Carboxylation as an initial reaction in the anaerobic metabolism of naphthalene and phenanthrene by sulfidogenic consortia.

PubMed Central

Zhang, X; Young, L Y

1997-01-01

The anaerobic biodegradation of naphthalene (NAP) and phenanthrene (PHE) was investigated by using sediment collected from the Arthur Kill in New York/New Jersey harbor. The initial cultures were composed of 10% sediment and 90% mineral medium containing 20 mM sulfate. Complete loss of NAP and PHE (150 to 200 muM) was observed after 150 days of incubation. Upon refeeding, NAP and PHE were utilized within 14 days. The utilization of both compounds was inhibited in the presence of 20 mM molybdate. [14C]NAP and [14C]PHE were mineralized to 14CO2. The activities could be maintained and propagated by subculturing in mineral medium. In the presence of halogenated analogs, 2-naphthoate was detected in NAP-utilizing enrichments. The mass spectrum of the derivatized 2-napththoate from the enrichment supplemented with both [13C]bicarbonate and NAP indicates the incorporation of 13CO2 into NAP. In the PHE-utilizing enrichment, a metabolite was detected by both high-pressure liquid chromatography and gas chromatography-mass spectrometry analyses. The molecular ion and fragmentation pattern of its mass spectrum indicate that it was phenanthrenecarboxylic acid. The results obtained with [13C] bicarbonate indicate that 13CO2 was incorporated into PHE. It appears, therefore, that carboxylation is an initial key reaction for the anaerobic metabolism and NAP and PHE. To our knowledge, this is the first report providing evidence for intermediates of PAH degradation under anaerobic conditions. PMID:9471963

HR-TEM and FT-Raman dataset of the caffeine interacted Phe-Phe peptide nanotube for possible sensing applications.

PubMed

Narayanan, A Lakshmi; Dhamodaran, M; Solomon, J Samu; Karthikeyan, B; Govindhan, R

2018-02-01

Sensing ability of caffeine interaction with Phe-Phe annotates (PNTs), is presented (Govindhan et al., 2017; Karthikeyan et al., 2014; Tavagnacco et al., 2013; Kennedy et al., 2011; Wang et al., 2017) [1-5] in this data set. Investigation of synthesized caffeine carrying peptide nanotubes are carried out by FT-Raman spectral analysis and high resolution transmission electron microscopy (HR-TEM). Particle size of the caffeine loaded PNTs is < 40 nm. The FT-Raman spectrum signals are enhanced in the region of 400-1700 cm -1 . These data are ideal tool for the applications like biosensing and drug delivery research (DDS).

Photon and radiowave emission from peeling pressure sensitive adhesives in air

NASA Technical Reports Server (NTRS)

Donaldson, E. E.; Shen, X. A.; Dickinson, J. T.

1985-01-01

During separation of an adhesive from a polymer substrate in air, intense bursts of photons ('phE', for photon emission) and long wavelength electromagnetic radiation ('RE', for radiowave emission), similar to those reported earlier by Deryagin, et al. (1978) have been observed. In this paper, careful measurements of the phE time distributions, as well as time correlations between bursts of phE and RE, are reported. These results support the view that patches of electrical charge produced by charge separation between dissimilar materials lead to microdischarges in and near the crack tip. The role of these discharges in producing sustained phE after the discharge has been extinguished is also discussed.

Didehydrophenylalanine, an abundant modification in the beta subunit of plant polygalacturonases.

PubMed

Sergeant, Kjell; Printz, Bruno; Gutsch, Annelie; Behr, Marc; Renaut, Jenny; Hausman, Jean-Francois

2017-01-01

The structure and the activity of proteins are often regulated by transient or stable post- translational modifications (PTM). Different from well-known, abundant modifications such as phosphorylation and glycosylation some modifications are limited to one or a few proteins across a broad range of related species. Although few examples of the latter type are known, the evolutionary conservation of these modifications and the enzymes responsible for their synthesis suggest an important physiological role. Here, the first observation of a new, fold-directing PTM is described. During the analysis of alfalfa cell wall proteins a -2Da mass shift was observed on phenylalanine residues in the repeated tetrapeptide FxxY of the beta-subunit of polygalacturonase. This modular protein is known to be involved in developmental and stress-responsive processes. The presence of this modification was confirmed using in-house and external datasets acquired by different commonly used techniques in proteome studies. Based on these analyses it was found that all identified phenylalanine residues in the sequence FxxY of this protein were modified to α,β-didehydro-Phe (ΔPhe). Besides showing the reproducible identification of ΔPhe in different species arguments that substantiate the fold-determining role of ΔPhe are given.

Effects of stingless bee propolis on experimental asthma.

PubMed

de Farias, José Hidelbland Cavalcante; Reis, Aramys Silva; Araújo, Marcio Antonio Rodrigues; Araújo, Maria José Abigail Mendes; Assunção, Anne Karine Martins; de Farias, Jardel Cavalcante; Fialho, Eder Magalhães Silva; Silva, Lucilene Amorim; Costa, Graciomar Conceição; Guerra, Rosane Nassar Meireles; Ribeiro, Maria Nilce Sousa; do Nascimento, Flávia Raquel Fernandes

2014-01-01

Bee products have been used empirically for centuries, especially for the treatment of respiratory diseases. The present study evaluated the effect of treatment with a propolis hydroalcoholic extract (PHE) produced by Scaptotrigona aff. postica stingless bee in a murine asthma model. BALB/c mice were immunized twice with ovalbumin (OVA) subcutaneously. After 14 days, they were intranasally challenged with OVA. Groups P50 and P200 received PHE by gavage at doses of 50 and 200 mg/kg, respectively. The DEXA group was treated with intraperitoneal injection of dexamethasone. The OVA group received only water. The mice were treated daily for two weeks and then they were immunized a second time with intranasal OVA. The treatment with PHE decreased the cell number in the bronchoalveolar fluid (BAL). Histological analysis showed reduced peribronchovascular inflammation after treatment with PHE especially the infiltration of polymorphonuclear cells. In addition, the concentration of interferon- γ (IFN- γ ) in the serum was decreased. These results were similar to those obtained with dexamethasone. Treatment with S. aff postica propolis reduced the pathology associated with murine asthma due an inhibition of inflammatory cells migration to the alveolar space and the systemic progression of the allergic inflammation.

Mechanism and kinetic properties for the OH-initiated atmospheric oxidation degradation of 9,10-Dichlorophenanthrene.

PubMed

Dang, Juan; Shi, Xiangli; Zhang, Qingzhu; Hu, Jingtian; Wang, Wenxing

2015-02-01

Chlorinated polycyclic aromatic hydrocarbons (ClPAHs) have become a serious environmental concern due to their widespread occurrence and dioxin-like toxicities. In this work, the mechanism of the OH-initiated atmospheric oxidation degradation of 9,10-dichlorophenanthrene (9,10-Cl₂Phe) was investigated by using high-accuracy quantum chemistry calculations. The rate constants of the crucial elementary reactions were determined by the Rice-Ramsperger-Kassel-Marcus (RRKM) theory. The theoretical results were compared with the available experimental data. The main oxidation products are a group of ring-retaining and ring-opening compounds including chlorophenanthrols, 9,10-dichlorophenanthrene-3,4-dione, dialdehydes, chlorophenanthrenequinones, nitro-9,10-Cl₂Phe and epoxides et al. The overall rate constant of the OH addition reaction is 2.35 × 10(-12)cm(3) molecule(-1)s(-1) at 298 K and 1 atm. The atmospheric lifetime of 9,10-Cl₂Phe determined by OH radicals is about 5.05 days. This study provides a comprehensive investigation of the OH-initiated oxidation degradation of 9,10-Cl₂Phe and should contribute to clarifying its atmospheric fate. Copyright © 2014 Elsevier B.V. All rights reserved.

Effect of Al on stability of DHMS up to the uppermost lower mantle

NASA Astrophysics Data System (ADS)

Xu, C.; Inoue, T.

2017-12-01

Water plays an important role on Earth. It influences the physical and chemical property of minerals and melts, which further effects the evolution of the Earth. A series of dense hydrous magnesium silicate (DHMS) phases such as phase A (PhA), phase E (PhE), superhydrous phase B (SUB) and phase D (PhD) have been suggested as potential water carriers to transition zone and even to the lower mantle under the conditions present in the cold subducting slabs [e.g. Kawamoto, 2004; Komabayashi and Omori, 2006]. Because of its importance, the DHMS have been widely studied by using different starting materials in MgO-SiO2-H2O system. Recently, the newly reported Al-PhD is stable at temperatures up to 2,000 °C at 26 GPa, which indicates aluminum increases stability regions of DHMS [e.g. Pamato et al., 2015]. To systematically study the effect of Al on the stability of hydrous phases, we use Kawai-type high pressure apparatus to investigate nature clinochlore, which contains about 15 wt% H2O and about 14 wt% Al2O3. The Al-bearing hydrous PhE, SUB and PhD were observed with P-T increasing. Following the P-T path of cold subduction, the phase assemblage PhE + PhD is stable at 14-23 GPa, and even a trace of PhE is detected at 1150°C and 25 GPa coexisting with PhD. The phase SUB is stable between 16-22 GPa coexisting with PhE + PhD. Following the P-T path of hot subduction, the phase assemblage PhE + Gt is observed at 14-18 GPa coexisting with fluid or melt. The phase assemblage SUB + PhD is stable at 18-25 GPa, which may extend to higher pressures and temperatures. Therefore, it is obvious that Al enhances the stabilities of these three hydrous minerals, which are stable even in the hot subducting conditions. On the other hand, the Al substitution mechanism in PhE, SUB and PhD were clarified according to chemical compositional relationship between Mg, Si, Al. This shows that they can hold a significant amount of H (water) in their structure. Our results may indicate that the wide stabilities of Al-bearing DHMS increase the chance of water transportation to deeper mantle after antigorite (serpentine) decomposition at the shallow region of the subduction zone.

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.

Cinacalcet Rectifies Hypercalcemia in a Patient With Familial Hypocalciuric Hypercalcemia Type 2 (FHH2) Caused by a Germline Loss‐of‐Function Gα11 Mutation

PubMed Central

Gorvin, Caroline M; Hannan, Fadil M; Cranston, Treena; Valta, Helena; Makitie, Outi; Schalin‐Jantti, Camilla

2017-01-01

ABSTRACT G‐protein subunit α‐11 (Gα11) couples the calcium‐sensing receptor (CaSR) to phospholipase C (PLC)‐mediated intracellular calcium (Ca2+ i) and mitogen‐activated protein kinase (MAPK) signaling, which in the parathyroid glands and kidneys regulates parathyroid hormone release and urinary calcium excretion, respectively. Heterozygous germline loss‐of‐function Gα11 mutations cause familial hypocalciuric hypercalcemia type 2 (FHH2), for which effective therapies are currently not available. Here, we report a novel heterozygous Gα11 germline mutation, Phe220Ser, which was associated with hypercalcemia in a family with FHH2. Homology modeling showed the wild‐type (WT) Phe220 nonpolar residue to form part of a cluster of hydrophobic residues within a highly conserved cleft region of Gα11, which binds to and activates PLC; and predicted that substitution of Phe220 with the mutant Ser220 polar hydrophilic residue would disrupt PLC‐mediated signaling. In vitro studies involving transient transfection of WT and mutant Gα11 proteins into HEK293 cells, which express the CaSR, showed the mutant Ser220 Gα11 protein to impair CaSR‐mediated Ca2+ i and extracellular signal‐regulated kinase 1/2 (ERK) MAPK signaling, consistent with diminished activation of PLC. Furthermore, engineered mutagenesis studies demonstrated that loss of hydrophobicity within the Gα11 cleft region also impaired signaling by PLC. The loss‐of‐function associated with the Ser220 Gα11 mutant was rectified by treatment of cells with cinacalcet, which is a CaSR‐positive allosteric modulator. Furthermore, in vivo administration of cinacalcet to the proband harboring the Phe220Ser Gα11 mutation, normalized serum ionized calcium concentrations. Thus, our studies, which report a novel Gα11 germline mutation (Phe220Ser) in a family with FHH2, reveal the importance of the Gα11 hydrophobic cleft region for CaSR‐mediated activation of PLC, and show that allosteric CaSR modulation can rectify the loss‐of‐function Phe220Ser mutation and ameliorate the hypercalcemia associated with FHH2. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc. PMID:28833550

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

Intramolecular addition of cysteine thiyl radical to phenylalanine and tyrosine in model peptides, Phe (CysS•) and Tyr(CysS•): a computational study

PubMed Central

Naumov, Sergej; Schöneich, Christian

2009-01-01

Density Functional Theory (DFT) and ab initio calculations were carried out to evaluate the potential for intramolecular addition of cysteine (Cys) thiyl radicals (CysS•) to aromatic amino acids (Phe and Tyr) in water. These calculations yielded stable cyclic conformations, in which π-complexes were more stable than cyclohexadienyl radicals in water. In these π-complexes, the C2-S distances were significantly shorter compared to the C1-S and C3-S distances. Comparable results on the relative stabilities were obtained for model calculations for the addition of HS•/CH3S• to toluene and para-hydroxytoluene. The adduct of thiyl radicals with Phe was more stable than that with Tyr, and the stabilization energies depended on the C-terminal substituents. PMID:19309133

Enhancing effects of saccharin on gustatory responses to D-phenylalanine in monkey single chorda tympani fibers.

PubMed

Ninomiya, Y; Hellekant, G

1994-01-28

Taste enhancing effects of sodium saccharin (Sac) on D-phenylalanine (D-Phe), first found in mice, were examined by comparing single fiber responses to various taste stimuli in the monkey chorda tympani nerve. Fifteen fibers sampled were divided into the following 5 groups according to their responsiveness to 5 prototypical taste stimuli; 8 sucrose-, 2 quinine-, 2 acid-, 2 NaCl- and one monosodium glutamate (MSG)-best fibers. Out of 8 sucrose-best fibers, 5 fibers showed enhancement of D-Phe responses after the stimulation with Sac, but neither the remaining 3 sucrose-best fibers nor other fibers showed the enhancement. These results suggest that (1) the enhancement of D-Phe responses by Sac also occurs in the monkey peripheral taste system, and (2) there exist distinct receptor sites for D-Phe responsible for occurrence of the enhancement, and (3) taste cells possessing the D-Phe receptor site are innervated by a limited subpopulation of sucrose-best fibers.

Economic impact of feeding a phenylalanine-restricted diet to adults with previously untreated phenylketonuria.

PubMed

Brown, M C; Guest, J F

1999-02-01

The aim of the present study was to estimate the direct healthcare cost of managing adults with previously untreated phenylketonuria (PKU) for one year before any dietary restrictions and for the first year after a phenylalanine- (PHE-) restricted diet was introduced. The resource use and corresponding costs were estimated from medical records and interviews with health care professionals experienced in caring for adults with previously untreated PKU. The mean annual cost of caring for a client being fed an unrestricted diet was estimated to be 83 996 pound silver. In the first year after introducing a PHE-restricted diet, the mean annual cost was reduced by 20 647 pound silver to 63 348 pound silver as a result of a reduction in nursing time, hospitalizations, outpatient clinic visits and medications. However, the economic benefit of the diet depended on whether the clients were previously high or low users of nursing care. Nursing time was the key cost-driver, accounting for 79% of the cost of managing high users and 31% of the management cost for low users. In contrast, the acquisition cost of a PHE-restricted diet accounted for up to 6% of the cost for managing high users and 15% of the management cost for low users. Sensitivity analyses showed that introducing a PHE-restricted diet reduces the annual cost of care, provided that annual nursing time was reduced by more than 8% or more than 5% of clients respond to the diet. The clients showed fewer negative behaviours when being fed a PHE-restricted diet, which may account for the observed reduction in nursing time needed to care for these clients. In conclusion, feeding a PHE-restricted diet to adults with previously untreated PKU leads to economic benefits to the UK's National Health Service and society in general.

Wide-Field Variability Survey of the Globular Cluster M 79 and a New Period-Luminosity Relation for SX Phe Stars

NASA Astrophysics Data System (ADS)

Kopacki, G.

2015-03-01

We present the results of a search for variable stars in a 26×39 arcmin2 field around globular cluster M 79 (NGC 1904). The search was made by means of an extended version of image subtraction, which allows us to analyze in a uniform manner CCD frames obtained with different telescopes and cameras of different sizes and resolutions. The search resulted in finding 20 new variable stars, among which 13 are cluster members. The members include one new RR Lyr star of subtype c, three SX Phe stars, and nine variable red giants. We also show that V7 is a W Vir star with a period of 13.985 d. Revised mean periods of RRab and RRc stars, ab=0.71 d and c=0.34 d, respectively, and relative percentage of RRc stars, Nc/(Nab+Nc)=45% confirm that M 79 belongs to the Oosterhoff II group of globular clusters. The mean V magnitude of the horizontal branch of M 79 based on ten RR Lyr stars has been estimated to be VHB=RR=16.11±0.03 mag. In one RRc star, V9, light changes with three close frequencies were detected, indicating excitation of nonradial modes. An SX Phe star, V18, is a double-mode pulsator with two radial modes excited, fundamental and first overtone. Moreover, we have discovered two SX Phe or δ Sct stars and one W UMa type system, all likely field objects. We also studied the period-luminosity relation for SX Phe stars. Using 62 fundamental and fundamentalized periods of radial double-mode and high-amplitude SX Phe stars known in Galactic globular clusters, we have derived the slope and zero point of this relation to be, -3.3±0.27 and 2.68±0.03 mag (at log(P/d)=-1.24), respectively.

New antagonists of LHRH. II. Inhibition and potentiation of LHRH by closely related analogues.

PubMed

Bajusz, S; Csernus, V J; Janaky, T; Bokser, L; Fekete, M; Schally, A V

1988-12-01

Modifications of the previously described LHRH antagonists, [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Trp3, D-Cit6, D-Ala10]LHRH and the corresponding D-Hci6 analogue, have been made to alter the hydrophobicity of the N-terminal acetyl-tripeptide portion. Substitution of D-Trp3 with the less hydrophobic D-Pal(3) had only marginal effects on the antagonistic activities and receptor binding potencies of the D-Cit/D-Hci6 analogues, but it appeared to further improve the toxicity lowering effect of D-Cit/D-Hci6 substitution. Antagonists containing D-Pal(3)3 and D-Cit/D-Hci6 residues, i.e. [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Pal(3)3, D-Cit6, D-Ala10]LHRH (SB-75) and [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Pal(3)3, D-Hci6, D-Ala10]LHRH (SB-88), were completely free of the toxic effects, such as cyanosis and respiratory depression leading to death, which have been observed in rats with the D-Trp3, D-Arg6 antagonist and related antagonists. Replacement of the N-acetyl group with the hydrophilic carbamoyl group caused a slight decrease in antagonistic activities, particularly in vitro. Introduction of urethane type acyl group such as methoxycarbonyl (Moc) or t-butoxycarbonyl (Boc) led to analogues that showed LHRH-potentiating effect. The increase in potency induced by these analogues, e.g. [Moc-D-Nal(2)1, D-Phe(4Cl)2, D-Trp3, D-Cit6, D-Ala10]LHRH and [Boc-D-Phe1, D-Phe(4Cl)2, D-Pal(3)3, D-Cit6, D-Ala10]LHRH, was 170-260% and persisted for more than 2 h when studied in a superfused rat pituitary system.

Cyclopeptide Dmt-[D-Lys-p-CF3-Phe-Phe-Asp]NH2, a novel G protein-biased agonist of the mu opioid receptor.

PubMed

Piekielna-Ciesielska, Justyna; Ferrari, Federica; Calo', Girolamo; Janecka, Anna

2018-03-01

Opioid peptides and alkaloid drugs such as morphine, mediate their analgesic effects, but also undesired side effects, mostly through activation of the mu opioid receptor which belongs to the G protein-coupled receptor (GPCR) family. A new important pharmacological concept in the field of GPCRs is biased agonism. Two mu receptor ligands, Dmt-c[D-Lys-Phe-Phe-Asp]NH 2 (C-36) and Dmt-c[D-Lys-Phe-p-CF 3 -Phe-Asp]NH 2 (F-81), were evaluated in terms of their ability to promote or block mu receptor/G protein and mu receptor/β-arrestin interactions. Using the bioluminescence resonance energy transfer (BRET) assay it was shown that C-36 activated both, G protein and β-arrestin pathways. Incorporation of trifluoromethyl group into the aromatic ring of phenylalanine in the sequence of F-81 led to activation of G-protein pathway rather than β-arrestin recruitment. Opioid cyclopeptide F-81 turned out to be a biased G protein mu receptor agonist. Such biased ligands are able to separate the biological actions of an activated receptor and have the potential to become more effective drug candidates with fewer side effects. Copyright © 2017 Elsevier Inc. All rights reserved.

The power of power: electrokinetic control of PAH interactions with exfoliated graphite.

PubMed

Qin, Jinyi; Moustafa, Ahmed; Harms, Hauke; El-Din, Mohamed Gamal; Wick, Lukas Y

2015-05-15

Exfoliated graphite (EG) exhibits exceptional sorption capacity for petroleum and dissolved hydrocarbons owing to its highly hydrophobic surface and wide pore size distribution. The high price of preparing EG, however, restricts its application. Methods which increase the rate or extent of sorption to EG even further are thus longed for. Here, we assess the effects of weak direct current (DC) fields on the sorption of the polycyclic aromatic hydrocarbon phenanthrene (PHE) to EG. DC applied to an ionic solution in a solid matrix invokes electroosmotic flow (EOF), i.e., the surface charge-induced movement of the solution. EG was exposed to weak DC fields in the presence of dissolved PHE to test if EOF increases transport of PHE to poorly accessible sorption sites. DC fields increased PHE sorption rates in EG sevenfold and reduced the desorption rate of sorbed PHE by >99%. EOF thus appeared to be highly effective in translocating PHE into pores, which contribute most of the sorption sites, but are difficult to access in the absence of EOF by molecular diffusion only. The observed 'power of power' may be used to kinetically regulate the interaction of sorbates with EG or other porous sorbents in environmental (bio-) technology. Copyright © 2015 Elsevier B.V. All rights reserved.

Phenylalanine ammonia lyase, enzyme substitution therapy for phenylketonuria, where are we now?

PubMed

Sarkissian, Christineh N; Gámez, Alejandra

2005-12-01

Phenylketonuria (PKU) is an autosomal recessive genetic disorder in which mutations in the phenylalanine-4-hydroxylase (PAH) gene result in an inactive enzyme (PAH, EC 1.14.16.1). The effect is an inability to metabolize phenylalanine (Phe), translating into elevated levels of Phe in the bloodstream (hyperphenylalaninemia). If therapy is not implemented at birth, mental retardation can occur. PKU patients respond to treatment with a low-phenylalanine diet, but compliance with the diet is difficult, therefore the development of alternative treatments is desirable. Enzyme substitution therapy with a recombinant phenylalanine ammonia lyase (PAL) is currently being explored. This enzyme converts Phe to the harmless metabolites, trans-cinnamic acid and trace ammonia. Taken orally and when non-absorbable and protected, PAL lowers plasma Phe in mutant hyperphenylalaninemic mouse models. Subcutaneous administration of PAL results in more substantial lowering of plasma and significant reduction in brain Phe levels, however the metabolic effect is not sustained following repeated injections due to an immune response. We have chemically modified PAL by pegylation to produce a protected form of PAL that possesses better specific activity, prolonged half-life, and reduced immunogenicity in vivo. Subcutaneous administration of pegylated molecules to PKU mice has the desired metabolic response (prolonged reduction in blood Phe levels) with greatly attenuated immunogenicity.

Small constrained SP1-7 analogs bind to a unique site and promote anti-allodynic effects following systemic injection in mice.

PubMed

Jonsson, A; Fransson, R; Haramaki, Y; Skogh, A; Brolin, E; Watanabe, H; Nordvall, G; Hallberg, M; Sandström, A; Nyberg, F

2015-07-09

Previous results have shown that the substance P (SP) N-terminal fragment SP1-7 may attenuate hyperalgesia and produce anti-allodynia in animals using various experimental models for neuropathic pain. The heptapeptide was found to induce its effects through binding to and activating specific sites apart from any known neurokinin or opioid receptor. Furthermore, we have applied a medicinal chemistry program to develop lead compounds mimicking the effect of SP1-7. The present study was designed to evaluate the pharmacological effect of these compounds using the mouse spared nerve injury (SNI) model of chronic neuropathic pain. Also, as no comprehensive screen with the aim to identify the SP1-7 target has yet been performed we screened our lead compound H-Phe-Phe-NH2 toward a panel of drug targets. The extensive target screen, including 111 targets, did not reveal any hit for the binding site among a number of known receptors or enzymes involved in pain modulation. Our animal studies confirmed that SP1-7, but also synthetic analogs thereof, possesses anti-allodynic effects in the mouse SNI model of neuropathic pain. One of the lead compounds, a constrained H-Phe-Phe-NH2 analog, was shown to exhibit a significant anti-allodynic effect. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

Exploring the Potential of (99m)Tc(CO)3-Labeled Triazolyl Peptides for Tumor Diagnosis.

PubMed

Gaonkar, Raghuvir H; Ganguly, Soumya; Baishya, Rinku; Dewanjee, Saikat; Sinha, Samarendu; Gupta, Amit; Ganguly, Shantanu; Debnath, Mita C

2016-04-01

In recent years the authors have reported on (99m)Tc(CO)3-labeled peptides that serve as carriers for biomolecules or radiopharmaceuticals to the tumors. In continuation of that work they report the synthesis of a pentapeptide (Met-Phe-Phe-Gly-His; pep-1), a hexapeptide (Met-Phe-Phe-Asp-Gly-His; pep-2), and a tetrapeptide (Asp-Gly-Arg-His; pep-3) and the attachment of 3-amino-1,2,4-triazole to the β carboxylic function of the aspartic acid unit of pep-2 and pep-3. The pharmacophores were radiolabeled in high yields with [(99m)Tc(CO)3(H2O)3](+) metal aqua ion, characterized for their stability in serum and saline, as well as in His solution, and found to be substantially stable. B16F10 cell line binding studies showed favorable uptake and internalization. In vivo behavior of the radiolabeled triazolyl peptides was assessed in mice bearing induced tumor. The (99m)Tc(CO)3-triazolyl pep-3 demonstrated rapid urinary clearance and comparatively better tumor uptake. Imaging studies showed visualization of the tumor using (99m)Tc(CO)3-triazolyl pep-3, but due to high abdominal background, low delineation occurred. Based on the results further experiments will be carried out for targeting tumor with triazolyl peptides.

Structural Mimicry of Receptor Interaction by Antagonistic Interleukin-6 (IL-6) Antibodies.

PubMed

Blanchetot, Christophe; De Jonge, Natalie; Desmyter, Aline; Ongenae, Nico; Hofman, Erik; Klarenbeek, Alex; Sadi, Ava; Hultberg, Anna; Kretz-Rommel, Anke; Spinelli, Silvia; Loris, Remy; Cambillau, Christian; de Haard, Hans

2016-06-24

Interleukin 6 plays a key role in mediating inflammatory reactions in autoimmune diseases and cancer, where it is also involved in metastasis and tissue invasion. Neutralizing antibodies against IL-6 and its receptor have been approved for therapeutic intervention or are in advanced stages of clinical development. Here we describe the crystal structures of the complexes of IL-6 with two Fabs derived from conventional camelid antibodies that antagonize the interaction between the cytokine and its receptor. The x-ray structures of these complexes provide insights into the mechanism of neutralization by the two antibodies and explain the very high potency of one of the antibodies. It effectively competes for binding to the cytokine with IL-6 receptor (IL-6R) by using side chains of two CDR residues filling the site I cavities of IL-6, thus mimicking the interactions of Phe(229) and Phe(279) of IL-6R. In the first antibody, a HCDR3 tryptophan binds similarly to hot spot residue Phe(279) Mutation of this HCDR3 Trp residue into any other residue except Tyr or Phe significantly weakens binding of the antibody to IL-6, as was also observed for IL-6R mutants of Phe(279) In the second antibody, the side chain of HCDR3 valine ties into site I like IL-6R Phe(279), whereas a LCDR1 tyrosine side chain occupies a second cavity within site I and mimics the interactions of IL-6R Phe(229). © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Phenylketonuria in adulthood: a collaborative study.

PubMed

Koch, R; Burton, B; Hoganson, G; Peterson, R; Rhead, W; Rouse, B; Scott, R; Wolff, J; Stern, A M; Guttler, F; Nelson, M; de la Cruz, F; Coldwell, J; Erbe, R; Geraghty, M T; Shear, C; Thomas, J; Azen, C

2002-09-01

During 1967-1983, the Maternal and Child Health Division of the Public Health Services funded a collaborative study of 211 newborn infants identified on newborn screening as having phenylketonuria (PKU). Subsequently, financial support was provided by the National Institute of Child Health and Human Development (NICHD). The infants were treated with a phenylalanine (Phe)-restricted diet to age 6 years and then randomized either to continue the diet or to discontinue dietary treatment altogether. One hundred and twenty-five of the 211 children were then followed until 10 years of age. In 1998, NICHD scheduled a Consensus Development Conference on Phenylketonuria and initiated a study to follow up the participants from the original Collaborative Study to evaluate their present medical, nutritional, psychological, and socioeconomic status. Fourteen of the original clinics (1967-1983) participated in the Follow-up Study effort. Each clinic director was provided with a list of PKU subjects who had completed the original study (1967-1983), and was asked to evaluate as many as possible using a uniform protocol and data collection forms. In a subset of cases, magnetic resonance imaging and spectroscopy (MRI/MRS) were performed to study brain Phe concentrations. The medical evaluations revealed that the subjects who maintained a phenylalanine-restricted diet reported fewer problems than the diet discontinuers, who had an increased rate of eczema, asthma, mental disorders, headache, hyperactivity and hypoactivity. Psychological data showed that lower intellectual and achievement test scores were associated with dietary discontinuation and with higher childhood and adult blood Phe concentrations. Abnormal MRI results were associated with higher brain Phe concentrations. Early dietary discontinuation for subjects with PKU is associated with poorer outcomes not only in intellectual ability, but also in achievement test scores and increased rates of medical and behavioural problems.

Mechanism of Interaction between the General Anesthetic Halothane and a Model Ion Channel Protein, II: Fluorescence and Vibrational Spectroscopy Using a Cyanophenylalanine Probe

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

Liu, J.; Strzalka, J; Tronin, A

2009-01-01

We demonstrate that cyano-phenylalanine (PheCN) can be utilized to probe the binding of the inhalational anesthetic halothane to an anesthetic-binding, model ion channel protein hbAP-PheCN. The Trp to PheCN mutation alters neither the a-helical conformation nor the 4-helix bundle structure. The halothane binding properties of this PheCN mutant hbAP-PheCN, based on fluorescence quenching, are consistent with those of the prototype, hbAP1. The dependence of fluorescence lifetime as a function of halothane concentration implies that the diffusion of halothane in the nonpolar core of the protein bundle is one-dimensional. As a consequence, at low halothane concentrations, the quenching of the fluorescencemore » is dynamic, whereas at high concentrations the quenching becomes static. The 4-helix bundle structure present in aqueous detergent solution and at the air-water interface, is preserved in multilayer films of hbAP-PheCN, enabling vibrational spectroscopy of both the protein and its nitrile label (-CN). The nitrile groups' stretching vibration band shifts to higher frequency in the presence of halothane, and this blue-shift is largely reversible. Due to the complexity of this amphiphilic 4-helix bundle model membrane protein, where four PheCN probes are present adjacent to the designed cavity forming the binding site within each bundle, all contributing to the infrared absorption, molecular dynamics (MD) simulation is required to interpret the infrared results. The MD simulations indicate that the blue-shift of -CN stretching vibration induced by halothane arises from an indirect effect, namely an induced change in the electrostatic protein environment averaged over the four probe oscillators, rather than a direct interaction with the oscillators. hbAP-PheCN therefore provides a successful template for extending these investigations of the interactions of halothane with the model membrane protein via vibrational spectroscopy, using cyano-alanine residues to form the anesthetic binding cavity.« less

Combination of virtual screening protocol by in silico towards the discovery of novel 4-hydroxyphenylpyruvate dioxygenase inhibitors

NASA Astrophysics Data System (ADS)

Fu, Ying; Sun, Yi-Na; Yi, Ke-Han; Li, Ming-Qiang; Cao, Hai-Feng; Li, Jia-Zhong; Ye, Fei

2018-02-01

4-Hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27, HPPD) is a potent new bleaching herbicide target. Therefore, in silico structure-based virtual screening was performed in order to speed up the identification of promising HPPD inhibitors. In this study, an integrated virtual screening protocol by combining 3D-pharmacophore model, molecular docking and molecular dynamics (MD) simulation was established to find novel HPPD inhibitors from four commercial databases. 3D-pharmacophore Hypo1 model was applied to efficiently narrow potential hits. The hit compounds were subsequently submitted to molecular docking studies, showing four compounds as potent inhibitor with the mechanism of the Fe(II) coordination and interaction with Phe360, Phe403 and Phe398. MD result demonstrated that nonpolar term of compound 3881 made great contributions to binding affinities. It showed an IC50 being 2.49 µM against AtHPPD in vitro. The results provided useful information for developing novel HPPD inhibitors, leading to further understanding of the interaction mechanism of HPPD inhibitors.

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.

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

Insights into the catalytic mechanisms of phenylalanine and tryptophan hydroxylase from kinetic isotope effects on aromatic hydroxylation.

PubMed

Pavon, Jorge Alex; Fitzpatrick, Paul F

2006-09-12

Phenylalanine hydroxylase (PheH) and tryptophan hydroxylase (TrpH) catalyze the aromatic hydroxylation of phenylalanine and tryptophan, forming tyrosine and 5-hydroxytryptophan, respectively. The reactions of PheH and TrpH have been investigated with [4-(2)H]-, [3,5-(2)H(2)]-, and (2)H(5)-phenylalanine as substrates. All (D)k(cat) values are normal with Delta117PheH, the catalytic core of rat phenylalanine hydroxylase, ranging from 1.12-1.41. In contrast, for Delta117PheH V379D, a mutant protein in which the stoichiometry between tetrahydropterin oxidation and amino acid hydroxylation is altered, the (D)k(cat) value with [4-(2)H]-phenylalanine is 0.92 but is normal with [3,5-(2)H(2)]-phenylalanine. The ratio of tetrahydropterin oxidation to amino acid hydroxylation for Delta117PheH V379D shows a similar inverse isotope effect with [4-(2)H]-phenylalanine. Intramolecular isotope effects, determined from the deuterium contents of the tyrosine formed from [4-(2)H]-and [3,5(2)H(2)]-phenylalanine, are identical for Delta117PheH and Delta117PheH V379D, suggesting that steps subsequent to oxygen addition are unaffected in the mutant protein. The inverse effects are consistent with the reaction of an activated ferryl-oxo species at the para position of the side chain of the amino acid to form a cationic intermediate. The normal effects on the (D)k(cat) value for the wild-type enzyme are attributed to an isotope effect of 5.1 on the tautomerization of a dienone intermediate to tyrosine with a rate constant 6- to7-fold that for hydroxylation. In addition, there is a slight ( approximately 34%) preference for the loss of the hydrogen originally at C4 of phenylalanine. With (2)H(5)-indole-tryptophan as a substrate for Delta117PheH, the (D)k(cat) value is 0.89, consistent with hydroxylation being rate-limiting in this case. When deuterated phenylalanines are used as substrates for TrpH, the (D)k(cat) values are within error of those for Delta117PheH V379D. Overall, these results are consistent with the aromatic amino acid hydroxylases all sharing the same chemical mechanism, but with the isotope effect for hydroxylation by PheH being masked by tautomerization of an enedione intermediate to tyrosine.

The role of phenylalanine-119 of the reverse transcriptase of mouse mammary tumour virus in DNA synthesis, ribose selection and drug resistance.

PubMed

Entin-Meer, Michal; Sevilya, Ziv; Hizi, Amnon

2002-10-15

Phe-119 in the reverse transcriptase (RT) of mouse mammary tumour virus (MMTV) is homologous with Tyr-115 in HIV type 1 (HIV-1) RT and to Phe-155 in murine leukaemia virus (MLV) RT. By mutating these residues in HIV-1 and MLV RTs (which are strict DNA polymerases) the enzymes were shown to function also as RNA polymerases. Owing to the uniqueness of MMTV as a type B retrovirus, we have generated a Phe-119-Val mutant of MMTV RT to study the involvement of this residue in affecting the catalytic features of this RT. The data presented here show that the mutant MMTV RT can incorporate both deoxyribonucleosides and ribonucleosides while copying either RNA or DNA. In addition, this mutant RT shows resistance to nucleoside analogues and an enhanced fidelity of DNA synthesis; all relative to the wild-type enzyme. The Phe-119-Val mutant is also different from the wild-type enzyme in its preference for most template primers tested and in its ability to synthesize DNA under non-processive and processive conditions. Overall, it is likely that the aromatic side chain of Phe-119 is located at the dNTP-binding site of MMTV RT and thus might be part of a putative "steric gate" that prevents the incorporation of nucleoside triphosphates. Since the only three-dimensional structures of RTs published so far are those of HIV-1 and MLV, it is likely that MMTV RT folds quite similarly to these RTs.

Beta-endorphin. Synthesis and biological activity of analogs with disulfide bridges.

PubMed

Blake, J; Helmeste, D M; Li, C H

1985-06-01

Two analogs of human beta-endorphin (beta-EP) which contain cystine bridges, [Cys15-Cys26,Phe27,Gly31]-beta-EP (I) and [Cys16-Cys26,Phe27,Gly31]-beta-EP (II), were synthesized by the solid-phase method. Peptides I and II were shown to contain 2-2.5 times the opiate receptor binding activity of beta-endorphin. We also synthesized two analogs with reduced alkylated cysteine residues and these peptides, [Arg9,19,24,28,29 Cys(Cam)11,26,Phe27,Gly31] and [Arg9,19,24,28,29,Cys-(Cam)12,26,Phe27,Gly31], were shown to have approximately the same opiate receptor activity as beta-endorphin.

High capacity for extracellular acid-base regulation in the air-breathing fish Pangasianodon hypophthalmus.

PubMed

Damsgaard, Christian; Gam, Le Thi Hong; Tuong, Dang Diem; Thinh, Phan Vinh; Huong Thanh, Do Thi; Wang, Tobias; Bayley, Mark

2015-05-01

The evolution of accessory air-breathing structures is typically associated with reduction of the gills, although branchial ion transport remains pivotal for acid-base and ion regulation. Therefore, air-breathing fishes are believed to have a low capacity for extracellular pH regulation during a respiratory acidosis. In the present study, we investigated acid-base regulation during hypercapnia in the air-breathing fish Pangasianodon hypophthalmus in normoxic and hypoxic water at 28-30°C. Contrary to previous studies, we show that this air-breathing fish has a pronounced ability to regulate extracellular pH (pHe) during hypercapnia, with complete metabolic compensation of pHe within 72 h of exposure to hypoxic hypercapnia with CO2 levels above 34 mmHg. The high capacity for pHe regulation relies on a pronounced ability to increase levels of HCO3(-) in the plasma. Our study illustrates the diversity in the physiology of air-breathing fishes, such that generalizations across phylogenies may be difficult. © 2015. Published by The Company of Biologists Ltd.

Dynamics of an Active-Site Flap Contributes to Catalysis in a JAMM Family Metallo Deubiquitinase.

PubMed

Bueno, Amy N; Shrestha, Rashmi K; Ronau, Judith A; Babar, Aditya; Sheedlo, Michael J; Fuchs, Julian E; Paul, Lake N; Das, Chittaranjan

2015-10-06

The endosome-associated deubiquitinase (DUB) AMSH is a member of the JAMM family of zinc-dependent metallo isopeptidases with high selectivity for Lys63-linked polyubiquitin chains, which play a key role in endosomal-lysosomal sorting of activated cell surface receptors. The catalytic domain of the enzyme features a flexible flap near the active site that opens and closes during its catalytic cycle. Structural analysis of its homologues, AMSH-LP (AMSH-like protein) and the fission yeast counterpart, Sst2, suggests that a conserved Phe residue in the flap may be critical for substrate binding and/or catalysis. To gain insight into the contribution of this flap in substrate recognition and catalysis, we generated mutants of Sst2 and characterized them using a combination of enzyme kinetics, X-ray crystallography, molecular dynamics simulations, and isothermal titration calorimetry (ITC). Our analysis shows that the Phe residue in the flap contributes key interactions during the rate-limiting step but not to substrate binding, since mutants of Phe403 exhibit a defect only in kcat but not in KM. Moreover, ITC studies show Phe403 mutants have similar KD for ubiquitin compared to the wild-type enzyme. The X-ray structures of both Phe403Ala and the Phe403Trp, in both the free and ubiquitin bound form, reveal no appreciable structural change that might impair substrate or alter product binding. We observed that the side chain of the Trp residue is oriented identically with respect to the isopeptide moiety of the substrate as the Phe residue in the wild-type enzyme, so the loss of activity seen in this mutant cannot be explained by the absence of a group with the ability to provide van der Waals interactions that facilitate the hyrdolysis of the Lys63-linked diubiquitin. Molecular dynamics simulations indicate that the flap in the Trp mutant is quite flexible, allowing almost free rotation of the indole side chain. Therefore, it is possible that these different dynamic properties of the flap in the Trp mutant, compared to the wild-type enzyme, manifest as a defect in interactions that facilitate the rate-limiting step. Consistent with this notion, the Trp mutant was able to cleave Lys48-linked and Lys11-linked diubiquitin better than the wild-type enzyme, indicating altered mobility and hence reduced selectivity.

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.

Ring size of somatostatin analogues (ODT-8) modulates receptor selectivity and binding affinity

PubMed Central

Erchegyi, Judit; Grace, Christy Rani R.; Samant, Manoj; Cescato, Renzo; Piccand, Veronique; Riek, Roland; Reubi, Jean Claude; Rivier, Jean E.

2009-01-01

The synthesis, biological testing and NMR studies of several analogues of H-c[Cys3-Phe6-Phe7-dTrp8-Lys9-Thr10-Phe11-Cys14]-OH (ODT-8, a pan-somatostatin analogue) (1), have been performed to assess the effect of changing the stereochemistry and the number of the atoms in the disulfide bridge on binding affinity. Cysteine at positions 3 and/or 14 (SRIF numbering) were/was substituted with d-cysteine, Nor-cysteine, d-Nor-cysteine, Homo-cysteine and/or d-Homo-cysteine. The 3D structures of selected partially selective, bioactive analogues (3, 18, 19 and 21) were carried out in DMSO. Interestingly and not unexpectedly, the 3D structures of these analogues comprised the pharmacophore for which the analogues had the highest binding affinities (i.e., sst4 in all cases). PMID:18410084

The physiological cost of wearing the propellant handler's ensemble at the Kennedy Space Center

NASA Technical Reports Server (NTRS)

Schonfeld, Brian R.; Doerr, Donald F.; Tomaselli, Clare Marie

1990-01-01

The potential for exposure to toxins used in the propulsion systems of spacecraft dictates the use of a whole body protective suit, the Propellant Handler's Ensemble (PHE) during preflight preparation and launching. The weight, structure, and operating parameters of the PHE may be expected to have a significant impact upon the metabolic, cardiovascular, and thermal responses of the user, especially during ambient temperature extremes and high workload situations. Four male subjects participated in tests in -7, 23, and 43 C (20, 74, and 110 F) environments in two versions of the PHE, the autonomous backpack (BP) and the hoseline (HL) supplied configuration. Measurements included heart rate (HR) rectal temperature, four skin temperatures, oxygen (O2), and carbon dioxide (CO2) in the helmet area, interior suit temperature, and suit pressure. Exercise metabolism was estimated from HR, PHE weight, and treadmill speed and grade. The HR responses between each PHE configuration were not statistically different. As a percentage of HR maximum, the mean values were 79 percent (COLD), 84 percent (LAB), and 90 percent (HOT). Helmet O2 and CO2 levels were correlated with percent HR max (P less than 0.001). Rectal temperatures were similar for each PHE configuration, except in the HOT exposure where the BP version exceeded the HL configuration (P less than 0.05). In nearly every instance the HR was driven to moderately high levels, the supplied respiratory gases were not optimum, and thermal adversity was a primary stressor. Our findings suggest that medical and physical fitness standards, along with operational restrictions, should be imposed upon PHE users to avoid situations that could adversely affect the worker.

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.

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.

Seed and Foliar Application of Amino Acids Improve Variables of Nitrogen Metabolism and Productivity in Soybean Crop.

PubMed

Teixeira, Walquíria F; Fagan, Evandro B; Soares, Luis H; Soares, Jérssica N; Reichardt, Klaus; Neto, Durval D

2018-01-01

The application of amino acids in crops has been a common practice in recent years, although most of the time they are associated with products based on algae extracts or on fermented animal or vegetable wastes. However, little is known about the isolated effect of amino acids on the development of crops. Therefore, the objective of this research was to evaluate the effect of the application of isolated amino acids on the in some steps of the soybean nitrogen metabolism and on productivity. Experiments were carried out in a greenhouse and in the field with the application of the amino acids glutamate (Glu), phenylalanine (Phe), cysteine (Cys) and glycine (Gly) and as a set (Glu+Phe+Cys+Gly), as seed treatment (ST), as foliar application (FA) and both (ST+FA), at the V 4 growth stage. Evaluations consisted of nitrate reductase and urease activities, nitrate, ureide, total amino acids and total nitrogen content in leaves, and productivity. The application of Glu to leaves, Cys as ST and a mixture of Glu+Cys+Phe+Gly as ST+FA in the greenhouse experiment increased the total amino acids content. In the field experiment all treatments increased the amino acid content in leaves. At the V 6 stage in the field experiment, all modes of Gly application, Glu as ST and FA, Cys and Phe as ST+FA and Glu+Cys+Phe+Gly as FA increased the nitrate content in leaves. In the greenhouse, application of Cys and Phe as ST increased the production of soybean plants by at least 21%. The isolated application of Cys, Phe, Gly, Glu and the set of these amino acids as ST increased the productivity of soybean plants in the field experiment by at least 22%.

Seed and Foliar Application of Amino Acids Improve Variables of Nitrogen Metabolism and Productivity in Soybean Crop

PubMed Central

Teixeira, Walquíria F.; Fagan, Evandro B.; Soares, Luis H.; Soares, Jérssica N.; Reichardt, Klaus; Neto, Durval D.

2018-01-01

The application of amino acids in crops has been a common practice in recent years, although most of the time they are associated with products based on algae extracts or on fermented animal or vegetable wastes. However, little is known about the isolated effect of amino acids on the development of crops. Therefore, the objective of this research was to evaluate the effect of the application of isolated amino acids on the in some steps of the soybean nitrogen metabolism and on productivity. Experiments were carried out in a greenhouse and in the field with the application of the amino acids glutamate (Glu), phenylalanine (Phe), cysteine (Cys) and glycine (Gly) and as a set (Glu+Phe+Cys+Gly), as seed treatment (ST), as foliar application (FA) and both (ST+FA), at the V4 growth stage. Evaluations consisted of nitrate reductase and urease activities, nitrate, ureide, total amino acids and total nitrogen content in leaves, and productivity. The application of Glu to leaves, Cys as ST and a mixture of Glu+Cys+Phe+Gly as ST+FA in the greenhouse experiment increased the total amino acids content. In the field experiment all treatments increased the amino acid content in leaves. At the V6 stage in the field experiment, all modes of Gly application, Glu as ST and FA, Cys and Phe as ST+FA and Glu+Cys+Phe+Gly as FA increased the nitrate content in leaves. In the greenhouse, application of Cys and Phe as ST increased the production of soybean plants by at least 21%. The isolated application of Cys, Phe, Gly, Glu and the set of these amino acids as ST increased the productivity of soybean plants in the field experiment by at least 22%. PMID:29643860

Measuring patient engagement: development and psychometric properties of the Patient Health Engagement (PHE) Scale

PubMed Central

Graffigna, Guendalina; Barello, Serena; Bonanomi, Andrea; Lozza, Edoardo

2015-01-01

Beyond the rhetorical call for increasing patients' engagement, policy makers recognize the urgency to have an evidence-based measure of patients' engagement and capture its effect when planning and implementing initiatives aimed at sustaining the engagement of consumers in their health. In this paper, authors describe the Patient Health Engagement Scale (PHE-scale), a measure of patient engagement that is grounded in rigorous conceptualization and appropriate psychometric methods. The scale was developed based on our previous conceptualization of patient engagement (the PHE-model). In particular, the items of the PHE-scale were developed based on the findings from the literature review and from interviews with chronic patients. Initial psychometric analysis was performed to pilot test a preliminary version of the items. The items were then refined and administered to a national sample of chronic patients (N = 382) to assess the measure's psychometric performance. A final phase of test-retest reliability was performed. The analysis showed that the PHE Scale has good psychometric properties with good correlation with concurrent measures and solid reliability. Having a valid and reliable measure to assess patient engagement is the first step in understanding patient engagement and its role in health care quality, outcomes, and cost containment. The PHE Scale shows a promising clinical relevance, indicating that it can be used to tailor intervention and assess changes after patient engagement interventions. PMID:25870566

Measuring patient engagement: development and psychometric properties of the Patient Health Engagement (PHE) Scale.

PubMed

Graffigna, Guendalina; Barello, Serena; Bonanomi, Andrea; Lozza, Edoardo

2015-01-01

Beyond the rhetorical call for increasing patients' engagement, policy makers recognize the urgency to have an evidence-based measure of patients' engagement and capture its effect when planning and implementing initiatives aimed at sustaining the engagement of consumers in their health. In this paper, authors describe the Patient Health Engagement Scale (PHE-scale), a measure of patient engagement that is grounded in rigorous conceptualization and appropriate psychometric methods. The scale was developed based on our previous conceptualization of patient engagement (the PHE-model). In particular, the items of the PHE-scale were developed based on the findings from the literature review and from interviews with chronic patients. Initial psychometric analysis was performed to pilot test a preliminary version of the items. The items were then refined and administered to a national sample of chronic patients (N = 382) to assess the measure's psychometric performance. A final phase of test-retest reliability was performed. The analysis showed that the PHE Scale has good psychometric properties with good correlation with concurrent measures and solid reliability. Having a valid and reliable measure to assess patient engagement is the first step in understanding patient engagement and its role in health care quality, outcomes, and cost containment. The PHE Scale shows a promising clinical relevance, indicating that it can be used to tailor intervention and assess changes after patient engagement interventions.

Metal-Polycyclic Aromatic Hydrocarbon Mixture Toxicity in Hyalella azteca. 1. Response Surfaces and Isoboles To Measure Non-additive Mixture Toxicity and Ecological Risk.

PubMed

Gauthier, Patrick T; Norwood, Warren P; Prepas, Ellie E; Pyle, Greg G

2015-10-06

Mixtures of metals and polycyclic aromatic hydrocarbons (PAHs) occur ubiquitously in aquatic environments, yet relatively little is known regarding their potential to produce non-additive toxicity (i.e., antagonism or potentiation). A review of the lethality of metal-PAH mixtures in aquatic biota revealed that more-than-additive lethality is as common as strictly additive effects. Approaches to ecological risk assessment do not consider non-additive toxicity of metal-PAH mixtures. Forty-eight-hour water-only binary mixture toxicity experiments were conducted to determine the additive toxic nature of mixtures of Cu, Cd, V, or Ni with phenanthrene (PHE) or phenanthrenequinone (PHQ) using the aquatic amphipod Hyalella azteca. In cases where more-than-additive toxicity was observed, we calculated the possible mortality rates at Canada's environmental water quality guideline concentrations. We used a three-dimensional response surface isobole model-based approach to compare the observed co-toxicity in juvenile amphipods to predicted outcomes based on concentration addition or effects addition mixtures models. More-than-additive lethality was observed for all Cu-PHE, Cu-PHQ, and several Cd-PHE, Cd-PHQ, and Ni-PHE mixtures. Our analysis predicts Cu-PHE, Cu-PHQ, Cd-PHE, and Cd-PHQ mixtures at the Canadian Water Quality Guideline concentrations would produce 7.5%, 3.7%, 4.4% and 1.4% mortality, respectively.

Surface decoration of red blood cells with maleimidophenyl-polyethylene glycol facilitated by thiolation with iminothiolane: an approach to mask A, B, and D antigens to generate universal red blood cells.

PubMed

Nacharaju, Parimala; Boctor, Fouad N; Manjula, Belur N; Acharya, Seetharama A

2005-03-01

The surface decoration of red blood cells (RBCs) by polyethylene glycol (PEG) chains has been an approach developed to camouflage the blood group antigens from their antibodies. A PEGylation protocol, however, that can mask the antigens appropriately to inhibit the agglutination of RBCs with the respective antibodies is not available so far. A new approach for PEGylation of RBC membrane proteins has been designed with thiolation-mediated maleimide chemistry. The accessibility of the surface lysine residues of membrane proteins to bulky PEG reagents was increased by linking an extension arm carrying a thiol group. RBCs have been PEGylated by thiolation-mediated chemistry with maleimidophenyl-PEG (Mal-Phe-PEG) reagents of different chain lengths. Mal-Phe-PEG-5000 chains alone masked the most important antigens of the Rh system (C, c, E, e, and D) from their antibodies. The masking of the A and B antigens needed a combination of Mal-Phe-PEG-5000 and Mal-Phe-PEG-20000 chains to inhibit the agglutination of RBCs completely with anti-A or anti-B. Thiolation-mediated PEGylation of RBCs with Mal-Phe-PEG-5000 and Mal-Phe-PEG-20000 converts Group A Rh(D)+ and B Rh(D)+ RBCs into RBCs with serologic behavior comparable to Group O Rh(D)- RBCs that are considered as universal RBCs for transfusion.

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.

Crystallization and preliminary crystallographic analysis of the catechol 2,3-dioxygenase PheB from Bacillus stearothermophilus BR219

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

Sugimoto, Keisuke; Matsufuzi, Kazuki; Ohnuma, Hiroaki

2006-02-01

PheB, an extradiol-cleaving catecholic dioxygenase, was crystallized by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant. The crystal belongs to the orthorhombic system, space group P2{sub 1}2{sub 1}2{sub 1}, and diffracts to 2.3 Å resolution. Class II extradiol-cleaving catecholic dioxygenase, a key enzyme of aromatic compound degradation in bacteria, cleaves the aromatic ring of catechol by adding two O atoms. PheB is one of the class II extradiol-cleaving catecholic dioxygenases and shows a high substrate specificity for catechol derivatives, which have one aromatic ring. In order to reveal the mechanism of the substrate specificity of PheB, PheB hasmore » been crystallized by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant. The space group of the obtained crystal was P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 65.5, b = 119.2, c = 158.7 Å. The crystal diffracted to 2.3 Å resolution.« less

Assessment of microplastic-sorbed contaminant bioavailability through analysis of biomarker gene expression in larval zebrafish.

PubMed

Sleight, Victoria A; Bakir, Adil; Thompson, Richard C; Henry, Theodore B

2017-03-15

Microplastics (MPs) are prevalent in marine ecosystems. Because toxicants (termed here "co-contaminants") can sorb to MPs, there is potential for MPs to alter co-contaminant bioavailability. Our objective was to demonstrate sorption of two co-contaminants with different physicochemistries [phenanthrene (Phe), log 10 K ow =4.57; and 17α-ethinylestradiol (EE2), log 10 K ow =3.67] to MPs; and assess whether co-contaminant bioavailability was increased after MP settlement. Bioavailability was indicated by gene expression in larval zebrafish. Both Phe and EE2 sorbed to MPs, which reduced bioavailability by a maximum of 33% and 48% respectively. Sorption occurred, but was not consistent with predictions based on co-contaminant physicochemistry (Phe having higher log 10 K ow was expected to have higher sorption). Contaminated MPs settled to the bottom of the exposures did not lead to increased bioavailability of Phe or EE2. Phe was 48% more bioavailable than predicted by a linear sorption model, organism-based measurements therefore contribute unique insight into MP co-contaminant bioavailability. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

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.

Pointing History Engine for the Spitzer Space Telescope

NASA Technical Reports Server (NTRS)

Bayard, David; Ahmed, Asif; Brugarolas, Paul

2007-01-01

The Pointing History Engine (PHE) is a computer program that provides mathematical transformations needed to reconstruct, from downlinked telemetry data, the attitude of the Spitzer Space Telescope (formerly known as the Space Infrared Telescope Facility) as a function of time. The PHE also serves as an example for development of similar pointing reconstruction software for future space telescopes. The transformations implemented in the PHE take account of the unique geometry of the Spitzer telescope-pointing chain, including all data on relative alignments of components, and all information available from attitude-determination instruments. The PHE makes it possible to coordinate attitude data with observational data acquired at the same time, so that any observed astronomical object can be located for future reference and re-observation. The PHE is implemented as a subroutine used in conjunction with telemetry-formatting services of the Mission Image Processing Laboratory of NASA s Jet Propulsion Laboratory to generate the Boresight Pointing History File (BPHF). The BPHF is an archival database designed to serve as Spitzer s primary astronomical reference documenting where the telescope was pointed at any time during its mission.

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.

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.

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.

Individuals with chronic low back pain demonstrate delayed onset of the back muscle activity during prone hip extension.

PubMed

Suehiro, Tadanobu; Mizutani, Masatoshi; Ishida, Hiroshi; Kobara, Kenichi; Osaka, Hiroshi; Watanabe, Susumu

2015-08-01

Prone hip extension (PHE) is commonly used in the evaluation of the stability of the lumbopelvic region. There is little evidence of difference in muscle activity onset timing between healthy individuals and individuals with chronic low back pain (CLBP) during PHE. The purpose of this study was to determine if individuals with and without CLBP differ in the onset time of the trunk and hip extensor muscles activity during PHE. The participants were 20 patients with CLBP and 20 healthy individuals. Electromyography data of the erector spinae, multifidus, gluteus maximus, and semitendinosus were collected during PHE using a surface electromyograph. Relative differences in the onset times between each muscle and the prime mover (i.e., the semitendinosus) were calculated. The onsets of the bilateral multifidus and contralateral erector spinae were significantly delayed in the CLBP group compared with the healthy group (p<0.001), despite the onset timings of leg movement not being significantly different between the groups. The onset times of the gluteus maximus and ipsilateral erector spinae showed no significant differences between the groups. These results suggest that individuals with CLBP use an altered, and possibly inadequate, trunk muscle recruitment pattern. Copyright © 2015 Elsevier Ltd. All rights reserved.

Prone Hip Extension Muscle Recruitment is Associated with Hamstring Injury Risk in Amateur Soccer.

PubMed

Schuermans, Joke; Van Tiggelen, Damien; Witvrouw, Erik

2017-09-01

'Core stability' is considered essential in rehabilitation and prevention. Particularly with respect to hamstring injury prevention, assessment and training of lumbo-pelvic control is thought to be key. However, supporting scientific evidence is lacking. To explore the importance of proximal neuromuscular function with regard to hamstring injury susceptibility, this study investigated the association between the Prone Hip Extension (PHE) muscle activation pattern and hamstring injury incidence in amateur soccer players. 60 healthy male soccer players underwent a comprehensive clinical examination, comprising a range of motion assessments and the investigation of the posterior chain muscle activation pattern during PHE. Subsequently, hamstring injury incidence was recorded prospectively throughout a 1.5-season monitoring period. Players who were injured presented a PHE activation pattern that differed significantly from those who did not. Contrary to the controls, hamstring activity onset was significantly delayed (p=0.018), resulting in a shifted activation sequence. Players were 8 times more likely to get injured if the hamstring muscles were activated after the lumbar erector spinae instead of vice versa (p=0.009). Assessment of muscle recruitment during PHE demonstrated to be useful in injury prediction, suggesting that neuromuscular coordination in the posterior chain influences hamstring injury vulnerability. © Georg Thieme Verlag KG Stuttgart · New York.

A comparison of the effects of substance P and shorter analogues on the synaptosomal ATPases activities in the rat brain.

PubMed

Lachowicz, L; Janiszewska, G

1987-01-01

The influence in vitro of SP and C-terminal fragments of analogues SP(5-11) (pyroGlu5, Tyr8); SP(6-11) (pyroGlu6, Tyr8); SP(6-11) (pyroGlu6, D-Phe7); SP(6-11) (pyroGlu6, D-Phe8) on the (Ca, Mg) and (Na, K) ATPases activities from synaptosomal membranes of cerebral cortex and hippocampus of rat brain were compared. The data obtained in this study indicate the following: 1. Substance P stimulates the activities of (Na, K) and (Ca, Mg) ATPases more effectively in synaptosomal membranes from hippocampus than cerebral cortex. 2. Heptapeptide SP(5-11) (pyroGlu5, Tyr8) causes a more distinct increase of (Ca, Mg) ATPase activity in cortical synaptosomal membranes than SP does. 3. The change of L-Phe conformation to D in position 7 in hexapeptide induces reduction of enzymes activities in hippocampus. 4. Especially important for the maintenance of biological activity of drugs is the replacement of Gln5 with pyroGlu6 and conformation of Phe residues. 5. SP and shorter analogues of fragments SP C-terminal SP regulate the active cation transport in synaptosomal membranes of cerebral cortex and hippocampus.

A Cyclic Tetrapeptide (“Cyclodal”) and Its Mirror-Image Isomer Are Both High-Affinity μ Opioid Receptor Antagonists

PubMed Central

Weltrowska, Grazyna; Nguyen, Thi M.-D.; Chung, Nga N.; Wood, JodiAnne; Ma, Xiaoyu; Guo, Jason; Wilkes, Brian C.; Ge, Yang; Laferrière, André; Coderre, Terence J.; Schiller, Peter W.

2016-01-01

Head-to-tail cyclization of the μ opioid receptor (MOR) agonist [Dmt1]DALDA (H-Dmt-d-Arg-Phe-Lys-NH2 (9; Dmt = 2′,6′-dimethyltyrosine) resulted in a highly active, selective MOR antagonist, c[-d-Arg-Phe-Lys-Dmt-] (1) (“cyclodal”), with subnanomolar binding affinity. A docking study of cyclodal using the crystal structure of MOR in the inactive form showed a unique binding mode with the two basic residues of the ligand forming salt bridges with the Asp127 and Glu229 receptor residues. Cyclodal showed high plasma stability and was able to cross the blood–brain barrier to reverse morphine-induced, centrally mediated analgesia when given intravenously. Surprisingly, the mirror-image isomer (optical antipode) of cyclodal, c[-Arg-d-Phe-d-Lys-d-Dmt-] (2), also turned out to be a selective MOR antagonist with 1 nM binding affinity, and thus, these two compounds represent the first example of mirror image opioid receptor ligands with both optical antipodes having high binding affinity. Reduction of the Lys-Dmt peptide bond in cyclodal resulted in an analogue, c[-d-Arg-Phe-LysΨ[CH2NH]Dmt-] (8), with MOR agonist activity. PMID:27676089

SERS study of transformation of phenylalanine to tyrosine under particle irradiation

NASA Astrophysics Data System (ADS)

Zhang, Jingjing; Huang, Qing; Yao, Guohua; Ke, Zhigang; Zhang, Hong; Lu, Yilin

2014-08-01

Surface enhanced Raman scattering or spectroscopy (SERS) is a very powerful analytical tool which has been widely applied in many scientific research and application fields. It is therefore also very intriguing for us to introduce SERS technique in the radiobiological research, where in many cases only a very few of biomolecules are subjected to changes which however can lead to significant biological effects. The radiation induced biochemical reactions are normally very sophisticated with different substances produced in the system, so currently it is still a big challenge for SERS to analyze such a mixture system which contains multiple analytes. In this context, this work aimed to establish and consolidate the feasibility of SERS as an effective tool in radiation chemistry, and this purpose, we employed SERS as a sensitive probe to a known process, namely, the oxidation of phenylalanine (Phe) under particle irradiation, where the energetic particles were obtained from either plasma discharge or electron-beam. During the irradiation, three types of tyrosine (Tyr), namely, p-Tyr, m-Tyr and o-Tyr were produced, and all these tyrosine isomers together with Phe could be identified and measured based on the SERS spectral analysis of the corresponding enhanced characteristic signals, namely, 1002 cm-1 for Phe, 1161 cm-1 for p-Tyr, 990 cm-1 for m-Tyr, and 970 cm-1 for o-Tyr, respectively. The estimation of the quantities of different tyrosine isomers were also given and verified by conventional method such as high performance liquid chromatography (HPLC). As for comparison of different ways of particle irradiation, our results also indicated that electron-beam irradiation was more efficient for converting Phe into Tyr than plasma discharge treatment, confirming the role of hydroxyl radicals in the Phe-Tyr conformation. Therefore, our work has not only demonstrated that SERS can be successfully applied in the radiobiological study, but also given insights into the mechanism for the interaction between particle radiation and biological systems.

Effects of side-chain orientation on the backbone conformation of the dehydrophenylalanine residue. Theoretical and X-ray study.

PubMed

Buczek, Aneta; Siodłak, Dawid; Bujak, Maciej; Broda, Małgorzata A

2011-04-21

Two E isomers of α,β-dehydro-phenylalanine, Ac-(E)-ΔPhe-NHMe (1a) and Ac-(E)-ΔPhe-NMe(2) (2a), have been synthesized and their low temperature structures determined by single-crystal X-ray diffraction. A systematic theoretical analysis was performed on these molecules and their Z isomers (1b and 2b). The ϕ,ψ potential energy surfaces were calculated at the MP2/6-31+G(d,p) and B3LYP/6-31+G(d,p) levels in the gas phase and at the B3LYP/6-31+G(d,p) level in the chloroform and water solutions with the SCRF-PCM method. All minima were fully optimized by the MP2 and DFT methods, and their relative stabilities were analyzed in terms of π-conjugation, internal H-bonds, and dipole-dipole interactions between carbonyl groups. The results indicate that all the studied compounds can adopt the conformation H (ϕ, ψ ≈ ±40°, ∓120°) which is atypical for standard amino acids residues. A different arrangement of the side chain in the E and Z isomers causes them to have different conformational preferences. In the presence of a polar solvent both Z isomers of ΔPhe (1b and 2b) are found to adopt the 3(10)-helical conformation (left- and right-handed are equally likely). On the other hand, this conformation is not accessible or highly energetic for E isomers of ΔPhe (1a and 2a). Those isomers have an intrinsic inclination to have an extended conformation. The conformational space of the Z isomers is much more restricted than that of the E derivative both in the gas phase and in solution. In the gas phase the E isomers of ΔPhe have lower energies than the Z ones, but in the aqueous solution the energy order is reversed.

Therapeutic brain modulation with targeted large neutral amino acid supplements in the Pah-enu2 phenylketonuria mouse model.

PubMed

van Vliet, Danique; Bruinenberg, Vibeke M; Mazzola, Priscila N; van Faassen, Martijn Hjr; de Blaauw, Pim; Pascucci, Tiziana; Puglisi-Allegra, Stefano; Kema, Ido P; Heiner-Fokkema, M Rebecca; van der Zee, Eddy A; van Spronsen, Francjan J

2016-11-01

Phenylketonuria treatment consists mainly of a Phe-restricted diet, which leads to suboptimal neurocognitive and psychosocial outcomes. Supplementation of large neutral amino acids (LNAAs) has been suggested as an alternative dietary treatment strategy to optimize neurocognitive outcome in phenylketonuria and has been shown to influence 3 brain pathobiochemical mechanisms in phenylketonuria, but its optimal composition has not been established. In order to provide additional pathobiochemical insight and develop optimal LNAA treatment, several targeted LNAA supplements were investigated with respect to all 3 biochemical disturbances underlying brain dysfunction in phenylketonuria. Pah-enu2 (PKU) mice received 1 of 5 different LNAA-supplemented diets beginning at postnatal day 45. Control groups included phenylketonuria mice receiving an isonitrogenic and isocaloric high-protein diet or the AIN-93M diet, and wild-type mice receiving the AIN-93M diet. After 6 wk, brain and plasma amino acid profiles and brain monoaminergic neurotransmitter concentrations were measured. Brain Phe concentrations were most effectively reduced by supplementation of LNAAs, such as Leu and Ile, with a strong affinity for the LNAA transporter type 1. Brain non-Phe LNAAs could be restored on supplementation, but unbalanced LNAA supplementation further reduced brain concentrations of those LNAAs that were not (sufficiently) included in the LNAA supplement. To optimally ameliorate brain monoaminergic neurotransmitter concentrations, LNAA supplementation should include Tyr and Trp together with LNAAs that effectively reduce brain Phe concentrations. The requirement for Tyr supplementation is higher than it is for Trp, and the relative effect of brain Phe reduction is higher for serotonin than it is for dopamine and norepinephrine. The study shows that all 3 biochemical disturbances underlying brain dysfunction in phenylketonuria can be targeted by specific LNAA supplements. The study thus provides essential information for the development of optimal LNAA supplementation as an alternative dietary treatment strategy to optimize neurocognitive outcome in patients with phenylketonuria. © 2016 American Society for Nutrition.

Predicting the safety and efficacy of buffer therapy to raise tumour pHe: an integrative modelling study

PubMed Central

Martin, N K; Robey, I F; Gaffney, E A; Gillies, R J; Gatenby, R A; Maini, P K

2012-01-01

Background: Clinical positron emission tomography imaging has demonstrated the vast majority of human cancers exhibit significantly increased glucose metabolism when compared with adjacent normal tissue, resulting in an acidic tumour microenvironment. Recent studies demonstrated reducing this acidity through systemic buffers significantly inhibits development and growth of metastases in mouse xenografts. Methods: We apply and extend a previously developed mathematical model of blood and tumour buffering to examine the impact of oral administration of bicarbonate buffer in mice, and the potential impact in humans. We recapitulate the experimentally observed tumour pHe effect of buffer therapy, testing a model prediction in vivo in mice. We parameterise the model to humans to determine the translational safety and efficacy, and predict patient subgroups who could have enhanced treatment response, and the most promising combination or alternative buffer therapies. Results: The model predicts a previously unseen potentially dangerous elevation in blood pHe resulting from bicarbonate therapy in mice, which is confirmed by our in vivo experiments. Simulations predict limited efficacy of bicarbonate, especially in humans with more aggressive cancers. We predict buffer therapy would be most effectual: in elderly patients or individuals with renal impairments; in combination with proton production inhibitors (such as dichloroacetate), renal glomular filtration rate inhibitors (such as non-steroidal anti-inflammatory drugs and angiotensin-converting enzyme inhibitors), or with an alternative buffer reagent possessing an optimal pK of 7.1–7.2. Conclusion: Our mathematical model confirms bicarbonate acts as an effective agent to raise tumour pHe, but potentially induces metabolic alkalosis at the high doses necessary for tumour pHe normalisation. We predict use in elderly patients or in combination with proton production inhibitors or buffers with a pK of 7.1–7.2 is most promising. PMID:22382688

Identification of residues in the insulin molecule important for binding to insulin-degrading enzyme.

PubMed

Affholter, J A; Cascieri, M A; Bayne, M L; Brange, J; Casaretto, M; Roth, R A

1990-08-21

Insulin-degrading enzyme (IDE) hydrolyzes insulin at a limited number of sites. Although the positions of these cleavages are known, the residues of insulin important in its binding to IDE have not been defined. To this end, we have studied the binding of a variety of insulin analogues to the protease in a solid-phase binding assay using immunoimmobilized IDE. Since IDE binds insulin with 600-fold greater affinity than it does insulin-like growth factor I (25 nM and approximately 16,000 nM, respectively), the first set of analogues studied were hybrid molecules of insulin and IGF I. IGF I mutants [insB1-17,17-70]IGF I, [Tyr55,Gln56]IGF I, and [Phe23,Phe24,Tyr25]IGF I have been synthesized and share the property of having insulin-like amino acids at positions corresponding to primary sites of cleavage of insulin by IDE. Whereas the first two exhibit affinities for IDE similar to that of wild type IGF I, the [Phe23,Phe24,Tyr25]IGF I analogue has a 32-fold greater affinity for the immobilized enzyme. Replacement of Phe-23 by Ser eliminates this increase. Removal of the eight amino acid D-chain region of IGF I (which has been predicted to interfere with binding to the 23-25 region) results in a 25-fold increase in affinity for IDE, confirming the importance of residues 23-25 in the high-affinity recognition of IDE. A similar role for the corresponding (B24-26) residues of insulin is supported by the use of site-directed mutant and semisynthetic insulin analogues. Insulin mutants [B25-Asp]insulin and [B25-His]insulin display 16- and 20-fold decreases in IDE affinity versus wild-type insulin.(ABSTRACT TRUNCATED AT 250 WORDS)

A false positive newborn screening result due to a complex allele carrying two frequent CF-causing variants.

PubMed

Bergougnoux, Anne; Boureau-Wirth, Amandine; Rouzier, Cécile; Altieri, Jean-Pierre; Verneau, Fanny; Larrieu, Lise; Koenig, Michel; Claustres, Mireille; Raynal, Caroline

2016-05-01

The detection of two frequent CFTR disease-causing variations in the context of a newborn screening program (NBS) usually leads to the diagnosis of cystic fibrosis (CF) and a relevant genetic counseling in the family. In the present study, CF-causing variants p.Phe508del (F508del) and c.3140-26A>G (3272-26A>G) were identified on a neonate with positive ImmunoReactive Trypsinogen test by the Elucigene™ CF30 kit. The CF diagnosis initially suggested, despite three inconclusive Sweat Chloride Tests (SCT), was finally ruled out after the familial segregation study combined with a negative SCT. Haplotype studies, based on the comparison of 80 p.Phe508del haplotypes, suggested a probable de novo occurrence of c.3140-26A>G on the p.Phe508del ancestral allele in this family. This false positive case emphasizes the importance of SCT in the NBS strategy. Moreover, it raises the need for familial segregation studies in CF and in overall molecular diagnosis strategy of autosomal recessive diseases. Copyright © 2016 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

A conformation-selective IR-UV study of the dipeptides Ac-Phe-Ser-NH2 and Ac-Phe-Cys-NH2: probing the SH···O and OH···O hydrogen bond interactions.

PubMed

Yan, Bin; Jaeqx, Sander; van der Zande, Wim J; Rijs, Anouk M

2014-06-14

The conformational preferences of peptides are mainly controlled by the stabilizing effect of intramolecular interactions. In peptides with polar side chains, not only the backbone but also the side chain interactions determine the resulting conformations. In this paper, the conformational preferences of the capped dipeptides Ac-Phe-Ser-NH2 (FS) and Ac-Phe-Cys-NH2 (FC) are resolved under laser-desorbed jet cooling conditions using IR-UV ion dip spectroscopy and density functional theory (DFT) quantum chemistry calculations. As serine (Ser) and cysteine (Cys) only differ in an OH (Ser) or SH (Cys) moiety; this subtle alteration allows us to study the effect of the difference in hydrogen bonding for an OH and SH group in detail, and its effect on the secondary structure. IR absorption spectra are recorded in the NH stretching region (3200-3600 cm(-1)). In combination with quantum chemical calculations the spectra provide a direct view of intramolecular interactions. Here, we show that both FS as FC share a singly γ-folded backbone conformation as the most stable conformer. The hydrogen bond strength of OH···O (FS) is stronger than that of SH···O (FC), resulting in a more compact gamma turn structure. A second conformer is found for FC, showing a β turn interaction.

The role of aromatic side-chains in amyloid growth and membrane interaction of the islet amyloid polypeptide fragment LANFLVH.

PubMed

Milardi, Danilo; Sciacca, Michele F M; Pappalardo, Matteo; Grasso, Domenico M; La Rosa, Carmelo

2011-01-01

Human islet amyloid polypeptide (hIAPP) is known to misfold and aggregate into amyloid deposits that may be found in pancreatic tissues of patients affected by type 2 diabetes. Recent studies have shown that the highly amyloidogenic peptide LANFLVH, corresponding the N-terminal 12-18 region of IAPP, does not induce membrane damage. Here we assess the role played by the aromatic residue Phe in driving both amyloid formation and membrane interaction of LANFLVH. To this aim, a set of variant heptapeptides in which the aromatic residue Phe has been substituted with a Leu and Ala is studied. Differential scanning calorimetry (DSC) and membrane-leakage experiments demonstrated that Phe substitution noticeably affects the peptide-induced changes in the thermotropic properties of the lipid bilayer but not its membrane damaging potential. Atomic force microscopy (AFM), ThT fluorescence and Congo red birefringence assays evidenced that the Phe residue is not required for fibrillogenesis, but it can influence the self-assembling kinetics. Molecular dynamics simulations have paralleled the outcome of the experimental trials also providing informative details about the structure of the different peptide assemblies. These results support a general theory suggesting that aromatic residues, although capable of affecting the self-assembly kinetics of small peptides and peptide-membrane interactions, are not essential either for amyloid formation or membrane leakage, and indicate that other factors such as β-sheet propensity, size and hydrophobicity of the side chain act synergistically to determine peptide properties.

Improving the selection efficiency of the counter-selection marker pheS* for the genetic engineering of Bacillus amyloliquefaciens.

PubMed

Kharchenko, Maria S; Teslya, Petr N; Babaeva, Maria N; Zakataeva, Natalia P

2018-05-01

Bacillus subtilis pheS was genetically modified to obtain a counter-selection marker with high selection efficiency in Bacillus amyloliquefaciens. The application of the new replication-thermosensitive integrative vector pNZTM1, containing this marker, pheS BsT255S/A309G , with a two-step replacement recombination procedure provides an effective tool for the genetic engineering of industrially important Bacillus species. Copyright © 2018. Published by Elsevier B.V.

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

A Conserved Hydrophobic Core in Gαi1 Regulates G Protein Activation and Release from Activated Receptor.

PubMed

Kaya, Ali I; Lokits, Alyssa D; Gilbert, James A; Iverson, T M; Meiler, Jens; Hamm, Heidi E

2016-09-09

G protein-coupled receptor-mediated heterotrimeric G protein activation is a major mode of signal transduction in the cell. Previously, we and other groups reported that the α5 helix of Gαi1, especially the hydrophobic interactions in this region, plays a key role during nucleotide release and G protein activation. To further investigate the effect of this hydrophobic core, we disrupted it in Gαi1 by inserting 4 alanine amino acids into the α5 helix between residues Gln(333) and Phe(334) (Ins4A). This extends the length of the α5 helix without disturbing the β6-α5 loop interactions. This mutant has high basal nucleotide exchange activity yet no receptor-mediated activation of nucleotide exchange. By using structural approaches, we show that this mutant loses critical hydrophobic interactions, leading to significant rearrangements of side chain residues His(57), Phe(189), Phe(191), and Phe(336); it also disturbs the rotation of the α5 helix and the π-π interaction between His(57) and Phe(189) In addition, the insertion mutant abolishes G protein release from the activated receptor after nucleotide binding. Our biochemical and computational data indicate that the interactions between α5, α1, and β2-β3 are not only vital for GDP release during G protein activation, but they are also necessary for proper GTP binding (or GDP rebinding). Thus, our studies suggest that this hydrophobic interface is critical for accurate rearrangement of the α5 helix for G protein release from the receptor after GTP binding. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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.

Biodegradation of Mixed PAHs by PAH-Degrading Endophytic Bacteria.

PubMed

Zhu, Xuezhu; Ni, Xue; Waigi, Michael Gatheru; Liu, Juan; Sun, Kai; Gao, Yanzheng

2016-08-09

Endophytic bacteria can promote plant growth, induce plant defence mechanisms, and increase plant resistance to organic contaminants. The aims of the present study were to isolate highly PAH-degrading endophytic bacteria from plants growing at PAH-contaminated sites and to evaluate the capabilities of these bacteria to degrade polycyclic aromatic hydrocarbons (PAHs) in vitro, which will be beneficial for re-colonizing target plants and reducing plant PAH residues through the inoculation of plants with endophytic bacteria. Two endophytic bacterial strains P₁ (Stenotrophomonas sp.) and P₃ (Pseudomonas sp.), which degraded more than 90% of phenanthrene (PHE) within 7 days, were isolated from Conyza canadensis and Trifolium pretense L., respectively. Both strains could use naphthalene (NAP), PHE, fluorene (FLR), pyrene (PYR), and benzo(a)pyrene (B(a)P) as the sole sources of carbon and energy. Moreover, these bacteria reduced the contamination of mixed PAHs at high levels after inoculation for 7 days; strain P₁ degraded 98.0% NAP, 83.1% FLR, 87.8% PHE, 14.4% PYR, and 1.6% B(a)P, and strain P₃ degraded 95.3% NAP, 87.9% FLR, 90.4% PHE, 6.9% PYR, and negligible B(a)P. Notably, the biodegradation of PAHs could be promoted through additional carbon and nitrogen nutrients; therein, beef extract was suggested as the optimal co-substrate for the degradation of PAHs by these two strains (99.1% PHE was degraded within 7 days). Compared with strain P₁, strain P₃ has more potential for the use in the removal of PAHs from plant tissues. These results provide a novel perspective in the reduction of plant PAH residues in PAH-contaminated sites through inoculating plants with highly PAH-degrading endophytic bacteria.

Ascorbic Acid Enhances the Accumulation of Polycyclic Aromatic Hydrocarbons (PAHs) in Roots of Tall Fescue (Festuca arundinacea Schreb.)

PubMed Central

Gao, Yanzheng; Li, Hui; Gong, Shuaishuai

2012-01-01

Plant contamination by polycyclic aromatic hydrocarbons (PAHs) is crucial to food safety and human health. Enzyme inhibitors are commonly utilized in agriculture to control plant metabolism of organic components. This study revealed that the enzyme inhibitor ascorbic acid (AA) significantly reduced the activities of peroxidase (POD) and polyphenol oxidase (PPO), thus enhancing the potential risks of PAH contamination in tall fescue (Festuca arundinacea Schreb.). POD and PPO enzymes in vitro effectively decomposed naphthalene (NAP), phenanthrene (PHE) and anthracene (ANT). The presence of AA reduced POD and PPO activities in plants, and thus was likely responsible for enhanced PAH accumulation in tall fescue. This conclusion is supported by the significantly enhanced uptake of PHE in plants in the presence of AA, and the positive correlation between enzyme inhibition efficiencies and the rates of metabolism of PHE in tall fescue roots. This study provides a new perspective, that the common application of enzyme inhibitors in agricultural production could increase the accumulation of organic contaminants in plants, hence enhancing risks to food safety and quality. PMID:23185628

Effect of surfactant amendment to PAHs-contaminated soil for phytoremediation by maize (Zea mays L.).

PubMed

Liao, Changjun; Liang, Xujun; Lu, Guining; Thai, Truonggiang; Xu, Wending; Dang, Zhi

2015-02-01

Understanding the uptake of organic pollutants by plants is an important part of the assessment of risks from crops grown on contaminated soils. This study was an investigation of the effects of surfactants added to PAHs-contaminated soil on the uptake and accumulation of PAHs in maize tissues during phytoremediation. The accumulation of phenanthrene (PHE) and pyrene (PYR) by maize plant was not influenced significantly by the surfactant amendment to the soil. The distribution of PHE and PYR in maize tissues was not positively correlated with the corresponding lipid contents. Remarkably, the concentrations of PHE (20.9 ng g(-1)) and PYR (0.9 ng g(-1)) in maize grain were similar to or even much lower than those in some foods. Moreover, surfactants could enhance the removal of pollutants from contaminated soil during phytoremediation, which might be due to surfactant desorption ability and microbial activity in soil. The study suggests that use of maize plant with surfactant is an alternative technology for remediation of PAHs-contaminated soils. Copyright © 2014 Elsevier Inc. All rights reserved.

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.

Cloning and sequencing of the pheP gene, which encodes the phenylalanine-specific transport system of Escherichia coli.

PubMed Central

Pi, J; Wookey, P J; Pittard, A J

1991-01-01

The phenylalanine-specific permease gene (pheP) of Escherichia coli has been cloned and sequenced. The gene was isolated on a 6-kb Sau3AI fragment from a chromosomal library, and its presence was verified by complementation of a mutant lacking the functional phenylalanine-specific permease. Subcloning from this fragment localized the pheP gene on a 2.7-kb HindIII-HindII fragment. The nucleotide sequence of this 2.7-kb region was determined. An open reading frame was identified which extends from a putative start point of translation (GTG at position 636) to a termination signal (TAA at position 2010). The assignment of the GTG as the initiation codon was verified by site-directed mutagenesis of the initiation codon and by introducing a chain termination mutation into the pheP-lacZ fusion construct. A single initiation site of transcription 30 bp upstream of the start point of translation was identified by the primer extension analysis. The pheP structural gene consists of 1,374 nucleotides specifying a protein of 458 amino acid residues. The PheP protein is very hydrophobic (71% nonpolar residues). A topological model predicted from the sequence analysis defines 12 transmembrane segments. This protein is highly homologous with the AroP (general aromatic transport) system of E. coli (59.6% identity) and to a lesser extent with the yeast permeases CAN1 (arginine), PUT4 (proline), and HIP1 (histidine) of Saccharomyces cerevisiae. Images PMID:1711024

Superactive amidated COOH-terminal glucagon analogues with no methionine or tryptophan.

PubMed

Murphy, W A; Coy, D H; Lance, V A

1986-01-01

The functions of the Trp-25 and Met-27 residues and the free carboxy terminus of glucagon have been debated for many years. Despite some semi-synthetic data to the contrary, comparison of the glucagon sequence with the other 5 members of this family of peptides, all of them amides and particularly growth hormone-releasing factor(1-29) amide and its recently described analogues, suggests that alterations to these positions should be quite well tolerated in terms of biological activity. To test this prediction, [Phe-25,Leu-27]-glucagon amide was synthesized in high yield and was found to actually have superior glycogenolytic activity (196%) to glucagon in the rat. Replacement of Gly-4 by D-Phe, which has been shown to give much enhanced glycogenolytic activity than glucagon itself, also increased the activity of [D-Phe-4,Phe-25,Leu-27]-glucagon amide (518%). The L-Phe-4-analogue, [Phe-4,25,Leu-27]-glucagon amide, in contrast, was 20 times less active (30%), strongly suggesting the presence of a beta-bend in this N-terminal region of glucagon. This was supported by Chou-Fasman structural predictions which indicate extensive folding in the 1-15 region. Indeed, additional conformational restriction by substitution of D-Ser in position 2 of glucagon also increased activity to 226%. [D-Gln-3]-glucagon was slightly less active (74%) than glucagon. Chou-Fasman calculations on glucagon were compared to similar treatments of the VIP, secretin, PHI, and GRF(1-29) sequences.

Simultaneous identification/determination system for phentolamine and sildenafil as adulterants in soft drinks advertising roborant nutrition.

PubMed

Mikami, Eiichi; Ohno, Tsutomu; Matsumoto, Hiroshi

2002-12-04

An easily available, simultaneous identification/determination procedure for phentolamine (PHE) and sildenafil (SIL) in adulterated dietary supplements was established by using a combination of three different analytical methods; thin-layer chromatography (TLC), liquid chromatography-mass spectrometry (LC/MS) and a high-performance liquid chromatography (HPLC)/photo-diode-array. The sample solution for TLC was applied to silica gel 60 F(254) plates with chloroform/ammonia solution (28)/methanol (70:5:3, lower layer) and chloroform/diethylamine/methanol (15:3:2) as the developing solvent. Spots were located under UV radiation at 254 nm. Mass spectra of PHE and SIL by LC/MS were investigated with electrospray ionization (ESI) interface, under both positive and negative ion mode. The HPLC analysis was performed on a column of Wakosil 5C18 (4.6 mm x 150 mm, 5 microm) with water/methanol/acetonitrile/triethylamine (580:250:170:1) adjusted with phosphoric acid to pH 3.0 as the mobile phase, and the effluent was monitored with a photo-diode-array detector. Quantitative HPLC analysis of PHE and SIL were detected at 280 nm. When this procedure was applied to commercial soft drinks, PHE and SIL were identified and determined at a concentration of 17 mg PHE and 44 mg SIL per bottle, respectively. The procedure described here is available for the screening of PHE and SIL in adulterated supplements. Copyright 2002 Elsevier Science Ireland Ltd.

Kirromycin, an Inhibitor of Protein Biosynthesis that Acts on Elongation Factor Tu

PubMed Central

Wolf, Heinz; Chinali, Gianni; Parmeggiani, Andrea

1974-01-01

Kirromycin, a new inhibitor of protein synthesis, is shown to interfere with the peptide transfer reaction by acting on elongation factor Tu (EF-Tu). All the reactions associated with this elongation factor are affected. Formation of the EF-Tu·GTP complex is strongly stimulated. Peptide bond formation is prevented only when Phe-tRNAPhe is bound enzymatically to ribosomes, presumably because GTP hydrolysis associated with enzymatic binding of Phe-tRNAPhe is not followed by release of EF-Tu·GDP from the ribosome. This antibiotic also enables EF-Tu to catalyze the binding of Phe-tRNAPhe to the poly(U)·ribosome complex even in the absence of GTP. EF-Tu activity in the GTPase reaction is dramatically affected by kirromycin: GTP hydrolysis, which normally requires ribosomes and aminoacyl-tRNA, takes place with the elongation factor alone. This GTPase shows the same Km for GTP as the one dependent on Phe-tRNAPhe and ribosomes in the absence of the antibiotic. Ribosomes and Phe-tRNAPhe, but not tRNAPhe or Ac-Phe-tRNAPhe, stimulate the kirromycin-induced EF-Tu GTPase. These results indicate that the catalytic center of EF-Tu GTPase that is dependent upon aminoacyl-tRNA and ribosomes is primarily located on the elongation factor. In conclusion, kirromycin can substitute for GTP, aminoacyl-tRNA, or ribosomes in various reactions involving EF-Tu, apparently by affecting the allosteric controls between the sites on the EF-Tu molecule interacting with these components. PMID:4373734

In vivo amino acid transport of subacute and chronic cerebral infarction evaluated by 12-18F-phenylalanine

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

Shimosegawa, E.; Miura, S.; Murakami, M.

1994-05-01

On the basis of previous validation of kinetic two-compartment model and the determination of normal values of three parameters (k{sub 1}:influx rate constant, k{sub 2}:outflux rate constant, Vd:distribution volume), PET measurements of in vivo amino acid transport from blood to brain using L-(2-18F)-fluorophenylalanine ({sup 18}F-Phe) were undergone in the patients with cerebral infarction. The purposes of this study are to evaluate the alteration of amino acid transport in subacute and chronic stage of cerebral infarction and to compare with cerebral blood flow (CBF) and oxygen metabolism. Dynamic {sup 18}F-Phe PET studies for 50 minutes were performed in 7 patients withmore » cerebral infarction. The input function was obtained by 27 points of arterial sampling. In all patients, measurements of CBF, cerebral blood volume (CBV), cerebral metabolic rate of oxygen (CMRO{sub 2}), and oxygen extraction fraction (OEF) were made on the same day of {sup 18}F-Phe PET measurement. Each patient was studied twice, within 2 weeks of the onset and 3 months later. Weighted integration technique with table look-up method was applied for the reconstruction of parametric images of {sup 18}F-Phe and ROI analysis of k{sub 1}, k{sub 2}, and Vd. In subacute stage, significant reduction of k{sub 2} value in infarct area was observed when compared to that in periinfarct area (p<0.05) and in normal cortices (p<0.001). k{sub 1} value in this stage showed only slightly decrease in infarct area, therefore, Vd value in infarct area increased significantly compared to normal cortices (p<0.001). In chronic stage, both k{sub 1} and k{sub 2} values in infarct area were significantly lower than that in normal cortices (p<0.001), and corresponding Vd value reduced to normal level. Correlativity between kinetic parameters of {sup 18}F-Phe and CBF or oxygen metabolism was not observed both in subacute and chronic stage of infarction.« less

Characterization of the Role of a Highly Conserved Sequence in ATP Binding Cassette Transporter G (ABCG) Family in ABCG1 Stability, Oligomerization, and Trafficking

PubMed Central

2013-01-01

ATP-binding cassette transporter G1 (ABCG1) mediates cholesterol and oxysterol efflux onto lipidated lipoproteins and plays an important role in macrophage reverse cholesterol transport. Here, we identified a highly conserved sequence present in the five ABCG transporter family members. The conserved sequence is located between the nucleotide binding domain and the transmembrane domain and contains five amino acid residues from Asn at position 316 to Phe at position 320 in ABCG1 (NPADF). We found that cells expressing mutant ABCG1, in which Asn316, Pro317, Asp319, and Phe320 in the conserved sequence were replaced with Ala simultaneously, showed impaired cholesterol efflux activity compared with wild type ABCG1-expressing cells. A more detailed mutagenesis study revealed that mutation of Asn316 or Phe 320 to Ala significantly reduced cellular cholesterol and 7-ketocholesterol efflux conferred by ABCG1, whereas replacement of Pro317 or Asp319 with Ala had no detectable effect. To confirm the important role of Asn316 and Phe320, we mutated Asn316 to Asp (N316D) and Gln (N316Q), and Phe320 to Ile (F320I) and Tyr (F320Y). The mutant F320Y showed the same phenotype as wild type ABCG1. However, the efflux of cholesterol and 7-ketocholesterol was reduced in cells expressing ABCG1 mutant N316D, N316Q, or F320I compared with wild type ABCG1. Further, mutations N316Q and F320I impaired ABCG1 trafficking while having no marked effect on the stability and oligomerization of ABCG1. The mutant N316Q and F320I could not be transported to the cell surface efficiently. Instead, the mutant proteins were mainly localized intracellularly. Thus, these findings indicate that the two highly conserved amino acid residues, Asn and Phe, play an important role in ABCG1-dependent export of cellular cholesterol, mainly through the regulation of ABCG1 trafficking. PMID:24320932

Nervus terminalis projection to the retina in the 'four-eyed' fish, Anableps anableps.

PubMed

Meyer, D L; Malz, C R; Jadhao, A G

1996-08-02

The eye of the surface dwelling 'four-eyed' fish, Anableps possesses an aquatic and an aerial optical system. The aerial system is strongly hyperopic when the animal dives, i.e. during mating, and the dorsal pupil is submerged. We studied the retino-petal nervus terminals projection to the aerial and to the aquatic retina by Phe-Met-Arg-Phe-NH2 (FMRF) immunocytochemistry and found both to be equally innervated. This finding sheds doubt on the proposed functional significance of this projection for reproductive behaviour.

"Trampoline" ejection of organic molecules from graphene and graphite via keV cluster ions impacts.

PubMed

Verkhoturov, Stanislav V; Gołuński, Mikołaj; Verkhoturov, Dmitriy S; Geng, Sheng; Postawa, Zbigniew; Schweikert, Emile A

2018-04-14

We present the data on ejection of molecules and emission of molecular ions caused by single impacts of 50 keV C 60 2+ on a molecular layer of deuterated phenylalanine (D8Phe) deposited on free standing, 2-layer graphene. The projectile impacts on the graphene side stimulate the abundant ejection of intact molecules and the emission of molecular ions in the transmission direction. To gain insight into the mechanism of ejection, Molecular Dynamic simulations were performed. It was found that the projectile penetrates the thin layer of graphene, partially depositing the projectile's kinetic energy, and molecules are ejected from the hot area around the hole that is made by the projectile. The yield, Y, of negative ions of deprotonated phenylalanine, (D8Phe-H) - , emitted in the transmission direction is 0.1 ions per projectile impact. To characterize the ejection and ionization of molecules, we have performed the experiments on emission of (D8Phe-H) - from the surface of bulk D8Phe (Y = 0.13) and from the single molecular layer of D8Phe deposited on bulk pyrolytic graphite (Y = 0.15). We show that, despite the similar yields of molecular ions, the scenario of the energy deposition and ejection of molecules is different for the case of graphene due to the confined volume of projectile-analyte interaction. The projectile impact on the graphene-D8Phe sample stimulates the collective radial movement of analyte atoms, which compresses the D8Phe layer radially from the hole. At the same time, this compression bends and stretches the graphene membrane around the hole thus accumulating potential energy. The accumulated potential energy is transformed into the kinetic energy of correlated movement upward for membrane atoms, thus the membrane acts as a trampoline for the molecules. The ejected molecules are effectively ionized; the ionization probability is ∼30× higher compared to that obtained for the bulk D8Phe target. The proposed mechanism of ionization involves tunneling of electrons from the vibrationally excited area around the hole to the molecules. Another proposed mechanism is a direct proton transfer exchange, which is suitable for a bulk target: ions of molecular fragments (i.e., CN - ) generated in the impact area interact with intact molecules from the rim of this area. There is a direct proton exchange process for the system D8Phe molecule + CN - .

"Trampoline" ejection of organic molecules from graphene and graphite via keV cluster ions impacts

NASA Astrophysics Data System (ADS)

Verkhoturov, Stanislav V.; Gołuński, Mikołaj; Verkhoturov, Dmitriy S.; Geng, Sheng; Postawa, Zbigniew; Schweikert, Emile A.

2018-04-01

We present the data on ejection of molecules and emission of molecular ions caused by single impacts of 50 keV C602+ on a molecular layer of deuterated phenylalanine (D8Phe) deposited on free standing, 2-layer graphene. The projectile impacts on the graphene side stimulate the abundant ejection of intact molecules and the emission of molecular ions in the transmission direction. To gain insight into the mechanism of ejection, Molecular Dynamic simulations were performed. It was found that the projectile penetrates the thin layer of graphene, partially depositing the projectile's kinetic energy, and molecules are ejected from the hot area around the hole that is made by the projectile. The yield, Y, of negative ions of deprotonated phenylalanine, (D8Phe-H)-, emitted in the transmission direction is 0.1 ions per projectile impact. To characterize the ejection and ionization of molecules, we have performed the experiments on emission of (D8Phe-H)- from the surface of bulk D8Phe (Y = 0.13) and from the single molecular layer of D8Phe deposited on bulk pyrolytic graphite (Y = 0.15). We show that, despite the similar yields of molecular ions, the scenario of the energy deposition and ejection of molecules is different for the case of graphene due to the confined volume of projectile-analyte interaction. The projectile impact on the graphene-D8Phe sample stimulates the collective radial movement of analyte atoms, which compresses the D8Phe layer radially from the hole. At the same time, this compression bends and stretches the graphene membrane around the hole thus accumulating potential energy. The accumulated potential energy is transformed into the kinetic energy of correlated movement upward for membrane atoms, thus the membrane acts as a trampoline for the molecules. The ejected molecules are effectively ionized; the ionization probability is ˜30× higher compared to that obtained for the bulk D8Phe target. The proposed mechanism of ionization involves tunneling of electrons from the vibrationally excited area around the hole to the molecules. Another proposed mechanism is a direct proton transfer exchange, which is suitable for a bulk target: ions of molecular fragments (i.e., CN-) generated in the impact area interact with intact molecules from the rim of this area. There is a direct proton exchange process for the system D8Phe molecule + CN-.

The metabolism of 5-hydroxytryptamine and beta-phenylethylamine in perfused rat lung and in vitro.

PubMed Central

Bakhle, Y S; Youdim, M B

1979-01-01

1 Metabolism of 5-hydroxytryptamine (5-HT) and beta-phenylethylamine (PHE) by monoamine oxidase (MAO) was investigated in rat isolated lungs and in mitochondrial preparations from rat lung. 2. In perfused lungs 5-HT metabolism had an apparent Km of 2 microgram and PHE metaoblism a Km of 54 microgram, whereas in vitro the Km values were 330 microgram and 28 microgram respectively. 3 In vitro, MAO activity had substrate and inhibitor specificities compatible with the presence of A and B types of MAO. 4 In perfused lung, metabolism of 5-HT but not that of PHE was inhibited by desmethylimipramine. 5 These results show that PHE metabolism in perfused lung, unlike that of other metabolized amines, is not limited by transport and the transport process for PHE is unlike that of 5-HT or noradrenaline. 6 These results also show that the kinetic parameters obtained for MAO activity in vitro do not generally apply to the isolated lung where transport of substrate can be the deciding factor. This discrepancy emphasizes that the enzymic properties of the whole organ cannot relaibly be deduced from its enzymic content. PMID:32944

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.

Total enzymatic synthesis of cholecystokinin CCK-5.

PubMed

Xiang, H; Xiang, G Y; Lu, Z M; Guo, L; Eckstein, H

2004-08-01

This paper describes the enzymatic synthesis of the C-terminal fragment H-Gly-Trp-Met-Asp-Phe-NH2 of cholecystokinin. Immobilized enzymes were used for the formation of all peptide bonds except thermolysin. Beginning the synthesis with phenylacetyl (PhAc) glycine carboxamidomethyl ester (OCam) and H-Trp-OMe by using immobilized papain as biocatalyst in buffered ethyl acetate, the dipeptide methyl ester was then coupled directly with Met-OEt.HCl by alpha-chymotrypsin/Celite 545 in a solvent free system. For the 3+2 coupling PhAc-Gly-Trp-Met-OEt had to be converted into its OCam ester. The other fragment H-Asp(OMe)-Phe-NH2 resulted from the coupling of Cbo-Asp(OMe)-OH with H-Phe-NH2.HCl and thermolysin as catalyst, followed by catalytic hydrogenation. Finally PhAc-Gly-Trp-Met-Asp-Phe-NH2 was obtained in a smooth reaction from PhAc-Gly-Trp-Met-OCam and H-Asp(OMe)-Phe-NH2 with alpha-chymotrypsin/Celite 545 in acetonitrile, followed by basic hydrolysis of the beta-methyl ester. The PhAc-group is removed with penicillin G amidase and CCK-5 is obtained in an overall isolated yield of 19.6%.

[Study on the secondary metabolites from the marine sponge Phakellia fusca fungi PF18].

PubMed

Tang, Li-Dan; Liang, Yuan-Wei; Liao, Xiao-Jian; Liang, Qiu; Xu, Shi-Hai; Li, Zhi-Yong

2011-12-01

To study the secondary metabolites from the marine sponge Phakellia fusca epiphytic fungi. The compounds were isolated by column chromatography over silica gel and purified by Sephadex LH-20 column chromatography and preparative TLC. The structures were elucidated by means of physiochemical properties and spectroscopic analyses. Four compounds were separated and identified as: cyclo-(L-Val-L-Pro) (1), cyclo-(L-Phe-L-Pro) (2), cyclo-(L-Tyr-L-Pro) (3), cyclo-(3-hydroxy-4-methyldecanoyl-Gly-L-Val-D-Leu-L-Ala-L-Phe) (4). Compounds 1-4 are obtained from the marine sponge Phakellia fusca epiphytic fungi for the first time.

Tablet Splitting of Antiepileptic Drugs in Pediatric Epilepsy: Potential Effect on Plasma Drug Concentrations.

PubMed

Nidanapu, Ravi Prasad; Rajan, Sundaram; Mahadevan, Subramanian; Gitanjali, Batmanabane

2016-12-01

Tablet splitting is the process of dividing a tablet into portions to obtain a prescribed dose of medication. Very few studies have investigated whether split parts of a tablet deliver the expected amount of drug to patients. Our objectives were to evaluate the split parts of adult-dose tablet formulations for percentage of weight deviation, weight uniformity, weight loss, drug content, and the content uniformity of four antiepileptic drugs (AEDs) prescribed to pediatric patients. We also measured AED plasma concentrations in the children. We chose to study first-line AEDs (phenytoin sodium [PHE], sodium valproate [SVA], carbamazepine, and phenobarbitone) as they are routinely prescribed in India. We asked caregivers to perform the same splitting process they follow in their homes on three whole tablets during their routine visit to the outpatient department. After caregivers split the tablets, we studied the weight and content of the split parts. We also used high-performance liquid chromatography to study plasma drug concentrations in children who had received split AEDs for at least 4 months. A total of 168 caregivers participated in the study, and we analyzed 1098 split tablet parts. In total, 539 (49.0 %) split parts were above the specified limit of the 2010 Indian Pharmacopeia (IP) acceptable percentage weight deviation (PHE 169 [48.8 %], SVA 187 [51.9 %], carbamazepine 56 [41.1 %], phenobarbitone 127 [49.6 %]); 456 (41.5 %) split parts were outside the proxy IP specification for drug content (PHE 135 [39.0 %], SVA 140 [38.8 %], carbamazepine 51 [37.5 %], phenobarbitone 130 [50.7 %]), and 253 split parts were outside the acceptable content uniformity range of <85 % and >115 % (PHE 85 [24.5 %], SVA 98 [27.2 %], carbamazepine 14 [10.2 %], phenobarbitone 56 [21.8 %]). In total, 130 (72.2 %) patients had plasma drug concentrations outside the therapeutic range (PHE 36 [72.0 %], SVA 39 [78.0 %], carbamazepine 34 [68.0 %], phenobarbitone 21 [70.0 %]). Splitting adult-dosage formulations of AEDs results in patients not receiving the optimal dose. Plasma drug concentrations are also not optimal. Pediatric dosage formulations should be preferred to splitting adult-dosage formulations in pediatric epilepsy.

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.

[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.

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.

Mutations at the S1 sites of methionine aminopeptidases from Escherichia coli and Homo sapiens reveal the residues critical for substrate specificity.

PubMed

Li, Jing-Ya; Cui, Yong-Mei; Chen, Ling-Ling; Gu, Min; Li, Jia; Nan, Fa-Jun; Ye, Qi-Zhuang

2004-05-14

Methionine aminopeptidase (MetAP) catalyzes the removal of methionine from newly synthesized polypeptides. MetAP carries out this cleavage with high precision, and Met is the only natural amino acid residue at the N terminus that is accepted, although type I and type II MetAPs use two different sets of residues to form the hydrophobic S1 site. Characteristics of the S1 binding pocket in type I MetAP were investigated by systematic mutation of each of the seven S1 residues in Escherichia coli MetAP type I (EcMetAP1) and human MetAP type I (HsMetAP1). We found that Tyr-65 and Trp-221 in EcMetAP1, as well as the corresponding residues Phe-197 and Trp-352 in HsMetAP1, were essential for the hydrolysis of a thiopeptolide substrate, Met-S-Gly-Phe. Mutation of Phe-191 to Ala in HsMetAP1 caused inactivity in contrast to the full activity of EcMetAP1(Y62A), which may suggest a subtle difference between the two type I enzymes. The more striking finding is that mutation of Cys-70 in EcMetAP1 or Cys-202 in HsMetAP1 opens up the S1 pocket. The thiopeptolides Leu-S-Gly-Phe and Phe-S-Gly-Phe, with previously unacceptable Leu or Phe as the N-terminal residue, became efficient substrates of EcMetAP1(C70A) and HsMetAP1(C202A). The relaxed specificity shown in these S1 site mutants for the N-terminal residues was confirmed by hydrolysis of peptide substrates and inhibition by reaction products. The structural features at the enzyme active site will be useful information for designing specific MetAP inhibitors for therapeutic applications.

Validation of amino-acids measurement in dried blood spot by FIA-MS/MS for PKU management.

PubMed

Bruno, C; Dufour-Rainfray, D; Patin, F; Vourc'h, P; Guilloteau, D; Maillot, F; Labarthe, F; Tardieu, M; Andres, C R; Emond, P; Blasco, H

2016-09-01

Phenylketonuria (PKU) is a metabolic disorder leading to high concentrations of phenylalanine (Phe) and low concentrations of tyrosine (Tyr) in blood and brain that may be neurotoxic. This disease requires a regular monitoring of plasma Phe and Tyr as well as branched-chain amino-acids concentrations to adapt the Phe-restricted diet and other therapy that may be prescribed in PKU. We validated a Flow Injection Analysis tandem Mass Spectrometry (FIA-MS/MS) to replace the enzymatic method routinely used for neonatal screening in order to monitor in parallel to Phe, Tyr and branched-chain amino-acids not detected by the enzymatic method. We ascertained the performances of the method: linearity, detection and quantification limits, contamination index, accuracy. We cross validated the FIA-MS/MS and enzymatic methods and we evaluated our own reference ranges to monitor Phe, Tyr, Leu, Val on 59 dried blood spots of normal controls. We also evaluated Tyr, Leu and Val concentrations in PKU patients to detect some potential abnormalities, not evaluated by the enzymatic method. We developed a rapid method with excellent performances including precision and accuracy <15%. We noted an excellent correlation of Phe concentrations between FIA-MS/MS and enzymatic methods (p<0.0001) based on our database which are similar to references ranges published. We observed that 50% of PKU patients had lower concentrations of Tyr, Leu and/or Val that could not be detected by the enzymatic method. Based on laboratory accreditation recommendations, we validated a robust, rapid and reliable FIA-MS/MS method to monitor plasma Phe concentrations but also Tyr, Leu and Val concentrations, suitable for PKU management. We evaluated our own reference ranges of concentration for a routine application of this method. Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Functional Characterization of a Robust Marine Microbial Esterase and Its Utilization in the Stereo-Selective Preparation of Ethyl (S)-3-Hydroxybutyrate.

PubMed

Wang, Yilong; Zhang, Yun; Hu, Yunfeng

2016-11-01

One novel microbial esterase PHE21 was cloned from the genome of Pseudomonas oryzihabitans HUP022 identified from the deep sea of the Western Pacific. PHE21 was heterologously expressed and functionally characterized to be a robust esterase which behaved high resistance to various metal ions, organic solvents, surfactants, and NaCl. Despite the fact that the two enantiomers of ethyl 3-hydroxybutyrate were hard to be enzymatically resolved before, we successfully resolved racemic ethyl 3-hydroxybutyrate through direct hydrolysis reactions and generated chiral ethyl (S)-3-hydroxybutyrate using esterase PHE21. After process optimization, the enantiomeric excess, the conversion rate, and the yield of desired product ethyl (S)-3-hydroxybutyrate could reach 99, 65, and 87 %, respectively. PHE21 is a novel marine microbial esterase with great potential in asymmetric synthesis as well as in other industries.

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.

Mitochondrial transfer RNA(Phe) mutation associated with a progressive neurodegenerative disorder characterized by psychiatric disturbance, dementia, and akinesia-rigidity.

PubMed

Young, Tim M; Blakely, Emma L; Swalwell, Helen; Carter, Janet E; Kartsounis, Luke D; O'Donovan, Dominic G; Turnbull, Douglass M; Taylor, Robert W; de Silva, Rajith N

2010-11-01

Mitochondrial diseases are characterized by wide phenotypic and genetic variability, but presentations in adults with akinetic rigidity and hyperkinetic movement disorders are rare. To describe clinically a subject with progressive neurodegeneration characterized by psychosis, dementia, and akinesia-rigidity, and to associate this phenotype with a novel mitochondrial transfer RNA(Phe) (tRNA(Phe)) (MTTF) mutation. Case description and detailed laboratory investigations of a 57-year-old woman at a university teaching hospital and a specialist mitochondrial diagnostic laboratory. Histopathological findings indicated that an underlying mitochondrial abnormality was responsible for the subject's progressive neurological disorder, with mitochondrial genome sequencing revealing a novel m.586G>A MTTF mutation. The clinical phenotypes associated with mitochondrial disorders may include akinesia-rigidity and psychosis. Our findings further broaden the spectrum of neurological disease associated with mitochondrial tRNA(Phe) mutations.

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.

Transcriptome profiling and physiological studies reveal a major role for aromatic amino acids in mercury stress tolerance in rice seedlings.

PubMed

Chen, Yun-An; Chi, Wen-Chang; Trinh, Ngoc Nam; Huang, Li-Yao; Chen, Ying-Chih; Cheng, Kai-Teng; Huang, Tsai-Lien; Lin, Chung-Yi; Huang, Hao-Jen

2014-01-01

Mercury (Hg) is a serious environmental pollution threat to the planet. The accumulation of Hg in plants disrupts many cellular-level functions and inhibits growth and development, but the mechanism is not fully understood. To gain more insight into the cellular response to Hg, we performed a large-scale analysis of the rice transcriptome during Hg stress. Genes induced with short-term exposure represented functional categories of cell-wall formation, chemical detoxification, secondary metabolism, signal transduction and abiotic stress response. Moreover, Hg stress upregulated several genes involved in aromatic amino acids (Phe and Trp) and increased the level of free Phe and Trp content. Exogenous application of Phe and Trp to rice roots enhanced tolerance to Hg and effectively reduced Hg-induced production of reactive oxygen species. Hg induced calcium accumulation and activated mitogen-activated protein kinase. Further characterization of the Hg-responsive genes we identified may be helpful for better understanding the mechanisms of Hg in plants.

Propellant Handler's Ensemble (PHE) Aka Self-Contained Atmospheric Protective Ensemble (SCAPE), Ventilator Improvement Study Project

NASA Technical Reports Server (NTRS)

Oliva-Buisson, Yvette J. (Compiler)

2014-01-01

The overall objective for this project is to evaluate two candidate alternatives for the existing Propellant Handler's Ensemble (PHE) escape ventilator. The new candidate ventilators use newer technology with similar quantities of air at approximately half the weight of the current ventilator. Ventilators are typically used to ingress/egress a hazardous work area when hard line air is provided at the work area but the hose is not long enough to get the operator to and from the staging area to the work area. The intent of this test is to verify that the new ventilators perform as well as or better than the current ventilators in maintaining proper oxygen (O2) and carbon dioxide (CO2) levels in the PHE during a typical use for the rated time period (10 minutes). We will evaluate two new units comparing them to the existing unit. Subjects will wear the Category I version of the Propellant Handler's Ensemble with the rear suit pouch snapped.

Spectrum of small-bowel mucosal abnormalities identified by capsule endoscopy in patients with portal hypertension of varied etiology.

PubMed

Chandrasekar, T S; Janakan, Gokul Bollu; Chandrasekar, Viveksandeep Thoguluva; Kalamegam, Raja Yogesh; Suriyanarayanan, Sathiamoorthy; Sanjeevaraya, Prasad Menta

2017-01-01

Bleeding from small intestinal ectopic varices and persistent anemia caused by portal hypertensive enteropathy (PHE) can be very challenging. Capsule endoscopy (CE) is one of the best noninvasive modalities in identifying such lesions. The aims of this study are to study the prevalence of small-bowel changes related to portal hypertension (PHT) and to correlate them with the observations related to the effects of portal hypertension in the esophagus, stomach, and colon. Thirty-two patients with various etiologies of PHT with either anemia or gastrointestinal bleed were included along with age- and sex-matched controls without PHT. All patients underwent blood tests, gastroscopy, colonoscopy, and CE. The small-bowel findings by CE were categorized as inflammatory-like and vascular lesions. The small-bowel changes were analyzed to find out any association with various demographic, clinical, and endoscopic variables. Thirty-one out of 32 patients with PHT (96.8%) had PHE identified by CE. Of them, 31 (96.8%) had inflammatory-like appearance, 11 (34.4%) had vascular lesions, and 2 (6.2%) had small-bowel varices. Inflammatory-like appearance was noted in eight (25%) and angiodysplastic lesions in two (6.2%) controls. Findings compatible with PHE were detected in 96.8% of the patients and 25% of the controls (X 2 =34.72, p=0.000).The presence of PHE was not associated with any of the above-mentioned variables. Small-bowel mucosal changes were seen in significantly higher number of patients with PHT with anemia.

Application of SAIL phenylalanine and tyrosine with alternative isotope-labeling patterns for protein structure determination.

PubMed

Takeda, Mitsuhiro; Ono, Akira M; Terauchi, Tsutomu; Kainosho, Masatsune

2010-01-01

The extensive collection of NOE constraint data involving the aromatic ring signals is essential for accurate protein structure determination, although it is often hampered in practice by the pervasive signal overlapping and tight spin couplings for aromatic rings. We have prepared various types of stereo-array isotope labeled phenylalanines (epsilon- and zeta-SAIL Phe) and tyrosine (epsilon-SAIL Tyr) to overcome these problems (Torizawa et al. 2005), and proven that these SAIL amino acids provide dramatic spectral simplification and sensitivity enhancement for the aromatic ring NMR signals. In addition to these SAIL aromatic amino acids, we recently synthesized delta-SAIL Phe and delta-SAIL Tyr, which allow us to observe and assign delta-(13)C/(1)H signals very efficiently. Each of the various types of SAIL Phe and SAIL Tyr yields well-resolved resonances for the delta-, epsilon- or zeta-(13)C/(1)H signals, respectively, which can readily be assigned by simple and robust pulse sequences. Since the delta-, epsilon-, and zeta-proton signals of Phe/Tyr residues give rise to complementary NOE constraints, the concomitant use of various types of SAIL-Phe and SAIL-Tyr would generate more accurate protein structures, as compared to those obtained by using conventional uniformly (13)C, (15)N-double labeled proteins. We illustrated this with the case of an 18.2 kDa protein, Escherichia coli peptidyl-prolyl cis-trans isomerase b (EPPIb), and concluded that the combined use of zeta-SAIL Phe and epsilon-SAIL Tyr would be practically the best choice for protein structural determinations.

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.

Spectroscopic characterization of the SH2- and active site-directed peptide sequences of a bivalent Src kinase inhibitor.

PubMed

Desamero, Ruel Z B; Kang, Jeonghee; Dol, Chrystel; Chinwong, Justina; Walters, Karim; Sivarajah, Thulashie; Profit, Adam A

2009-07-01

The spectral properties of the SH2 and active site-directed sequences of the bivalent Src kinase inhibitor Ac-EELL(F5)Phe-(GABA)3-pYEEIE-amide (1) have been determined. Ac-pYEEIE-amide (2) and AcEELL(F5)Phe-amide (3), as well as the amino acids phosphotyrosine (pTyr) and pentafluorophenylalanine (F5)Phe, have been characterized by electronic absorption, fluorescence, and vibrational spectroscopy. Specific and unique marker bands that originate from the phosphate group of pTyr and the fluorinated aromatic ring of (F5)Phe have been identified, with the latter showing some solvent dependence. Peptide 2 was found to have excitation and emission wavelengths emanating from pTyr at 268 and 295 nm, respectively, whereas peptide 3 displayed excitation and emission peaks attributable to (F5)Phe at 274 and 315 nm, respectively. Fourier transform infrared (FT-IR) analysis of the amino acid pTyr identified distinct marker bands at approximately 930, 1090, and 1330 cm(-1) that could be attributed to the phosphate group. These markers were also observed in the IR spectrum of peptide 2. Likewise, peptide 3 displayed a characteristic C-F stretching mode at 961 cm(-1) due to the presence of (F5)Phe, including two C-F reporting ring modes at 1509 and 1527 cm(-1). Identifying and monitoring spectroscopic changes in these marker bands may afford a means to observe the molecular interactions that occur when peptides 1-3 bind to the Src kinase.

Monodispersed calcium carbonate nanoparticles modulate local pH and inhibit tumor growth in vivo

NASA Astrophysics Data System (ADS)

Som, Avik; Raliya, Ramesh; Tian, Limei; Akers, Walter; Ippolito, Joseph E.; Singamaneni, Srikanth; Biswas, Pratim; Achilefu, Samuel

2016-06-01

The acidic extracellular environment of tumors potentiates their aggressiveness and metastasis, but few methods exist to selectively modulate the extracellular pH (pHe) environment of tumors. Transient flushing of biological systems with alkaline fluids or proton pump inhibitors is impractical and nonselective. Here we report a nanoparticles-based strategy to intentionally modulate the pHe in tumors. Biochemical simulations indicate that the dissolution of calcium carbonate nanoparticles (nano-CaCO3) in vivo increases pH asymptotically to 7.4. We developed two independent facile methods to synthesize monodisperse non-doped vaterite nano-CaCO3 with distinct size range between 20 and 300 nm. Using murine models of cancer, we demonstrate that the selective accumulation of nano-CaCO3 in tumors increases tumor pH over time. The associated induction of tumor growth stasis is putatively interpreted as a pHe increase. This study establishes an approach to prepare nano-CaCO3 over a wide particle size range, a formulation that stabilizes the nanomaterials in aqueous solutions, and a pH-sensitive nano-platform capable of modulating the acidic environment of cancer for potential therapeutic benefits.The acidic extracellular environment of tumors potentiates their aggressiveness and metastasis, but few methods exist to selectively modulate the extracellular pH (pHe) environment of tumors. Transient flushing of biological systems with alkaline fluids or proton pump inhibitors is impractical and nonselective. Here we report a nanoparticles-based strategy to intentionally modulate the pHe in tumors. Biochemical simulations indicate that the dissolution of calcium carbonate nanoparticles (nano-CaCO3) in vivo increases pH asymptotically to 7.4. We developed two independent facile methods to synthesize monodisperse non-doped vaterite nano-CaCO3 with distinct size range between 20 and 300 nm. Using murine models of cancer, we demonstrate that the selective accumulation of nano-CaCO3 in tumors increases tumor pH over time. The associated induction of tumor growth stasis is putatively interpreted as a pHe increase. This study establishes an approach to prepare nano-CaCO3 over a wide particle size range, a formulation that stabilizes the nanomaterials in aqueous solutions, and a pH-sensitive nano-platform capable of modulating the acidic environment of cancer for potential therapeutic benefits. Electronic supplementary information (ESI) available: Summary of experiments, theoretical schema of effect, synthesis schema, X-Ray diffraction results, TEM of effects of different solvents on particles in various solvents. See DOI: 10.1039/c5nr06162h

Structural basis for highly effective HIV-1 neutralization by CD4-mimetic miniproteins revealed by 1.5 Å co-crystal structure of gp120 and M48U1

PubMed Central

Acharya, Priyamvada; Luongo, Timothy; Louder, Mark K.; McKee, Krisha; Yang, Yongping; Kwon, Young Do; Mascola, John R.; Kessler, Pascal; Martin, Loïc; Kwong, Peter D.

2014-01-01

The interface between HIV-1 gp120 envelope glycoprotein and CD4 receptor contains an unusual interfacial cavity, the “Phe43 cavity”, which miniprotein mimetics of CD4 with non-natural extensions can potentially utilize to enhance their neutralization of HIV-1. Here we report co-crystal structures of HIV-1 gp120 with miniproteins M48U1 and M48U7, which insert cyclohexylmethoxy and 5-hydroxypentylmethoxy extensions, respectively, into the Phe43 cavity. Both inserts displayed flexibility and hydrophobic interactions, but the M48U1 insert showed better shape complementarity with the Phe43 cavity than the M48U7 insert. Subtle alteration in gp120 conformation played a substantial role in optimizing fit. With M48U1, these translated into a YU2-gp120 affinity of 0.015 nM and neutralization of all 180-circulating HIV-1 strains tested, except clade-A/E isolates with non-canonical Phe43 cavities. Ligand chemistry, shape complementary, surface burial, and gp120 conformation act in concert to modulate binding of ligands to the gp120-Phe43 cavity and, when optimized, can effect near pan-neutralization of HIV-1. PMID:23707685

Mg-Al and Zn-Fe layered double hydroxides used for organic species storage and controlled release.

PubMed

Seftel, E M; Cool, P; Lutic, D

2013-12-01

Layered double hydroxides (LDH) containing (Mg and Al) or (Zn and Fe) were prepared by coprecipitation at constant pH, using NaOH and urea as precipitation agents. The most pure LDH phase in the Zn/Fe system was obtained with urea and in Mg/Al system when using NaOH. The incorporation of phenyl-alanine (Phe) anions in the interlayer of the LDH was performed by direct coprecipitation, ionic exchange and structure reconstruction of the mixed oxide obtained by the calcination of the coprecipitated product at 400°C. The reconstruction method and the direct coprecipitation in a medium containing Phe in the initial mixture were less successful in terms of high yields of organic-mineral composite than the ionic exchange method. A spectacular change in sample morphology and yield in exchanged solid was noticed for the Zn3Fe sample obtained by ionic exchange for 6h with Phe solution. A delivery test in PBS of pH=7.4 showed the release of the Phe in several steps up to 25 h indicating different host-guest interactions between the Phe and the LDH matrix. This behavior makes the preparation useful to obtain late delivery drugs, by the incorporation of the anion inside the LDH layer. © 2013.

Capsaicin Synthesis Requires in Situ Phenylalanine and Valine Formation in in Vitro Maintained Placentas from Capsicum chinense.

PubMed

Baas-Espinola, Fray M; Castro-Concha, Lizbeth A; Vázquez-Flota, Felipe A; Miranda-Ham, María L

2016-06-21

Capsaicinoids (CAP) are nitrogenous metabolites formed from valine (Val) and phenylalanine (Phe) in the placentas of hot Capsicum genotypes. Placentas of Habanero peppers can incorporate inorganic nitrogen into amino acids and have the ability to secure the availability of the required amino acids for CAP biosynthesis. In order to determine the participation of the placental tissue as a supplier of these amino acids, the effects of blocking the synthesis of Val and Phe by using specific enzyme inhibitors were analyzed. Isolated placentas maintained in vitro were used to rule out external sources' participation. Blocking Phe synthesis, through the inhibition of arogenate dehydratase, significantly decreased CAP accumulation suggesting that at least part of Phe required in this process has to be produced in situ. Chlorsulfuron inhibition of acetolactate synthase, involved in Val synthesis, decreased not only Val accumulation but also that of CAP, pointing out that the requirement for this amino acid can also be fulfilled by this tissue. The presented data demonstrates that CAP accumulation in in vitro maintained placentas can be accomplished through the in situ availability of Val and Phe and suggests that the synthesis of the fatty acid chain moiety may be a limiting factor in the biosynthesis of these alkaloids.

Beta-methyl substitution of cyclohexylalanine in Dmt-Tic-Cha-Phe peptides results in highly potent delta opioid antagonists.

PubMed

Tóth, Géza; Ioja, Eniko; Tömböly, Csaba; Ballet, Steven; Tourwé, Dirk; Péter, Antal; Martinek, Tamás; Chung, Nga N; Schiller, Peter W; Benyhe, Sándor; Borsodi, Anna

2007-01-25

The opioid peptide TIPP (H-Tyr-Tic-Phe-Phe-OH, Tic:1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) was substituted with Dmt (2',6'-dimethyltyrosine) and a new unnatural amino acid, beta-MeCha (beta-methyl-cyclohexylalanine). This double substitution led to a new series of opioid peptides displaying subnanomolar delta antagonist activity and mu agonist or antagonist properties depending on the configuration of the beta-MeCha residue. The most promising analog, H-Dmt-Tic-(2S,3S)-beta-MeCha-Phe-OH was a very selective delta antagonist both in the mouse vas deferens (MVD) assay (Ke = 0.241 +/- 0.05 nM) and in radioligand binding assay (K i delta = 0.48 +/- 0.05 nM, K i mu/K i delta = 2800). The epimeric peptide H-Dmt-Tic-(2S,3R)-beta-MeCha-Phe-OH and the corresponding peptide amide turned out to be mixed partial mu agonist/delta antagonists in the guinea pig ileum and MVD assays. Our results constitute further examples of the influence of Dmt and beta-methyl substitution as well as C-terminal amidation on the potency, selectivity, and signal transduction properties of TIPP related peptides. Some of these compounds represent valuable pharmacological tools for opioid research.

Structure-activity relationship of linear peptide Bu-His6-DPhe7-Arg8-Trp9-Gly10-NH2 at the human melanocortin-1 and -4 receptors: DPhe7 and Trp9 substitution.

PubMed

Danho, Waleed; Swistok, Joseph; Cheung, Adrian Wai-Hing; Kurylko, Grazyna; Franco, Lucia; Chu, Xin-Jie; Chen, Li; Yagaloff, Keith

2003-02-24

A series of pentapeptides, based on hMC4R pentapeptide agonist (Bu-His(6)-DPhe(7)-Arg(8)-Trp(9)-Gly(10)-NH(2)), was prepared in which either DPhe(7) or Trp(9) residue was systematically substituted. A number of interesting DPhe surrogates (D-Thi, D-3-CF(3)Phe, D-2-Nal and D-3,4-diClPhe) as well as Trp surrogates (2-Nal and Bta) were identified in this study.

The millimagnitude variability of the HgMn star φ Phe

NASA Astrophysics Data System (ADS)

Prvák, M.; Krtička, J.; Korhonen, H.

2018-01-01

The horizontally inhomogeneous chemical composition of the atmospheres of the chemically peculiar stars causes wavelength redistribution of the spectral energy in areas with increased abundance of heavier elements. Due to the rotation of the star, this usually leads to strictly periodic photometric variability in some spectral regions. We used abundance maps of the HgMn star φ Phe (HD 11753), obtained by means of the Doppler imaging, to model its photometric variability. Comparing the light curves derived from abundance maps obtained at different times, we also study how the time evolution of the surface spots affects this variability.

Truncation studies of alpha-melanotropin peptides identify tripeptide analogues exhibiting prolonged agonist bioactivity.

PubMed

Haskell-Luevano, C; Sawyer, T K; Hendrata, S; North, C; Panahinia, L; Stum, M; Staples, D J; Castrucci, A M; Hadley, M F; Hruby, V J

1996-01-01

Truncation studies of alpha-melanotropin peptides identify tripeptide analogues exhibiting prolonged agonist bioactivity: PEPTIDES 17(6) 995-1002, 1996.-Systematic analysis of fragment derivatives of the superpotent alpha-MSH analogue. Ac-Ser.Tyr-Ser-Nle4-Glu- His-DPhe7-Arg-Trp-Gly-Lys-Pro-Val-NH2(NDP-MSH), led to the discovery of tripeptide agonists possessing prolonged bioactivity in the frog skin assay. Of particular significance to this discovery was Ac-DPhe-Arg-DTrp-NH2, which was the most potent tripeptide in this series exhibiting sustained melanotropic activity. Different pharmacophore models appear to exist that are dependent on the substructure and stereochemistry of the MSH(6-9) "active site." The tripeptides Ac-DPhe-Arg-Trp-NH2, Ac-DPhe-Arg-DTrp-NH2, and Ac-DPhe-DArg-Trp-NH2 stereo-chemical combinations require only Phe7-Xaa8-Trp9, whereas Ac-DPhe-DArg-DTrp-NH2, Ac-Phe-Arg-DTrp-NH2, and Ac-Phe-Arg-Trp-NH2 additionally require His4 for minimal biological activity. Ac-DPhe-Arg-DTrp-NH2 represents a novel prototype lead for the development of MSH-based peptidomimetic agonists.

Curcumin-Loaded Blood-Stable Polymeric Micelles for Enhancing Therapeutic Effect on Erythroleukemia.

PubMed

Gong, Feirong; Chen, Dan; Teng, Xin; Ge, Junhua; Ning, Xianfeng; Shen, Ya-Ling; Li, Jian; Wang, Shanfeng

2017-08-07

Curcumin has high potential in suppressing many types of cancer and overcoming multidrug resistance in a multifaceted manner by targeting diverse molecular targets. However, the rather low systemic bioavailability resulted from its poor solubility in water and fast metabolism/excretion in vivo has hampered its applications in cancer therapy. To increase the aqueous solubility of curcumin while retaining the stability in blood circulation, here we report curcumin-loaded copolymer micelles with excellent in vitro and in vivo stability and antitumor efficacy. The two copolymers used for comparison were methoxy-poly(ethylene glycol)-block-poly(ε-caprolactone) (mPEG-PCL) and N-(tert-butoxycarbonyl)-l-phenylalanine end-capped mPEG-PCL (mPEG-PCL-Phe(Boc)). In vitro cytotoxicity evaluation against human pancreatic SW1990 cell line showed that the delivery of curcumin in mPEG-PCL-Phe(Boc) micelles to cancer cells was efficient and dosage-dependent. The pharmacokinetics in ICR mice indicated that intravenous (i.v.) administration of curcumin/mPEG-PCL-Phe(Boc) micelles could retain curcumin in plasma much better than curcumin/mPEG-PCL micelles. Biodistribution results in Sprague-Dawley rats also showed higher uptake and slower elimination of curcumin into liver, lung, kidney, and brain, and lower uptake into heart and spleen of mPEG-PCL-Phe(Boc) micelles, as compared with mPEG-PCL micelles. Further in vivo efficacy evaluation in multidrug-resistant human erythroleukemia K562/ADR xenograft model revealed that i.v. administration of curcumin-loaded mPEG-PCL-Phe(Boc) micelles significantly delayed tumor growth, which was attributed to the improved stability of curcumin in the bloodstream and increased systemic bioavailability. The mPEG-PCL-Phe(Boc) micellar system is promising in overcoming the key challenge of curcumin's to promote its applications in cancer therapy.

Predicting the distribution of whey protein fouling in a plate heat exchanger using the kinetic parameters of the thermal denaturation reaction of β-lactoglobulin and the bulk temperature profiles.

PubMed

Blanpain-Avet, P; André, C; Khaldi, M; Bouvier, L; Petit, J; Six, T; Jeantet, R; Croguennec, T; Delaplace, G

2016-12-01

Fouling of plate heat exchangers (PHE) is a severe problem in the dairy industry, notably because the relationship between the build-up of protein fouling deposits and the chemical reactions taking place in the fouling solution has not yet been fully elucidated. Experiments were conducted at pilot scale in a corrugated PHE, and fouling deposits were generated using a model β-lactoglobulin (β-LG) fouling solution for which the β-LG thermal denaturation reaction constants had been previously determined experimentally. Then 18 different bulk temperature profiles within the PHE were imposed. Analysis of the fouling runs shows that the dry deposit mass per channel versus the ratio R=k unf /k agg (with k unf and k agg representing, respectively, the unfolding and aggregation rate constants computed from both the identification of the β-LG thermal denaturation process and knowledge of the imposed bulk temperature profile into the PHE channel) is able to gather reasonably well the experimental fouling mass data into a unique master curve. This type of representation of the results clearly shows that the heat-induced reactions (unfolding and aggregation) of the various β-LG molecular species in the bulk fluid are essential to capture the trend of the fouling mass distribution inside a PHE. This investigation also illustrates unambiguously that the release of the unfolded β-LG (also called β-LG molten globule) within the bulk fluid (and the absence of its consumption in the form of aggregates) is a key phenomenon that controls the extent of protein fouling as well as its location inside the PHE. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Carbohydrate protease conjugates: Stabilized proteases for peptide synthesis

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

Wartchow, C.A.; Wang, Peng; Bednarski, M.D.

1995-12-31

The synthesis of oligopeptides using stable carbohydrate protease conjugates (CPCs) was examined in acetonitrile solvent systems. CPC[{alpha}-chymotrypsin] was used for the preparation of peptides containing histidine, phenylalanine, tryptophan in the P{sub 1} position in 60-93% yield. The CPC[{alpha}-chymotrypsin]-catalyzed synthesis of octamer Z-Gly-Gly-Phe-Gly-Gly-Phe-Gly-Gly-OEt from Z-Gly-Gly-Phe-Gly-Gly-Phe-OMe was achieved in 71% yield demonstrating that synthesis peptides containing both hydrophylic and hydrophobic amino acids. The P{sub 2} specificity of papain for aromatic residues was utilized for the 2 + 3 coupling of Z-Tyr-Gly-OMe to H{sub 2}N-Gly-Phe-Leu-OH to generate the leucine enkephalin derivative in 79% yield. Although papain is nonspecific for the hydrolysis of N-benzyloxycarbonylmore » amino acid methyl esters in aqueous solution, the rates of synthesis for these derivitives with nucleophile leucine tert-butyl ester differed by nearly 2 orders of magnitude. CPC[thermolysin] was used to prepare the aspartame precursor Z-Asp-Phe-OMe in 90% yield. The increased stability of CPCs prepared from periodate-modified poly(2-methacryl- amido-2-deoxy-D-glucose), poly(2-methacrylamido-2-deoxy-D-galactose), and poly(5-methacryl-amido-5-deoxy-D-ribose), carbohydrate materials designed to increase the aldehyde concentration in aqueous solution, suggests that the stability of CPCs is directly related to the aldehyde concentration of the carbohydrate material. Periodate oxidation of poly(2-methacrylamido-2-deoxy-D-glucose) followed by covalent attachment to {alpha}-chymotrypsin gave a CPC with catalytic activity in potassium phosphate buffer at 90{degrees}C for 2 h. 1 fig., 1 tab., 40 refs.« less

Transport of persistent organic pollutants by microplastics in estuarine conditions

NASA Astrophysics Data System (ADS)

Bakir, Adil; Rowland, Steven J.; Thompson, Richard C.

2014-03-01

Microplastics represent an increasing source of anthropogenic contamination in aquatic environments, where they may also act as scavengers and transporters of persistent organic pollutants. As estuaries are amongst the most productive aquatic systems, it is important to understand sorption behaviour and transport of persistent organic pollutants (POPs) by microplastics along estuarine gradients. The effects of salinity sorption equilibrium kinetics on the distribution coefficients (Kd) of phenanthrene (Phe) and 4,4‧-DDT, onto polyvinyl chloride (PVC) and onto polyethylene (PE) were therefore investigated. A salinity gradient representing freshwater, estuarine and marine conditions, with salinities corresponding to 0 (MilliQ water, 690 μS/cm), 8.8, 17.5, 26.3 and 35 was used. Salinity had no significant effect on the time required to reach equilibrium onto PVC or PE and neither did it affect desorption rates of contaminants from plastics. Although salinity had no effect on sorption capacity of Phe onto plastics, a slight decrease in sorption capacity was observed for DDT with salinity. Salinity had little effect on sorption behaviour and POP/plastic combination was shown to be a more important factor. Transport of Phe and DDT from riverine to brackish and marine waters by plastic is therefore likely to be much more dependent on the aqueous POP concentration than on salinity. The physical characteristics of the polymer and local environmental conditions (e.g. plastic density, particle residence time in estuaries) will affect the physical transport of contaminated plastics. A transport model of POPs by microplastics under estuarine conditions is proposed. Transport of Phe and DDT by PVC and PE from fresh and brackish water toward fully marine conditions was the most likely net direction for contaminant transport and followed the order: Phe-PE >> DDT-PVC = DDT-PE >> Phe-PVC.

Strategy of Pseudomonas pseudoalcaligenes C70 for effective degradation of phenol and salicylate

PubMed Central

Heinaru, Eeva; Naanuri, Eve; Mehike, Maris; Leito, Ivo; Heinaru, Ain

2017-01-01

Phenol- and naphthalene-degrading indigenous Pseudomonas pseudoalcaligenes strain C70 has great potential for the bioremediation of polluted areas. It harbours two chromosomally located catechol meta pathways, one of which is structurally and phylogenetically very similar to the Pseudomonas sp. CF600 dmp operon and the other to the P. stutzeri AN10 nah lower operon. The key enzymes of the catechol meta pathway, catechol 2,3-dioxygenase (C23O) from strain C70, PheB and NahH, have an amino acid identity of 85%. The metabolic and regulatory phenotypes of the wild-type and the mutant strain C70ΔpheB lacking pheB were evaluated. qRT-PCR data showed that in C70, the expression of pheB- and nahH-encoded C23O was induced by phenol and salicylate, respectively. We demonstrate that strain C70 is more effective in the degradation of phenol and salicylate, especially at higher substrate concentrations, when these compounds are present as a mixture; i.e., when both pathways are expressed. Moreover, NahH is able to substitute for the deleted PheB in phenol degradation when salicylate is also present in the growth medium. The appearance of a yellow intermediate 2-hydroxymuconic semialdehyde was followed by the accumulation of catechol in salicylate-containing growth medium, and lower expression levels and specific activities of the C23O of the sal operon were detected. However, the excretion of the toxic intermediate catechol to the growth medium was avoided when the growth medium was supplemented with phenol, seemingly due to the contribution of the second meta pathway encoded by the phe genes. PMID:28257519

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.

Identification of chemical markers in Cordyceps sinensis by HPLC-MS/MS.

PubMed

Hu, Hankun; Xiao, Ling; Zheng, Baogen; Wei, Xin; Ellis, Alexis; Liu, Yi-Ming

2015-10-01

Authentication and quality assessment of Cordyceps sinensis, a precious and pricey natural product that offers a variety of health benefits, is highly significant. To identify effective chemical markers, authentic C. sinensis was thoroughly screened by using HPLC-MS/MS. In addition to many previously reported ingredients, two glycosides, i.e., cyclo-Ala-Leu-rhamnose and Phe-o-glucose, were detected for the first time in this material. Six ingredients detected, including cordycepin, D-mannitol, Phe, Phe-o-glucose, cyclo-Gly-Pro, and cyclo-Ala-Leu-rhamnose, were selected as a collection of chemical markers. An HPLC-MS/MS method was developed to simultaneously quantify them with sensitivity and specificity. The method had limits of detection ranging from 0.008 μg mL(-1) for cordycepin to 0.75 μg mL(-1) for cyclo-Gly-Pro. Recovery was found between 96 and 103 % in all tests. To evaluate the effectiveness of the marker collection proposed, five authentic C. sinensis samples and five samples of its substitutes were analyzed. Cordycepin, D-mannitol, and Phe were found present in all samples. The contents ranged from 0.0076 to 0.029 % (w/w) for cordycepin, 0.33 to 18.9 % for mannitol, and 0.0013 to 0.642 % for Phe. Interestingly, the two glycosides, Phe-o-glucose and cyclo-Ala-Leu-rhamnose, were detected only in authentic C. sinensis samples. These results indicated that the proposed protocol based on HPLC-MS/MS quantification of the markers might have a great potential in authentication and quality assessment of C. sinensis. Graphical abstract Chemical markers of C. sinensis identified in this work.

Gatekeeping versus Promiscuity in the Early Stages of the Andrimid Biosynthetic Assembly Line

PubMed Central

Magarvey, Nathan A.; Fortin, Pascal D.; Thomas, Paul M.; Kelleher, Neil L.; Walsh, Christopher T.

2009-01-01

The antibiotic andrimid, a nanomolar inhibitor of bacterial acetyl coenzyme A carboxylase, is generated on an unusual polyketide/nonribosomal pep-tide enzyme assembly line in that all thiolation (T) domains/small-molecule building stations are on separate proteins. In addition, a transglutaminase homologue is used to condense andrimid building blocks together on the andrimid assembly line. The first two modules of the andrimid assembly line yields an octatrienoyl-β-Phe-thioester tethered to the AdmI T domain, with amide bond formation carried out by a free-standing transglutaminase homologue AdmF. Analysis of the aminomutase AdmH reveals its specific conversion from l-Phe to (S)-β-Phe, which in turn is activated by AdmJ and ATP to form (S)-β-Phe-aminoacyl-AMP. AdmJ then transfers the (S)-β-Phe moiety to one of the free-standing T domains, AdmI, but not AdmA, which instead gets loaded with an octatrienoyl group by other enzymes. AdmF, the amide synthase, will accept a variety of acyl groups in place of the octatrienoyl donor if presented on either AdmA or AdmI. AdmF will also use either stereoisomer of phenylalanine or β-Phe when presented on AdmA and AdmI, but not when placed on noncognate T domains. Further, we show the polyketide synthase proteins responsible for the polyunsaturated acyl cap can be bypassed in vitro with N-acetylcysteamine as a low-molecular-weight acyl donor to AdmF and also in vivo in an Escherichia coli strain bearing the andrimid biosynthetic gene cluster with a knockout in admA. PMID:18652473

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.

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

A Potential Yeast Actin Allosteric Conduit Dependent on Hydrophobic Core Residues Val-76 and Trp-79*

PubMed Central

Wen, Kuo-Kuang; McKane, Melissa; Stokasimov, Ema; Fields, Jonathon; Rubenstein, Peter A.

2010-01-01

Intramolecular allosteric interactions responsible for actin conformational regulation are largely unknown. Previous work demonstrated that replacing yeast actin Val-76 with muscle actin Ile caused decreased nucleotide exchange. Residue 76 abuts Trp-79 in a six-residue linear array beginning with Lys-118 on the surface and ending with His-73 in the nucleotide cleft. To test if altering the degree of packing of these two residues would affect actin dynamics, we constructed V76I, W79F, and W79Y single mutants as well as the Ile-76/Phe-79 and Ile-76/Tyr-79 double mutants. Tyr or Phe should decrease crowding and increase protein flexibility. Subsequent introduction of Ile should restore packing and dampen changes. All mutants showed decreased growth in liquid medium. W79Y alone was severely osmosensitive and exhibited vacuole abnormalities. Both properties were rescued by Ile-76. Phe-79 or Tyr decreased the thermostability of actin and increased its nucleotide exchange rate. These effects, generally greater for Tyr than for Phe, were reversed by introduction of Ile-76. HD exchange showed that the mutations caused propagated conformational changes to all four subdomains. Based on results from phosphate release and light-scattering assays, single mutations affected polymerization in the order of Ile, Phe, and Tyr from least to most. Introduction of Ile-76 partially rescued the polymerization defects caused by either Tyr-79 or Phe-79. Thus, alterations in crowding of the 76–79 residue pair can strongly affect actin conformation and behavior, and these results support the theory that the amino acid array in which they are located may play a central role in actin regulation. PMID:20442407

Guinea Pig Chymase Is Leucine-specific

PubMed Central

Caughey, George H.; Beauchamp, Jeremy; Schlatter, Daniel; Raymond, Wilfred W.; Trivedi, Neil N.; Banner, David; Mauser, Harald; Fingerle, Jürgen

2008-01-01

To explore guinea pigs as models of chymase biology, we cloned and expressed the guinea pig ortholog of human chymase. In contrast to rats and mice, guinea pigs appear to express just one chymase, which belongs to the α clade, like primate chymases and mouse mast cell protease-5. The guinea pig enzyme autolyzes at Leu residues in the loop where human chymase autolyzes at Phe. In addition, guinea pig α-chymase selects P1 Leu in a combinatorial peptide library and cleaves Ala-Ala-Pro-Leu-4-nitroanilide but has negligible activity toward substrates with P1 Phe and does not cleave angiotensin I. This contrasts with human chymase, which cleaves after Phe or Tyr, prefers P1 Phe in peptidyl 4-nitroanilides, and avidly hydrolyzes angiotensin I at Phe8 to generate bioactive angiotensin II. The guinea pig enzyme also is inactivated more effectively by α1-antichymotrypsin, which features P1 Leu in the reactive loop. Unlike mouse, rat, and hamster α-chymases, guinea pig chymase lacks elastase-like preference for P1 Val or Ala. Partially humanized A216G guinea pig chymase acquires human-like P1 Phe- and angiotensin-cleaving capacity. Molecular models suggest that the wild type active site is crowded by the Ala216 side chain, which potentially blocks access by bulky P1 aromatic residues. On the other hand, the guinea pig pocket is deeper than in Val-selective chymases, explaining the preference for the longer aliphatic side chain of Leu. These findings are evidence that chymase-like peptidase specificity is sensitive to small changes in structure and provide the first example of a vertebrate Leu-selective peptidase. PMID:18353771

Changing Incidence of Cystic Fibrosis in Wisconsin, USA

PubMed Central

Bersie, Rachel; Hoffman, Gary; Rock, Michael; Baker, Mei; Farrell, Philip M.; Simpson, Pippa; Levy, Hara

2015-01-01

Summary Rationale Previous investigations of cystic fibrosis (CF) incidence in Massachusetts, Colorado, and Minnesota (USA) yielded contradictory results, particularly regarding allele p.Phe508del; the racial compositions of the cohorts were not reported. Objectives To clarify discrepancies in reported incidence with the ultimate goal of improving screening and quality of care, we assessed CF incidence, stratified by race and mutations in cystic fibrosis transmembrane conductance regulator (CFTR), in Wisconsin (USA) from 1994 to 2011. Methods Data on patients diagnosed with CF (N=283), CFTR genotypes, CF carriers, and birth rate were collected. All data were categorized by racial background of the birth mother and the incidence of CF births was accordingly adjusted. Spearman’s nonparametric rank correlation and Fisher’s exact test were performed for continuous and categorical variables, respectively. Trends over time were fitted with a cubic spline. Results We detected a trending increase in CF cases (range within all data 1.67–2.98 per 10,000 births per year), homozygous p.Phe508del cases (0.57–1.79 per 10,000), heterozygous p.Phe508del cases (0.29–1.55 per 10,000), and cases lacking p. Phe508del (0–0.45 per 10,000). Both the number of cases lacking the p.Phe508del mutation per year and the number of cases lacking p.Phe508del per 10,000 births significantly increased (P=0.05) from 1994 to 2011; the increase in overall incidence was not significant. The number of carriers identified through newborn screening significantly increased within the non-Hispanic Black (P=0.0.021) and Hispanic (P=0.003) populations. Conclusion The racial composition of the CF cohort is changing in Wisconsin, possibly influencing disease detection, care, and outcome. PMID:26258862

Changing incidence of cystic fibrosis in Wisconsin, USA.

PubMed

Parker-McGill, Katelyn; Nugent, Melodee; Bersie, Rachel; Hoffman, Gary; Rock, Michael; Baker, Mei; Farrell, Philip M; Simpson, Pippa; Levy, Hara

2015-11-01

Previous investigations of cystic fibrosis (CF) incidence in Massachusetts, Colorado, and Minnesota (USA) yielded contradictory results, particularly regarding allele p.Phe508del; the racial compositions of the cohorts were not reported. To clarify discrepancies in reported incidence with the ultimate goal of improving screening and quality of care, we assessed CF incidence, stratified by race and mutations in cystic fibrosis transmembrane conductance regulator (CFTR), in Wisconsin (USA) from 1994 to 2011. Data on patients diagnosed with CF (N = 283), CFTR genotypes, CF carriers, and birth rate were collected. All data were categorized by racial background of the birth mother and the incidence of CF births was accordingly adjusted. Spearman's nonparametric rank correlation and Fisher's exact test were performed for continuous and categorical variables, respectively. Trends over time were fitted with a cubic spline. We detected a trending increase in CF cases (range within all data 1.67-2.98 per 10,000 births per year), homozygous p.Phe508del cases (0.57-1.79 per 10,000), heterozygous p.Phe508del cases (0.29-1.55 per 10,000), and cases lacking p.Phe508del (0-0.45 per 10,000). Both the number of cases lacking the p.Phe508del mutation per year and the number of cases lacking p.Phe508del per 10,000 births significantly increased (P = 0.05) from 1994 to 2011; the increase in overall incidence was not significant. The number of carriers identified through newborn screening significantly increased within the non-Hispanic Black (P = 0.0.021) and Hispanic (P = 0.003) populations. The racial composition of the CF cohort is changing in Wisconsin, possibly influencing disease detection, care, and outcome. © 2015 Wiley Periodicals, Inc.

Secondary Structures in Phe-Containing Isolated Dipeptide Chains: Laser Spectroscopy vs Quantum Chemistry.

PubMed

Loquais, Yohan; Gloaguen, Eric; Habka, Sana; Vaquero-Vara, Vanesa; Brenner, Valérie; Tardivel, Benjamin; Mons, Michel

2015-06-11

The intrinsic conformational landscape of two phenylalanine-containing protein chain models (-Gly-Phe- and -Ala-Phe- sequences) has been investigated theoretically and experimentally in the gas phase. The near UV spectroscopy (first ππ* transition of the Phe ring) is obtained experimentally under jet conditions where the conformational features can be resolved. Single-conformation IR spectroscopy in the NH stretch region is then obtained by IR/UV double resonance in the ground state, leading to resolved vibrational spectra that are assigned in terms of conformation and H-bonding content from comparison with quantum chemistry calculations. For the main conformer, whose UV spectrum exhibits a significant Franck-Condon activity in low frequency modes involving peptide backbone motions relative to the Phe chromophore, excited state IR spectroscopy has also been recorded in a UV/IR/UV experiment. The NH stretch spectral changes observed in such a ππ* labeling experiment enable us to determine those NH bonds that are coupled to the phenyl ring; they are compared to CC2 excited state calculations to quantify the geometry change upon ππ* excitation. The complete and consistent series of data obtained enable us to propose an unambiguous assignment for the gallery of conformers observed and to demonstrate that, in these two sequences, three conceptually important local structural motifs of proteins (β-strands, 27 ribbons, and β-turns) are represented. The satisfactory agreement between the experimental conformational distribution and the predicted landscape anticipated from the DFT-D approach demonstrates the capabilities of a theoretical method that accounts for dispersive interactions. It also shows that the flaws, inherent to a resonant two-photon ionization detection scheme, often evoked for aromatic chromophores, do not seem to be significant in the case of Phe.

Albumin Redhill (-1 Arg, 320 Ala----Thr): a glycoprotein variant of human serum albumin whose precursor has an aberrant signal peptidase cleavage site.

PubMed

Brennan, S O; Myles, T; Peach, R J; Donaldson, D; George, P M

1990-01-01

Albumin Redhill is an electrophoretically slow genetic variant of human serum albumin that does not bind 63Ni2+ and has a molecular mass 2.5 kDa higher than normal albumin. Its inability to bind Ni2+ was explained by the finding of an additional residue of Arg at position -1. This did not explain the molecular basis of the genetic variation (since proalbumin contains adjacent Arg residues at -1 and -2) or the increase in apparent molecular mass. Fractionation of tryptic digests on concanavalin A-Sepharose followed by peptide mapping of the bound and unbound fractions and sequence analysis of the glycopeptides identified a mutation of 320 Ala----Thr. This introduces an Asn-Tyr-Thr oligosaccharide attachment sequence centered on Asn-318 and explains the increase in molecular mass. This, however, did not satisfactorily explain the presence of the additional Arg residue at position -1. DNA sequencing of polymerase chain reaction-amplified genomic DNA encoding the prepro sequence of albumin indicated an additional mutation of -2 Arg----Cys. This introduces a prepro sequence, Met-Lys-Trp-Val-Thr-Phe-Ile-Ser-Leu-Leu-Phe-Leu-Phe-Ser-Ser-Ala-Tyr- Ser-Arg-Gly-Val-Phe-Cys-Arg (cf.-Tyr-Ser-Arg-Gly-Val-Phe-Arg-Arg- in normal human pre-proalbumin). We propose that the new Phe-Cys-Arg sequence in the propeptide is an aberrant signal peptidase cleavage site and that the signal peptidase cleaves the propeptide of albumin Redhill in the lumen of the endoplasmic reticulum before it reaches the Golgi vesicles, the site of the diarginyl-specific proalbumin convertase.

A cGMP kinase mutant with increased sensitivity to the protein kinase inhibitor peptide PKI(5-24).

PubMed

Ruth, P; Kamm, S; Nau, U; Pfeifer, A; Hofmann, F

1996-01-01

Synthetic peptides corresponding to the active domain of the heat-stable inhibitor protein PKI are very potent inhibitors of cAMP-dependent protein kinase, but are extremely weak inhibitors of cGMP-dependent protein kinase. In this study, we tried to confer PKI sensitivity to cGMP kinase by site-directed mutagenesis. The molecular requirements for high affinity inhibition by PKI were deduced from the crystal structure of the cAMP kinase/PKI complex. A prominent site of interaction are residues Tyr235 and Phe239 in the catalytic subunit, which from a sandwich-like structure with Phe10 of the PKI(5-24) peptide. To increase the sensitivity for PKI, the cGMP kinase codons at the corresponding sites, Ser555 and Ser559, were changed to Tyr and Phe. The mutant cGMP kinase was stimulated half maximally by cGMP at 3-fold higher concentrations (240 nM) than the wild type (77 nM). Wild type and mutant cGMP kinase did not differ significantly in their Km and Vmax for three different substrate peptides. The PKI(5-24) peptide inhibited phosphotransferase activity of the mutant cGMP kinase with higher potency than that of wild type, with Ki values of 42 +/- .3 microM and 160 +/- .7 microM, respectively. The increased affinity of the mutant cGMP kinase was specific for the PKI(5-24) peptide. Mutation of the essential Phe10 in the PKI(5-24) sequence to an Ala yielded a peptide that inhibited mutant and wild type cGMP kinase with similar potency, with Ki values of 160 +/- 11 and 169 +/- 27 microM, respectively. These results suggest that the mutations Ser555Tyr and Ser559Phe are required, but not sufficient, for high affinity inhibition of cGMP kinase by PKI.

Molecular dynamics study of biodegradation of azo dyes via their interactions with AzrC azoreductase.

PubMed

Haghshenas, Hamed; Kay, Maryam; Dehghanian, Fariba; Tavakol, Hossein

2016-01-01

Azo dyes are one of the most important class of dyes, which have been widely used in industries. Because of the environmental pollution of azo dyes, many studies have been performed to study their biodegradation using bacterial systems. In present work, the AzrC of mesophilic gram-positive Bacillus sp. B29 has been considered to study its interaction with five common azo dyes (orange G, acid red 88, Sudan I, orange I, and methyl red). The molecular dynamics simulations have been employed to study the interaction between AzrC and azo dyes. The trajectory was confirmed using root mean square deviation and the root mean square fluctuation analyses. Then, the hydrogen bond and alanine scanning analyses were performed to reveal active site residues. Phe105 (A), Phe125 (B), Phe172 (B), and Pro132 (B) have been found as the most important hydrophobic residues whereas Asn104 (A), Tyr127 (B), and Asn187 (A) have key role in making hydrogen bond. The results of molecular mechanics Poisson-Boltzmann surface area and molecular mechanics generalized Born surface area calculations proved that the hydrophobic azo dyes like Acid red 88 binds more tightly to the AzrC protein. The calculated data suggested MR A 121 (B) I as a potential candidate for improving the AzrC-MR interactions.

[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.

Products of cholecystokinin (CCK)-octapeptide proteolysis interact with central CCK receptors.

PubMed

Steardo, L; Knight, M; Tamminga, C A; Chase, T N

1985-03-15

Peptidases present in central nervous system (CNS) synaptic membranes, hydrolyze the neuroactive peptide cholecystokinin-octapeptide (CCK-8; Asp-Tyr-SO3H-Met-Gly-Trp-Met-Asp-Phe-NH2). In order to determine the pathway of degradation, synthetic CCK-8 was incubated at 37 degrees C with purified synaptic membranes; at various intervals reaction samples were removed from the reaction mixture and analysed by high-performance liquid chromatography to identify and quantify the peptide fragments. The results indicate an initial endopeptidase cleavage at the Met-Gly bond producing CCK-5 (Gly-Trp-Met-Asp-Phe-NH2). The carboxyl-terminal pentapeptide is further proteolysed to CCK-4 (Trp-Met-Asp-Phe-NH2) by a puromycin-sensitive aminopeptidase and to CCK-3 (Met-Asp-Phe-NH2) and Gly-Trp by an endopeptidase action. CCK-3 and CCK-2 appear to be relatively stable end-products. Moreover, these proteolytic fragments are shown to bind to the CCK receptor in brain with varying potencies.

Numerical investigation of cryogen re-gasification in a plate heat exchanger

NASA Astrophysics Data System (ADS)

Malecha, Ziemowit; Płuszka, Paweł; Brenk, Arkadiusz

2017-12-01

The efficient re-gasification of cryogen is a crucial process in many cryogenic installations. It is especially important in the case of LNG evaporators used in stationary and mobile applications (e.g. marine and land transport). Other gases, like nitrogen or argon can be obtained at highest purity after re-gasification from their liquid states. Plate heat exchangers (PHE) are characterized by a high efficiency. Application of PHE for liquid gas vaporization processes can be beneficial. PHE design and optimization can be significantly supported by numerical modelling. Such calculations are very challenging due to very high computational demands and complexity related to phase change modelling. In the present work, a simplified mathematical model of a two phase flow with phase change was introduced. To ensure fast calculations a simplified two-dimensional (2D) numerical model of a real PHE was developed. It was validated with experimental measurements and finally used for LNG re-gasification modelling. The proposed numerical model showed to be orders of magnitude faster than its full 3D original.

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.

Novel selective human melanocortin-3 receptor ligands: Use of the 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one (Aba) scaffold

PubMed Central

Ballet, Steven; Mayorov, Alexander V.; Cai, Minying; Tymecka, Dagmara; Chandler, Kevin B.; Palmer, Erin S.; Van Rompaey, Karolien; Misicka, Aleksandra; Tourwé, Dirk; Hruby, Victor J.

2008-01-01

In search of new selective antagonists and/or agonists for the human melanocortin receptor subtypes hMC1R to hMC5R to elucidate the specific biological roles of each GPCR, we modified the structures of the superagonist MT-II (Ac-Nle-c[Asp-His-D-Phe-Arg-Trp-Lys]-NH2) and the hMC3R/hMC4R antagonist SHU9119 (Ac-Nle-c[Asp-His-D-Nal(2′)-Arg-Trp-Lys]-NH2) by replacing the His-D-Phe and His-D-Nal(2′) fragments in MT-II and SHU9119, respectively, with Aba-Xxx (4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one-Xxx) dipeptidomimetics (Xxx = D-Phe/pCl-D-Phe/D-Nal(2′)). Employment of the Aba mimetic yielded novel selective high affinity hMC3R and hMC3R/hMC5R antagonists. PMID:17314042

Angiotensin converting enzyme (ACE) and neprilysin hydrolyze neuropeptides: a brief history, the beginning and follow-ups to early studies.

PubMed

Skidgel, Randal A; Erdös, Ervin G

2004-03-01

Our investigations started when synthetic bradykinin became available and we could characterize two enzymes that cleaved it: kininase I or plasma carboxypeptidase N and kininase II, a peptidyl dipeptide hydrolase that we later found to be identical with the angiotensin I converting enzyme (ACE). When we noticed that ACE can cleave peptides without a free C-terminal carboxyl group (e.g., with a C-terminal nitrobenzylamine), we investigated inactivation of substance P, which has a C-terminal Met(11)-NH(2). The studies were extended to the hydrolysis of the neuropeptide, neurotensin and to compare hydrolysis of the same peptides by neprilysin (neutral endopeptidase 24.11, CD10, NEP). Our publication in 1984 dealt with ACE and NEP purified to homogeneity from human kidney. NEP cleaved substance P (SP) at Gln(6)-Phe(7), Phe(7)[see text]-Phe(8), and Gly(9)-Leu(10) and neurotensin (NT) at Pro(10)-Tyr(11) and Tyr(11)-Ile(12). Purified ACE also rapidly inactivated SP as measured in bioassay. HPLC analysis showed that ACE cleaved SP at Phe(8)-Gly(9) and Gly(9)-Leu(10) to release C-terminal tri- and dipeptide (ratio = 4:1). The hydrolysis was Cl(-) dependent and inhibited by captopril. ACE released only dipeptide from SP free acid. ACE hydrolyzed NT at Tyr(11)-Ile(12) to release Ile(12)-Leu(13). Then peptide substrates were used to inhibit ACE hydrolyzing Fa-Phe-Gly-Gly and NEP cleaving Leu(5)-enkephalin. The K(i) values in microM were as follows: for ACE, bradykinin = 0.4, angiotensin I = 4, SP = 25, SP free acid = 2, NT = 14, and Met(5)-enkephalin = 450, and for NEP, bradykinin = 162, angiotensin I = 36, SP = 190, NT = 39, Met(5)-enkephalin = 22. These studies showed that ACE and NEP, two enzymes widely distributed in the body, are involved in the metabolism of SP and NT. Below we briefly survey how NEP and ACE in two decades have gained the reputation as very important factors in health and disease. This is due to the discovery of more endogenous substrates of the enzymes and to the very broad and beneficial therapeutic applications of ACE inhibitors.

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.

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.

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

(D-Phe/sup 12/)bombesin analogues: a new class of bombesin receptor antagonists

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

Heinz-Erian, P.; Coy, D.H.; Tamura, M.

1987-03-01

Previous attempts to develop analogues of bombesin that function as specific receptor antagonists have been unsuccessful. Alteration of the histidine in luteinizing hormone releasing factor has resulted in analogues that function as competitive antagonists. In the present study the authors have used a similar strategy and altered the histidine in bombesin. (D-Phe/sup 12/)bombesin, (D-Phe/sup 12/,Leu/sup 14/)bombesin, and (Try/sup 4/, D-)je/sup 12/) bombesin did not stimulate amylase release from guinea pig pancreatic acini when present alone, but each analog inhibited bombesin-stimulated secretion. For each analog, detectable inhibition occurred at 1 ..mu..M and half-maximal inhibition at 4 ..mu..M. Each analog inhibited amylasemore » release by bombesin and other agonists that stimulate secretion by interacting with bombesin receptors. The analogues of bombesin did not alter stimulation by substance P or other agonists that interact with other receptors. The inhibition of the action of bombesin was competitive with Schild plots having slopes of 1.0. Each analog also inhibited binding of /sup 125/I-labeled (Try/sup 4/) bombesin but not /sup 125/I-labeled substance P. These results demonstrate that (D-Phe/sup 12/) analogues of bombesin function as bombesin receptor antagonists and are the only bombesin receptor antagonists that interact only with the bombesin receptor. Because of their specificity, these analogues may prove useful for defining the role of bombesin in various physiological or pathological processes.« less

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.

Quorum-sensing inhibitory compounds from extremophilic microorganisms isolated from a hypersaline cyanobacterial mat.

PubMed

Abed, Raeid M M; Dobretsov, Sergey; Al-Fori, Marwan; Gunasekera, Sarath P; Sudesh, Kumar; Paul, Valerie J

2013-07-01

In this study, extremely halophilic and moderately thermophilic microorganisms from a hypersaline microbial mat were screened for their ability to produce antibacterial, antidiatom, antialgal, and quorum-sensing (QS) inhibitory compounds. Five bacterial strains belonging to the genera Marinobacter and Halomonas and one archaeal strain belonging to the genus Haloterrigena were isolated from a microbial mat. The strains were able to grow at a maximum salinity of 22-25 % and a maximum temperature of 45-60 °C. Hexanes, dichloromethane, and butanol extracts from the strains inhibited the growth of at least one out of nine human pathogens. Only butanol extracts of supernatants of Halomonas sp. SK-1 inhibited growth of the microalga Dunaliella salina. Most extracts from isolates inhibited QS of the acyl homoserine lactone producer and reporter Chromobacterium violaceum CV017. Purification of QS inhibitory dichloromethane extracts of Marinobacter sp. SK-3 resulted in isolation of four related diketopiperazines (DKPs): cyclo(L-Pro-L-Phe), cyclo(L-Pro-L-Leu), cyclo(L-Pro-L-isoLeu), and cyclo(L-Pro-D-Phe). QS inhibitory properties of these DKPs were tested using C. violaceum CV017 and Escherichia coli-based QS reporters (pSB401 and pSB1075) deficient in AHL production. Cyclo(L-Pro-L-Phe) and cyclo(L-Pro-L-isoLeu) inhibited QS-dependent production of violacein by C. violaceum CV017. Cyclo(L-Pro-L-Phe), cyclo(L-Pro-L-Leu), and cyclo(L-Pro-L-isoLeu) reduced QS-dependent luminescence of the reporter E. coli pSB401 induced by 3-oxo-C6-HSL. Our study demonstrated the ability of halophilic and moderately thermophilic strains from a hypersaline microbial mat to produce biotechnologically relevant compounds that could be used as antifouling agents.

Novel kinin B1 receptor agonists with improved pharmacological profiles.

PubMed

Côté, Jérôme; Savard, Martin; Bovenzi, Veronica; Bélanger, Simon; Morin, Josée; Neugebauer, Witold; Larouche, Annie; Dubuc, Céléna; Gobeil, Fernand

2009-04-01

There is some evidence to suggest that inducible kinin B1 receptors (B1R) may play beneficial and protecting roles in cardiovascular-related pathologies such as hypertension, diabetes, and ischemic organ diseases. Peptide B1R agonists bearing optimized pharmacological features (high potency, selectivity and stability toward proteolysis) hold promise as valuable therapeutic agents in the treatment of these diseases. In the present study, we used solid-phase methodology to synthesize a series of novel peptide analogues based on the sequence of Sar[dPhe(8)]desArg(9)-bradykinin, a relatively stable peptide agonist with moderate affinity for the human B1R. We evaluated the pharmacological properties of these peptides using (1) in vitro competitive binding experiments on recombinant human B1R and B2R (for index of selectivity determination) in transiently transfected human embryonic kidney 293 cells (HEK-293T cells), (2) ex vivo vasomotor assays on isolated human umbilical veins expressing endogenous human B1R, and (3) in vivo blood pressure tests using anesthetized lipopolysaccharide-immunostimulated rabbits. Key chemical modifications at the N-terminus, the positions 3 and 5 on Sar[dPhe(8)]desArg(9)-bradykinin led to potent analogues. For example, peptides 18 (SarLys[Hyp(3),Cha(5), dPhe(8)]desArg(9)-bradykinin) and 20 (SarLys[Hyp(3),Igl(5), dPhe(8)]desArg(9)-bradykinin) outperformed the parental molecule in terms of affinity, functional potency and duration of action in vitro and in vivo. These selective agonists should be valuable in future animal and human studies to investigate the potential benefits of B1R activation.

N-terminal fatty acylated His-dPhe-Arg-Trp-NH(2) tetrapeptides: influence of fatty acid chain length on potency and selectivity at the mouse melanocortin receptors and human melanocytes.

PubMed

Todorovic, Aleksandar; Holder, Jerry Ryan; Bauzo, Rayna M; Scott, Joseph Walker; Kavanagh, Renny; Abdel-Malek, Zalfa; Haskell-Luevano, Carrie

2005-05-05

The melanocortin system is involved in the regulation of a diverse number of physiologically important pathways including pigmentation, feeding behavior, weight and energy homeostasis, inflammation, and sexual function. All the endogenous melanocortin agonist ligands possess the conserved His-Phe-Arg-Trp tetrapeptide sequence that is postulated to be important for melanocortin receptor molecular recognition and stimulation. Previous studies by our laboratory resulted in the discovery that increasing alkyl chain length at the N-terminal "capping" region of the His-dPhe-Arg-Trp-NH(2) tetrapeptide resulted in a 100-fold increased melanocortin receptor agonist potency. This study was undertaken to systematically evaluate the pharmacological effects of increasing N-capping alkyl chain length of the CH(3)(CH(2))(n)CO-His-dPhe-Arg-Trp-NH(2) (n = 6-16) tetrapeptide template. Twelve analogues were synthesized and pharmacologically characterized at the mouse melanocortin receptors MC1R and MC3R-MC5R and human melanocytes known to express the MC1R. These peptides demonstrated melanocortin receptor selectivity profiles different from those of previously published tetrapeptides. The most notable results of enhanced ligand potency (20- to 200-fold) and receptor selectivity were observed at the MC1R. Tetrapeptides that possessed greater than nine alkyl groups were superior to alpha-MSH in terms of the stimulation of human melanocyte tyrosinase activity. Additionally, the n-pentadecanoyl derivative had a residual effect on tyrosinase activity that existed for at least 4 days after the peptide was removed from the human melanocyte culture medium. These data demonstrate the utility, potency, and residual effect of melanocortin tetrapeptides by adding N-terminal fatty acid moieties.

Introduction of a mutation in the shutter region of antithrombin (Phe77 --> Leu) increases affinity for heparin and decreases thermal stability.

PubMed

Quinsey, Noelene S; Fitton, Hazel L; Coughlin, Paul; Whisstock, James C; Dafforn, Timothy R; Carrell, Robin W; Bottomley, Stephen P; Pike, Robert N

2003-09-02

The shutter region of serpins consists of a number of highly conserved residues that are critical for both stability and function. Several variants of antithrombin with substitutions in this region are unstable and predispose the carrier to thrombosis. Although most mutations in the shutter region investigated to date are deleterious with respect to serpin stability and function, the substitution of Phe51 by Leu in alpha(1)-antitrypsin results in enhanced stability. Here, we have investigated the effects of introducing an analogous mutation into antithrombin (Phe 77 to Leu). The mutation did not affect the kinetics of interaction with proteases. Strikingly, however, the thermostability of the protein was markedly decreased, with the serpin displaying a 13 degrees C decrease in melting temperature as compared to wild-type recombinant antithrombin. Further studies revealed that in contrast to wild-type antithrombin, the mutant adopted the latent (inactive) conformation upon mild heating. Previous studies on shutter region mutations that destabilize antithrombin revealed that such variants possess enhanced affinity for both heparin pentasaccharide and full-length heparin. The N135A/F77L mutant had unchanged affinity for heparin pentasaccharide, but the affinity for full-length heparin was increased. We suggest that the Phe77Leu mutation causes conformational changes around the top of the D-helix in antithrombin, in particular, to the arginine 132 and 133 residues that may mediate additional antithrombin/heparin interactions. This paper also demonstrates that there are major differences between the shutter regions of antithrombin and alpha(1)-antitrypsin since a stabilizing mutation in antitrypsin has the converse effect in antithrombin.

Electrostatic Evaluation of the Propellant Handlers Ensemble

NASA Technical Reports Server (NTRS)

Hogue, Michael D.; Calle, Carlos I.; Buhler, Charles

2006-01-01

The Self-Contained Atmospheric Protective Ensemble (SCAPE) used in propellant handling at NASA's Kennedy Space Center (KSC) has recently completed a series of tests to determine its electrostatic properties of the coverall fabric used in the Propellant Handlers Ensemble (PHE). Understanding these electrostatic properties are fundamental to ensuring safe operations when working with flammable rocket propellants such as hydrazine, methyl hydrazine, and unsymmetrical dimethyl hydrazine. These tests include surface resistivity, charge decay, triboelectric charging, and flame incendivity. In this presentation, we will discuss the results of these tests on the current PHE as well as new fabrics and materials being evaluated for the next generation of PHE.

Emergency Preparedness & Recovery News Releases - PHE

Science.gov Websites

and reload this page. Skip over global navigation links U.S. Department of Health and Human Services Health Emergency - Leading a Nation Prepared Search Search PHE Home > Emergency Emergency Preparedness necesitan medicamentos HHS Acting Secretary Declares Public Health Emergency to Address National Opioid

40 CFR 721.7620 - Alkyl(heterocyclicyl) phe-nyl-azohetero monocyclic polyone, ((alkylimidazolyl) methyl) derivative...

Code of Federal Regulations, 2010 CFR

2010-07-01

...-azohetero monocyclic polyone, ((alkylimidazolyl) methyl) derivative (generic name). 721.7620 Section 721... Alkyl(heterocyclicyl) phe-nyl-azohetero monocyclic polyone, ((alkylimidazolyl) methyl) derivative...) derivative (PMN P-86-136) is subject to reporting under this section for the significant new uses described...

40 CFR 721.7620 - Alkyl(heterocyclicyl) phe-nyl-azohetero monocyclic polyone, ((alkylimidazolyl) methyl) derivative...

Code of Federal Regulations, 2011 CFR

2011-07-01

...-azohetero monocyclic polyone, ((alkylimidazolyl) methyl) derivative (generic name). 721.7620 Section 721... Alkyl(heterocyclicyl) phe-nyl-azohetero monocyclic polyone, ((alkylimidazolyl) methyl) derivative...) derivative (PMN P-86-136) is subject to reporting under this section for the significant new uses described...

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.

Topography of tyrosine residues and their involvement in peroxidation of polyunsaturated cardiolipin in cytochrome c/cardiolipin peroxidase complexes

PubMed Central

Kapralov, Alexandr A.; Yanamala, Naveena; Tyurina, Yulia Y.; Castro, Laura; Arias, Alejandro Samhan; Vladimirov, Yuri A.; Maeda, Akihiro; Weitz, Andrew A.; Peterson, Jim; Mylnikov, Danila; Demicheli, Verónica; Tortora, Verónica; Klein-Seetharaman, Judith; Radi, Rafael; Kagan, Valerian E.

2011-01-01

Formation of cytochrome c (cyt c)/cardiolipin (CL) peroxidase complex selective towards peroxidation of polyunsaturated CLs is a pre-requisite for mitochondrial membrane permeabilization. Tyrosine residues – via the generation of tyrosyl radicals (Tyr•) - are likely reactive intermediates of the peroxidase cycle leading to CL peroxidation. We used mutants of horse heart cyt c in which each of the four Tyr residues was substituted for Phe and assessed their contribution to the peroxidase catalysis. Tyr67Phe mutation was associated with a partial loss of the oxygenase function of the cyt c/CL complex and the lowest concentration of H2O2-induced Tyr radicals in electron paramagnetic resonance (EPR) spectra. Our MS experiments directly demonstrated decreased production of CL-hydroperoxides (CL-OOH) by Tyr67Phe mutant. Similarly, oxidation of a phenolic substrate, Amplex Red, was affected to a greater extent in Tyr67Phe than in three other mutants. Tyr67Phe mutant exerted high resistance to H2O2-induced oligomerization. Measurements of Tyr fluorescence, hetero-nuclear magnetic resonance (NMR) and computer simulations position Tyr67 in close proximity to the porphyrin ring heme iron and one of the two axial heme-iron ligand residues, Met80. Thus, the highly conserved Tyr67 is a likely electron-donor (radical acceptor) in the oxygenase half-reaction of the cyt c/CL peroxidase complex. PMID:21550335

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.

Effects of introducing a single charged residue into the phenylalanine clamp of multimeric anthrax protective antigen.

PubMed

Janowiak, Blythe E; Fischer, Audrey; Collier, R John

2010-03-12

Multimeric pores formed in the endosomal membrane by the Protective Antigen moiety of anthrax toxin translocate the enzymatic moieties of the toxin to the cytosolic compartment of mammalian cells. There is evidence that the side chains of the Phe(427) residues come into close proximity with one another in the lumen of the pore and form a structure, termed the Phe clamp, that catalyzes the translocation process. In this report we describe the effects of replacing Phe(427) in a single subunit of the predominantly heptameric pore with a basic or an acidic amino acid. Incorporating any charged residue at this position inhibited cytotoxicity >or=1,000-fold in our standard assay and caused strong inhibition of translocation in a planar phospholipid bilayer system. His and Glu were the most strongly inhibitory residues, ablating both cytotoxicity and translocation. Basic residues at position 427 prevented the Phe clamp from interacting with a translocation substrate to form a seal against the passage of ions and accelerated dissociation of the substrate from the pore. Acidic residues, in contrast, allowed the seal to form and the substrate to remain firmly bound, but blocked its passage, perhaps via electrostatic interactions with the positively charged N-terminal segment. Our findings are discussed in relation to the role of the Phe clamp in a Brownian ratchet model of translocation.

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

Li, T. S.; Balbinot, E.; Mondrik, N.

We report the discovery of an excess of main sequence turn-off stars in the direction of the constellations of Eridanus and Phoenix from the first year data of the Dark Energy Survey (DES). The Eridanus-Phoenix (EriPhe) overdensity is centered around l~285 deg and b~-60 deg and spans at least 30 deg in longitude and 10 deg in latitude. The Poisson significance of the detection is at least 9 sigma. The stellar population in the overdense region is similar in brightness and color to that of the nearby globular cluster NGC 1261, indicating that the heliocentric distance of EriPhe is aboutmore » d~16 kpc. The extent of EriPhe in projection is therefore at least ~4 kpc by ~3 kpc. On the sky, this overdensity is located between NGC 1261 and a new stellar stream discovered by DES at a similar heliocentric distance, the so-called Phoenix Stream. Given their similar distance and proximity to each other, it is possible that these three structures may be kinematically associated. Alternatively, the EriPhe overdensity is morphologically similar to the Virgo overdensity and the Hercules-Aquila cloud, which also lie at a similar Galactocentric distance. These three overdensities lie along a polar plane separated by ~120 deg and may share a common origin. Spectroscopic follow-up observations of the stars in EriPhe are required to fully understand the nature of this overdensity.« less

Hierarchical self-assembly of di-, tri- and tetraphenylalanine peptides capped with two fluorenyl functionalities: from polymorphs to dendrites.

PubMed

Mayans, Enric; Ballano, Gema; Casanovas, Jordi; Del Valle, Luis J; Pérez-Madrigal, Maria M; Estrany, Francesc; Jiménez, Ana I; Puiggalí, Jordi; Cativiela, Carlos; Alemán, Carlos

2016-06-28

Homopeptides with 2, 3 and 4 phenylalanine (Phe) residues and capped with fluorenylmethoxycarbonyl and fluorenylmethyl esters at the N-terminus and C-terminus, respectively, have been synthesized to examine their self-assembly capabilities. Depending on the conditions, the di- and triphenylalanine derivatives self-organize into a wide variety of stable polymorphic structures, which have been characterized: stacked braids, doughnut-like shapes, bundled arrays of nanotubes, corkscrew-like shapes and spherulitic microstructures. These highly aromatic Phe-based peptides also form incipient branched dendritic microstructures, even though they are highly unstable, making their manipulation very difficult. Conversely, the tetraphenylalanine derivative spontaneously self-assembles into stable dendritic microarchitectures made of branches growing from nucleated primary frameworks. The fractal dimension of these microstructures is ∼1.70, which provides evidence for self-similarity and two-dimensional diffusion controlled growth. DFT calculations at the M06L/6-31G(d) level have been carried out on model β-sheets since this is the most elementary building block of Phe-based peptide polymorphs. The results indicate that the antiparallel β-sheet is more stable than the parallel one, with the difference between them growing with the number of Phe residues. Thus, the cooperative effects associated with the antiparallel disposition become more favorable when the number of Phe residues increases from 2 to 4, while those of the parallel disposition remained practically constant.

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

ZnO-dotted porous ZnS cluster microspheres for high efficient, Pt-free photocatalytic hydrogen evolution.

PubMed

Wu, Aiping; Jing, Liqiang; Wang, Jianqiang; Qu, Yang; Xie, Ying; Jiang, Baojiang; Tian, Chungui; Fu, Honggang

2015-03-09

The Pt-free photocatalytic hydrogen evolution (PHE) has been the focus in the photocatalysis field. Here, the ZnO-dotted porous ZnS cluster microsphere (PCMS) is designed for high efficient, Pt-free PHE. The PCMS is designed through an easy "controlling competitive reaction" strategy by selecting the thiourea as S(2-) source and Zn(Ac)₂·2H₂O as Zn source in ethylene glycol medium. Under suitable conditions, one of the PCMS, named PCMS-1, with high SBET specific area of 194 m(2)g(-1), microsphere size of 100 nm and grain size of 3 nm can be obtained. The formation of PCMS is verified by TEM, XAES, XPS, Raman and IR methods. Importantly, a series of the experiments and theoretical calculation demonstrate that the dotting of ZnO not only makes the photo-generated electrons/hole separate efficiently, but also results in the formation of the active catalytic sites for PHE. As a result, the PCMS-1 shows the promising activity up to 367 μmol h(-1) under Pt-free condition. The PHE activity has no obvious change after addition 1 wt.% Pt, implying the presence of active catalytic sites for hydrogen evolution in the PCMS-1. The easy synthesis process, low preparation cost of the PCMS makes their large potential for Pt-free PHE.

ZnO-dotted porous ZnS cluster microspheres for high efficient, Pt-free photocatalytic hydrogen evolution

NASA Astrophysics Data System (ADS)

Wu, Aiping; Jing, Liqiang; Wang, Jianqiang; Qu, Yang; Xie, Ying; Jiang, Baojiang; Tian, Chungui; Fu, Honggang

2015-03-01

The Pt-free photocatalytic hydrogen evolution (PHE) has been the focus in the photocatalysis field. Here, the ZnO-dotted porous ZnS cluster microsphere (PCMS) is designed for high efficient, Pt-free PHE. The PCMS is designed through an easy ``controlling competitive reaction'' strategy by selecting the thiourea as S2- source and Zn(Ac)2.2H2O as Zn source in ethylene glycol medium. Under suitable conditions, one of the PCMS, named PCMS-1, with high SBET specific area of 194 m2g-1, microsphere size of 100 nm and grain size of 3 nm can be obtained. The formation of PCMS is verified by TEM, XAES, XPS, Raman and IR methods. Importantly, a series of the experiments and theoretical calculation demonstrate that the dotting of ZnO not only makes the photo-generated electrons/hole separate efficiently, but also results in the formation of the active catalytic sites for PHE. As a result, the PCMS-1 shows the promising activity up to 367 μmol h-1 under Pt-free condition. The PHE activity has no obvious change after addition 1 wt.% Pt, implying the presence of active catalytic sites for hydrogen evolution in the PCMS-1. The easy synthesis process, low preparation cost of the PCMS makes their large potential for Pt-free PHE.

Comparing Urinary Biomarkers of Airborne and Dermal Exposure to Polycyclic Aromatic Compounds in Asphalt-Exposed Workers

PubMed Central

Sobus, Jon R.; McClean, Michael D.; Herrick, Robert F.; Waidyanatha, Suramya; Nylander-French, Leena A.; Kupper, Lawrence L.; Rappaport, Stephen M.

2009-01-01

When working with hot mix asphalt, road pavers are exposed to polycyclic aromatic hydrocarbons (PAHs) through the inhalation of vapors and particulate matter (PM) and through dermal contact with PM and contaminated surfaces. Several PAHs with four to six rings are potent carcinogens which reside in these particulate emissions. Since urinary biomarkers of large PAHs are rarely detectable in asphalt workers, attention has focused upon urinary levels of the more volatile and abundant two-ring and three-ring PAHs as potential biomarkers of PAH exposure. Here, we compare levels of particulate polycyclic aromatic compounds (P-PACs, a group of aromatic hydrocarbons containing PAHs and heterocyclic compounds with four or more rings) in air and dermal patch samples from 20 road pavers to the corresponding urinary levels of naphthalene (U-Nap) (two rings), phenanthrene (U-Phe) (three rings), monohydroxylated metabolites of naphthalene (OH-Nap) and phenanthrene (OH-Phe), and 1-hydroxypyrene (OH-Pyr) (four rings), the most widely used biomarker of PAH exposure. For each worker, daily breathing-zone air (n = 55) and dermal patch samples (n = 56) were collected on three consecutive workdays along with postshift, bedtime, and morning urine samples (n = 149). Measured levels of P-PACs and the urinary analytes were used to statistically model exposure–biomarker relationships while controlling for urinary creatinine, smoking status, age, body mass index, and the timing of urine sampling. Levels of OH-Phe in urine collected postshift, at bedtime, and the following morning were all significantly associated with levels of P-PACs in air and dermal patch samples. For U-Nap, U-Phe, and OH-Pyr, both air and dermal patch measurements of P-PACs were significant predictors of postshift urine levels, and dermal patch measurements were significant predictors of bedtime urine levels (all three analytes) and morning urine levels (U-Nap and OH-Pyr only). Significant effects of creatinine concentration were observed for all analytes, and modest effects of smoking status and body mass index were observed for U-Phe and OH-Pyr, respectively. Levels of OH-Nap were not associated with P-PAC measurements in air or dermal patch samples but were significantly affected by smoking status, age, day of sample collection, and urinary creatinine. We conclude that U-Nap, U-Phe, OH-Phe, and OH-Pyr can be used as biomarkers of exposure to particulate asphalt emissions, with OH-Phe being the most promising candidate. Indications that levels of U-Nap, U-Phe, and OH-Pyr were significantly associated with dermal patch measurements well into the evening after a given work shift, combined with the small ratios of within-person variance components to between-person variance components at bedtime, suggest that bedtime measurements may be useful for investigating dermal PAH exposures. PMID:19602502

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

The Phe105 loop of Alix Bro1 domain plays a key role in HIV-1 release

PubMed Central

Sette, Paola; Mu, Ruiling; Dussupt, Vincent; Jiang, Jiansheng; Snyder, Greg; Smith, Patrick; Xiao, Tsan. Sam; Bouamr, Fadila

2011-01-01

Summary Alix and cellular paralogs HD-PTP and Brox contain N-terminal Bro1 domains that bind ESCRT-III CHMP4. In contrast to HD-PTP and Brox, expression of the Bro1 domain of Alix alleviates HIV-1 release defects due to interrupted access to ESCRT. In an attempt to elucidate this functional discrepancy, we solved the crystal structures of the Bro1 domains of HD-PTP and Brox. They revealed typical “boomerang” folds they share with the Bro1 Alix domain. However, they each contain unique structural features that may be relevant to their specific function(s). In particular, phenylalanine residue in position 105 (Phe105) of Alix belongs to a long loop that is unique to its Bro1 domain. Concurrently mutation of Phe105 and surrounding residues at the tip of the loop compromises the function of Alix in HIV-1 budding without affecting its interactions with Gag or CHMP4. These studies identify a new functional determinant in the Bro1 domain of Alix. PMID:21889351

Carboxylate-Containing Two-Photon Probe for the Simultaneous Detection of Extra- and Intracellular pH Values in Colon Cancer Tissue.

PubMed

Si, Ho Young; Cho, Myoung Ki; Kang, Ji Su; Noh, Choong-Kyun; Shin, Sung Jae; Lim, Chang Su; Kim, Hwan Myung

2018-06-11

Acidified extracellular pH (pHe) is directly related to various disorders such as tumor invasion and the resistance to drugs. In this study, we developed two-photon-excitable emission ratiometric probes (XBH1-3) for the in situ measurement of pHe. These probes, based on benzimidazole and polar solubilizing groups, exhibited a strong two-photon-induced fluorescence and sensitive blue-to-green emission color changes with p K a values of 5.1-5.7. XBH1, containing a carboxylic acid, stained the extracellular region in neutral media; it entered the cell under acidic media, thereby allowing a precise measurement of the extra- and intra-cellular pH values in the acidified tissue. XBH2, containing the sulfonate peripheral unit, facilitated the monitoring of the pHe value only. Ratiometric two-photon microscopy imaging revealed that XBH1 can directly monitor the pH values both inside and outside the cells in colon cancer tissue; there is also the morphological aspect. This could be useful for cancer analyses and drug development.

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.

Conformation-Specific Spectroscopy of a Prototypical γ-PEPTIDE-WATER Complex: Ac-γ2-hPhe-NHMe-(H2O)1

NASA Astrophysics Data System (ADS)

Buchanan, Evan G.; James, William H., III; Zwier, Timothy S.; Guo, Li; Gellman, Samuel H.

2010-06-01

The prototypical γ-peptide, Ac-γ2-hPhe-NHMe, has been previously studied in a supersonic jet expansion, with three different conformers observed. Two of the monomers form nine atom, intramolecular hydrogen bonded rings, which differ by the position of the aromatic chromophore relative to the backbone. The third monomer conformer has no intramolecular H-bonds, but forms instead an intramolecular, amide-amide stacked structure unique to the γ-peptide backbone. This talk focuses attention on the conformation-specific IR spectra of the Ac-γ2-hPhe-NHMe-(H2O)1 complex, which is observed to form six unique conformational isomers, all of which preserve the two distinct monomer structural motifs. Three conformers are assigned to the nine atom intramolecular hydrogen bond family with the water hydrogen bonded to it as donor in different locations. The other three belong to the amide-amide stacking family with the water forming a bridge between the two amide planes. Infrared photodissocation of the water molecule from the complex to form γ-peptide monomer conformations will also be discussed.

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.

Investigation of chromatography and polymer/salt aqueous two-phase processes for downstream processing development of recombinant phenylalanine dehydrogenase.

PubMed

Omidinia, Eskandar; Shahbaz Mohamadi, Hamid; Dinarvand, Rassoul; Taherkhani, Heshmat-Allah

2010-03-01

This work presents a comprehensive study between the polymer/salt aqueous two-phase systems (ATPS) and chromatography process for downstream processing of recombinant Bacillus badius phenylalanine dehydrogenase (PheDH). First, the partitioning behavior of recombinant PheDH in polyethylene glycol (PEG)/K2HPO4 ATPS was examined. For comparative purpose, a classical chromatographic protocol was performed as well. Investigation of chromatography and ATPS procedures revealed that the ATPS comprising of 9% (w/w) PEG-6000, 16% (w/w) K2HPO4 and 16% (w/w) KCl with pH of 8.0, volume ratio (V ( R )) of 0.25, temperature of 25 degrees C and 40% (w/w) cell lysate ensured the most favorable approach for PheDH downstream process. A specific activity of 4,231.4 U/mg, a yield of 96.7% and a recovery of 162.0% were obtained. Furthermore, the shorter process time (4 vs. 48 h) and the lower total cost (4 vs. 20 euro) were additionally features that confirmed the suitability of proposed technique.

40 CFR 721.1850 - Toluene sulfonamide bis-phe-nol A epoxy adduct.

Code of Federal Regulations, 2014 CFR

2014-07-01

... epoxy adduct. 721.1850 Section 721.1850 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.1850 Toluene sulfonamide bis-phe-nol A epoxy adduct. (a) Chemical... as toluene sulfonamide bisphenol A epoxy adduct (PMN P-90-113) is subject to reporting under this...

40 CFR 721.1850 - Toluene sulfonamide bis-phe-nol A epoxy adduct.

Code of Federal Regulations, 2012 CFR

2012-07-01

... epoxy adduct. 721.1850 Section 721.1850 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.1850 Toluene sulfonamide bis-phe-nol A epoxy adduct. (a) Chemical... as toluene sulfonamide bisphenol A epoxy adduct (PMN P-90-113) is subject to reporting under this...

40 CFR 721.1850 - Toluene sulfonamide bis-phe-nol A epoxy adduct.

Code of Federal Regulations, 2013 CFR

2013-07-01

... epoxy adduct. 721.1850 Section 721.1850 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.1850 Toluene sulfonamide bis-phe-nol A epoxy adduct. (a) Chemical... as toluene sulfonamide bisphenol A epoxy adduct (PMN P-90-113) is subject to reporting under this...

40 CFR 721.1850 - Toluene sulfonamide bis-phe-nol A epoxy adduct.

Code of Federal Regulations, 2011 CFR

2011-07-01

... epoxy adduct. 721.1850 Section 721.1850 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.1850 Toluene sulfonamide bis-phe-nol A epoxy adduct. (a) Chemical... as toluene sulfonamide bisphenol A epoxy adduct (PMN P-90-113) is subject to reporting under this...

Assessment of polycyclic aromatic hydrocarbons (PAHs) in mussels (Mytilus galloprovincialis) of Prince Islands, Marmara Sea.

PubMed

Balcıoğlu, Esra Billur

2016-08-15

In this study, PAH analyses have been conducted on indigenous mussels. Mussel samples (Mytilus galloprovincialis) have been collected from seven stations of Prince Islands during September 2015. Concentrations of total determined PAHs (sum of 16 compounds) ranged between 664 and 9083ngg(-1). The origin of PAHs has been found to be pyrolytic according to the PHE/ANT and FA/PYR ratios in Büyükada. For other islands, PAH origins have been observed as pyrolytic and petrogenic together according to the PHE/ANT, FA/PYR and BaA/CHR ratios. Copyright © 2016 Elsevier Ltd. All rights reserved.

Breast Cancer Risk Associated with Genotype Polymorphisms of the Aurora Kinase a Gene (AURKA): a Case-Control Study in a High Altitude Ecuadorian Mestizo Population.

PubMed

López-Cortés, Andrés; Cabrera-Andrade, Alejandro; Oña-Cisneros, Fabián; Rosales, Felipe; Ortiz, Malena; Tejera, Eduardo; Paz-Y-Miño, César

2018-07-01

Breast cancer (BC) is the leading cause of cancer related death among women in 2014. The AURKA gene that encodes the protein called Aurora kinase A plays an important role in the progression of the cell cycle, by controlling and promoting the entry into the phase of mitosis. The single nucleotide polymorphism AURKA T91A (rs2273535) (Phe21Ile) has been identified as functional alternator of this kinase, the Ile allele is associated with the occurrence of chromosome segregation errors and tumor progression. Therefore, it is essential to know how BC risk is associated with histopathological characteristics, immunohistochemical characteristics, and genotype polymorphism in a high altitude Ecuadorian mestizo population. In this retrospective case-control study 200 individuals were analyzed. DNA was extracted from 100 healthy and 100 affected women. Genotypes were determined by genomic sequencing. We found significant association between the AURKA T91A (rs2273535) (Phe21Ile) genotype and an increased risk of BC development: Phe/Ile (odds ratio [OR] = 2.6; 95% confidence interval [CI] = 1.4-4.9; P = 0.004), Ile/Ile (OR = 3.8; 95% CI = 1.6-9.0; P = 0.002), and Phe/Ile + Ile/Ile (OR = 2.9; 95% CI = 1.6-5.2; P = 0.001). Additionally, the rs2273535 variant was associated with the tumor grade SBR III (OR = 9.6; 95% CI = 1.0-91.9; P = 0.048) and the Ki-67 ≥ 20 (OR = 16.5; 95% CI = 2.7-101.3; P = 0.002). In brief, this study provides the first evidence where the Ile allele of the AURKA gene could act as potentially predictive biomarker of BC in the high altitude Ecuadorian mestizo population that lives at 2800 m above sea level (masl).

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.

Small molecule inhibitors of Ca 2+-S100B reveal two protein conformations

DOE PAGES

Cavalier, Michael C.; Ansari, Mohd. Imran; Pierce, Adam D.; ...

2016-01-04

The drug pentamidine inhibits calcium-dependent complex formation with p53 ( CaS100B·p53) in malignant melanoma (MM) and restores p53 tumor suppressor activity in vivo. However, off-target effects associated with this drug were problematic in MM patients. Structure–activity relationship (SAR) studies were therefore completed in this study with 23 pentamidine analogues, and X-ray structures of CaS100B·inhibitor complexes revealed that the C-terminus of S100B adopts two different conformations, with location of Phe87 and Phe88 being the distinguishing feature and termed the “FF-gate”. For symmetric pentamidine analogues ( CaS100B· 5a, CaS100B· 6b) a channel between sites 1 and 2 on S100B was occluded bymore » residue Phe88, but for an asymmetric pentamidine analogue ( CaS100B· 17), this same channel was open. Finally, the CaS100B· 17 structure illustrates, for the first time, a pentamidine analog capable of binding the “open” form of the “FF-gate” and provides a means to block all three “hot spots” on CaS100B, which will impact next generation CaS100B·p53 inhibitor design.« less

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.

Small molecule inhibitors of Ca 2+-S100B reveal two protein conformations

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

Cavalier, Michael C.; Ansari, Mohd. Imran; Pierce, Adam D.

The drug pentamidine inhibits calcium-dependent complex formation with p53 ( CaS100B·p53) in malignant melanoma (MM) and restores p53 tumor suppressor activity in vivo. However, off-target effects associated with this drug were problematic in MM patients. Structure–activity relationship (SAR) studies were therefore completed in this study with 23 pentamidine analogues, and X-ray structures of CaS100B·inhibitor complexes revealed that the C-terminus of S100B adopts two different conformations, with location of Phe87 and Phe88 being the distinguishing feature and termed the “FF-gate”. For symmetric pentamidine analogues ( CaS100B· 5a, CaS100B· 6b) a channel between sites 1 and 2 on S100B was occluded bymore » residue Phe88, but for an asymmetric pentamidine analogue ( CaS100B· 17), this same channel was open. Finally, the CaS100B· 17 structure illustrates, for the first time, a pentamidine analog capable of binding the “open” form of the “FF-gate” and provides a means to block all three “hot spots” on CaS100B, which will impact next generation CaS100B·p53 inhibitor design.« less

Polyurethane foam (PUF) passive samplers for monitoring phenanthrene in stormwater.

PubMed

Dou, Yueqin; Zhang, Tian C; Zeng, Jing; Stansbury, John; Moussavi, Massoum; Richter-Egger, Dana L; Klein, Mitchell R

2016-04-01

Pollution from highway stormwater runoff has been an increasing area of concern. Many structural Best Management Practices (BMPs) have been implemented for stormwater treatment and management. One challenge for these BMPs is to sample stormwater and monitor BMP performance. The main objective of this study was to evaluate the feasibility of using polyurethane foam (PUF) passive samplers (PSs) for sampling phenanthrene (PHE) in highway stormwater runoff and BMPs. Tests were conducted using batch reactors, glass-tube columns, and laboratory-scale BMPs (bioretention cells). Results indicate that sorption for PHE by PUF is mainly linearly relative to time, and the high sorption capacity allows the PUF passive sampler to monitor stormwater events for months or years. The PUF passive samplers could be embedded in BMPs for monitoring influent and effluent PHE concentrations. Models developed to link the results of batch and column tests proved to be useful for determining removal or sorption parameters and performance of the PUF-PSs. The predicted removal efficiencies of BMPs were close to the real values obtained from the control columns with errors ranging between -8.46 and 1.52%. This research showed that it is possible to use PUF passive samplers for sampling stormwater and monitoring the performance of stormwater BMPs, which warrants the field-scale feasibility studies in the future.

An eco-friendly in situ activatable antibiotic via cucurbit[8]uril-mediated supramolecular crosslinking of branched polyethylenimine.

PubMed

Li, Shengke; Jiang, Nan; Zhao, Wenxuan; Ding, Yuan-Fu; Zheng, Ying; Wang, Lian-Hui; Zheng, Jun; Wang, Ruibing

2017-05-30

We report an unprecedented, eco-friendly, in situ activatable model antibiotic, phenylalanyl-polyethylenimine (PhePEI), to potentially diminish antibiotic pollution of the environment and associated antibiotic resistance. The inactive PhePEI can be reversibly activated upon supramolecular crosslinking by cucurbit[8]uril, conferring potent antibacterial activity.

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.

Functional characterization of salt-tolerant microbial esterase WDEst17 and its use in the generation of optically pure ethyl (R)-3-hydroxybutyrate.

PubMed

Wang, Yilong; Xu, Yongkai; Zhang, Yun; Sun, Aijun; Hu, Yunfeng

2018-06-01

The two enantiomers of ethyl 3-hydroxybutyrate are important intermediates for the synthesis of a great variety of valuable chiral drugs. The preparation of chiral drug intermediates through kinetic resolution reactions catalyzed by esterases/lipases has been demonstrated to be an efficient and environmentally friendly method. We previously functionally characterized microbial esterase PHE21 and used PHE21 as a biocatalyst to generate optically pure ethyl (S)-3-hydroxybutyrate. Herein, we also functionally characterized one novel salt-tolerant microbial esterase WDEst17 from the genome of Dactylosporangium aurantiacum subsp. Hamdenensis NRRL 18085. Esterase WDEst17 was further developed as an efficient biocatalyst to generate (R)-3-hydroxybutyrate, an important chiral drug intermediate, with the enantiomeric excess being 99% and the conversion rate being 65.05%, respectively, after process optimization. Notably, the enantio-selectivity of esterase WDEst17 was opposite than that of esterase PHE21. The identification of esterases WDEst17 and PHE21 through genome mining of microorganisms provides useful biocatalysts for the preparation of valuable chiral drug intermediates. © 2018 Wiley Periodicals, Inc.

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.

Ultra-high resolution X-ray structures of two forms of human recombinant insulin at 100 K.

PubMed

Lisgarten, David R; Palmer, Rex A; Lobley, Carina M C; Naylor, Claire E; Chowdhry, Babur Z; Al-Kurdi, Zakieh I; Badwan, Adnan A; Howlin, Brendan J; Gibbons, Nicholas C J; Saldanha, José W; Lisgarten, John N; Basak, Ajit K

2017-08-01

The crystal structure of a commercially available form of human recombinant (HR) insulin, Insugen (I), used in the treatment of diabetes has been determined to 0.92 Å resolution using low temperature, 100 K, synchrotron X-ray data collected at 16,000 keV (λ = 0.77 Å). Refinement carried out with anisotropic displacement parameters, removal of main-chain stereochemical restraints, inclusion of H atoms in calculated positions, and 220 water molecules, converged to a final value of R = 0.1112 and R free  = 0.1466. The structure includes what is thought to be an ordered propanol molecule (POL) only in chain D(4) and a solvated acetate molecule (ACT) coordinated to the Zn atom only in chain B(2). Possible origins and consequences of the propanol and acetate molecules are discussed. Three types of amino acid representation in the electron density are examined in detail: (i) sharp with very clearly resolved features; (ii) well resolved but clearly divided into two conformations which are well behaved in the refinement, both having high quality geometry; (iii) poor density and difficult or impossible to model. An example of type (ii) is observed for the intra-chain disulphide bridge in chain C(3) between Sγ6-Sγ11 which has two clear conformations with relative refined occupancies of 0.8 and 0.2, respectively. In contrast the corresponding S-S bridge in chain A(1) shows one clearly defined conformation. A molecular dynamics study has provided a rational explanation of this difference between chains A and C. More generally, differences in the electron density features between corresponding residues in chains A and C and chains B and D is a common observation in the Insugen (I) structure and these effects are discussed in detail. The crystal structure, also at 0.92 Å and 100 K, of a second commercially available form of human recombinant insulin, Intergen (II), deposited in the Protein Data Bank as 3W7Y which remains otherwise unpublished is compared here with the Insugen (I) structure. In the Intergen (II) structure there is no solvated propanol or acetate molecule. The electron density of Intergen (II), however, does also exhibit the three types of amino acid representations as in Insugen (I). These effects do not necessarily correspond between chains A and C or chains B and D in Intergen (II), or between corresponding residues in Insugen (I). The results of this comparison are reported. Graphical abstract Conformations of PheB25 and PheD25 in three insulin structures: implications for biological activity? Insulin residues PheB25 and PheD25 are considered to be important for insulin receptor binding and changes in biological activity occur when these residues are modified. In porcine insulin and Intergen (II) PheB25 adopts conformation B and PheD25 conformation D. However, unexpectedly PheB25 in Insugen (I) human recombinant insulin adopts two distinct conformations corresponding to B and D, Figure 1 and PheD25 adopts a single conformation corresponding to B not D, Figure 2. Conformations of this residue in the ultra-high resolution structure of Insugen (I) are therefore unique within this set. Figures were produced with Biovia, Discovery Studio 2016.

Exploring the inter-molecular interactions in amyloid-β protofibril with molecular dynamics simulations and molecular mechanics Poisson-Boltzmann surface area free energy calculations.

PubMed

Liu, Fu-Feng; Liu, Zhen; Bai, Shu; Dong, Xiao-Yan; Sun, Yan

2012-04-14

Aggregation of amyloid-β (Aβ) peptides correlates with the pathology of Alzheimer's disease. However, the inter-molecular interactions between Aβ protofibril remain elusive. Herein, molecular mechanics Poisson-Boltzmann surface area analysis based on all-atom molecular dynamics simulations was performed to study the inter-molecular interactions in Aβ(17-42) protofibril. It is found that the nonpolar interactions are the important forces to stabilize the Aβ(17-42) protofibril, while electrostatic interactions play a minor role. Through free energy decomposition, 18 residues of the Aβ(17-42) are identified to provide interaction energy lower than -2.5 kcal/mol. The nonpolar interactions are mainly provided by the main chain of the peptide and the side chains of nine hydrophobic residues (Leu17, Phe19, Phe20, Leu32, Leu34, Met35, Val36, Val40, and Ile41). However, the electrostatic interactions are mainly supplied by the main chains of six hydrophobic residues (Phe19, Phe20, Val24, Met35, Val36, and Val40) and the side chains of the charged residues (Glu22, Asp23, and Lys28). In the electrostatic interactions, the overwhelming majority of hydrogen bonds involve the main chains of Aβ as well as the guanidinium group of the charged side chain of Lys28. The work has thus elucidated the molecular mechanism of the inter-molecular interactions between Aβ monomers in Aβ(17-42) protofibril, and the findings are considered critical for exploring effective agents for the inhibition of Aβ aggregation.

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.

Exploring the inter-molecular interactions in amyloid-β protofibril with molecular dynamics simulations and molecular mechanics Poisson-Boltzmann surface area free energy calculations

NASA Astrophysics Data System (ADS)

Liu, Fu-Feng; Liu, Zhen; Bai, Shu; Dong, Xiao-Yan; Sun, Yan

2012-04-01

Aggregation of amyloid-β (Aβ) peptides correlates with the pathology of Alzheimer's disease. However, the inter-molecular interactions between Aβ protofibril remain elusive. Herein, molecular mechanics Poisson-Boltzmann surface area analysis based on all-atom molecular dynamics simulations was performed to study the inter-molecular interactions in Aβ17-42 protofibril. It is found that the nonpolar interactions are the important forces to stabilize the Aβ17-42 protofibril, while electrostatic interactions play a minor role. Through free energy decomposition, 18 residues of the Aβ17-42 are identified to provide interaction energy lower than -2.5 kcal/mol. The nonpolar interactions are mainly provided by the main chain of the peptide and the side chains of nine hydrophobic residues (Leu17, Phe19, Phe20, Leu32, Leu34, Met35, Val36, Val40, and Ile41). However, the electrostatic interactions are mainly supplied by the main chains of six hydrophobic residues (Phe19, Phe20, Val24, Met35, Val36, and Val40) and the side chains of the charged residues (Glu22, Asp23, and Lys28). In the electrostatic interactions, the overwhelming majority of hydrogen bonds involve the main chains of Aβ as well as the guanidinium group of the charged side chain of Lys28. The work has thus elucidated the molecular mechanism of the inter-molecular interactions between Aβ monomers in Aβ17-42 protofibril, and the findings are considered critical for exploring effective agents for the inhibition of Aβ aggregation.

Double-tagged fluorescent bacterial bioreporter for the study of polycyclic aromatic hydrocarbon diffusion and bioavailability.

PubMed

Tecon, Robin; Binggeli, Olivier; van der Meer, Jan R

2009-09-01

Bacterial degradation of polycyclic aromatic hydrocarbons (PAHs), ubiquitous contaminants from oil and coal, is typically limited by poor accessibility of the contaminant to the bacteria. In order to measure PAH availability in complex systems, we designed a number of diffusion-based assays with a double-tagged bacterial reporter strain Burkholderia sartisoli RP037-mChe. The reporter strain is capable of mineralizing phenanthrene (PHE) and induces the expression of enhanced green fluorescent protein (eGFP) as a function of the PAH flux to the cell. At the same time, it produces a second autofluorescent protein (mCherry) in constitutive manner. Quantitative epifluorescence imaging was deployed in order to record reporter signals as a function of PAH availability. The reporter strain expressed eGFP proportionally to dosages of naphthalene or PHE in batch liquid cultures. To detect PAH diffusion from solid materials the reporter cells were embedded in 2 cm-sized agarose gel patches, and fluorescence was recorded over time for both markers as a function of distance to the PAH source. eGFP fluorescence gradients measured on known amounts of naphthalene or PHE served as calibration for quantifying PAH availability from contaminated soils. To detect reporter gene expression at even smaller diffusion distances, we mixed and immobilized cells with contaminated soils in an agarose gel. eGFP fluorescence measurements confirmed gel patch diffusion results that exposure to 2-3 mg lampblack soil gave four times higher expression than to material contaminated with 10 or 1 (mg PHE) g(-1).

Phe71 in Type III Trypanosomal Protein Arginine Methyltransferase 7 (TbPRMT7) Restricts the Enzyme to Monomethylation.

PubMed

Cáceres, Tamar B; Thakur, Abhishek; Price, Owen M; Ippolito, Nicole; Li, Jun; Qu, Jun; Acevedo, Orlando; Hevel, Joan M

2018-02-27

Protein arginine methyltransferase 7 (PRMT7) is unique within the PRMT family as it is the only isoform known to exclusively make monomethylarginine (MMA). Given its role in epigenetics, the mechanistic basis for the strict monomethylation activity is under investigation. It is thought that PRMT7 enzymes are unable to add a second methyl group because of steric hindrance in the active site that restricts them to monomethylation. To test this, we probed the active site of trypanosomal PRMT7 (TbPRMT7) using accelerated molecular dynamics, site-directed mutagenesis, kinetic, binding, and product analyses. Both the dynamics simulations and experimental results show that the mutation of Phe71 to Ile converts the enzyme from a type III methyltransferase into a mixed type I/II, that is, an enzyme that can now perform dimethylation. In contrast, the serine and alanine mutants of Phe71 preserve the type III behavior of the native enzyme. These results are inconsistent with a sterics-only model to explain product specificity. Instead, molecular dynamics simulations of these variants bound to peptides show hydrogen bonding between would-be substrates and Glu172 of TbPRMT7. Only in the case of the Phe71 to Ile mutation is this interaction between MMA and the enzyme maintained, and the geometry for optimal S N 2 methyl transfer is obtained. The results of these studies highlight the benefit of combined computational and experimental methods in providing a better understanding for how product specificity is dictated by PRMTs.

[Association between platelet-activating factor acetylhydrolase gene polymorphisms and gastrointestinal bleeding in children with Henoch-Schönlein purpura].

PubMed

Wang, Bao-Xiang; Mei, Hong; Peng, Han-Ming; Gao, Yuan; Ding, Yan

2017-04-01

To study the association between the single nucleotide polymorphisms (SNPs) of the ninth exon Val279Phe of platelet-activating factor acetylhydrolase (PAF-AH) gene and gastrointestinal bleeding in children with Henoch-Schönlein purpura (HSP). A total 516 children with HSP were enrolled, among whom 182 had gastrointestinal bleeding and 334 had no gastrointestinal bleeding. PCR was used to investigate the distribution of genotypes and alleles in the SNPs of Val97Phe. The plasma PAF-AH activity was measured, as well as the levels of platelet-activating factor (PAF), granular membrane protein-140 (GMP-140), β-thromboglobulin (β-TG), and platelet factor 4 (PF4). The Val279Phe genotype and allele frequencies were in Hardy-Weinberg equilibrium, and the homozygous genotype TT and heterozygotes accounted for 0.97% and 6.05% respectively. The gastrointestinal bleeding group had a significantly higher allele frequency than the control group (5.22% vs 3.33%; P<0.01). The HSP patients with GG genotype in the gastrointestinal bleeding group had significantly higher levels of plasma PAF and GMP-140 than those in the non-gastrointestinal bleeding group (P<0.05), while the non-gastrointestinal bleeding group had a significantly higher PAF-AH activity than the gastrointestinal bleeding group (P<0.05). There were no significant differences in β-TG and PF4 between the two groups (P>0.05). Val279Phe gene polymorphisms in PAF-AH are associated with PAF-AH activity and PAF and GMP-140 levels and may be a risk factor for HSP with gastrointestinal bleeding.

Attenuation of phenanthrene and pyrene adsorption by sewage sludge-derived biochar in biochar-amended soils.

PubMed

Zielińska, Anna; Oleszczuk, Patryk

2016-11-01

The aim of this study was to evaluate the effect of soils on the sorption of phenanthrene (PHE) and pyrene (PYR) by sewage sludge-derived biochars (SS-derived biochars). The SS-derived biochars were added to soils with varying properties as well as with a different degree and source of polycyclic aromatic hydrocarbons (PAHs) contamination. The biochars (BCs) were produced from sewage sludge during pyrolysis at temperatures of 500 °C (BC500) and 700 °C (BC700). The addition of biochars to the soils (5 %, w/w) increased the sorption of PHE from 8.3 to 20.3 % and PYR from 14.5 to 31.7 % by amended soil. BC700 biochar was characterized by better sorption capacity than BC500 biochar. Nevertheless, the presence of soil reduces the effectiveness of biochars in binding the compounds studied. The sorption capacity of the biochars decreased several times after they had been mixed with the soil compared to pure biochars. The study found dissolved organic carbon (DOC) and clay minerals present in the soils to have a significant effect on reducing the efficiency of PHE and PYR sorption by biochar. A greater impact of fouling was observed in the case of BC500 biochar characterized by lower porosity than BC700 biochar.

Serum ischemia modified albumin is a possible new marker of oxidative stress in phenylketonuria.

PubMed

Keshavarzi, Fatemeh; Rastegar, Mohsen; Vessal, Mahmood; Rafiei Dehbidi, Gholamreza; Khorsand, Marjan; Ganjkarimi, Amir Hossein; Takhshid, Mohammad Ali

2018-06-01

The role of oxidative stress in the pathogenesis of phenylketonuria (PKU)-associated disorders has been implicated. Ischemia modified albumin (IMA) is a modified form of serum albumin, which is produced under the conditions of oxidative stress. The aim of this study was to measure the serum level of IMA in the PKU patients and to investigate its ability in predicting the status of oxidative stress in these patients. Fifty treated-PKU patients and fifty age- and sex-matched healthy subjects were included in the study. The blood samples were obtained and the serum level of phenylalanine (Phe) was measured using reverse phase HPLC method. The levels of IMA, malondialdehyde (MDA), gamma-glutamyl transferase (GGT) activity, and uric acid (UA) were determined using colorimetric methods. The levels of serum Phe, IMA, and MDA were significantly higher (p < 0.001) and the level of UA (p < 0.05) was lower in the PKU patients compared to control group. Serum IMA level was positively correlated with MDA (r = 0.585, p < 0.001) and UA (r = 0.6, p < 0.001). An inverse relationship was observed between the serum level of IMA and Phe (r = - 0.410, p < 0. 01). Results of the present study suggest that serum IMA level could be used as a novel marker for the evaluation of oxidative stress in the PKU patients.

Amino acid specific stable nitrogen isotope values in avian tissues: Insights from captive American kestrels and wild herring gulls

USGS Publications Warehouse

Hebert, Craig E.; Popp, B.N.; Fernie, K.J.; Ka'apu-Lyons, C.; Rattner, Barnett A.; Wallsgrove, N.

2016-01-01

Through laboratory and field studies, the utility of amino acid compound-specific nitrogen isotope analysis (AA-CSIA) in avian studies is investigated. Captive American kestrels (Falco sparverius) were fed an isotopically characterized diet and patterns in δ15N values of amino acids (AAs) were compared to those in their tissues (muscle and red blood cells) and food. Based upon nitrogen isotope discrimination between diet and kestrel tissues, AAs could mostly be categorized as source AAs (retaining baseline δ15N values) and trophic AAs (showing 15N enrichment). Trophic discrimination factors based upon the source (phenylalanine, Phe) and trophic (glutamic acid, Glu) AAs were 4.1 (muscle) and 5.4 (red blood cells), lower than those reported for metazoan invertebrates. In a field study involving omnivorous herring gulls (Larus argentatus smithsonianus), egg AA isotopic patterns largely retained those observed in the laying female’s tissues (muscle, red blood cells, and liver). Realistic estimates of gull trophic position were obtained using bird Glu and Phe δ15N values combined with β values (difference in Glu and Phe δ15N in primary producers) for aquatic and terrestrial food webs. Egg fatty acids were used to weight β values for proportions of aquatic and terrestrial food in gull diets. This novel approach can be applied to generalist species that feed across ecosystem boundaries.

Stabilizing interactions between aromatic and basic side chains in alpha-helical peptides and proteins. Tyrosine effects on helix circular dichroism.

PubMed

Andrew, Charles D; Bhattacharjee, Samita; Kokkoni, Nicoleta; Hirst, Jonathan D; Jones, Gareth R; Doig, Andrew J

2002-10-30

Here we investigate the structures and energetics of interactions between aromatic (Phe or Tyr) and basic (Lys or Arg) amino acids in alpha-helices. Side chain interaction energies are measured using helical peptides, by quantifying their helicities with circular dichroism at 222 nm and interpreting the results with Lifson-Roig-based helix/coil theory. A difficulty in working with Tyr is that the aromatic ring perturbs the CD spectrum, giving an incorrect helicity. We calculated the effect of Tyr on the CD at 222 nm by deriving the intensities of the bands directly from the electronic and magnetic transition dipole moments through the rotational strengths corresponding to each excited state of the polypeptide. This gives an improved value of the helix preference of Tyr (from 0.48 to 0.35) and a correction to the helicity for the peptides containing Tyr. We find that Phe-Lys, Lys-Phe, Phe-Arg, Arg-Phe, and Tyr-Lys are all stabilizing by -0.10 to -0.18 kcal.mol-1 when placed i, i + 4 on the surface of a helix in aqueous solution, despite the great difference in polarity between these residues. Interactions between these side chains have previously been attributed to cation-pi bonds. A survey of protein structures shows that they are in fact predominantly hydrophobic interactions between the CH2 groups of Lys or Arg and the aromatic rings.

Discovery of a stellar overdensity in Eridanus-Phoenix in the dark energy survey

DOE PAGES

Li, T. S.; Balbinot, E.; Mondrik, N.; ...

2016-01-27

We report the discovery of an excess of main sequence turn-off stars in the direction of the constellations of Eridanus and Phoenix from the first year data of the Dark Energy Survey (DES). The Eridanus-Phoenix (EriPhe) overdensity is centered around l~285 deg and b~-60 deg and spans at least 30 deg in longitude and 10 deg in latitude. The Poisson significance of the detection is at least 9 sigma. The stellar population in the overdense region is similar in brightness and color to that of the nearby globular cluster NGC 1261, indicating that the heliocentric distance of EriPhe is aboutmore » d~16 kpc. The extent of EriPhe in projection is therefore at least ~4 kpc by ~3 kpc. On the sky, this overdensity is located between NGC 1261 and a new stellar stream discovered by DES at a similar heliocentric distance, the so-called Phoenix Stream. Given their similar distance and proximity to each other, it is possible that these three structures may be kinematically associated. Alternatively, the EriPhe overdensity is morphologically similar to the Virgo overdensity and the Hercules-Aquila cloud, which also lie at a similar Galactocentric distance. These three overdensities lie along a polar plane separated by ~120 deg and may share a common origin. Spectroscopic follow-up observations of the stars in EriPhe are required to fully understand the nature of this overdensity.« less

ACE-like hydrolysis of gastrin analogs and CCK-8 by fundic mucosal cells of different species with release of the amidated C-terminal dipeptide.

PubMed

Dubreuil, P; Fulcrand, P; Rodriguez, M; Laur, J; Bali, J P; Martinez, J

1990-06-19

Various gastrin analogues and CCK-8 (Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2) are hydrolyzed in vitro by angiotensin-converting enzyme (ACE), the main and initial cleavage occurring at the Met-Asp (or Leu-Asp) bond, releasing the C-terminal dipeptide amide Asp-Phe-NH2. Tetragastrin analogues (e.g., Boc-Trp-Leu-Asp-Phe-NH2) are degraded by a vesicular membrane fraction from rat gastric mucosa, yielding the C-terminal dipeptide Asp-Phe-NH2. We report here on the degradation of gastrin analogues and CCK-8 by a gastric mucosal cell preparation containing specific gastrin receptors. We have shown that gastrin analogues were specifically degraded by gastric mucosal cells from different species (e.g., rabbit and dog) at 37 degrees C (pH 7.4), releasing the C-terminal dipeptide Asp-Phe-NH2, similarly to ACE. This cleavage was found to be temperature and pH sensitive, and was inhibited by metalloproteinase inhibitors and by captopril, strongly suggesting that this enzymatic system closely resembles ACE. We have also demonstrated that a close correlation seems to exist between the apparent affinity of the gastrin analogues for gastrin receptors on gastric mucosal cells, and their ability of being hydrolyzed by this cell preparation. Moreover, all gastrin analogues which have been demonstrated to act as gastrin antagonists remained unaffected in the incubation conditions.

Diversity and distribution of 16S rRNA and phenol monooxygenase genes in the rhizosphere and endophytic bacteria isolated from PAH-contaminated sites

NASA Astrophysics Data System (ADS)

Peng, Anping; Liu, Juan; Ling, Wanting; Chen, Zeyou; Gao, Yanzheng

2015-07-01

This is the first investigation of the diversity and distribution of 16S rRNA and phenol monooxygenase (PHE) genes in endophytic and rhizosphere bacteria of plants at sites contaminated with different levels of PAHs. Ten PAHs at concentrations from 34.22 to 55.29 and 45.79 to 97.81 mg·kg-1 were measured in rhizosphere soils of Alopecurus aequalis Sobol and Oxalis corniculata L., respectively. The diversity of 16S rRNA and PHE genes in rhizosphere soils or plants changed with varying PAH pollution levels, as shown based on PCR-DGGE data. Generally, higher Shannon-Weiner indexes were found in mild or moderate contaminated areas. A total of 82 different bacterial 16S rRNA gene sequences belonging to five phyla; namely, Acfinobacteria, Proteobacteria, Chloroflexi, Cyanophyta, and Bacteroidetes, were obtained from rhizosphere soils. For the 57 identified PHE gene sequences, 18 were excised from rhizosphere bacteria and 39 from endophytic bacteria. The copy numbers of 16S rRNA and PHE genes in rhizosphere and endophytic bacteria varied from 3.83 × 103 to 2.28 × 106 and 4.17 × 102 to 1.99 × 105, respectively. The copy numbers of PHE genes in rhizosphere bacteria were significantly higher than in endophytic bacteria. Results increase our understanding of the diversity of rhizosphere and endophytic bacteria from plants grown in PAH-contaminated sites.

Embryonic common snapping turtles (Chelydra serpentina) preferentially regulate intracellular tissue pH during acid-base challenges.

PubMed

Shartau, Ryan B; Crossley, Dane A; Kohl, Zachary F; Brauner, Colin J

2016-07-01

The nests of embryonic turtles naturally experience elevated CO2 (hypercarbia), which leads to increased blood PCO2  and a respiratory acidosis, resulting in reduced blood pH [extracellular pH (pHe)]. Some fishes preferentially regulate tissue pH [intracellular pH (pHi)] against changes in pHe; this has been proposed to be associated with exceptional CO2 tolerance and has never been identified in amniotes. As embryonic turtles may be CO2 tolerant based on nesting strategy, we hypothesized that they preferentially regulate pHi, conferring tolerance to severe acute acid-base challenges. This hypothesis was tested by investigating pH regulation in common snapping turtles (Chelydra serpentina) reared in normoxia then exposed to hypercarbia (13 kPa PCO2 ) for 1 h at three developmental ages: 70% and 90% of incubation, and yearlings. Hypercarbia reduced pHe but not pHi, at all developmental ages. At 70% of incubation, pHe was depressed by 0.324 pH units while pHi of brain, white muscle and lung increased; heart, liver and kidney pHi remained unchanged. At 90% of incubation, pHe was depressed by 0.352 pH units but heart pHi increased with no change in pHi of other tissues. Yearlings exhibited a pHe reduction of 0.235 pH units but had no changes in pHi of any tissues. The results indicate common snapping turtles preferentially regulate pHi during development, but the degree of response is reduced throughout development. This is the first time preferential pHi regulation has been identified in an amniote. These findings may provide insight into the evolution of acid-base homeostasis during development of amniotes, and vertebrates in general. © 2016. Published by The Company of Biologists Ltd.

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.

Effect of phenylephrine bolus administration on left ventricular function during postural hypotension in anesthetized patients.

PubMed

Goertz, A W; Schmidt, M; Lindner, K H; Seefelder, C; Georgieff, M

1993-01-01

To investigate the effect of intravenous (IV) phenylephrine (PHE) bolus administration on left ventricular function in patients who developed postural hypotension during isoflurane anesthesia in the head-up tilt (reverse Trendelenburg) position. Prospective "before-after" trial. Operation theater of a university medical center. 15 ASA physical status I and II patients without cardiovascular disorders. The anesthetized patients were tilted from a supine horizontal to a 30-degree reverse-Trendelenburg position. Once a steady state was achieved, PHE 3 micrograms/kg was administered as an IV bolus dose. Transesophageal echocardiography was used to assess left ventricular function. We measured blood pressure (BP); heart rate; left ventricular end-systolic and end-diastolic area, diameter, and wall thickness; and ejection time at baseline and after tilt, immediately before and for a period of 3 minutes after PHE injection. We calculated fractional area change (FAC), mean velocity of circumferential fiber shortening (mVcf), and end-systolic wall stress. Head-up tilt caused a reduction of mean arterial pressure [from 68 to 54 mmHg (mean)], end-systolic and end-diastolic left ventricular area (from 9.7 to 6.5 cm2 and from 19.2 to 13.1 cm2, respectively) and end-systolic wall stress (from 56 to 33 10(3).dyne/cm2). FAC and mVcf remained unaltered. PHE administration restored baseline values or overcompensated the changes caused by tilt. FAC slightly decreased in response to PHE (from 0.51 to 0.43), end-systolic wall stress increased to 83 10(3).dyne/cm2, and mVcf remained unchanged. PHE bolus administration effectively restored BP and cardiac filling, which were reduced after head-up tilt, without causing a relevant impairment of left ventricular function or an increase in end-systolic wall stress above the upper normal limit.

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.

Immunohistochemical localization of FMRFamide-containing neurons and nerve fibers in the ganglia and the gonad wall of the scallop, Pecten maximus (L).

PubMed

Henry, M; Benlinmame, N; Belhsen, O K; Jule, Y; Mathieu, M

1995-02-01

The Phe-Met-Arg-Phe NH2 (FMRFamide)-like immunoreactivity was detected in neurons of the cerebro-pedal and visceral ganglia of the scallop Pecten maximus using immunohistochemical techniques. FMRFamide-like immunoreactivity was also found in nerve fibers localized in the connective tissue and the epithelial wall of the gonad. Electron microscopy study carried out on the gonads indicates the existence of numerous nerve fibers crossing the connective tissue; nerve terminals apposed to highly secretory cells were seen in the gonad wall. All in all, the present immunohistochemical and electron microscopic data suggest that FMRFamide might play an unusual secretagogue role in the gonad wall.

Breakthrough Therapies: Cystic Fibrosis (CF) Potentiators and Correctors

PubMed Central

Solomon, George M.; Marshall, Susan G.; Ramsey, Bonnie W.; Rowe, Steven M.

2015-01-01

Cystic Fibrosis is caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene resulting in abnormal protein function. Recent advances of targeted molecular therapies and high throughput screening have resulted in multiple drug therapies that target many important mutations in the CFTR protein. In this review, we provide the latest results and current progress of CFTR modulators for the treatment of cystic fibrosis, focusing on potentiators of CFTR channel gating and Phe508del processing correctors for the Phe508del CFTR mutation. Special emphasis is placed on the molecular basis underlying these new therapies and emerging results from the latest clinical trials. The future directions for augmenting the rescue of Phe508del with CFTR modulators is also emphasized. PMID:26097168

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.

Replacement of RNA hairpins by in vitro selected tetranucleotides.

PubMed Central

Dichtl, B; Pan, T; DiRenzo, A B; Uhlenbeck, O C

1993-01-01

An in vitro selection method based on the autolytic cleavage of yeast tRNA(Phe) by Pb2+ was applied to obtain tRNA derivatives with the anticodon hairpin replaced by four single-stranded nucleotides. Based on the rates of the site-specific cleavage by Pb2+ and the presence of a specific UV-induced crosslink, certain tetranucleotide sequences allow proper folding of the rest of the tRNA molecule, whereas others do not. One such successful tetramer sequence was also used to replace the acceptor stem of yeast tRNA(Phe) and the anticodon hairpin of E.coli tRNA(Phe) without disrupting folding. These experiments suggest that certain tetramers may be able to replace structurally nonessential hairpins in any RNA. Images PMID:7680121

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.

Non-invasive prenatal diagnosis (NIPD) of cystic fibrosis: an optimized protocol using MEMO fluorescent PCR to detect the p.Phe508del mutation.

PubMed

Guissart, C; Dubucs, C; Raynal, C; Girardet, A; Tran Mau Them, F; Debant, V; Rouzier, C; Boureau-Wirth, A; Haquet, E; Puechberty, J; Bieth, E; Dupin Deguine, D; Khau Van Kien, P; Brechard, M P; Pritchard, V; Koenig, M; Claustres, M; Vincent, M C

2017-03-01

Analysis of cell-free foetal DNA (cff-DNA) in maternal plasma is very promising for early diagnosis of monogenic diseases; in particular, cystic fibrosis (CF). However, NIPD of single-gene disorders has been limited by the availability of suitable technical platforms and the need to set up patient or disease-specific custom-made approaches. To make research applications more readily accessible to the clinic, we offer a simple assay combining two independent methods to determine the presence or absence of paternally inherited foetal allele p.Phe508del (the most frequent mutation in CF patients worldwide). The first method detects the presence or absence of a p.Phe508del allele by Mutant Enrichment with 3'-Modified Oligonucleotide PCR coupled to Fragment Length Analysis (MEMO-PCR-FLA). The second method detects the p.Phe508del allele with classical Multiplex Fluorescent PCR including five intragenic and extragenic STR markers of the CFTR locus and a specific SRY sequence. We collected 24 plasma samples from 23 women carrying foetuses at risk for CF and tested each sample using both methods. Our new procedures were successfully applied to 10 couples where fathers carried the p.Phe508del mutation and mothers were carrying a different mutation in the CFTR gene. These simple tests provided clear positive or negative results from the maternal plasma of the pregnant women. We confirmed the presence of cff-DNA in the studied samples by the identification of a tri-allelic DNA profile using a miniSTR kit. All results were correlated with chorionic villus sampling or amniocentesis analyses. This NIPD approach, easily set up in any clinical laboratory where prenatal diagnosis is routinely performed, offers many advantages over current methods: it is simple, rapid, and cost-effective. It opens up the possibility for testing a large number of couples with offspring at risk for CF. Copyright © 2016 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Removal of phenanthrene in aqueous solution containing photon competitors by TiO2-C-Ag film supported on fiberglass.

PubMed

González-Ramírez, Denisse Fabiola; Ávila-Pérez, Pedro; Torres-Bustillos, Luis G; Aguilar-López, Ricardo; Montes-Horcasitas, María C; Esparza-García, Fernando J; Rodríguez-Vázquez, Refugio

2017-07-03

Surface interactions with pollutants and photons are key factors that affect the applications of TiO 2 in environmental remediation. In this study, the solubilizing agents dimethylsulfoxide and polyoxyethylene sorbitan monooleate, which act as photon competitors, had no effect on the photocatalytic activity of TiO 2 -C-Ag film in phenanthrene (PHE) removal. Fiberglass with TiO 2 -C-Ag coating removed 91.1 ± 5.2 and 99.7 ± 0.4% of PHE in treatments using UVA (365-465 nm) and UVC (254 nm) irradiation, respectively. The use of fiberglass as a support increased the superficial area, thus allowing PHE sorption. C and Ag, which are electrically active impurities in TiO 2 , enhanced its photocatalytic activity and thus the attraction of the pollutant to its surface. The use of high-frequency UV light (UVC) decreased the amount of carbon species deposited on the TiO 2 CAg film surface. X-ray photoelectron spectroscopy of the TiO 2 -C-Ag film revealed extensive oxidation of the carbon deposited on the film under UVC light and loss of electrons from Ag clusters by conversion of Ag 0 to Ag 3+ .

Organic Pollutant Penetration through Fruit Polyester Skin: A Modified Three-compartment Diffusion Model

NASA Astrophysics Data System (ADS)

Li, Yungui; Li, Qingqing; Chen, Baoliang

2016-03-01

The surface of plants is covered by a continuous but heterogeneous cuticular membrane (CM). Serving as the first protective barrier, the uptake and transport behavior of organic pollutants at this interface continue to engage the research efforts of environmental chemist. To date, the contributions of cuticular components as a defense against the organic pollutants penetration remain unresolved. In this study, the unsteady-state penetration characteristics of phenanthrene (PHE) through isolated fruit CM was investigated. PHE penetration was differentiated by three cuticular compartments: epicuticular waxes (EW), cuticle proper (CP) and cuticular layer (CL). The driving force for PHE penetration was ascribed to the sharp concentration gradient built up endogenously by cuticular compartments with different lipophilic affinities. A modified penetration model was established and verified in terms of its general suitability for the hydrophobic chemicals and CMs of various plant species (apple, tomato and potato). The new three-compartment model demonstrates much higher accuracy in characterizing the uptake and transport behavior of semivolatile chemicals with fewer limitations in terms of environmental conditions and complexity (e.g., coexisting contaminants and temperature). This model could contribute to a more comprehensive understanding on the role of polymeric lipids in the organic pollutant sorption and transport into plants.

Driving reproduction: RFamide peptides behind the wheel.

PubMed

Kriegsfeld, Lance J

2006-12-01

The availability of tools for probing the genome and proteome more efficiently has allowed for the rapid discovery of novel genes and peptides that play important, previously uncharacterized roles in neuroendocrine regulation. In this review, the role of a class of neuropeptides containing the C-terminal Arg-Phe-NH(2) (RFamide) in regulating the reproductive axis will be highlighted. Neuropeptides containing the C-terminal Phe-Met-Arg-Phe-NH(2) (FMRFamide) were first identified as cardioregulatory elements in the bi-valve mollusk Macrocallista nimbosa. During the past two decades, numerous studies have shown the presence of structurally similar peptides sharing the RFamide motif across taxa. In vertebrates, RFamide peptides have pronounced influences on opiatergic regulation and neuroendocrine function. Two key peptides in this family are emerging as important regulators of the reproductive axis, kisspeptin and gonadotropin-inhibitory hormone (GnIH). Kisspeptin acts as the accelerator, directly driving gonadotropin-releasing hormone (GnRH) neurons, whereas GnIH acts as the restraint. Recent evidence suggests that both peptides play a role in mediating the negative feedback effects of sex steroids. This review presents the hypothesis that these peptides share complementary roles by responding to internal and external stimuli with opposing actions to precisely regulate the reproductive axis.

Driving Reproduction: RFamide Peptides Behind the Wheel

PubMed Central

Kriegsfeld, Lance J.

2012-01-01

The availability of tools for probing the genome and proteome more efficiently has allowed for the rapid discovery of novel genes and peptides that play important, previously-uncharacterized roles in neuroendocrine regulation. In this review, the role of a class of neuropeptides containing the C-terminal Arg-Phe-NH2 (RFamide) in regulating the reproductive axis will be highlighted. Neuropeptides containing the C-terminal Phe- Met-Arg-Phe-NH2 (FMRFamide) were first identified as cardioregulatory elements in the bi-valve mollusk, Macrocallista nimbosa. During the past two decades, numerous studies have shown the presence of structurally-similar peptides sharing the RFamide motif across taxa. In vertebrates, RFamide peptides have pronounced influences on opiatergic regulation and neuroendocrine function. Two key peptides in this family are emerging as important regulators of the reproductive axis, kisspeptin and gonadotropin-inhibitory hormone (GnIH). Kisspeptin acts as the accelerator, directly driving gonadotropin-releasing hormone (GnRH) neurons, whereas GnIH acts as the restraint. Recent evidence suggests that both peptides play a role in mediating the negative feedback effects of sex steroids. This review presents the hypothesis that these peptides share complementary roles by responding to internal and external stimuli with opposing actions to precisely regulate the reproductive axis. PMID:16876801

Organic Pollutant Penetration through Fruit Polyester Skin: A Modified Three-compartment Diffusion Model.

PubMed

Li, Yungui; Li, Qingqing; Chen, Baoliang

2016-03-24

The surface of plants is covered by a continuous but heterogeneous cuticular membrane (CM). Serving as the first protective barrier, the uptake and transport behavior of organic pollutants at this interface continue to engage the research efforts of environmental chemist. To date, the contributions of cuticular components as a defense against the organic pollutants penetration remain unresolved. In this study, the unsteady-state penetration characteristics of phenanthrene (PHE) through isolated fruit CM was investigated. PHE penetration was differentiated by three cuticular compartments: epicuticular waxes (EW), cuticle proper (CP) and cuticular layer (CL). The driving force for PHE penetration was ascribed to the sharp concentration gradient built up endogenously by cuticular compartments with different lipophilic affinities. A modified penetration model was established and verified in terms of its general suitability for the hydrophobic chemicals and CMs of various plant species (apple, tomato and potato). The new three-compartment model demonstrates much higher accuracy in characterizing the uptake and transport behavior of semivolatile chemicals with fewer limitations in terms of environmental conditions and complexity (e.g., coexisting contaminants and temperature). This model could contribute to a more comprehensive understanding on the role of polymeric lipids in the organic pollutant sorption and transport into plants.

Neurotensin analogs [D-TYR11] and [D-PHE11]neurotensin resist degradation by brain peptidases in vitro and in vivo.

PubMed

Checler, F; Vincent, J P; Kitabgi, P

1983-12-01

The present study was designed to compare the susceptibility of neurotensin (NT), [3H]NT, [D-Tyr11]NT and [D-Phe11]NT to degradation by 1) rat brain synaptic membranes in vitro and 2) after i.c.v. administration in the rat in vivo. Degradation was assessed by purifying the peptides using reverse phase high-performance liquid chromatography and by measuring the amount of radioactive or absorbing (OD 230) material under each peptide peak. In contrast to NT, [D-Tyr11]NT and [D-Phe11]NT were resistant to degradation by brain synaptic peptidases in vitro. Furthermore, NT was rapidly metabolized in brain tissues after i.c.v. administration, whereas [D-Tyr11]NT was metabolically stable. The present data confirm the central role of NT residue Tyr11 in the mechanisms of NT inactivation by brain synaptic peptidases. They account for the higher in vivo potency of [D-Tyr11]NT as compared with its in vitro potency. Finally, they explain, at least in part, the need to administer large doses of NT in the brain in order to observe neurobehavioral and neuropharmacological effects.

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.

Factors associated with the intention of health care personnel to respond to a disaster.

PubMed

Connor, Susan B

2014-12-01

Over the past decade, numerous groups of researchers have studied the willingness of health care personnel (HCP) to respond when a disaster threatens the health of a community. Not one of those studies reported that 100% of HCP were willing to work during a public-health event (PHE). The objective of this study was to explore factors associated with the intent of HCP to respond to a future PHE. The theory of planned behavior (TPB) framed this cross-sectional study. Data were obtained via a web-based survey from 305 HCP. Linear associations between the TPB-based predictor and outcome variables were examined using Pearson's correlations. Differences between two groups of HCP were calculated using independent t tests. A model-generating approach was used to develop and assess a series of TBP-based observed variable structural equation models for prediction of intent to respond to a future PHE and to explore moderating and mediating effects. The beginning patterns of relationships identified by the correlation matrix and t tests were evident in the final structural equation model, even though the patterns of prediction differed from those posited by the theory. Outcome beliefs had both a significant, direct effect on intention and an indirect effect on intention that was mediated by perceived behavioral control. Control beliefs appeared to influence intention through perceived behavioral control, as posited by the TPB, and unexpectedly through subjective norm. Subjective norm not only mediated the relationship between control beliefs and intention, but also the relationship between referent beliefs and intention. Additionally, professional affiliation seemed to have a moderating effect on intention. The intention to respond was influenced primarily by normative and control factors. The intent of nurses to respond to a future PHE was influenced most by the control factors, whereas the intent of other HCP was shaped more by the normative factors. Health care educators can bolster the normative and control factors through education by focusing on team building and knowledge related to accessing supplies and support needed to respond when a disaster occurs.

Analysis of Cytochrome P450 CYP119 Ligand-dependent Conformational Dynamics by Two-dimensional NMR and X-ray Crystallography*

PubMed Central

Basudhar, Debashree; Madrona, Yarrow; Kandel, Sylvie; Lampe, Jed N.; Nishida, Clinton R.; de Montellano, Paul R. Ortiz

2015-01-01

Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. We used two-dimensional 1H,15N HSQC chemical shift perturbation mapping of 15N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop with various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. The results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states. PMID:25670859

Attempts to locate residues in complementarity-determining regions of antibody combining sites that make contact with antigen.

PubMed

Kabat, E A; Wu, T T; Bilofsky, H

1976-02-01

From collected data on variable region sequences of heavy chains of immunoglobulins, the probability of random associations of any two amino-acid residues in the complementarity-determining segments was computed, and pairs of residues occurring significantly more frequently than expected were selected by computer. Significant associations between Phe 32 and Tyr 33, Phe 32 and Glu 35, and Tyr 33 and Glu 35 were found in six proteins, all of which were mouse myeloma proteins which bound phosphorylcholine (= phosphocholine). From the x-ray structure of McPC603, Tyr 33 and Glu 35 are contacting residues; a seventh phosphorylcholine-binding mouse myeloma protein also contained Phe 32 and Tyr 33 but position 35 had only been determined as Glx and thus this position had not been selected. Met 34 occurred in all seven phosphorylcholine-binding myeoma proteins but was also present at this position in 29 other proteins and thus was not selected; it is seen in the x-ray structure not to be a contacting residue. The role of Phe 32 is not obvious but it could have some conformational influence. A human phosphorylcholine-binding myeloma protien also had Phe, Tyr, and Met at positions 32, 33, and 34, but had Asp instead of Glu at position 35 and showed a lower binding constant. The ability to use sequence data to locate residues in complementarity-determing segments making contact with antigenic determinants and those playing essentially a structural role would contribute substantially to the understanding of antibody specificity.

Cellular pH measurements in Emiliania huxleyi reveal pronounced membrane proton permeability.

PubMed

Suffrian, K; Schulz, K G; Gutowska, M A; Riebesell, U; Bleich, M

2011-05-01

• To understand the influence of changing surface ocean pH and carbonate chemistry on the coccolithophore Emiliania huxleyi, it is necessary to characterize mechanisms involved in pH homeostasis and ion transport. • Here, we measured effects of changes in seawater carbonate chemistry on the fluorescence emission ratio of BCECF (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein) as a measure of intracellular pH (pH(i)). Out of equilibrium solutions were used to differentiate between membrane permeation pathways for H(+), CO(2) and HCO(3)(-). • Changes in fluorescence ratio were calibrated in single cells, resulting in a ratio change of 0.78 per pH(i) unit. pH(i) acutely followed the pH of seawater (pH(e)) in a linear fashion between pH(e) values of 6.5 and 9 with a slope of 0.44 per pH(e) unit. pH(i) was nearly insensitive to changes in seawater CO(2) at constant pH(e) and HCO(3)(-). An increase in extracellular HCO(3)(-) resulted in a slight intracellular acidification. In the presence of DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid), a broad-spectrum inhibitor of anion exchangers, E. huxleyi acidified irreversibly. DIDS slightly reduced the effect of pH(e) on pH(i). • The data for the first time show the occurrence of a proton permeation pathway in E. huxleyi plasma membrane. pH(i) homeostasis involves a DIDS-sensitive mechanism. © 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.

Cardiac Teratogenicity in Mouse Maternal Phenylketonuria: Defining phenotype parameters and genetic background influences

PubMed Central

Seagraves, Nikki J.; McBride, Kim L.

2012-01-01

Maternal phenylketonuria (MPKU) is a syndrome including cardiovascular malformations (CVMs), microcephaly, intellectual impairment, and small for gestational age, caused by in-utero exposure to elevated serum phenylalanine (Phe) due to PKU in the mother. It is becoming a public health concern as more women with PKU reach child bearing age. Although a mouse model of PKU, BTBR Pahenu2, has been available for 20 years, it has not been well utilized for studying MPKU. We used this model to delineate critical parameters in Phe cardiovascular teratogenicity and study the effect of genetic background. Dosing and timing experiments were performed with the BTBR Pahenu2 mouse. A dose response curve was noted, with CVM rates at maternal serum Phe levels <360 μM (control), 360 – 600 μM (low), 600 – 900 μM (mid), and >900μM (high) of 11.86%, 16.67%, 30.86%, and 46.67% respectively. A variety of CVMs were noted on the BTBR background, including double outlet right ventricle (DORV), aortic arch artery (AAA)abnormalities, and ventricular septal defects (VSDs). Timed exposure experiments identified a teratogenic window from embryonic day 8.5-13.5, with higher rates of conotruncal and valve defects occurring in early exposure time and persistent truncus arteriosus (PTA) and aortic arch branching abnormalities occurring with late exposure. Compared to the BTBR strain, N10+ Pahenu2 congenics on the C3H/HeJ background had higher rates of CVMs in general and propensity to left ventricular outflow tract (LVOT) malformations, while the C57B/L6 background had similar CVM rates but predominately AAA abnormalities. We have delineated key parameters of Phe cardiovascular teratogenicity, demonstrated the utility of this MPKU model on different mouse strains, and shown how genetic background profoundly affects the phenotype. PMID:22951387

Synthesis and antioxidant activity of peptide-based ebselen analogues.

PubMed

Satheeshkumar, Kandhan; Mugesh, Govindasamy

2011-04-18

A series of di- and tripeptide-based ebselen analogues has been synthesized. The compounds were characterized by (1)H, (13)C, and (77)Se NMR spectroscopy and mass spectral techniques. The glutathione peroxidase (GPx)-like antioxidant activity has been studied by using H(2)O(2) , tert-butyl hydroperoxide (tBuOOH), and cumene hydroperoxide (Cum-OOH) as substrates, and glutathione (GSH) as a cosubstrate. Although all the peptide-based compounds have a selenazole ring similar to that of ebselen, the GPx activity of these compounds highly depends on the nature of the peptide moiety attached to the nitrogen atom of the selenazole ring. It was observed that the introduction of a phenylalanine (Phe) amino acid residue in the N-terminal reduces the activity in all three peroxide systems. On the other hand, the introduction of aliphatic amino acid residues such as valine (Val) significantly enhances the GPx activity of the ebselen analogues. The difference in the catalytic activity of dipeptide-based ebselen derivatives can be ascribed mainly to the change in the reactivity of these compounds toward GSH and peroxide. Although the presence of the Val-Ala-CO(2) Me moiety facilitates the formation of a catalytically active selenol species, the reaction of ebselen analogues that has a Phe-Ile-CO(2) Me residue with GSH does not generate the corresponding selenol. To understand the antioxidant activity of the peptide-based ebselen analogues in the absence of GSH, these compounds were studied for their ability to inhibit peroxynitrite (PN)-mediated nitration of bovine serum albumin (BSA) and oxidation of dihydrorhodamine 123. In contrast to the GPx activity, the PN-scavenging activity of the Phe-based peptide analogues was found to be comparable to that of the Val-based compounds. However, the introduction of an additional Phe residue to the ebselen analogue that had a Val-Ala dipeptide significantly reduced the potency of the parent compound in PN-mediated nitration. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effects of perilymphatic pressure, sodium nitroprusside, and bupivacaine on cochlear fluid pH of guinea pigs.

PubMed

Suzuki, Masaaki; Kotani, Ryosuke

2015-01-01

Hydrostatic positive pressure and vasoconstrictor acidified the cochlear fluids, whereas the vasodilator made the fluids alkaline. CBF might play a role in regulating cochlea fluid pH. Cochlea fluid pH is highly dependent on the HCO3(-)/CO2 buffer system. Cochlear blood flow (CBF) supplies O2 and removes CO2. It is speculated that cochlear blood flow changes might affect the balance of the HCO3(-)/CO2 buffer system in the cochlea. It is known that the elevation of inner ear pressure decreases the CBF, and local application of vasodilating or vasoconstricting agents directly to the cochlea changes the CBF. The purpose of this study was to elucidate the effect of positive hydrostatic inner ear pressure and application of a vasodilator and vasoconstrictor of cochlear vessels on the pH of the endolymph and perilymph. The authors performed animal physiological experiments on 30 guinea pigs. Hydrostatic positive pressure was infused through a glass capillary tube inserted into the scala tympani of the basal turn. The vasodilator, nitric oxide donor (sodium nitroprusside; SNP), and the vasoconstrictor, bupivacaine, were placed topically onto the round window of the guinea pig cochlea. Endolymph pH (pHe) and endocochlear potential (EP) were monitored by double-barreled ion-selective microelectrodes in the second turn of the guinea pig cochlea. During the topical application study, scala vestibuli perilymph pH (pHv) was also measured simultaneously in the second turn. The application of hydrostatic positive pressure caused a decrease in pHe and EP. Positive perilymphatic pressure caused the endolymph to become acidic pressure-dependently. Application of 3.0% SNP evoked an increase in both the pHe and pHv, following by a gradual recovery to baseline levels. On the other hand, 0.5% bupivacaine caused a decrease in both the pHe and pHv. The EP during topical application showed slight, non-significant changes.

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…

Toward the 21st Century: Preparing Proactive Visionary Transformational Leaders for Building Learning Communities. Human Resource Development. Tampa Cluster. Winter 1994.

ERIC Educational Resources Information Center

Groff, Warren H.

This document describes the Tampa Cluster human resources development (HRD) seminar that was conducted as part of Nova University's distance education program in higher education (PHE). Discussed first are HRD in the agricultural and business industrial eras and changing HRD practices/needs, Nova University's PHE and HRD program, the proceedings…

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

Variation of the net charge, lipophilicity, and side chain flexibility in Dmt(1)-DALDA: Effect on Opioid Activity and Biodistribution.

PubMed

Novoa, Alexandre; Van Dorpe, Sylvia; Wynendaele, Evelien; Spetea, Mariana; Bracke, Nathalie; Stalmans, Sofie; Betti, Cecilia; Chung, Nga N; Lemieux, Carole; Zuegg, Johannes; Cooper, Matthew A; Tourwé, Dirk; De Spiegeleer, Bart; Schiller, Peter W; Ballet, Steven

2012-11-26

The influence of the side chain charges of the second and fourth amino acid residues in the peptidic μ opioid lead agonist Dmt-d-Arg-Phe-Lys-NH(2) ([Dmt(1)]-DALDA) was examined. Additionally, to increase the overall lipophilicity of [Dmt(1)]-DALDA and to investigate the Phe(3) side chain flexibility, the final amide bond was N-methylated and Phe(3) was replaced by a constrained aminobenzazepine analogue. The in vitro receptor binding and activity of the peptides, as well as their in vivo transport (brain in- and efflux and tissue biodistribution) and antinociceptive properties after peripheral administration (ip and sc) in mice were determined. The structural modifications result in significant shifts of receptor binding, activity, and transport properties. Strikingly, while [Dmt(1)]-DALDA and its N-methyl analogue, Dmt-d-Arg-Phe-NMeLys-NH(2), showed a long-lasting antinociceptive effect (>7 h), the peptides with d-Cit(2) generate potent antinociception more rapidly (maximal effect at 1h postinjection) but also lose their analgesic activity faster when compared to [Dmt(1)]-DALDA and [Dmt(1),NMeLys(4)]-DALDA.

Variation of the net charge, lipophilicity and side chain flexibility in Dmt1-DALDA: effect on opioid activity and biodistribution

PubMed Central

Novoa, Alexandre; Van Dorpe, Sylvia; Wynendaele, Evelien; Spetea, Mariana; Bracke, Nathalie; Stalmans, Sofie; Betti, Cecilia; Chung, Nga N.; Lemieux, Carole; Zuegg, Johannes; Cooper, Matthew A.; Tourwé, Dirk; De Spiegeleer, Bart; Schiller, Peter W.; Ballet, Steven

2012-01-01

The influence of the side chain charges of the second and fourth amino acid residues in the peptidic μ opioid lead agonist Dmt-D-Arg-Phe-Lys-NH2 ([Dmt1]-DALDA) was examined. Additionally, to increase the overall lipophilicity of [Dmt1]-DALDA and to investigate the Phe3 side chain flexibility, the final amide bond was N-methylated and Phe3 was replaced by a constrained aminobenzazepine analogue. The in vitro receptor binding and activity of the peptides, as well as their in vivo transport (brain in- and efflux and tissue biodistribution) and antinociceptive properties after peripheral administration (i.p. and s.c.) in mice were determined. The structural modifications result in significant shifts of receptor binding, activity and transport properties. Strikingly, while [Dmt1]-DALDA and its N-methyl analogue, Dmt-D-Arg-Phe-NMeLys-NH2, showed a long-lasting antinociceptive effect (>7h), the peptides with D-Cit2 generate potent antinociception more rapidly (maximal effect at 1h post-injection) but also lose their analgesic activity faster, when compared to [Dmt1]-DALDA and [Dmt1,NMeLys4]-DALDA. PMID:23102273