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.

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

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.

The action of a synthetic derivative of Met5-enkephalin-Arg6-Phe7 on behavioral and endocrine responses.

PubMed

Csabafi, Krisztina; Jászberényi, Miklós; Bagosi, Zsolt; Tóth, Géza; Wollemann, Mária; Telegdy, Gyula

2011-08-01

The neuroendocrine and behavioral effects of Tyr-D-Ala-Gly-Phe-D-Nle-Arg-Phe (DADN), a more stable derivative of the endogenous opiate Met-enkephalin related peptide Met(5)-enkephalin-Arg(6)-Phe(7) were investigated in mice. The behavioral experiments consisted of monitoring the horizontal (square crossing) and vertical (rearing) locomotion in the open field system. To evaluate the effect of the heptapeptide on the hypothalamo-pituitary-adrenal (HPA) axis, the plasma corticosterone level was measured. DADN induced dose-dependent increases in locomotion and rearing 30 min after intracerebroventricular injection and also elicited marked activation of the hormonal stress response. To elucidate the receptors involved in the mediation of these actions, animals were pretreated with the nonselective opioid antagonist naloxone, the selective κ-receptor antagonist nor-binaltorphimine or the μ(1)-receptor blocker naloxonazine. Both the HPA activation and the behavioral responses were diminished by the preadministration of naloxone. Nor-binaltorphimine did not display a significant effect, while naloxonazine completely abolished the hyperactivity and the corticosterone elevation elicited by the analog. These findings suggest that μ-receptors predominate in the mediation of the neuroendocrine actions of DADN, while κ-receptors do not play a significant role. Copyright © 2011. Published by Elsevier Inc.

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

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.

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

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

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.

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.

Inhibition of rat prostate tumor growth by an octapeptide analog of somatostatin.

PubMed

Murphy, W A; Lance, V A; Moreau, S; Moreau, J P; Coy, D H

1987-06-29

Analogs of a potent octapeptide analog of somatostatin (SRIF) H-(D)Phe-Cys-Tyr-(D)Trp-Lys-Val-Cys-Thr(NH2) were synthesized. Aromatic substitutions for Tyr resulted in little change in inhibitory potency on growth hormone (GH) secretion in the rat. Substitutions for Val or (D)Trp resulted in analogs with diminished activity. Substitution of (D)Nal for (D)Phe increased duration of GH inhibition. Final weights of subcutaneously implanted prostate tumors (R3327) were 41% lower in rats treated with an N-terminal 4-chloro-(D)phenylalanyl analog as compared to vehicle treated controls. The analog had no effect on testicular weight or final plasma testosterone levels. SRIF analogs may represent an alternative treatment for prostate cancer that would be free of the untoward reproductive effects of other treatments (e.g. LH-RH or castration).

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

PubMed Central

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

2011-01-01

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

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.

Amino acid metabolism during exercise in trained rats: the potential role of carnitine in the metabolic fate of branched-chain amino acids.

PubMed

Ji, L L; Miller, R H; Nagle, F J; Lardy, H A; Stratman, F W

1987-08-01

The influence of endurance training and an acute bout of exercise on plasma concentrations of free amino acids and the intermediates of branched-chain amino acid (BCAA) metabolism were investigated in the rat. Training did not affect the plasma amino acid levels in the resting state. Plasma concentrations of alanine (Ala), aspartic acid (Asp), asparagine (Asn), arginine (Arg), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), and valine (Val) were significantly lower, whereas glutamate (Glu), glycine (Gly), ornithine (Orn), tryptophan (Trp), tyrosine (Tyr), creatinine, urea, and ammonia levels were unchanged, after one hour of treadmill running in the trained rats. Plasma concentration of glutamine (Glu), the branched-chain keto acids (BCKA) and short-chain acyl carnitines were elevated with exercise. Ratios of plasma BCAA/BCKA were dramatically lowered by exercise in the trained rats. A decrease in plasma-free carnitine levels was also observed. These data suggest that amino acid metabolism is enhanced by exercise even in the trained state. BCAA may only be partially metabolized within muscle and some of their carbon skeletons are released into the circulation in forms of BCKA and short-chain acyl carnitines.

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.

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.

Two novel mutations in the fibrinogen γ nodule.

PubMed

Kotlín, Roman; Pastva, Ondřej; Stikarová, Jana; Hlaváčková, Alžběta; Suttnar, Jiří; Chrastinová, Leona; Riedel, Tomáš; Salaj, Peter; Dyr, Jan E

2014-10-01

Congenital dysfibrinogenemia and hypofibrinogenemia are rare diseases characterized by inherited abnormality in the fibrinogen molecule, resulting in functional defects (dysfibrinogenemia) or low fibrinogen plasma levels (hypofibrinogenemia). We have described two abnormal fibrinogens - fibrinogen Hranice (γ Phe204Val) and Praha IV (γ Ser313Gly). The carrier of the Hranice mutation was a 40-year-old female with low fibrinogen levels. The carrier of the Praha IV mutation was a 42-year-old man with a history of idiopathic thrombosis, low functional fibrinogen levels, and a prolonged thrombin time. Fibrin polymerization kinetics measurement was normal in both cases (after the addition of either thrombin or reptilase), as well as was fibrinolysis. Scanning electron microscopy and confocal microscopy revealed significantly wider fibers in both cases, when compared with fibers prepared from healthy control samples. Although both cases are situated in the γ-nodule, they manifested differently. While the γ Ser313Gly mutation manifested as dysfibrinogenemia with a thrombotic background, the γ Phe204Val mutation manifested as hypofibrinogenemia without clinical symptoms. The mutation sites of both fibrinogens are in highly conserved regions of the fibrinogen γ chains. γ Ser313 is situated in a class 16:18 β hairpin and is involved in hydrogen bonding with γ Asp320. γ Phe204 is situated in an inverse γ turn and may be involved in π-π interactions. Both mutations cause conformational changes in fibrinogen, which lead either to impaired fibrinogen assembly (fibrinogen Hranice) or abnormal fibrinogen function (fibrinogen Praha IV). Copyright © 2014 Elsevier Ltd. All rights reserved.

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.

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.

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

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

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

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

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.

Effect of experimentally increased protein supply to postpartum dairy cows on plasma protein synthesis, rumen tissue proliferation, and immune homeostasis.

PubMed

Larsen, M; Røntved, C M; Theil, P K; Khatun, M; Lauridsen, C; Kristensen, N B

2017-05-01

The effect of experimentally increasing the postpartum protein supply on plasma protein synthesis, rumen tissue proliferation, and immune homeostasis was studied using 8 periparturient Holstein cows in a complete randomized design. At calving, cows were assigned to abomasal infusion of water (CTRL) or casein (CAS) in addition to a lactation diet. Casein infusion was gradually decreased from 696 ± 1 g/d at +2 d relative to calving (DRTC) to 212 ± 10 g/d at +29 DRTC to avoid excessive supply. Synthesis rate of plasma proteins was measured at -14, +4, +15, and +29 DRTC by measuring [C]Phe isotopic enrichment in arterial plasma free Phe, total plasma proteins, and albumin after 3, 5, and 7 h of jugular ring[C]Phe infusion. Plasma volume was determined at +4 and +29 DRTC by dilution of a [I]BSA dose. Synthesis rate of tissue protein in biopsied rumen papillae was determined by measuring [C]Phe isotopic enrichment, and mRNA expression of selected genes was measured by real-time qPCR. Total and differential leukocyte counts were performed and immune responsiveness of monocytes was evaluated by tumor necrosis factor ɑ (TNFɑ) concentration on ex vivo whole blood stimulation with Escherichia coli lipopolysaccharide (LPS) and responsiveness of T-lymphocytes by interferon γ (IFNγ) concentration on stimulation with Staphylococcus aureus enterotoxin β (SEB). Further, ELISA plasma concentrations of IgM, IgA, and IgG were determined. The DRTC affected the majority of investigated parameters as expected. The CAS treatment increased milk protein yield (P = 0.04), and tended to lower TNFɑ (P = 0.06), and lowered IFNγ (P = 0.03) responsiveness per monocyte and lymphocyte, respectively, compared with CTRL. Further, fractional synthesis rate of albumin was greater at +4 DRTC for CAS compared with CTRL but did not differ by +29 DRTC (interaction: P = 0.01). In rumen papillae, synthesis rate of tissue protein was greater for CAS compared with CTRL (P < 0.01) and mRNA expression of genes for cell proliferation tended to be or were greater for CAS compared with CTRL (P ≤ 0.07). In conclusion, increased postpartum protein supply seem to enhance vital body functions as interpreted from increased liver synthesis of albumin, increased rumen papillae proliferation, and stabilized the ex vivo inflammatory responsiveness of leukocytes. Further studies are needed to enlighten the importance of increased postpartum protein supply in periparturient cows.

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

PubMed

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

2014-03-21

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

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.

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.

G673 could be a novel mutational hot spot for intragenic suppressors of pheS5 lesion in Escherichia coli.

PubMed

Ponmani, Thangaraj; Munavar, M Hussain

2014-06-01

The pheS5 Ts mutant of Escherichia coli defined by a G293 → A293 transition, which is responsible for thermosensitive Phenylalanyl-tRNA synthetase has been well studied at both biochemical and molecular level but genetic analyses pertaining to suppressors of pheS5 were hard to come by. Here we have systematically analyzed a spectrum of Temperature-insensitive derivatives isolated from pheS5 Ts mutant and identified two intragenic suppressors affecting the same base pair coordinate G673 (pheS19 defines G673 → T673 ; Gly225 → Cys225 and pheS28 defines G673 → C673 ; Gly225 → Arg225). In fact in the third derivative, the intragenic suppressor originally named pheS43 (G673 → C673 transversion) is virtually same as pheS28. In the fourth case, the very pheS5 lesion itself has got changed from A293 → T293 (named pheS40). Cloning of pheS(+), pheS5, pheS5-pheS19, pheS5-pheS28 alleles into pBR322 and introduction of these clones into pheS5 mutant revealed that excess of double mutant protein is not at all good for the survival of cells at 42°C. These results clearly indicate a pivotal role for Gly225 in the structural/functional integrity of alpha subunit of E. coli PheRS enzyme and it is proposed that G673 might define a hot spot for intragenic suppressors of pheS5. © 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

MAMP (microbe-associated molecular pattern)-induced changes in plasma membrane-associated proteins.

PubMed

Uhlíková, Hana; Solanský, Martin; Hrdinová, Vendula; Šedo, Ondrej; Kašparovský, Tomáš; Hejátko, Jan; Lochman, Jan

2017-03-01

Plant plasma membrane associated proteins play significant roles in Microbe-Associated Molecular Pattern (MAMP) mediated defence responses including signal transduction, membrane transport or energetic metabolism. To elucidate the dynamics of proteins associated with plasma membrane in response to cryptogein, a well-known MAMP of defence reaction secreted by the oomycete Phytophthora cryptogea, 2D-Blue Native/SDS gel electrophoresis of plasma membrane fractions was employed. This approach revealed 21 up- or down-regulated protein spots of which 15 were successfully identified as proteins related to transport through plasma membrane, vesicle trafficking, and metabolic enzymes including cytosolic NADP-malic enzyme and glutamine synthetase. Observed changes in proteins were also confirmed on transcriptional level by qRT-PCR analysis. In addition, a significantly decreased accumulation of transcripts observed after employment of a mutant variant of cryptogein Leu41Phe, exhibiting a conspicuous defect in induction of resistance, sustains the contribution of identified proteins in cryptogein-triggered cellular responses. Our data provide further evidence for dynamic MAMP-induced changes in plasma membrane associated proteins. Copyright © 2016 Elsevier GmbH. All rights reserved.

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

[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.

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.

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.

[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.

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.

Studies on the vascular permeability induced by intrathecal substance P and bradykinin in the rat.

PubMed

Jacques, L; Couture, R

1990-08-02

The effects of substance P (SP), SP fragments, neurokinin A (NKA), neurokinin B (NKB) and selective agonists for neurokinin receptors were assessed on cutaneous vascular permeability after intrathecal (i.t.) administration in rats. Dose-dependent increases in plasma extravasation were observed with the following rank orders of potency ([p-Glu6]SP-(6-11) greater than SP greater than or equal to SP-(4-11) greater than [p-Glu5,MePhe8,Sar9]SP-(5-11) = [p-Glu5]SP-(5-11) greater than SP-(7-11) and SP greater than NKA greater than NKB). The N-terminal fragments SP-(1-4), SP-(1-7) and SP-(1-9) were inactive up to 65 nmol. The NK-1 receptor selective agonists [( beta-Ala4,Sar9,Met(O2)11]SP-(4-11) and [Pro9,Met(O2)11]SP) were more potent than the NK-2 ([Nle10]NKA-(4-10] and NK-3 ([beta-Asp4,MePhe7]NKB-(4-10) and [MePhe7]NKB) receptor-selective agonists. Plasma extravasation was also increased by i.t. bradykinin (BK, 8.1 nmol) while the fragment BK-(1-8), a potent B1-receptor-selective agonist, produced only a slight effect at 81 nmol. When BK was given after prior i.t. administration of 6.1 nmol of [Thi5.8,D-Phe7]BK, an antagonist of BK at the B2-receptor, the increase in vascular permeability was significantly attenuated. The analogue [Leu8]BK-(1-8) (10.3 nmol), an antagonist of BK at the B1-receptor, failed to modify the BK-induced plasma extravasation. Plasma extravasation induced by SP (6.5 nmol) and BK (8.1 nmol) was abolished in cervically vagotomized rats, and significantly reduced in both spinal rats and in capsaicin-treated animals. Conversely, bilateral adrenalectomy (48 h earlier) and intercollicular decerebration (30 min earlier) had no major effect on the response elicited either by SP or BK. The response to SP remained unaffected by methysergide and hexamethonium but was significantly reduced by methylnitrate atropine and diphenhydramine. Indomethacin significantly enhanced the plasma extravasation induced by SP. These results suggest that SP and BK may play a role as spinal mediators in peripheral vascular permeability through a sensory and cholinergic vagal mechanism involving a spinobulbar pathway. The receptors mediating the response to SP and BK in the spinal cord are of the NK-1 and B2 subtypes, respectively.

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

Isolation and biological activity of [Trp5]bradykinin from the plasma of the phylogenetically ancient fish, the bowfin and the longnosed gar.

PubMed

Conlon, J M; Platzack, B; Marra, L E; Youson, J H; Olson, K R

1995-01-01

The holostean fish occupy an important position in vertebrate phylogeny as extant representatives of a ancient group of ray-finned fish with evolutionary connections to present-day teleosts. Incubation of heat-denatured plasma from the bowfin Amia calva with trypsin generated bradykinin-like immunoreactivity. The primary structure of bowfin bradykinin was established as Ala-Pro-Pro-Gly-Trp-Ser-Pro-Phe-Arg. This amino acid sequence contains one amino acid substitution (Phe5 --> Trp) compared with mammalian bradykinin. The same peptide was generated in heat-denatured plasma from the longnosed gar Lepisosteus osseus. Treatment of plasma from either the bowfin or gar with glass beads under conditions previously shown to activate Factor XII in the plasma of mammals and reptiles did not generate bradykinin. Bolus injections of synthetic bowfin bradykinin (0.1, 0.3, and 1.0 nmol/kg) into the bulbus arteriosus of unanesthetized bowfin resulted in an immediate fall in arterial blood pressure of 5-10 min duration that was followed by a dose-dependent rise in pressure that was sustained for 30-60 min. There was no change in heart rate following bradykinin administration. The data suggest that the kallikrein-kinin system may predate the appearance of teleosts and may play a role in cardiovascular regulation in holosteans.

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®.

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

DTIC Science & Technology

2012-01-01

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

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.

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

PubMed

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

2016-08-15

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

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

PubMed Central

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

2016-01-01

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

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.

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.

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.

Attenuated plasma extravasation to sensory neuropeptides in diabetic rats.

PubMed

Mathison, R; Davison, J S

1993-01-01

The effects of either substance P (SP) or a metabolically stable SP analogue, [pGlu5,Me-Phe8,Sar9]SP(5-11), alone or in combination with calcitonin gene-related peptide (CGRP) on blood pressure (BP) and extravasation of serum albumin were examined in normal and diabetic rats. CGRP (12 ng/kg) modified neither BP nor vascular permeability in control and diabetic rats. Both SP and its analogue (74 ng/kg) produced hypotension, and increased plasma extravasation in the respiratory tissues, urinary bladder and skin. The simultaneous injection of CGRP and SP resulted in modest potentiation of the vascular permeability actions of SP in control and diabetic rats. However, extravasation induced by [pGlu5,Me-Phe8,Sar9]SP(5-11) was potentiated by CGRP in control animals, but not in diabetic rats. Defective neurogenic inflammatory responses in diabetic rats may result from decreased responses in the effector tissues of diabetic rats to the neuropeptides released from sensory nerves.

Capillary permeability induced by intravenous neurokinins. Receptor characterization and mechanism of action.

PubMed

Jacques, L; Couture, R; Drapeau, G; Regoli, D

1989-08-01

The effects on plasma extravasation of three increasing doses from 6.5 pmol to 650 nmol/kg of substance P (SP), SP fragments, neurokinin A (NKA), neurokinin B (NKB) and selective agonists for neurokinin receptors were assessed in three cutaneous tissues (skin of hind paws, dorsal skin and ears) by intravenous (i.v.) administration in the pentobarbitone anaesthetized rat. Dose-dependent increases in plasma extravasation were observed with the following rank orders of potency (SP greater than NKA greater than NKB) for neurokinins and (SP greater than [p-Glu6]SP(6-11) greater than SP(4-11) greater than [p-Glu5]SP(5-11) greater than SP(7-11] for C-terminal SP fragments. The metabolically stable SP analogue [p-Glu5, MePhe8, Sar9]SP(5-11) was slightly more potent than [p-Glu5]SP(5-11). The N-terminal fragments SP(1-4), SP(1-7) and SP(1-9) were inactive up to 650 nmol/kg. The NK-1 receptor selective agonists [Sar9, Met(O2)11]SP and [beta-Ala4, Sar9, Met (O2)11]SP(4-11) were more potent than the NK-2 [( Nle10]NKA(4-10] and NK-3 [( MePhe7]NKB and [beta-Asp4, MePhe7]NKB(4-10] receptor selective agonists. Plasma extravasation induced by SP (6.5 nmol/kg) was unchanged in the presence of atropine, methysergide, diphenhydramine or during the i.v. and intra-arterial (i.a.) infusion of D-Arg0[Hyp3.D-Phe7]BK, an antagonist of bradykinin. Plasma extravasation induced by SP and [Sar9, Met(O2)11]SP was significantly reduced by indomethacin while that induced by NKA, NKB, [beta-Ala4, Sar9, Met(O2)11]SP(4-11), SP(4-11) and [p-Glu6]SP(6-11) was unaffected by the cyclooxygenase inhibitor. Compound 48/80 (0.75 mg/kg), histamine (10 mg/kg) and 5-HT (10 mg/kg) caused an increase in plasma extravasation, only the effect of compound 48/80 was abolished by indomethacin. Pretreatment with compound 48/80 prevented its own action on plasma extravasation and significantly reduced that induced by 6.5 nmol/kg of SP. These results rule out the involvement of acetylcholine (muscarinic receptors), 5-HT (5-HT1 and 5-HT2 receptors), histamine (H1 receptors) and kinins (B2 receptors) in the response to SP and indicate that the two positively charged amino acids (Arg, Lys) at the N-terminal end of the SP molecule are essential to trigger the release of prostaglandins from mast cells. This mechanism is responsible for the indirect effect of SP and related peptides on capillary permeability and does not appear to be mediated by a selective SP receptor. In addition, neurokinins may increase capillary permeability by direct activation of a NK-1 receptor type on the vascular endothelium.

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.

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

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.

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.

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

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.

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

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.

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.

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.

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

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.

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.

Multiple Click-Selective tRNA Synthetases Expand Mammalian Cell-Specific Proteomics.

PubMed

Yang, Andrew C; du Bois, Haley; Olsson, Niclas; Gate, David; Lehallier, Benoit; Berdnik, Daniela; Brewer, Kyle D; Bertozzi, Carolyn R; Elias, Joshua E; Wyss-Coray, Tony

2018-06-13

Bioorthogonal tools enable cell-type-specific proteomics, a prerequisite to understanding biological processes in multicellular organisms. Here we report two engineered aminoacyl-tRNA synthetases for mammalian bioorthogonal labeling: a tyrosyl ( ScTyr Y43G ) and a phenylalanyl ( MmPhe T413G ) tRNA synthetase that incorporate azide-bearing noncanonical amino acids specifically into the nascent proteomes of host cells. Azide-labeled proteins are chemoselectively tagged via azide-alkyne cycloadditions with fluorophores for imaging or affinity resins for mass spectrometric characterization. Both mutant synthetases label human, hamster, and mouse cell line proteins and selectively activate their azido-bearing amino acids over 10-fold above the canonical. ScTyr Y43G and MmPhe T413G label overlapping but distinct proteomes in human cell lines, with broader proteome coverage upon their coexpression. In mice, ScTyr Y43G and MmPhe T413G label the melanoma tumor proteome and plasma secretome. This work furnishes new tools for mammalian residue-specific bioorthogonal chemistry, and enables more robust and comprehensive cell-type-specific proteomics in live mammals.

Binding of peptides with basic and aromatic residues to bilayer membranes: phenylalanine in the myristoylated alanine-rich C kinase substrate effector domain penetrates into the hydrophobic core of the bilayer.

PubMed

Zhang, Wenyi; Crocker, Evan; McLaughlin, Stuart; Smith, Steven O

2003-06-13

Electrostatic interactions with positively charged regions of membrane-associated proteins such as myristoylated alanine-rich C kinase substrate (MARCKS) may have a role in regulating the level of free phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in plasma membranes. Both the MARCKS protein and a peptide corresponding to the effector domain (an unstructured region that contains 13 basic residues and 5 phenylalanines), MARCKS-(151-175), laterally sequester the polyvalent lipid PI(4,5)P2 in the plane of a bilayer membrane with high affinity. We used high resolution magic angle spinning NMR to establish the location of MARCKS-(151-175) in membrane bilayers, which is necessary to understand the sequestration mechanism. Measurements of cross-relaxation rates in two-dimensional nuclear Overhauser enhancement spectroscopy NMR experiments show that the five Phe rings of MARCKS-(151-175) penetrate into the acyl chain region of phosphatidylcholine bilayers containing phosphatidylglycerol or PI(4,5)P2. Specifically, we observed strong cross-peaks between the aromatic protons of the Phe rings and the acyl chain protons of the lipids, even for very short (50 ms) mixing times. The position of the Phe rings implies that the adjacent positively charged amino acids in the peptide are close to the level of the negatively charged lipid phosphates. The deep location of the MARCKS peptide in the polar head group region should enhance its electrostatic sequestration of PI(4,5)P2 by an "image charge" mechanism. Moreover, this location has interesting implications for membrane curvature and local surface pressure effects and may be relevant to a wide variety of other proteins with basic-aromatic clusters, such as phospholipase D, GAP43, SCAMP2, and the N-methyl-d-aspartate receptor.

Aminopeptidase activity in rat brain synaptosomes - 2-mercaptoethanol stimulation and Arg-vasopressin degradation

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

Simmons, W.H.; Orawski, A.T.

1986-03-05

Rat brain synaptic plasma membranes contain an amastatin-inhibited aminopeptidase activity which degrades Arg-vaso-pressin (AVP). The pH optimum for AVP cleavage was found to be 6.8, similar to that reported for oxytocin. The ability of other peptides and arylamides such as oxytocin, Tyr-Phe-Met-Arg-Phe-NH/sub 2/ and Arg-Arg-..beta..NA to inhibit cleavage of (/sup 3/H-Tyr/sup 2/)-AVP suggests that the enzyme may not be specific for AVP. The AVP-cleaving activity has been solubilized and partially characterized. Synaptosomes were lysed with hypotonic buffer, washed, and extracted with 1% Nonidet P-40 detergent. The solubilized protein was chromatographed by gel filtration HPLC on Superose 6. A single peakmore » of activity was found with a M.W. = 117,000 which could hydrolyze 1mM Ala-..beta..NA, Arg-..beta..NA, Arg-Arg-..beta..NA, Phe-Met and Phe-Arg as well as slowly cleave AVP with the ultimate release of /sup 3/H-Tyr. 2-Mercaptoethanol (3.9mM) (ME) stimulated activity 3.6 to 6.6-fold for arylamide and dipeptide substrates, but 35-fold for labelled AVP, possibly owing to reduction of the AVP disulfide bond. All activities in the presence of ME were completely inhibited by 0.2mM amastatin.« less

Design of potent substrate-analogue inhibitors of canine renin

NASA Technical Reports Server (NTRS)

Hui, K. Y.; Siragy, H. M.; Haber, E.

1992-01-01

Through a systematic study of structure-activity relationships, we designed potent renin inhibitors for use in dog models. In assays against dog plasma renin at neutral pH, we found that, as in previous studies of rat renin inhibitors, the structure at the P2 position appears to be important for potency. The substitution of Val for His at this position increases potency by one order of magnitude. At the P3 position, potency appears to depend on a hydrophobic side chain that does not necessarily have to be aromatic. Our results also support the approach of optimizing potency in a renin inhibitor by introducing a moiety that promotes aqueous solubility (an amino group) at the C-terminus of the substrate analogue. In the design of potent dog plasma renin inhibitors, the influence of the transition-state residue 4(S)-amino-3(S)-hydroxy-5-cyclohexylpentanoic acid (ACHPA)-commonly used as a substitute for the scissile-bond dipeptide to boost potency-is not obvious, and appears to be sequence dependent. The canine renin inhibitor Ac-paF-Pro-Phe-Val-statine-Leu-Phe-paF-NH2 (compound 15; IC50 of 1.7 nM against dog plasma renin at pH 7.4; statine, 4(S)-amino-3(S)-hydroxy-6-methylheptanoic acid; paF, para-aminophenylalanine) had a potent hypotensive effect when infused intravenously into conscious, sodium-depleted, normotensive dogs. Also, compound 15 concurrently inhibited plasma renin activity and had a profound diuretic effect.

Towards Longitudinal Mapping of Extracellular pH in Gliomas

PubMed Central

Huang, Yuegao; Coman, Daniel; Herman, Peter; Rao, Jyotsna U.; Maritim, Samuel; Hyder, Fahmeed

2016-01-01

Biosensor Imaging of Redundant Deviation in Shifts (BIRDS), an ultrafast chemical shift imaging technique, requires infusion of paramagnetic probes like 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylene phosphonate (DOTP8−) complexed with thulium (Tm3+) ion (i.e., TmDOTP5−), where the pH-sensitive resonances of hyperfine-shifted nonexchangeable protons contained within the paramagnetic magnetic resonance probe are detected. While imaging extracellular pH (pHe) with BIRDS meets an important cancer research need by mapping the intratumoral-peritumoral pHe gradient, the surgical intervention used to raise the probe’s plasma concentration limits longitudinal scans on the same subject. Here we describe using probenecid (i.e., an organic anion transporter inhibitor) to temporarily restrict renal clearance of TmDOTP5−, thereby facilitating molecular imaging by BIRDS without surgical intervention. Co-infusion of probenecid with TmDOTP5− increased the probe’s distribution into various organs, including the brain, compared with when infusing TmDOTP5− alone. In vivo BIRDS data using probenecid/TmDOTP5− co-infusion method in rats bearing RG2, 9L, and U87 brain tumors showed intratumoral-peritumoral pHe gradients that were unaffected by the probe dose. This co-infusion method can be used for pHe mapping with BIRDS in preclinical models for tumor characterization and therapeutic monitoring given the possibility of repeated scans with BIRDS (e.g., over days and even weeks) in the same subject. The longitudinal pHe readout by probenecid/TmDOTP5− co-infusion method for BIRDS adds translational value in tumor assessment and treatment. PMID:27472471

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.

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.

The involvement of sympathetic nerves in plasma extravasation induced by prostaglandin E2 and substance P.

PubMed

Mathison, R; Davison, J S

1994-05-02

The effects of intravenous injection of prostaglandin E2 (PGE2), substance P (SP) and a metabolically stable SP analogue, [pGlu5,Me-Phe8,Sar9]-SP (5-11) on plasma extravasation of albumin in the rat after blockade of prostaglandin synthesis with indomethacin or chemical sympathectomy with guanethidine were studied. Blood pressure was decreased by all agonists, but only the hypotensive effects of SP were enhanced by pretreatment with indomethacin and guanethidine. The increase in plasma extravasation induced by PGE2 in the tongue, skin and lungs was blocked by both guanethidine and indomethacin. Pretreatment of the rats with guanethidine or indomethacin increased extravasation induced by SP in the tongue-tip, dorsal skin and foot, but decreased the enhanced permeability in the pinna, and did not alter the actions of the peptide in other tissues. In contrast, both guanethidine and indomethacin pretreatment increased vascular permeability responses to [pGlu5,Me-Phe8,Sar9]-SP (5-11) administration in 9 and 14 of 16 tissues examined, respectively. Thus, intact sympathetic nerves and functional cycloxygenase activity exert inhibitory constraints on the vascular permeability effects of intravenously administered SP or its analogue. On the other hand the integrity of the sympathetic nerves and prostaglandin synthesis are required for PGE2-induced increases in vascular leak.

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.

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.

ICRF wall conditioning at TEXTOR-94 in the presence of a 2.25 T magnetic field

NASA Astrophysics Data System (ADS)

Esser, H. G.; Lyssoivan, A.; Freisinger, M.; Koch, R.; van Oost, G.; Weschenfelder, F.; Winter, J.; Textor-Icrh-Team

1997-02-01

To investigate alternative conditioning concepts for future fusion devices with permanent magnetic fields, plasmas produced by the coupling of ICRF power to He and gas mixtures of Helium + silane, have been analyzed in the presence of a 2.25 T toroidal magnetic field at TEXTOR-94. Their qualification for wall conditioning has been investigated for different He-pressures, PHe (1 × 10 -3 < PHe ( Pa) < 1 × 10 -1) and ICRF power, PICRF (100 < PICRF ( kW) < 800). Electron densities n e averaged along different radial lines of sight across the vacuum vessel from the top to the bottom have been obtained in the range 5 × 10 10 < ne ( cm-3) < 3 × 10 12. To study quantitatively the efficiency of hydrogen desorption from the first wall at different ICRF plasma conditions in a reproducible way, the first wall was presaturated by RG-glow discharges in H 2. The amount and the evolution of the H 2 desorption from rf discharge to rf discharge was determined by ion gauge measurements combined with mass spectrometry. To demonstrate the capability of the new method for plasma assisted thin film deposition, different amounts of silane (<50%) were added to the He gas. During the ICRF pulses, the silane molecules were dissociated in the plasma and the Si atoms stick to the wall. A good balance of the amount of Si disappearing from the gas phase and that measured by post mortem surface analyses of collector probes at the wall position was found.

Dietary Phenylalanine Requirements are Similar in Small, Medium, and Large Breed Adult Dogs using the Indicator Amino Acid Oxidation Technique.

PubMed

Mansilla, W D; Gorman, A; Fortener, L; Shoveller, A K

2018-05-26

We have previously determined Phenylalanine (Phe) requirements in mature dogs; however, little information is available on differences of Phe minimum requirements (MR) on different breed sizes. The objective of this study was to determine Phe requirements in adult dogs of 3 different breed sizes using the direct amino acid oxidation (DAAO) technique. In total, 12 adult dogs were used, 4 Miniature Dachshunds (5.3 ± 0.6 Kg BW; 1.8 ± 0.1 years old; mean ± SD), 4 Beagles (8.3 ± 0.7 Kg BW; 6.7 ± 0.2 years old; mean ± SD), and 4 Labrador Retrievers (34.9 ± 2.2 Kg BW; 4.4 ± 1.4 years old; mean ± SD). A basal Phe-deficient diet with excess of Tyrosine (Tyr) was formulated. Dogs were randomly fed the basal diet supplemented with increasing levels of Phe; the Phe content in the final experimental diets was 0.24, 0.29, 0.34, 0.44, 0.54, 0.64, and 0.74%. After 2 d of adaptation to the experimental diets, dogs underwent individual DAAO studies. During the DAAO studies, total daily feed was divided in 13 equal meals; at the sixth meal, dogs were fed a bolus of L-[1-13C]-Phe (9.40 mg/kg BW), and thereafter, L-[1-13C]-Phe (2.4 mg/kg BW) was supplied with every meal. Total production of 13CO2 (13CO2) during isotopic steady state was determined by enrichment of 13CO2 in breath samples and total production of CO2 measured using indirect calorimetry. The mean requirement for Phe and the 95% confidence interval (CI) were determined using a 2-phase linear regression model. To account for differences in feed intake, requirements were expressed in mg.kg BW-1.d-1. The mean requirement for Phe were 41.9, 41.3, and 42.6, and upper 95% CI of Phe requirements were 57.3, 58.4, and 64.8 mg.kg BW-1.d-1 for Miniature Dachshunds, Beagles, and Labrador Retrievers, respectively. The mean requirement and the upper 95% CI for the pooled data (all dogs) was 45.3 and 55.4 mg.kg BW-1.d-1, respectively. In conclusion, the Phe requirements for different breeds were similar among dog breeds studied. However, Phe recommendations proposed in this study are lower than those proposed by NRC and AAFCO (mg.kg BW-1.d-1).

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.

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

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.

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

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.

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.

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.

Determination of branched chain amino acids, methionine, phenylalanine, tyrosine and alpha-keto acids in plasma and dried blood samples using HPLC with fluorescence detection.

PubMed

Kand'ár, Roman; Záková, Pavla; Jirosová, Jana; Sladká, Michaela

2009-01-01

The determination of branched chain amino acids [BCAA; valine (Val), leucine (Leu), isoleucine (Ile)], alpha-keto acids derived from BCAA [BCKA; alpha-ketoisovaleric acid (KIV), alpha-ketoisocaproic acid (KIC), alpha-ketomethylvaleric acid (KMV)], methionine (Met), phenylalanine (Phe) and tyrosine (Tyr) is currently the most reliable approach for the diagnosis of maple syrup urine disease (MSUD), hypermethioninemia, phenylketonuria (PKU) and tyrosinemia. The aim of this study was to develop rapid and simple HPLC methods for measurement of BCAA, Met, Phe, Tyr and BCKA in plasma and dried blood samples. Samples of peripheral venous blood with EDTA as anticoagulant were obtained from a group of healthy blood donors (n=70, 35 females, 27-41 years of age and 35 males, 28-43 years of age). Blood-spot samples from a group of newborns (n=80, 40 girls and 40 boys 3-5 days of age) were collected onto #903 Specimen Collection Paper and allowed to dry for at least 24 h before analysis. Prior to separation, the amino acids (AA) were derivatized with o-phthaldialdehyde (OPA) and BCKA with o-phenylenediamine (OPD). Reverse phase column chromatography (LiChroCart 125-4 Purospher RP-18e, 5 microm) was used for separation and fluorescence detection used to monitoring of effluent. For AA analysis, 25 mmol/L sodium hydrogenphosphate-methanol (90:10, v/v), pH 6.5+/-0.1 was used as mobile phase A and 100% methanol was used as mobile phase B. Measurement of BCKA used a mixture of methanol and deionized water (55:45, v/v) as mobile phase A and mobile phase B consisted of 100% methanol. Analytical performance of these methods was satisfactory for the determination of all AA and BCKA. The intra-assay and inter-assay coefficients of variation were below 10% and recovery ranged from 90%-110%. We have developed simple, rapid and selective HPLC methods with fluorescence detection for the determination of BCAA, Met, Phe, Tyr and BCKA in plasma and dried blood samples.

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.

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.

Protein-bound tyrosine oxidation, nitration and chlorination by-products assessed by ultraperformance liquid chromatography coupled to tandem mass spectrometry.

PubMed

Torres-Cuevas, Isabel; Kuligowski, Julia; Cárcel, María; Cháfer-Pericás, Consuelo; Asensi, Miguel; Solberg, Rønnaug; Cubells, Elena; Nuñez, Antonio; Saugstad, Ola Didrik; Vento, Máximo; Escobar, Javier

2016-03-24

Free radicals cause alterations in cellular protein structure and function. Oxidized, nitrated, and chlorinated modifications of aromatic amino acids including phenylalanine and tyrosine are reliable biomarkers of oxidative stress and inflammation in clinical conditions. To develop, validate and apply a rapid method for the quantification of known hallmarks of tyrosine oxidation, nitration and chlorination in plasma and tissue proteins providing a snapshot of the oxidative stress and inflammatory status of the organism and of target organs respectively. The extraction and clean up procedure entailed protein precipitation, followed by protein re-suspension and enzymatic digestion with pronase. An Ultra Performance Liquid Chromatography-tandem Mass Spectrometry (UPLC-MS/MS) method was developed to quantify protein released ortho-tyrosine (o-Tyr), meta-tyrosine (m-Tyr), 3-nitrotyrosine (3NO2-Tyr) and 3-chlorotyrosine (3Cl-Tyr) as well as native phenylalanine (Phe) and tyrosine (p-Tyr) in plasma and tissue from a validated hypoxic newborn piglet experimental model. In plasma there was a significant increase in the 3NO2-Tyr/p-Tyr ratio. On the other hand m-Tyr/Phe and 3Cl-Tyr/p-Tyr ratios were significantly increased in liver of hypoxic compared with normoxic animals. Although no significant differences were found in brain tissue, a clear tendency to increased ratios was observed under hypoxic conditions. UPLC-MS/MS has proven suitable for the analysis of plasma and tissue samples from newborn piglets. The analysis of biomarkers of protein oxidation, nitration and chlorination will be applied in future studies aiming to provide a deeper insight into the mechanisms of oxidation-derived protein modification caused during neonatal asphyxia and resuscitation. Copyright © 2016 Elsevier B.V. All rights reserved.

[The effect of substance P on functional proteins in human neutrophil].

PubMed

Yang, Lin; Fa, Xiang-guang

2002-02-01

To explore the effect of substance P (SP) on the functional proteins on plasma membrane of neutrophil (Np). The response of Np to SP was examined by measuring the level of respiratory burst, the activities of ACP and ALP, the fluoroscopy intensity of CR3, CD45 and FM-LP. It was found that SP could increase respiratory burst of Np, decrease the activity of acid phosphatase (ACP), but had no effect on alkaline phosphatase (ALP). SP could also promote the amount of CD45, complement receptor type 3 (CR3) and N-Formyl-Met-Leu-Phe (FMLP) receptors. The results showed that the effects of SP on functional proteins in human Np membrane were universality and diversity. It implied that SP could affect various inflammation responses in Np.

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

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.

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.

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

PubMed

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

2018-06-20

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

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.

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

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.

Low effect of phenanthrene bioaccessibility on its biodegradation in diffusely contaminated soil.

PubMed

Crampon, M; Cébron, A; Portet-Koltalo, F; Uroz, S; Le Derf, F; Bodilis, J

2017-06-01

This study focused on the role of bioaccessibility in the phenanthrene (PHE) biodegradation in diffusely contaminated soil, by combining chemical and microbiological approaches. First, we determined PHE dissipation rates and PHE sorption/desorption isotherms for two soils (PPY and Pv) presenting similar chronic PAH contamination, but different physico-chemical properties. Our results revealed that the PHE dissipation rate was significantly higher in the Pv soil compared to the PPY soil, while PHE sorption/desorption isotherms were similar. Interestingly, increases of PHE desorption and potentially of PHE bioaccessibility were observed for both soils when adding rhamnolipids (biosurfactants produced by Pseudomonas aeruginosa). Second, using 13 C-PHE incubated in the same soils, we analyzed the PHE degrading bacterial communities. The combination of stable isotope probing (DNA-SIP) and 16S rRNA gene pyrosequencing revealed that Betaproteobacteria were the main PHE degraders in the Pv soil, while a higher bacterial diversity (Alpha-, Beta-, Gammaproteobacteria and Actinobacteria) was involved in PHE degradation in the PPY soil. The amendment of biosurfactants commonly used in biostimulation methods (i.e. rhamnolipids) to the two soils clearly modified the PHE sorption/desorption isotherms, but had no significant impact on PHE degradation rates and PHE-degraders identity. These results demonstrated that increasing the bioaccessibility of PHE has a low impact on its degradation and on the functional populations involved in this degradation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Detection and scavenging of hydroxyl radical via D-phenylalanine hydroxylation in human fluids.

PubMed

Biondi, Roberto; Brancorsini, Stefano; Poli, Giulia; Egidi, Maria Giulia; Capodicasa, Enrico; Bottiglieri, Livio; Gerli, Sandro; Brillo, Eleonora; Renzo, Gian Carlo Di; Cretoiu, Dragos; Micu, Romeo; Suciu, Nicolae

2018-05-01

Hydroxyl radical (.OH) is highly reactive, and therefore very short-lived. Finding new means to accurately detect .OH, and testing the ability of known .OH scavengers to neutralize them in human biological fluids would leverage our ability to more effectively counter oxidative (.OH) stress-mediated damage in human diseases. To achieve this, we pursued the evaluation of secondary products resulting from .OH attack, using a detection system based on Fenton reaction-mediated D-phenylalanine (D-Phe) hydroxylation. This reaction in turn generates o-tyrosine (o-tyr), m-tyrosine (m-tyr) and p-tyrosine (p-tyr). Here, these isomers were separated by HPLC, equipped with fluorescence detectors due to the natural fluorescence of these hydrotyrosines. By extension, we found that, adding radical scavengers competed with D-Phe on .OH attack, thus allowing to determine the .OH quenching capacity of a given compound expressed as inhibition ratio percent (IR%). Using a kinetic approach, we then tested the .OH scavenging capacity (OHSC) of well-known antioxidant molecules. In a test tube, N,N'-dimethylthiourea (DMTU) was the most efficient scavenger as compared to Trolox and N-Acethyl-L-cysteine, with NAC being the less effective. OHSC assay was then applied to biological fluid samples as seminal plasma, human serum from normal subjects and patients undergoing hemodialysis (HD), colostrum and human breast milk from mothers that received daily doses of 30g of chocolate (70% cocoa) during pregnancy. We found that a daily administration of dark chocolate during pregnancy almost doubled OHSC levels in breast milk (1.88 ± 0.12 times, p < 0.01). Furthermore, HD treatment determined a significant reduction of serum OHSC concentration (54.63 ± 2.82%, p < 0.001). Our results provide evidence that Fenton reaction-mediated D-Phe hydroxylation is a suitable method for routine and non-invasive evaluation of .OH detection and its scavenging in human biological fluids. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Superior visible light hydrogen evolution of Janus bilayer junctions via atomic-level charge flow steering

NASA Astrophysics Data System (ADS)

Li, Jie; Zhan, Guangming; Yu, Ying; Zhang, Lizhi

2016-05-01

Although photocatalytic hydrogen evolution (PHE) is ideal for solar-to-fuel conversion, it remains challenging to construct a highly efficient PHE system by steering the charge flow in a precise manner. Here we tackle this challenge by assembling 1T MoS2 monolayers selectively and chemically onto (Bi12O17) end-faces of Bi12O17Cl2 monolayers to craft two-dimensional (2D) Janus (Cl2)-(Bi12O17)-(MoS2) bilayer junctions, a new 2D motif different from van der Waals heterostructure. Electrons and holes from visible light-irradiated Bi12O17Cl2 are directionally separated by the internal electric field to (Bi12O17) and (Cl2) end-faces, respectively. The separated electrons can further migrate to MoS2 via Bi-S bonds formed between (Bi12O17) and MoS2 monolayers. This atomic-level directional charge separation endows the Janus bilayers with ultralong carrier lifetime of 3,446 ns and hence a superior visible-light PHE rate of 33 mmol h-1 g-1. Our delineated Janus bilayer junctions on the basis of the oriented assembly of monolayers presents a new design concept to effectively steer the charge flow for PHE.

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.

New archetypes in self-assembled Phe-Phe motif induced nanostructures from nucleoside conjugated-diphenylalanines.

PubMed

Datta, Dhrubajyoti; Tiwari, Omshanker; Ganesh, Krishna N

2018-02-15

During the last two decades, the molecular self-assembly of the short peptide diphenylalanine (Phe-Phe) motif has attracted increasing focus due to its unique morphological structure and utility for potential applications in biomaterial chemistry, sensors and bioelectronics. Due to the ease of their synthetic modifications and a plethora of available experimental tools, the self-assembly of free and protected diphenylalanine scaffolds (H-Phe-Phe-OH, Boc-Phe-Phe-OH and Boc-Phe-Phe-OMe) has unfurled interesting tubular, vesicular or fibrillar morphologies. Developing on this theme, here we attempt to examine the effect of structure and properties (hydrophobic and H-bonding) modifying the functional C-terminus conjugated substituents on Boc-Phe-Phe on its self-assembly process. The consequent self-sorting due to H-bonding, van der Waals force and π-π interactions, generates monodisperse nano-vesicles from these peptides characterized via their SEM, HRTEM, AFM pictures and DLS experiments. The stability of these vesicles to different external stimuli such as pH and temperature, encapsulation of fluorescent probes inside the vesicles and their release by external trigger are reported. The results point to a new direction in the study and applications of the Phe-Phe motif to rationally engineer new functional nano-architectures.

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

PubMed

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

2009-02-01

The clinical severity of phenylalanine hydroxylase deficiency is usually defined by either pre-treatment phenylalanine (Phe) concentration or Phe tolerance at 5 years of age. So far, little is known about the course of Phe tolerance or the ability of both pre-treatment Phe and Phe tolerance at early age to predict Phe tolerance at later age. This study was conducted to investigate the course of the individual Phe tolerance and to assess the predictive value of both the pre-treatment Phe concentration and Phe tolerance at 1 and 6 months and 1, 2, 3 and 5 years for Phe tolerance at 10 years of age. Data on blood Phe concentration, prescribed Phe intake and weight of 213 early and continuously treated Dutch PKU patients up to 10 years of age were collected. Data acquired under good metabolic control were used in the study. Tolerance was expressed in mg/day and mg/kg per day. Data at 1 and 6 months and at 1, 2, 3 and 5 years of 61, 58, 59, 57, 56 and 59 patients were included for comparison with the Phe tolerance at 10 years. Phe tolerances (mg/kg per day) at 2, 3 and 5 years showed a clear correlation with the tolerance at 10 years of age (r = 0.608, r = 0.725 and r = 0.661). Results for tolerance expressed as mg/day were comparable. Pre-treatment Phe concentrations did not correlate significantly with the tolerance. Pre-treatment Phe is unreliable but Phe tolerance is a reliable predictor of the tolerance at 10 years of age, starting at 2 years of age.

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.

Quantitative analysis of [Dmt(1)]DALDA in ovine plasma by capillary liquid chromatography-nanospray ion-trap mass spectrometry.

PubMed

Wan, Haibao; Umstot, Edward S; Szeto, Hazel H; Schiller, Peter W; Desiderio, Dominic M

2004-04-15

The synthetic opioid peptide analog Dmt-D-Arg-Phe-Lys-NH(2) ([Dmt(1)]DALDA; [Dmt= 2',6'-dimethyltyrosine) is a highly potent and selective mu opioid-receptor agonist. A very sensitive and robust capillary liquid chromatography/nanospray ion-trap (IT) mass spectrometry method has been developed to quantify [Dmt(1)]DALDA in ovine plasma, using deuterated [Dmt(1)]DALDA as the internal standard. The standard MS/MS spectra of d(0)- and d(5)-[Dmt(1)]DALDA were obtained, and the collision energy was experimentally optimized to 25%. The product ion [ M + 2H-NH(3)](2+) (m/z 312.2) was used to identify and to quantify the synthetic opioid peptide analog in ovine plasma samples. The MS/MS detection sensitivity for [Dmt(1)]DALDA was 625 amol. A calibration curve was constructed, and quantitative analysis was performed on a series of ovine plasma samples.

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.

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

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.

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.

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.

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.

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.

A novel natural mutation AαPhe98Ile in the fibrinogen coiled-coil affects fibrinogen function.

PubMed

Riedelová-Reicheltová, Zuzana; Kotlín, Roman; Suttnar, Jiří; Geierová, Véra; Riedel, Tomáš; Májek, Pavel; Dyr, Jan Evangelista

2014-01-01

The aim of this study was to investigate the structure and function of fibrinogen obtained from a patient with normal coagulation times and idiopathic thrombophilia. This was done by SDS-PAGE and DNA sequence analyses, scanning electron microscopy, fibrinopeptide release, fibrin polymerisation initiated by thrombin and reptilase, fibrinolysis, and platelet aggregometry. A novel heterozygous point mutation in the fibrinogen Aα chain, Phe98 to Ile, was found and designated as fibrinogen Vizovice. The mutation, which is located in the RGDF sequence (Aα 95-98) of the fibrinogen coiled-coil region, significantly affected fibrin clot morphology. Namely, the clot formed by fibrinogen Vizovice contained thinner and curled fibrin fibers with reduced length. Lysis of the clots prepared from Vizovice plasma and isolated fibrinogen were found to be impaired. The lysis rate of Vizovice clots was almost four times slower than the lysis rate of control clots. In the presence of platelets agonists the mutant fibrinogen caused increased platelet aggregation. The data obtained show that natural mutation of Phe98 to Ile in the fibrinogen Aα chain influences lateral aggregation of fibrin protofibrils, fibrinolysis, and platelet aggregation. They also suggest that delayed fibrinolysis, together with the abnormal fibrin network morphology and increased platelet aggregation, may be the direct cause of thrombotic complications in the patient associated with pregnancy loss.

Metabolomic Insights into the Nutritional Status of Adults and Adolescents with Phenylketonuria Consuming a Low-Phenylalanine Diet in Combination with Amino Acid and Glycomacropeptide Medical Foods

PubMed Central

Stroup, Bridget M.; Murali, Sangita G.; Rohr, Frances; Gleason, Sally T.; van Calcar, Sandra C.; Levy, Harvey L.

2017-01-01

Background Nutrient status in phenylketonuria (PKU) requires surveillance due to the restrictive low-Phe diet in combination with amino acid medical foods (AA-MF) or glycomacropeptide medical foods (GMP-MF). Micronutrient profiles of medical foods are diverse, and optimal micronutrient supplementation in PKU has not been established. Methods In a crossover design, 30 participants with PKU were randomized to consume AA-MF and Glytactin™ GMP-MF in combination with a low-Phe diet for 3 weeks each. Fasting venipunctures, medical food logs, and 3-day food records were obtained. Metabolomic analyses were completed in plasma and urine by Metabolon, Inc. Results The low-Phe diets in combination with AA-MF and GMP-MF were generally adequate based on Dietary Reference Intakes, clinical measures, and metabolomics. Without micronutrient supplementation of medical foods, >70% of participants would have inadequate intakes for 11 micronutrients. Despite micronutrient supplementation of medical foods, inadequate intakes of potassium in 93% of participants and choline in >40% and excessive intakes of sodium in >63% of participants and folic acid in >27% were observed. Sugar intake was excessive and provided 27% of energy. Conclusions Nutrient status was similar with AA-MF and Glytactin GMP-MF. More research related to micronutrient supplementation of medical foods for the management of PKU is needed. PMID:29464117

Importance of tetrahedral intermediate formation in the catalytic mechanism of the serine proteases chymotrypsin and subtilisin.

PubMed

Petrillo, Teodolinda; O'Donohoe, Catrina A; Howe, Nicole; Malthouse, J Paul G

2012-08-07

Two new inhibitors in which the terminal α-carboxyl groups of Z-Ala-Ala-Phe-COOH and Z-Ala-Pro-Phe-COOH have been replaced with a proton to give Z-Ala-Ala-Phe-H and Z-Ala-Pro-Phe-H, respectively, have been synthesized. Using these inhibitors, we estimate that for α-chymotrypsin and subtilisin Carlsberg the terminal carboxylate group decreases the level of inhibitor binding 3-4-fold while a glyoxal group increases the level of binding by 500-2000-fold. We show that at pH 7.2 the effective molarities of the catalytic hydroxyl group of the active site serine are 41000-229000 and 101000-159000 for α-chymotrypsin and subtilisin Carlsberg, respectively. It is estimated that oxyanion stabilization and the increased effective molarity of the catalytic serine hydroxyl group can account for the catalytic efficiency of the reaction. We argue that substrate binding induces the formation of a strong hydrogen bond or low-barrier hydrogen bond between histidine-57 and aspartate-102 that increases the pK(a) of the active site histidine, allowing it to be an effective general base catalyst for the formation of the tetrahedral intermediate and increasing the effective molarity of the catalytic hydroxyl group of serine-195. A catalytic mechanism for acyl intermediate formation in the serine proteases is proposed.

Optimal management of phenylketonuria: a centralized expert team is more successful than a decentralized model of care.

PubMed

Camfield, Carol S; Joseph, Marissa; Hurley, Teresa; Campbell, Karen; Sanderson, Susan; Camfield, Peter R

2004-07-01

To compare phenylketonuria (PKU) management by a centralized, expert team in the Province of Nova Scotia (NS) with the decentralized approach in New Brunswick (NB). Retrospective chart review documented frequency of outpatient visits, phenylalanine (Phe) concentration, and medical formula use. Structured telephone interviews with the 8 regional NB dietitians (NB-D) documented their knowledge and support in PKU management. Patients with PKU (n=108; age, birth to 42 years) reside in NB (n=69) and NS (n=39). More were lost to contact in NB than in NS (9/69 vs 1/39) and more were completely off diet in NB than in NS (24/60 vs 1/38, P=.05). All 15 children <2 years old followed by a PKU team in either NS or Saint John, NB had optimal Phe levels. Children 2 to 12 years of age in NS had better Phe control and more medical visits than in NB (P <.01). Older patients had more episodes of elevated Phe levels (P=.01). Formula was dispensed in appropriate yearly amounts to 52% in NB and >95% in NS. Mental handicap or borderline intelligence was common in both NB (44%) and NS (42%). All NB-D wished additional specialized medical, nursing, or social work assistance. PKU management appears to be more effective with an expert, coordinated team approach.

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.

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.

Terahertz spectra of L-phenylalanine and its monohydrate

NASA Astrophysics Data System (ADS)

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

2017-05-01

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

Recurrent somnolence in a 17-month-old infant: late-onset ornithine transcarbamylase (OTC) deficiency due to the novel hemizygous mutation c.535C > T (p.Leu179Phe).

PubMed

Fantur, Michaela; Karall, Daniela; Scholl-Buergi, Sabine; Häberle, Johannes; Rauchenzauner, Markus; Fruehwirth, Martin

2013-01-01

Herein, we describe a case of a now 28-month-old boy who presented at the age of 17 months with four episodes of recurrent vomiting and somnolence during a period of four months with increasing severity. A comprehensive clinical and metabolic evaluation revealed normal blood pH and blood glucose, normal cerebral computed tomography and electroencephalogram but an elevated plasma ammonia concentration, which raised the suspicion of a urea cycle disorder. The combination of elevated urinary orotic acid and plasma glutamine with normal citrulline suggested the diagnosis of ornithine transcarbamylase (OTC) deficiency, which was confirmed by molecular genetic testing revealing the novel hemizygous mutation c.535C > T (p.Leu179Phe) of the OTC gene. After restitution of anabolism by administration of parenteral glucose, substitution of citrulline and detoxification of ammonia with sodium benzoate, the patient recovered rapidly and is in a stable metabolic and neurological state since then. This case underlines that the diagnosis of a urea cycle defect should be considered in the differential diagnosis of recurrent idiopathic vomiting in combination with unexplained neurological symptoms also beyond the neonatal period due to the possibility of mild or atypical late-onset presentation (e.g. OTC deficiency in hemizygous males). Copyright © 2012 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.

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.

Salmonella typhimurium gyrA mutations associated with fluoroquinolone resistance.

PubMed Central

Reyna, F; Huesca, M; González, V; Fuchs, L Y

1995-01-01

Spontaneous quinolone-resistant mutants obtained from Salmonella typhimurium Su694 were screened for mutations by direct DNA sequencing of an amplified PCR gyrA fragment. Substitutions Ser-83-->Phe (Ser83Phe), Ser83Tyr, Asp87Tyr, and Asp87Asn and double mutation Ala67Pro-Gly81Ser, which resulted in decreased sensitivities to ciprofloxacin, enoxacin, pefloxacin, norfloxacin, ofloxacin, and nalidixic acid, were found. The levels of resistance to quinolones for each mutant were determined. PMID:7492118

Effects of phenylalanine, histidine, and leucine on basal and GHRH-stimulated GH secretion and on PRL, insulin, and glucose levels in short children. Comparison with the effects of arginine.

PubMed

Bellone, J; Valetto, M R; Aimaretti, G; Segni, M; Volta, C; Cardimale, G; Baffoni, C; Pasquino, A M; Bernasconi, S; Bartolotta, E; Mucci, M; Ghigo, E

1996-01-01

Of the amino acids arginine is the most potent GH secretagogue in man. It potentiates the GH response to GHRH, exerts a weaker PRL-releasing effect, stimulates insulin and glucagon and induces a biphasic glucose variation. The potency and effects of other amino acids on pituitary and pancreatic hormones need to be clarified. In 43 children with normal short stature (5.3-14.0 yr; 30 M and 13 F) the effects of the infusion of phenylalanine (Phe, 0.08 g/kg), histidine (His, 0.1 g/kg), and leucine (Leu, 0.08 g/kg) on basal and GHRH-stimulated GH secretion and on PRL, insulin and glucose levels were studied and compared with those of arginine at high (hArg, 0.5 g/kg) or low dose (lArg, 0.2 g/kg). Phe increased basal (p < 0.05) but not GHRH-stimulated GH levels, induced PRL and insulin rises (p < 0.03 and p < 0.03), and did not change glycemia. Though a trend toward an increase in basal GH levels was found after His, His and Leu did not significantly modify either basal or GHRH-induced GH secretion nor basal PRL, insulin and glucose levels. Both hArg and lArg increased basal (p < 0.0001 and p < 0.05, respectively) and GHRH-stimulated GH levels (p < 0.006 and p < 0.006). hArg increased both PRL (p < 0.002) and insulin levels (p < 0.005) more (p < 0.0005 and p < 0.004) than lArg (p < 0.005 and p < 0.005), while glucose levels showed a similar increase followed by a similar decrease. We conclude that in childhood: a) Phe significantly increases GH secretion but, differently from Arg, does not potentiate the response to GHRH, suggesting different mechanisms of action of these amino acids; b) differently from His and Leu, Phe is a PRL and insulin secretagogue but is less potent than Arg; c) Arg has the highest stimulatory effect on pituitary and pancreatic hormones.

Tryptophan Metabolism and Its Relationship with Depression and Cognitive Impairment Among HIV-infected Individuals.

PubMed

Keegan, Michael R; Chittiprol, Seetharamaiah; Letendre, Scott L; Winston, Alan; Fuchs, Dietmar; Boasso, Adriano; Iudicello, Jennifer; Ellis, Ronald J

2016-01-01

Cognitive impairment (CI) and major depressive disorder (MDD) remain prevalent in treated HIV-1 disease; however, the pathogenesis remains elusive. A possible contributing mechanism is immune-mediated degradation of tryptophan (TRP) via the kynurenine (KYN) pathway, resulting in decreased production of serotonin and accumulation of TRP degradation products. We explored the association of these biochemical pathways and their relationship with CI and MDD in HIV-positive (HIV+) individuals. In a cross-sectional analysis, concentrations of neopterin (NEO), tumor necrosis factor-alpha, TRP, KYN, KYN/TRP ratio, phenylalanine (PHE), tyrosine (TYR), PHE/TYR ratio, and nitrite were assessed in the cerebrospinal fluid (CSF) and plasma of HIV+ ( n = 91) and HIV-negative (HIV-) individuals ( n = 66). CI and MDD were assessed via a comprehensive neuropsychological test battery. A Global Deficit Score ≥0.5 was defined as CI. Nonparametric statistical analyses included Kruskal-Wallis and Mann-Whitney U tests, and multivariate logistic regression. Following Bonferroni correction, NEO concentrations were found to be greater in CSF and TRP concentration was found to be lower in the plasma of HIV+ versus HIV- individuals, including a subgroup of aviremic (defined as HIV-1 RNA <50 cps/mL) HIV+ participants receiving antiretroviral therapy ( n = 44). There was a nonsignificant trend toward higher KYN/TRP ratios in plasma in the HIV+ group ( P = 0.027; Bonferroni corrected α = 0.0027). In a logistic regression model, lower KYN/TRP ratios in plasma were associated with CI and MDD in the overall HIV+ group ( P = 0.038 and P = 0.063, respectively) and the aviremic subgroup ( P = 0.066 and P = 0.027, respectively), though this observation was not statistically significant following Bonferroni correction (Bonferroni corrected α = 0.0031). We observed a trend toward lower KYN/TRP ratios in aviremic HIV+ patients with CI and MDD.

Terahertz spectra of l-phenylalanine and its monohydrate.

PubMed

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

2017-05-05

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

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.

Ipamorelin, the first selective growth hormone secretagogue.

PubMed

Raun, K; Hansen, B S; Johansen, N L; Thøgersen, H; Madsen, K; Ankersen, M; Andersen, P H

1998-11-01

The development and pharmacology of a new potent growth hormone (GH) secretagogue, ipamorelin, is described. Ipamorelin is a pentapeptide (Aib-His-D-2-Nal-D-Phe-Lys-NH2), which displays high GH releasing potency and efficacy in vitro and in vivo. As an outcome of a major chemistry programme, ipamorelin was identified within a series of compounds lacking the central dipeptide Ala-Trp of growth hormone-releasing peptide (GHRP)-1. In vitro, ipamorelin released GH from primary rat pituitary cells with a potency and efficacy similar to GHRP-6 (ECs) = 1.3+/-0.4nmol/l and Emax = 85+/-5% vs 2.2+/-0.3nmol/l and 100%). A pharmacological profiling using GHRP and growth hormone-releasing hormone (GHRH) antagonists clearly demonstrated that ipamorelin, like GHRP-6, stimulates GH release via a GHRP-like receptor. In pentobarbital anaesthetised rats, ipamorelin released GH with a potency and efficacy comparable to GHRP-6 (ED50 = 80+/-42nmol/kg and Emax = 1545+/-250ng GH/ml vs 115+/-36nmol/kg and 1167+/-120ng GH/ml). In conscious swine, ipamorelin released GH with an ED50 = 2.3+/-0.03 nmol/kg and an Emax = 65+/-0.2 ng GH/ml plasma. Again, this was very similar to GHRP-6 (ED50 = 3.9+/-1.4 nmol/kg and Emax = 74+/-7ng GH/ml plasma). GHRP-2 displayed higher potency but lower efficacy (ED50 = 0.6 nmol/kg and Emax = 56+/-6 ng GH/ml plasma). The specificity for GH release was studied in swine. None of the GH secretagogues tested affected FSH, LH, PRL or TSH plasma levels. Administration of both GHRP-6 and GHRP-2 resulted in increased plasma levels of ACTH and cortisol. Very surprisingly, ipamorelin did not release ACTH or cortisol in levels significantly different from those observed following GHRH stimulation. This lack of effect on ACTH and cortisol plasma levels was evident even at doses more than 200-fold higher than the ED50 for GH release. In conclusion, ipamorelin is the first GHRP-receptor agonist with a selectivity for GH release similar to that displayed by GHRH. The specificity of ipamorelin makes this compound a very interesting candidate for future clinical development.

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

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

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.

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.

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

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

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.

Lactose carrier mutants of Escherichia coli with changes in sugar recognition (lactose versus melibiose).

PubMed

Varela, M F; Brooker, R J; Wilson, T H

1997-09-01

The purpose of this research was to identify amino acid residues that mediate substrate recognition in the lactose carrier of Escherichia coli. The lactose carrier transports the alpha-galactoside sugar melibiose as well as the beta-galactoside sugar lactose. Mutants from cells containing the lac genes on an F factor were selected by the ability to grow on succinate in the presence of the toxic galactoside beta-thio-o-nitrophenylgalactoside. Mutants that grew on melibiose minimal plates but failed to grow on lactose minimal plates were picked. In sugar transport assays, mutant cells showed the striking result of having low levels of lactose downhill transport but high levels of melibiose downhill transport. Accumulation (uphill) of melibiose was completely defective in all of the mutants. Kinetic analysis of melibiose transport in the mutants showed either no change or a greater than normal apparent affinity for melibiose. PCR was used to amplify the lacY DNA of each mutant, which was then sequenced by the Sanger method. The following six mutations were found in the lacY structural genes of individual mutants: Tyr-26-->Asp, Phe-27-->Tyr, Phe-29-->Leu, Asp-240-->Val, Leu-321-->Gln, and His-322-->Tyr. We conclude from these experiments that Tyr-26, Phe-27, Phe-29 (helix 1), Asp-240 (helix 7), Leu-321, and His-322 (helix 10) either directly or indirectly mediate sugar recognition in the lactose carrier of E. coli.

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.

Adsorption of phenanthrene on activated carbon increases mineralization rate by specific bacteria.

PubMed

Leglize, Pierre; Alain, Saada; Jacques, Berthelin; Corinne, Leyval

2008-03-01

Bioavailability of polycyclic aromatic hydrocarbons (PAH) in soil is affected by PAH sorption to solid phase. We studied the influence of activated carbon (AC) on phenanthrene (PHE) mineralization by five degrading bacterial strains isolated from contaminated soil. PHE adsorption on AC was important and reduced PHE aqueous concentration up to 90%. PHE degradation was improved in the presence of activated carbon with three of the bacterial strains, named NAH1, MATE3 and MATE7, which produced biofilms, whereas it was not improved with the two other ones, which did not produce biofilms, MATE10 and MATE12. Monitoring PHE distribution during incubation showed that aqueous PHE concentration was significantly higher with the biofilm-producing NAH1 than with MATE10. Bacterial adhesion on AC was also investigated. The precoating of AC with PHE increased NAH1 and MATE3 adhesion to the solid surface (>16 and >13%, respectively). Bacterial properties, such as biofilm production and adhesion to AC capacity seemed to be related to their ability to optimize PHE degradation by improving PHE diffusion and reducing diffusion path length.

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.

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.

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.

Mutation of the human mitochondrial phenylalanine-tRNA synthetase causes infantile-onset epilepsy and cytochrome c oxidase deficiency.

PubMed

Almalki, Abdulraheem; Alston, Charlotte L; Parker, Alasdair; Simonic, Ingrid; Mehta, Sarju G; He, Langping; Reza, Mojgan; Oliveira, Jorge M A; Lightowlers, Robert N; McFarland, Robert; Taylor, Robert W; Chrzanowska-Lightowlers, Zofia M A

2014-01-01

Mitochondrial aminoacyl-tRNA synthetases (aaRSs) are essential enzymes in protein synthesis since they charge tRNAs with their cognate amino acids. Mutations in the genes encoding mitochondrial aaRSs have been associated with a wide spectrum of human mitochondrial diseases. Here we report the identification of pathogenic mutations (a partial genomic deletion and a highly conserved p. Asp325Tyr missense variant) in FARS2, the gene encoding mitochondrial phenylalanyl-tRNA synthetase, in a patient with early-onset epilepsy and isolated complex IV deficiency in muscle. The biochemical defect was expressed in myoblasts but not in fibroblasts and associated with decreased steady state levels of COXI and COXII protein and reduced steady state levels of the mt-tRNA(Phe) transcript. Functional analysis of the recombinant mutant p. Asp325Tyr FARS2 protein showed an inability to bind ATP and consequently undetectable aminoacylation activity using either bacterial tRNA or human mt-tRNA(Phe) as substrates. Lentiviral transduction of cells with wildtype FARS2 restored complex IV protein levels, confirming that the p.Asp325Tyr mutation is pathogenic, causing respiratory chain deficiency and neurological deficits on account of defective aminoacylation of mt-tRNA(Phe). © 2013. Published by Elsevier B.V. All rights reserved.

Characterizations of a synthetic pituitary adenylate cyclase-activating polypeptide analog displaying potent neuroprotective activity and reduced in vivo cardiovascular side effects in a Parkinson's disease model.

PubMed

Lamine, Asma; Létourneau, Myriam; Doan, Ngoc Duc; Maucotel, Julie; Couvineau, Alain; Vaudry, Hubert; Chatenet, David; Vaudry, David; Fournier, Alain

2016-09-01

Parkinson's disease (PD) is characterized by a steady loss of dopamine neurons through apoptotic, inflammatory and oxidative stress processes. In that line of view, the pituitary adenylate cyclase-activating polypeptide (PACAP), with its ability to cross the blood-brain barrier and its anti-apoptotic, anti-inflammatory and anti-oxidative properties, has proven to offer potent neuroprotection in various PD models. Nonetheless, its peripheral actions, paired with low metabolic stability, hampered its clinical use. We have developed Ac-[Phe(pI)(6), Nle(17)]PACAP(1-27) as an improved PACAP-derived neuroprotective compound. In vitro, this analog stimulated cAMP production, maintained mitochondrial potential and protected SH-SY5Y neuroblastoma cells from 1-methyl-4-phenylpyridinium (MPP(+)) toxicity, as potently as PACAP. Furthermore, contrasting with PACAP, it is stable in human plasma and against dipeptidyl peptidase IV activity. When injected intravenously to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, PACAP and Ac-[Phe(pI)(6), Nle(17)]PACAP(1-27) restored tyrosine hydoxylase expression into the substantia nigra and modulated the inflammatory response. Albeit falls of mean arterial pressure (MAP) were observed with both PACAP- and Ac-[Phe(pI)(6), Nle(17)]PACAP(1-27)-treated mice, the intensity of the decrease as well as its duration were significantly less marked after iv injections of the analog than after those of the native polypeptide. Moreover, no significant changes in heart rate were measured with the animals for both compounds. Thus, Ac-[Phe(pI)(6), Nle(17)]PACAP(1-27) appears as a promising lead molecule for the development of PACAP-derived drugs potentially useful for the treatment of PD or other neurodegenerative diseases. Copyright © 2015 Elsevier Ltd. All rights reserved.

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.

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.

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).

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]).

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.

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.

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.

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.

Reciprocal moderation by Toxoplasma gondii seropositivity and blood phenylalanine - tyrosine ratio of their associations with trait aggression.

PubMed

Mathai, Ashwin Jacob; Lowry, Christopher A; Cook, Thomas B; Brenner, Lisa A; Brundin, Lena; Groer, Maureen W; Peng, Xiaoqing; Giegling, Ina; Hartmann, Annette M; Konte, Bettina; Friedl, Marion; Fuchs, Dietmar; Rujescu, Dan; Postolache, Teodor T

2016-12-01

We previously reported that trait aggression, proposed as an endophenotype for suicidal behavior, is positively associated with Toxoplasma gondii ( T. gondii ) seropositivity in females, but not in males. Additionally, older males seropositive for T. gondii had lower scores on measures of trait aggression, including self-aggression. Trait aggression may be influenced by dopaminergic signaling, which is known to be moderated by gender and age, and potentially enhanced in T. gondii positives through the intrinsic production of dopamine by the microorganism. Therefore, we investigated associations between trait aggression and interactions between T. gondii enzyme-linked immunoabsorbant assay (ELISA) IgG titer-determined seropositivity and high-performance liquid chromatography- (HPLC-) measured blood levels of dopamine precursors phenylalanine (Phe), tyrosine (Tyr), and their ratio in a sample of 1000 psychiatrically healthy participants. Aggressive traits were assessed using the questionnaire for measuring factors of aggression (FAF), the German version of the Buss-Durkee hostility questionnaire. We found that 1) the decrease in trait aggression scores in T. gondii -positive older males was only present in individuals with a low Phe:Tyr ratio, and 2) that there was a positive correlation between Phe:Tyr ratio and total aggression and selected subscales of aggression in T. gondii -positive males, but not in T. gondii -negative males. These findings point toward a gender-specific reciprocal moderation by Phe:Tyr ratio and T. gondii seropositivity of their associations with aggression scores, and lead to experimental interventions geared to manipulating levels of dopamine precursors in selected T. gondii positive individuals with increased propensity for aggression.

Kinetic studies on the oxidation of cytochrome b(5) Phe35 mutants with cytochrome c, plastocyanin and inorganic complexes.

PubMed

Yao, Ping; Wang, Yun-Hua; Sun, Bing-Yun; Xie, Yi; Hirota, Shun; Yamauchi, Osamu; Huang, Zhong-Xian

2002-04-01

To illustrate the functions of the aromatic residue Phe35 of cytochrome b(5) and to give further insight into the roles of the Phe35-containing hydrophobic patch and/or aromatic channel of cytochrome b(5), we studied electron transfer reactions of cytochrome b(5) and its Phe35Tyr and Phe35Leu variants with cytochrome c, with the wild-type and Tyr83Phe and Tyr83Leu variants of plastocyanin, and with the inorganic complexes [Fe(EDTA)](-), [Fe(CDTA)](-) and [Ru(NH(3))(6)](3+). The changes at Phe35 of cytochrome b(5) and Tyr83 of plastocyanin do not affect the second-order rate constants for the electron transfer reactions. These results show that the invariant aromatic residues and aromatic patch/channel are not essential for electron transfer in these systems.

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.

Enhancement of blood-tumor barrier permeability by Sar-[D-Phe8]des-Arg9BK, a metabolically resistant bradykinin B1 agonist, in a rat C6 glioma model

PubMed Central

Cardoso, Ronie Cleverson; Lobão-Soares, Bruno; Bianchin, Marino Muxfeldt; Carlotti, Carlos Gilberto; Walz, Roger; Alvarez-Silva, Márcio; Trentin, Andréa Gonçalves; Nicolau, Mauro

2004-01-01

Background While it is well known that bradykinin B2 agonists increase plasma protein extravasation (PPE) in brain tumors, the bradykinin B1 agonists tested thus far are unable to produce this effect. Here we examine the effect of the selective B1 agonist bradykinin (BK) Sar-[D-Phe8]des-Arg9BK (SAR), a compound resistant to enzymatic degradation with prolonged activity on PPE in the blood circulation in the C6 rat glioma model. Results SAR administration significantly enhanced PPE in C6 rat brain glioma compared to saline or BK (p < 0.01). Pre-administration of the bradykinin B1 antagonist [Leu8]-des-Arg (100 nmol/Kg) blocked the SAR-induced PPE in the tumor area. Conclusions Our data suggest that the B1 receptor modulates PPE in the blood tumor barrier of C6 glioma. A possible role for the use of SAR in the chemotherapy of gliomas deserves further study. PMID:15458573

Distinct Orai-coupling domains in STIM1 and STIM2 define the Orai-activating site

NASA Astrophysics Data System (ADS)

Wang, Xizhuo; Wang, Youjun; Zhou, Yandong; Hendron, Eunan; Mancarella, Salvatore; Andrake, Mark D.; Rothberg, Brad S.; Soboloff, Jonathan; Gill, Donald L.

2014-02-01

STIM1 and STIM2 are widely expressed endoplasmic reticulum (ER) Ca2+ sensor proteins able to translocate within the ER membrane to physically couple with and gate plasma membrane Orai Ca2+ channels. Although they are structurally similar, we reveal critical differences in the function of the short STIM-Orai-activating regions (SOAR) of STIM1 and STIM2. We narrow these differences in Orai1 gating to a strategically exposed phenylalanine residue (Phe-394) in SOAR1, which in SOAR2 is substituted by a leucine residue. Remarkably, in full-length STIM1, replacement of Phe-394 with the dimensionally similar but polar histidine head group prevents both Orai1 binding and gating, creating an Orai1 non-agonist. Thus, this residue is critical in tuning the efficacy of Orai activation. While STIM1 is a full Orai1-agonist, leucine-replacement of this crucial residue in STIM2 endows it with partial agonist properties, which may be critical for limiting Orai1 activation stemming from its enhanced sensitivity to store-depletion.

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

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

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.

Comparison of phenanthrene removal by Aspergillus niger ATC 16404 (filamentous fungi) and Pseudomonas putida KT2442 (bacteria) in enriched nutrient-liquid medium

NASA Astrophysics Data System (ADS)

Hamzah, N.; Kamil, N. A. F. M.; Singhal, N.; Padhye, L.; Swift, S.

2018-04-01

Polycyclic Aromatic Hydrocarbons (PAHs) is one of the persistent and carcinogenic pollutants that needs to be eliminated from the environment. The study on degradation of PAHs by bacteria is thoroughly discussed in literature. Many strains of bacteria were chosen in order to eliminate the PAHs compound in the environment. However, there are less study on the filamentous fungi although fungi appears to be an abundant population and as dominant group in PAHs contaminated soil habitats [1], [2]. This study was conducted to determine and compare the Phenanthrene (PHE) removal by fungi and bacteria in excessive nutrient-liquid culture. Then, the survival for both strains was investigated in the presence of PHE and finally, the analysis on the fungi-PHE interaction was carried out. In condition of excessive nutrient, the removal of PHE was evaluated for fungi and bacteria in batch experiment for 5 days. PHE removal for A.niger and P.putida were found to be 97% and 20% respectively after 5 days. The presence of PHE was negatively inhibits the grow of the bacteria and the fungus. The PHE uptake mechanism for A.niger was observed to be a passive transport mechanism with 45 μg per g fungus dry weight within 24 hr of incubation. As a conclusion, filamentous fungi have the potent role in the removal of PHE as well as bacteria but depending on the strains and the condition of the environment. Fungi is known to co-metabolize the PHE meanwhile, PHE can be used as sole carbon for bacteria. This preliminary result is significant in understanding the bacteria-fungi-PHE interaction to enhance the degradation of PAHs for co-culture study in the future.

Kinin B1 receptor antagonists containing alpha-methyl-L-phenylalanine: in vitro and in vivo antagonistic activities.

PubMed

Gobeil, F; Charland, S; Filteau, C; Perron, S I; Neugebauer, W; Regoli, D

1999-03-01

-To protect from metabolism and to improve potency of the AcLys-[D-betaNal7,Ile8]desArg9-bradykinin (BK) (R 715), we prepared and tested 3 analogues containing alpha-methyl-L-Phe ([alphaMe]Phe) in position 5: these are the AcLys-[(alphaMe)Phe5,D-betaNal7, Ile8]desArg9BK (R 892), Lys-Lys-[(alphaMe)Phe5,D-betaNal7, Ile8]desArg9BK (R 913), and AcLys-Lys-[(alphaMe)Phe5,D-betaNal7, Ile8]desArg9BK (R 914). The new compounds were tested against the contractile effect induced by desArg9BK on 2 B1 receptor bioassays, the human umbilical vein, and the rabbit aorta. Their antagonistic activities were compared with those of the early prototypes (Lys-[Leu8]desArg9BK and [Leu8]desArg9BK) and with other recently described peptide antagonists. The 3 (alphaMe)Phe analogues showed high antagonistic potencies (pA2) at both the human (8.8, 7.7, and 8. 7, respectively) and rabbit (8.6, 7.8, and 8.6, respectively) B1 receptors. No antagonistic effects (pA2<5) were observed on the B2 receptors that mediate the contractile effects of BK on the human umbilical vein, the rabbit jugular vein, and the guinea pig ileum. Moreover, these new B1 antagonists were found to be resistant to in vitro degradation by purified angiotensin-converting enzyme from rabbit lung. The Nalpha-acetylated forms, R 892 and R 914, were resistant to aminopeptidases from human plasma. In vivo antagonistic potencies (ID50) of B1 receptor antagonists were evaluated in anesthetized lipopolysaccharide-treated (for B1 receptor) and nontreated (for B2 receptor) rabbits against the hypotensive effects of exogenous desArg9BK and BK. R 892 efficiently inhibited (ID50 2.8 nmol/kg IV) hypotension induced by desArg9BK without affecting that evoked by BK (ID50 >600 nmol/kg IV). Conversely, the peptide antagonists Lys-Lys-[Hyp3,Igl5,D-Igl7,Oic8]desArg9BK (B 9858) and DArg-[Hyp3,Thi5,D-Tic7,Oic8] desArg9BK (S 0765) showed dual B1/B2 receptor antagonism in vitro and in vivo. It is concluded that R 892 and congeners provide selective, highly potent, and metabolically stable B1 kinin receptor antagonists that can be useful for the assessment of the physiological and pathological roles of kinin B1 receptors.

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

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.

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.

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.

Hydrogen peroxide-induced DNA damage is independent of nuclear calcium but dependent on redox-active ions.

PubMed Central

Jornot, L; Petersen, H; Junod, A F

1998-01-01

In cells undergoing oxidative stress, DNA damage may result from attack by .OH radicals produced by the Fenton reaction, and/or by nucleases activated by nuclear calcium. In the present study, the participation of these two mechanisms was investigated in HeLa cells. Nuclear-targeted aequorin was used for selectively monitoring Ca2+ concentrations within the nuclei ([Ca2+]n), in conjunction with the cell-permeant calcium chelator bis-(o-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid acetoxymethyl ester (BAPTA/AM), the lipid-soluble broad-spectrum metal chelator with low affinity for Ca2+ and Mg2+ N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), and the high-affinity iron/copper chelator 1, 10-phenanthroline (PHE). In Ca2+-containing medium, H2O2 induced extensive DNA strand breaks and an increase in [Ca2+]n that was almost identical to that observed in the cytosol ([Ca2+]c). In cells bathed in Ca2+-free/EGTA medium, in which the increases in [Ca2+]n and [Ca2+]c due to H2O2 were significantly reduced, similar levels of DNA fragmentation also occurred. In cells preloaded with BAPTA/AM or TPEN, the small increase of [Ca2+]n normally elicited by H2O2 in Ca2+-free medium was completely buffered, and DNA damage was largely prevented. On the other hand, pretreatment with PHE did not affect the calcium response in the nuclei, but completely prevented DNA strand breakage induced by H2O2. Re-addition of 100 microM CuSO4 and 100 microM FeSO4 to TPEN- and PHE-treated cells prior to H2O2 challenge reversed the effect of TPEN and PHE, whereas 1 mM was necessary to negate the effect of BAPTA/AM. The levels of DNA strand breakage observed, however, did not correlate with the amounts of 8-hydroxy 2'-deoxyguanosine (8-OHdG): H2O2 did not produce 8-OHdG, whereas PHE alone slightly increased 8-OHdG levels. CuSO4 and FeSO4 enhanced the effects of PHE, particularly in the presence of H2O2. Exposure of cells to a mixture of CuSO4/FeSO4 also resulted in a significant increase in 8-OHdG levels, which was prevented in cells preloaded with BAPTA/AM. Similar results were obtained in a cell-free system using isolated calf thymus DNA exposed to CuSO4/FeSO4, regardless of whether H2O2 was present or not. These results suggest that BAPTA/AM prevents H2O2-induced DNA damage by acting as an iron/copper chelator. These data also indicate that caution must be exercised in using Ca2+ chelating agents as evidence for a role in cellular Ca2+ levels in experimental conditions in which transition-metal-ion-mediated oxidant production is also occurring. PMID:9742216

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.

Solution structure of a small protein containing a fluorinated side chain in the core

PubMed Central

Cornilescu, Gabriel; Hadley, Erik B.; Woll, Matthew G.; Markley, John L.; Gellman, Samuel H.; Cornilescu, Claudia C.

2007-01-01

We report the first high-resolution structure for a protein containing a fluorinated side chain. Recently we carried out 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 either aryl–aryl or perfluoroaryl–perfluoroaryl interactions, and because perfluoroaryl units are more hydrophobic than are analogous aryl units. Only one mutation, Phe10 → F5-Phe, was found to provide enhanced tertiary structural stability relative to the native core (by ∼1 kcal/mol, according to guanidinium chloride denaturation studies). The NMR structure of this mutant, described here, reveals very little variation in backbone conformation or side chain packing relative to the wild type. Thus, although Phe → F5-Phe mutations offer the possibility of greater tertiary structural stability from side chain–side chain attraction and/or side chain desolvation, the constraints associated with the native c-VHP fold apparently prevent the modified polypeptide from taking advantage of this possibility. Our findings are important because they complement several studies that have shown that fluorination of saturated side chain carbon atoms can provide enhanced conformational stability. PMID:17123960

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.

Evaluation of an adsorbent based on agricultural waste (corn cobs) for removal of tyrosine and phenylalanine from aqueous solutions.

PubMed

Alves, Cibele C O; Franca, Adriana S; Oliveira, Leandro S

2013-01-01

Adsorption of phenolic amino acids, such as phenylalanine and tyrosine, is quite relevant for the production of protein hydrolysates used as dietary formulations for patients suffering from congenital disorders of amino acid metabolism, such as phenylketonuria. In this study, an adsorbent prepared from corn cobs was evaluated for the removal of tyrosine (Tyr) from both a single component solution and a binary aqueous solution with phenylalanine (Phe). The adsorption behavior of tyrosine was similar to that of phenylalanine in single component solutions, however, with a much lower adsorption capacity (14 mg g(-1) for Tyr compared to 109 mg g(-1) for Phe). Tyr adsorption kinetics was satisfactorily described by a pseudosecond-order model as it was for Phe. In adsorption equilibrium studies for binary mixtures, the presence of Tyr in Phe solutions favored Phe faster adsorption whereas the opposite behavior was observed for the presence of Phe in Tyr solutions. Such results indicate that, in binary systems, Phe will be adsorbed preferably to Tyr, and this is a welcome feature when employing the prepared adsorbent for the removal of Phe from protein hydrolysates to be used in dietary formulations for phenylketonuria treatment.

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.

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.

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.

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.

Active site-directed double mutants of dihydrofolate reductase.

PubMed

Ercikan-Abali, E A; Mineishi, S; Tong, Y; Nakahara, S; Waltham, M C; Banerjee, D; Chen, W; Sadelain, M; Bertino, J R

1996-09-15

Variants of dihydrofolate reductase (DHFR), which confer resistance to antifolates, are used as dominant selectable markers in vitro and in vivo and may be useful in the context of gene therapy. To identify improved mutant human DHFRs with increased catalytic efficiency and decreased binding to methotrexate, we constructed by site-directed mutagenesis four variants with substitutions at both Leu22 and Phe31 (i.e., Phe22-Ser31, Tyr22-Ser31, Phe22-Gly31, and Tyr22-Gly31). Antifolate resistance has been observed previously when individual changes are made at these active-site residues. Substrate and antifolate binding properties of these "double" mutants revealed that each have greatly diminished affinity for antifolates (> 10,000-fold) yet only slightly reduced substrate affinity. Comparison of in vitro measured properties with those of single-residue variants indicates that double mutants are indeed significantly superior. This was verified for one of the double mutants that provided high-level methotrexate resistance following retrovirus-mediated gene transfer in NIH3T3 cells.

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.

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.

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.

The effects of therapeutic hip exercise with abdominal core activation on recruitment of the hip muscles.

PubMed

Chan, Mandy Ky; Chow, Ka Wai; Lai, Alfred Ys; Mak, Noble Kc; Sze, Jason Ch; Tsang, Sharon Mh

2017-07-21

Core stabilization has been utilized for rehabilitation and prevention of lower limb musculoskeletal injuries. Previous studies showed that activation of the abdominal core muscles enhanced the hip muscle activity in hip extension and abduction exercises. However, the lack of the direct measurement and quantification of the activation level of the abdominal core muscles during the execution of the hip exercises affect the level of evidence to substantiate the proposed application of core exercises to promote training and rehabilitation outcome of the hip region. The aim of the present study was to examine the effects of abdominal core activation, which is monitored directly by surface electromyography (EMG), on hip muscle activation while performing different hip exercises, and to explore whether participant characteristics such as gender, physical activity level and contractile properties of muscles, which is assessed by tensiomyography (TMG), have confounding effect to the activation of hip muscles in enhanced core condition. Surface EMG of bilateral internal obliques (IO), upper gluteus maximus (UGMax), lower gluteus maximus (LGMax), gluteus medius (GMed) and biceps femoris (BF) of dominant leg was recorded in 20 young healthy subjects while performing 3 hip exercises: Clam, side-lying hip abduction (HABD), and prone hip extension (PHE) in 2 conditions: natural core activation (NC) and enhanced core activation (CO). EMG signals normalized to percentage of maximal voluntary isometric contraction (%MVIC) were compared between two core conditions with the threshold of the enhanced abdominal core condition defined as >20%MVIC of IO. Enhanced abdominal core activation has significantly promoted the activation level of GMed in all phases of clam exercise (P < 0.05), and UGMax in all phases of PHE exercise (P < 0.05), LGMax in eccentric phases of all 3 exercises (P < 0.05), and BF in all phases of all 3 exercises except the eccentric phase of PHE exercise (P < 0.05). The %MVIC of UGMax was significantly higher than that of LGMax in all phases of clam and HABD exercises under both CO and NC conditions (P < 0.001) while the %MVIC of LGMax was significantly higher than UGMax in concentric phase of PHE exercise under NC condition (P = 0.003). Gender, physical activity level and TMG parameters were not major covariates to activation of hip muscles under enhanced core condition. Abdominal core activation enhances the hip muscles recruitment in Clam, HABD and PHE exercises, and this enhancement is correlated with higher physical activity and stiffer hip muscle. Our results suggest the potential application of abdominal core activation for lower limb rehabilitation since the increased activation of target hip muscles may enhance the therapeutic effects of hip strengthening exercises.

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.

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.

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.

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

PubMed Central

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

2016-01-01

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

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

Acute but not chronic metabolic acidosis potentiates the acetylcholine-induced reduction in blood pressure: an endothelium-dependent effect

PubMed Central

Celotto, A.C.; Ferreira, L.G.; Capellini, V.K.; Albuquerque, A.A.S.; Rodrigues, A.J.; Evora, P.R.B.

2015-01-01

Metabolic acidosis has profound effects on vascular tone. This study investigated the in vivo effects of acute metabolic acidosis (AMA) and chronic metabolic acidosis (CMA) on hemodynamic parameters and endothelial function. CMA was induced by ad libitum intake of 1% NH4Cl for 7 days, and AMA was induced by a 3-h infusion of 6 M NH4Cl (1 mL/kg, diluted 1:10). Phenylephrine (Phe) and acetylcholine (Ach) dose-response curves were performed by venous infusion with simultaneous venous and arterial blood pressure monitoring. Plasma nitrite/nitrate (NOx) was measured by chemiluminescence. The CMA group had a blood pH of 7.15±0.03, which was associated with reduced bicarbonate (13.8±0.98 mmol/L) and no change in the partial pressure of arterial carbon dioxide (PaCO2). The AMA group had a pH of 7.20±0.01, which was associated with decreases in bicarbonate (10.8±0.54 mmol/L) and PaCO2 (47.8±2.54 to 23.2±0.74 mmHg) and accompanied by hyperventilation. Phe or ACh infusion did not affect arterial or venous blood pressure in the CMA group. However, the ACh infusion decreased the arterial blood pressure (ΔBP: -28.0±2.35 mm Hg [AMA] to -4.5±2.89 mmHg [control]) in the AMA group. Plasma NOx was normal after CMA but increased after AMA (25.3±0.88 to 31.3±0.54 μM). These results indicate that AMA, but not CMA, potentiated the Ach-induced decrease in blood pressure and led to an increase in plasma NOx, reinforcing the effect of pH imbalance on vascular tone and blood pressure control. PMID:26648089

Chemotactic peptide fMetLeuPhe induces translocation of the TRPV2 channel in macrophages.

PubMed

Nagasawa, Masahiro; Nakagawa, Yuko; Tanaka, Shigeyasu; Kojima, Itaru

2007-03-01

The present study was conducted to characterize the regulation and function of TRPV2 in macrophages. Among six members of the TRPV family channels, only the expression of TRPV2 was detected in macrophages. We then determined localization of TRPV2 using TtT/M87 macrophages transfected with TRPV2-EGFP. In serum-free condition, most of the TRPV2 signal was located in the cytoplasm and colocalized with the endoplasmic reticulum marker. Treatment with serum induced translocation of some of the TRPV2-EGFP to the plasma membrane. Serum-induced translocation was blocked by transfection of short-form TRPV2 (s-TRPV2) lacking a pore-forming region and the sixth transmembrane domain. Addition of a chemotactic peptide formyl Met-Leu-Phe (fMLP) also induced translocation of TRPV2-EGFP to the plasma membrane. The fMLP-induced translocation was blocked by an inhibitor of PI 3-kinase, LY294002, and pertussis toxin. Whole-cell patch clamp analysis showed a Cs+ current in the TtT/M87 cell, which was blocked by an addition of ruthenium red and transfection of either s-TRPV2 or siRNA for TRPV2. fMLP increased the Cs+ current. fMLP induced a rapid and sustained elevation of cytoplasmic Ca2+ ([Ca2+]C), the sustained phase of which was abolished by removal of extracellular calcium. The sustained elevation of [Ca2+]C was also blocked by ruthenium red, and transfection of either s-TRPV2 or siRNA. Finally, fMLP-induced migration of macrophage was blocked by ruthenium red or transfection of s-TRPV2. These results suggest that fMLP induces translocation of TRPV2 from intracellular compartment to the plasma membrane, and this translocation is critical for fMLP-induced calcium entry. Copyright 2006 Wiley-Liss, Inc.

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.

First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer

PubMed Central

Arturo, Emilia C.; Gupta, Kushol; Héroux, Annie; Stith, Linda; Cross, Penelope J.; Parker, Emily J.; Loll, Patrick J.; Jaffe, Eileen K.

2016-01-01

Improved understanding of the relationship among structure, dynamics, and function for the enzyme phenylalanine hydroxylase (PAH) can lead to needed new therapies for phenylketonuria, the most common inborn error of amino acid metabolism. PAH is a multidomain homo-multimeric protein whose conformation and multimerization properties respond to allosteric activation by the substrate phenylalanine (Phe); the allosteric regulation is necessary to maintain Phe below neurotoxic levels. A recently introduced model for allosteric regulation of PAH involves major domain motions and architecturally distinct PAH tetramers [Jaffe EK, Stith L, Lawrence SH, Andrake M, Dunbrack RL, Jr (2013) Arch Biochem Biophys 530(2):73–82]. Herein, we present, to our knowledge, the first X-ray crystal structure for a full-length mammalian (rat) PAH in an autoinhibited conformation. Chromatographic isolation of a monodisperse tetrameric PAH, in the absence of Phe, facilitated determination of the 2.9 Å crystal structure. The structure of full-length PAH supersedes a composite homology model that had been used extensively to rationalize phenylketonuria genotype–phenotype relationships. Small-angle X-ray scattering (SAXS) confirms that this tetramer, which dominates in the absence of Phe, is different from a Phe-stabilized allosterically activated PAH tetramer. The lack of structural detail for activated PAH remains a barrier to complete understanding of phenylketonuria genotype–phenotype relationships. Nevertheless, the use of SAXS and X-ray crystallography together to inspect PAH structure provides, to our knowledge, the first complete view of the enzyme in a tetrameric form that was not possible with prior partial crystal structures, and facilitates interpretation of a wealth of biochemical and structural data that was hitherto impossible to evaluate. PMID:26884182

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.

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.

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.

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.

Developing and validating a new national remote health advice syndromic surveillance system in England.

PubMed

Harcourt, S E; Morbey, R A; Loveridge, P; Carrilho, L; Baynham, D; Povey, E; Fox, P; Rutter, J; Moores, P; Tiffen, J; Bellerby, S; McIntosh, P; Large, S; McMenamin, J; Reynolds, A; Ibbotson, S; Smith, G E; Elliot, A J

2017-03-01

Public Health England (PHE) coordinates a suite of real-time national syndromic surveillance systems monitoring general practice, emergency department and remote health advice data. We describe the development and informal evaluation of a new syndromic surveillance system using NHS 111 remote health advice data. NHS 111 syndromic indicators were monitored daily at national and local level. Statistical models were applied to daily data to identify significant exceedances; statistical baselines were developed for each syndrome and area using a multi-level hierarchical mixed effects model. Between November 2013 and October 2014, there were on average 19 095 NHS 111 calls each weekday and 43 084 each weekend day in the PHE dataset. There was a predominance of females using the service (57%); highest percentage of calls received was in the age group 1-4 years (14%). This system was used to monitor respiratory and gastrointestinal infections over the winter of 2013-14, the potential public health impact of severe flooding across parts of southern England and poor air quality episodes across England in April 2014. This new system complements and supplements the existing PHE syndromic surveillance systems and is now integrated into the routine daily processes that form this national syndromic surveillance service. © Crown copyright 2016.

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.

ACE Phenotyping as a Guide Toward Personalized Therapy With ACE Inhibitors.

PubMed

Danilov, Sergei M; Tovsky, Stan I; Schwartz, David E; Dull, Randal O

2017-07-01

Angiotensin-converting enzyme (ACE) inhibitors (ACEI) are widely used in the management of cardiovascular diseases but with significant interindividual variability in the patient's response. To investigate whether interindividual variability in the response to ACE inhibitors is explained by the "ACE phenotype"-for example, variability in plasma ACE concentration, activity, and conformation and/or the degree of ACE inhibition in each individual. The ACE phenotype was determined in plasma of 14 patients with hypertension treated chronically for 4 weeks with 40 mg enalapril (E) or 20 mg E + 16 mg candesartan (EC) and in 20 patients with hypertension treated acutely with a single dose (20 mg) of E with or without pretreatment with hydrochlorothiazide. The ACE phenotyping included (1) plasma ACE concentration; (2) ACE activity (with 2 substrates: Hip-His-Leu and Z-Phe-His-Leu and calculation of their ratio); (3) detection of ACE inhibitors in patient's blood (indicator of patient compliance) and the degree of ACE inhibition (ie, adherence); and (4) ACE conformation. Enalapril reduced systolic and diastolic blood pressure in most patients; however, 20% of patients were considered nonresponders. Chronic treatment results in 40% increase in serum ACE concentrations, with the exception of 1 patient. There was a trend toward better response to ACEI among patients who had a higher plasma ACE concentration. Due to the fact that "20% of patients do not respond to ACEI by blood pressure drop," the initial blood ACE level could not be a predictor of blood pressure reduction in an individual patient. However, ACE phenotyping provides important information about conformational and kinetic changes in ACE of individual patients, and this could be a reason for resistance to ACE inhibitors in some nonresponders.

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.

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.

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

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.

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)

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

Apache Clinical Text and Knowledge Extraction System (cTAKES) | Informatics Technology for Cancer Research (ITCR)

Cancer.gov

The tool extracts deep phenotypic information from the clinical narrative at the document-, episode-, and patient-level. The final output is FHIR compliant patient-level phenotypic summary which can be consumed by research warehouses or the DeepPhe native visualization tool.

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.

Effects of acute combined serotonin and dopamine depletion on cue-induced drinking intention/desire and cognitive function in patients with alcohol dependence.

PubMed

Sun, Hong-Qiang; Liu, Yu; Li, Peng; Bao, Yan-Ping; Sheng, Li-Xia; Zhang, Rui-Ling; Cao, Yan-Jun; Di, Xiao-Lan; Yang, Fu-De; Wang, Fan; Luo, Yi-Xiao; Lu, Lin

2012-08-01

Alcohol cues can precipitate the desire to drink and cause relapse in recovering alcohol-dependent patients. Serotonin and dopamine may play a role in alcohol cue-induced craving. Acute combined tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe) depletion (CMD) in the diet attenuates the synthesis of serotonin and dopamine in the human brain. However, no study of the effects of acute CMD has been previously conducted. Therefore, we investigated whether the attenuation of serotonin and dopamine synthesis changes cue-induced alcohol craving in recently abstinent alcoholics. In this double-blind, randomized, placebo-controlled, crossover design, 12 male patients who met the Diagnostic and Statistical Manual of Mental Disorders, 4th edition, criteria for alcohol dependence were divided into two conditions: (1) monoamine depletion (i.e., consumption of a concentrated amino acid beverage that resulted in a rapid and significant decrease in plasma-free Tyr/Phe/Trp) and (2) balanced condition (i.e., consumption of a similar beverage that contained Tyr/Phe/Trp). The participants were scheduled for two experimental sessions, with an interval of ≥7 days. The cue-induced craving test session was conducted 6h after each amino acid beverage administration. Drinking urge, blood pressure, heart rate, working memory, and attention/psychomotor performance were assessed before and after administration. Compared with the balanced condition, the monoamine depletion condition significantly increased drinking intention/desire and diastolic blood pressure. Cognitive performance was not different between the two conditions. Acute combined serotonin and dopamine depletion may increase drinking intention/desire and diastolic blood pressure without influencing cognitive function. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

New Stable Isotope Method to Measure Protein Digestibility and Response to Pancreatic Enzyme Intake in Cystic Fibrosis

PubMed Central

Engelen, M.P.K.J.; Com, G.; Anderson, P.J.; Deutz, N.E.P.

2015-01-01

Background & Aims Adequate protein intake and digestion are necessary to prevent muscle wasting in cystic fibrosis (CF). Accurate and easy-to-use methodology to quantify protein maldigestion is lacking in CF. Objective To measure protein digestibility and the response to pancreatic enzyme intake in CF by using a new stable isotope methodology. Design In 19 CF and 8 healthy subjects, protein digestibility was quantified during continuous (sip) feeding for 6 hours by adding 15N-labeled spirulina protein and L-[ring-2H5]phenylalanine (PHE) to the nutrition and measuring plasma ratio [15N]PHE to [2H5]PHE. Pancreatic enzymes were ingested after 2 h in CF and the response in protein digestibility was assessed. To exclude difference in mucosal function, postabsorptive whole-body citrulline (CIT) production rate was measured by L-[5-13C-5,5-2H2]-CIT pulse and blood samples were taken to analyze tracer-tracee ratios. Results Protein digestibility was severely reduced in the CF group (47% of healthy subjects; P<0.001). Intake of pancreatic enzymes induced a slow increase in protein digestibility in CF until 90% of values obtained by healthy subjects. Maximal digestibility was reached at 100 min and maintained for 80 min. Stratification into CF children (n=10) and adults showed comparable values for protein digestibility and similar kinetic responses to pancreatic enzyme intake. Whole-body citrulline production was elevated in CF indicating preserved mucosal function. Conclusion Protein digestibility is severely compromised in patients with CF as measured by this novel and easy-to-use stable isotope approach. Pancreatic enzymes are able to normalize protein digestibility in CF, albeit with a severe delay. PMID:24268783

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

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.

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.

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.

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.

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

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.

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.

Comparison of the level of residual coagulant activity in different cheese varieties.

PubMed

Bansal, Nidhi; Fox, Patrick F; McSweeney, Paul L H

2009-08-01

The coagulant retained in cheese curd is a major contributor to proteolysis during ripening. The objective of this study was to quantify residual coagulant in 9 cheese varieties by measuring its activity on a synthetic heptapeptide (Pro-Thr-Glu-Phe-[NO2-Phe]-Arg-Leu) assayed using reversed-phase HPLC. The level of residual coagulant activity was highest in Camembert cheese, probably due to its low pH at whey drainage and the high moisture content of the cheese, followed in order by Feta=Port du Salut=Cheddar>Gouda>Emmental=Parmigiano Reggiano=low-moisture part-skim Mozzarella=Mozzarella di Bufala Campana. The high cooking temperature (50-54 degrees C) used during the manufacture of Emmental and Parmigiano Reggiano cheeses and the cooking and stretching step in hot water during the manufacture of Mozzarella cheese may be the reasons for the lowest residual coagulant activity in these cheeses. The level of residual coagulant activity was higher in Feta cheese made from milk concentrated by ultrafiltration than in conventional Feta.

Cerebral Artery Alpha-1 AR Subtypes: High Altitude Long-Term Acclimatization Responses

PubMed Central

Goyal, Ravi; Goyal, Dipali; Chu, Nina; Van Wickle, Jonathan; Longo, Lawrence D.

2014-01-01

In response to hypoxia and other stress, the sympathetic (adrenergic) nervous system regulates arterial contractility and blood flow, partly through differential activities of the alpha1 (α1) - adrenergic receptor (AR) subtypes (α1A-, α1B-, and α1D-AR). Thus, we tested the hypothesis that with acclimatization to long-term hypoxia (LTH), contractility of middle cerebral arteries (MCA) is regulated by changes in expression and activation of the specific α1-AR subtypes. We conducted experiments in MCA from adult normoxic sheep maintained near sea level (300 m) and those exposed to LTH (110 days at 3801 m). Following acclimatization to LTH, ovine MCA showed a 20% reduction (n = 5; P<0.05) in the maximum tension achieved by 10−5 M phenylephrine (PHE). LTH-acclimatized cerebral arteries also demonstrated a statistically significant (P<0.05) inhibition of PHE-induced contractility in the presence of specific α1-AR subtype antagonists. Importantly, compared to normoxic vessels, there was significantly greater (P<0.05) α1B-AR subtype mRNA and protein levels in LTH acclimatized MCA. Also, our results demonstrate that extracellular regulated kinase 1 and 2 (ERK1/2)-mediated negative feedback regulation of PHE-induced contractility is modulated by α1B-AR subtype. Overall, in ovine MCA, LTH produces profound effects on α1-AR subtype expression and function. PMID:25393740

Cerebral artery alpha-1 AR subtypes: high altitude long-term acclimatization responses.

PubMed

Goyal, Ravi; Goyal, Dipali; Chu, Nina; Van Wickle, Jonathan; Longo, Lawrence D

2014-01-01

In response to hypoxia and other stress, the sympathetic (adrenergic) nervous system regulates arterial contractility and blood flow, partly through differential activities of the alpha1 (α1) - adrenergic receptor (AR) subtypes (α1A-, α1B-, and α1D-AR). Thus, we tested the hypothesis that with acclimatization to long-term hypoxia (LTH), contractility of middle cerebral arteries (MCA) is regulated by changes in expression and activation of the specific α1-AR subtypes. We conducted experiments in MCA from adult normoxic sheep maintained near sea level (300 m) and those exposed to LTH (110 days at 3801 m). Following acclimatization to LTH, ovine MCA showed a 20% reduction (n = 5; P<0.05) in the maximum tension achieved by 10-5 M phenylephrine (PHE). LTH-acclimatized cerebral arteries also demonstrated a statistically significant (P<0.05) inhibition of PHE-induced contractility in the presence of specific α1-AR subtype antagonists. Importantly, compared to normoxic vessels, there was significantly greater (P<0.05) α1B-AR subtype mRNA and protein levels in LTH acclimatized MCA. Also, our results demonstrate that extracellular regulated kinase 1 and 2 (ERK1/2)-mediated negative feedback regulation of PHE-induced contractility is modulated by α1B-AR subtype. Overall, in ovine MCA, LTH produces profound effects on α1-AR subtype expression and function.

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.

The in vivo pH of the extravascular space of the lung

PubMed Central

Effros, Richard M.; Chinard, Francis P.

1969-01-01

The partition of 5,5-dimethyloxazolidine-2,4-dione (DMO) and of 11 amines between the vascular and extravascular spaces of the lung has been determined by the multiple indicator dilution technique. Four amines (nicotine, pentylamine, quinine, and benzylamine) were found to have pH-sensitive tissue to blood concentration ratios. Of these, tritiated nicotine appears to be the nost satisfactory indicator of tissue pH and values for the pH of the pulmonary extravascular space (pHe) have been calculated from the nicotine data. At an arterial pH (pHart) between 7.38 and 7.43 pHe averaged 6.69 ±0.07. Changes in pHe usually paralleled but were consistently less than concomitant changes in pHart. Alterations in PCO2 at constant pHart regularly produced relatively small, parallel changes in extravascular hydrogen ion concentrations. Local alterations in tissue pH due to PCO2 changes are apparently buffered quite rapidly and the pHe of the lung seems more closely linked to pHart than the cellular pH of other tissues. DMO, guanidine, methylamine, morphine, and atropine were confined to the vascular volume during the first circulation and could not be used to measure tissue pH. Histamine appeared to be bound to a pH-insensitive site. The extravascular distributions of antipyrine and aniline were unresponsive to alterations in arterial pH, presumably because they are essentially uncharged at pH levels found in the lung. PMID:4898722

Descriptive and hedonic analyses of low-Phe food formulations containing corn (Zea mays) seedling roots: toward development of a dietary supplement for individuals with phenylketonuria.

PubMed

Cliff, Margaret A; Law, Jessica R; Lücker, Joost; Scaman, Christine H; Kermode, Allison R

2016-01-15

Seedling roots of anthocyanin-rich corn (Zea mays) cultivars contain high levels of phenylalanine ammonia lyase (PAL) activity. The development of a natural dietary supplement containing corn roots could provide the means to improve the restrictive diet of phenylketonuria (PKU) patients by increasing their tolerance to dietary phenylalanine (Phe). Therefore this research was undertaken to explore the sensory characteristics of roots of four corn cultivars as well as to develop and evaluate food products (cereal bar, beverage, jam-like spread) to which roots had been added. Sensory profiles of corn roots were investigated using ten trained judges. Roots of Japanese Striped corn seedlings were more bitter, pungent and astringent than those of white and yellow cultivars, while roots from the Blue Jade cultivar had a more pronounced earthy/mushroom aroma. Consumer research using 24 untrained panelists provided hedonic (degree-of-liking) assessments for products with and without roots (controls). The former had lower mean scores than the controls; however, the cereal bar had scores above 5 on the nine-point scale for all hedonic assessments compared with the other treated products. By evaluating low-Phe food products containing corn roots, this research ascertained that the root-containing low-Phe cereal bar was an acceptable 'natural' dietary supplement for PKU-affected individuals. © 2015 Her Majesty the Queen in Right of Canada. Journal of the Science of Food and Agriculture © 2015 Society of Chemical Industry.

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

PubMed

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

2018-04-25

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

Evaluation of biological activities of new LH-RH antagonists (T-series) in male and female rats.

PubMed

Pinski, J; Yano, T; Janaky, T; Nagy, A; Juhasz, A; Bokser, L; Groot, K; Schally, A V

1993-01-01

A series of new highly potent LH-RH antagonists (T-series) has been synthesized in our laboratory. Among these analogs, antagonists [Ac-D-Nal(2), D-Phe(4Cl)2, D-Pal(3)3, D-Lys(A2pr(Car)2)6, D-Ala10]LH-RH (T-140); [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Pal(3)3, D-Lys(A2pr(Ac)2)6, D-Ala10]LH-RH (T-148); [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Pal(3)3, D-Lys(A2pr(For)2)6, D-Ala10]LH-RH (T-151) and [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Pal(3)3, D-Lys(A2bu(For)2)6, D-Ala10]LH-RH (T-159) were the most powerful. Antagonists T-140, T-148 and T-151 produced a complete blockade of ovulation in normal cycling rats at a dose of 1.5 micrograms/rat and antagonist T-159 at a dose of only 0.75 micrograms/rat. The inhibitory effects of compounds T-148, T-151 and T-159 on gonadotropin and sex steroid secretion were investigated in male and female rats. To determine their effect on LH levels in castrated male and ovariectomized female rats, T-148, T-151 and T-159 were injected subcutaneously in doses of 0.625 and 2.5 micrograms/rat. Blood samples were taken at different intervals for 48 h. All three compounds at either dose caused a significant (P < 0.01) decrease in LH levels for more than 6 h. Significant (P < 0.01) inhibition of LH lasted for more than 24 h following a dose of 2.5 micrograms sc of all 3 antagonists in both male and female rats. Serum FSH levels were also suppressed significantly for more than 48 h in castrated male rats by all three antagonists at a dose of 5 micrograms/rat sc.(ABSTRACT TRUNCATED AT 250 WORDS)

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

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.

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.

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.

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.

Effect of Hydrophobic Side Chains in the Induction of Immune Responses by Nanoparticle Adjuvants Consisting of Amphiphilic Poly(γ-glutamic acid).

PubMed

Shima, Fumiaki; Akagi, Takami; Akashi, Mitsuru

2015-05-20

The new generation vaccines are safe but poorly immunogenic, and thus they require the use of adjuvants. Adjuvants that can control the balance and induction level of cellular and humoral immunities are urgently required for the treatment of and/or protection from infectious diseases and cancers. However, there are no adjuvants which can achieve these requirements. In this study, amphiphilic poly(γ-glutamic acid) (γ-PGA) with various kinds of hydrophobic amino acid ethyl esters (AAE) was synthesized (γ-PGA-AAE) and used to prepare antigen-encapsulated nanoparticles (NPs). γ-PGA-graft-Leu (γ-PGA-Leu, where Leu = leucine ethyl ester), γ-PGA-graft-Phe (γ-PGA-Phe, where Phe = phenylalanine ethyl ester), and γ-PGA-graft-Trp (γ-PGA-Trp, where Trp = tryptophan ethyl ester) formed monodispersed NPs that encapsulated ovalbumin (OVA). The type and the induction level of the antigen-specific cellular and humoral immunities could be controlled by the kinds of hydrophobic segments and vaccine formulation (encapsulation or mixture) used. When OVA was encapsulated into NPs, the cellular immunity was dominantly induced, while humoral immunity was dominant when OVA was mixed with NPs. These results are a first report to demonstrate that the balance and induction level of cellular and humoral immunities could be controlled by modifying compositions of NPs and vaccine formulation. Our results suggest that γ-PGA-AAE NPs can provide safe and efficient nanoparticle-based vaccine adjuvants, and the results also provide guidelines in the rational design of amphiphilic polymers as vaccine adjuvants which can control the balance of immune responses.

Federal Public Health Actions - 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 > PHE Newsroom > Federal Public Health Actions Federal Public Health Actions Main Content April 20, 2018: Renewal of Determination that a

Metal-Catalyzed Cleavage of tRNA[superscript Phe

ERIC Educational Resources Information Center

Kirk, Sarah R.; Silverstein, Todd P.; McFarlane Holman, Karen L.

2008-01-01

This laboratory project is one component of a semester-long advanced biochemistry laboratory course that uses several complementary techniques to study tRNA[superscript Phe] conformational changes induced by ligand binding. In this article we describe a set of experiments in which students assay metal-catalyzed hydrolysis of tRNA[superscript Phe]…

Evaluation of extraction methods for the identification of proteins from date palm (Phoenix dactylifera L.) seed and flesh.

PubMed

Lee, Hooi Xian; Ahmad, Fisal; Saad, Bahruddin; Ismail, Mohd Nazri

2017-11-26

Date fruits are well known to be very nutritious. Nevertheless, the protein contents of the fruit, particularly the seed and flesh, are still understudied, largely due to their difficult physical characteristics. This study was conducted to compare three different protein extraction methods which were the trichloroacetic acid (TCA)-acetone (TCA-A), phenol (Phe), and TCA-acetone-phenol (TCA-A-Phe), and to perform proteomic analysis on date palm seed and flesh. Phe extraction method showed the highest protein yields for both seed (8.26 mg/g) and flesh (1.57 mg/g). Through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Phe, and TCA-A-Phe extraction methods were shown to be efficient in removing interfering compounds and gave well-resolved bands over a wide range of molecular weights. Following liquid chromatography-tandem mass spectrometry analysis, about 50-64% of extracted proteins were identified with known functions including those involved in glycolysis, Krebs cycle, defense, and storage. Phe protein extraction method was proven to be the optimal method for date flesh and seed.

Azidoethoxyphenylalanine as a Vibrational Reporter and Click Chemistry Partner in Proteins

PubMed Central

Tookmanian, Elise M.; Phillips-Piro, Christine M.; Fenlon, Edward E.; Brewer, Scott H.

2016-01-01

An unnatural amino acid, 4-(2-azidoethoxy)-l-phenylalanine (AePhe, 1), was designed and synthesized in three steps from known compounds in 54% overall yield. The sensitivity of the IR absorption of the azide of AePhe was established by comparison of the frequency of the asymmetric stretch vibration in water and dimethyl sulfoxide. AePhe was successfully incorporated into superfolder green fluorescent protein (sfGFP) at the 133 and 149 sites using the amber codon suppression method. The IR spectra of these sfGFP constructs indicated that the azide group at the 149 site was not fully solvated despite the location in sfGFP and the three-atom linker between the azido group and the aromatic ring of AePhe. An X-ray crystal structure of sfGFP-149-AePhe was solved at 1.45 Å resolution and provides an explanation for the IR data as the flexible linker adopts a conformation which partially buries the azide on the protein surface. Both sfGFP-AePhe constructs efficiently undergo a bioorthogonal strain-promoted click cycloaddition with a dibenzocyclooctyne derivative. PMID:26608683

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.

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+ .

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.

Binding Specificity of Two PBPs in the Yellow Peach Moth Conogethes punctiferalis (Guenée)

PubMed Central

Ge, Xing; Ahmed, Tofael; Zhang, Tiantao; Wang, Zhenying; He, Kanglai; Bai, Shuxiong

2018-01-01

Pheromone binding proteins (PBPs) play an important role in olfaction of insects by transporting sex pheromones across the sensillum lymph to odorant receptors. To obtain a better understanding of the molecular basis between PBPs and semiochemicals, we have cloned, expressed, and purified two PBPs (CpunPBP2 and CpunPBP5) from the antennae of Conogethes punctiferalis. Fluorescence competitive binding assays were used to investigate binding affinities of CpunPBP2 and CpunPBP5 to sex pheromone and volatiles. Results indicate both CpunPBP2 and CpunPBP5 bind sex pheromones E10-16:Ald, Z10-16:Ald and hexadecanal with higher affinities. In addition, CpunPBP2 and CpunPBP5 also could bind some odorants, such as 1-tetradecanol, trans-caryopyllene, farnesene, and β-farnesene. Homology modeling to predict 3D structure and molecular docking to predict key binding sites were used, to better understand interactions of CpunPBP2 and CpunPBP5 with sex pheromones E10-16:Ald and Z10-16:Ald. According to the results, Phe9, Phe33, Ser53, and Phe115 were key binding sites predicted for CpunPBP2, as were Ser9, Phe12, Val115, and Arg120 for CpunPBP5. Binding affinities of four mutants of CpunPBP2 and four mutants of CpunPBP5 with the two sex pheromones were investigated by fluorescence competitive binding assays. Results indicate that single nucleotides mutation may affect interactions between PBPs and sex pheromones. Expression levels of CpunPBP2 and CpunPBP5 in different tissues were evaluated using qPCR. Results show that CpunPBP2 and CpunPBP5 were largely amplified in the antennae, with low expression levels in other tissues. CpunPBP2 was expressed mainly in male antennae, whereas CpunPBP5 was expressed mainly in female antennae. These results provide new insights into understanding the recognition between PBPs and ligands. PMID:29666585

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.

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.

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.

UV Thermal Melting Curves of tRNA[superscript Phe] in the Presence of Ligands

ERIC Educational Resources Information Center

Kirk, Sarah R.; Silverstein, Todd P.; McFarlane Holman, Karen L.

2008-01-01

This laboratory project is one component of a semester-long advanced biochemistry laboratory course that uses several complementary techniques to study tRNA[superscript Phe] conformational changes induced by ligand binding. In this article we describe a set of experiments in which the thermal unfolding of tRNA[superscript Phe] is studied with…

Probing Changes in the Conformation of tRNA[superscript Phe]: An Integrated Biochemistry Laboratory Course

ERIC Educational Resources Information Center

Kirk, Sarah R.; Silverstein, Todd P.; McFarlane Holman, Karen L.; Taylor, Buck L. H.

2008-01-01

We have designed a new guided-inquiry laboratory for an advanced biochemistry course. This integrated laboratory focuses on the biomolecule tRNA[superscript Phe] and combines elements of bioorganic and bioinorganic chemistry with biochemistry. Throughout the semester students work together to study tRNA[superscript Phe] structure and ligand…

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

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

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.

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…

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.

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.

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.

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.

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.

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.

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

Development of a gradient elution high-performance liquid chromatography assay with ultraviolet detection for the determination in plasma of the anticancer peptide [Arg6, D-Trp7,9, mePhe8]-substance P (6-11) (antagonist G), its major metabolites and a C-terminal pyrene-labelled conjugate.

PubMed

Cummings, J; MacLellan, A J; Mark, M; Jodrell, D I

1999-09-24

[Arg6, D-Trp7,9 mePhe8]-substance P (6-11), code-named antagonist G, is a novel peptide currently undergoing early clinical trials as an anticancer drug. A sensitive, high efficiency high-performance liquid chromatography (HPLC) method is described for the determination in human plasma of antagonist G and its three major metabolites, deamidated-G (M1), G-minus Met11 (M2) and G[Met11(O)] (M3). Gradient elution was employed using 40 mM ammonium acetate in 0.15% trifluoroacetic acid as buffer A and acetonitrile as solvent B, with a linear gradient increasing from 30 to 100% B over 15 min, together with a microbore analytical column (microBondapak C18, 30 cm X 2 mm I.D.). Detection was by UV at 280 nm and the column was maintained at 40 degrees C. Retention times varied by <1% throughout the day and were as follows: G, 13.0 min; M1, 12.2 min; M2, 11.2 min; M3, 10.8 min, and 18.1 min for a pyrene conjugate of G (G-P). The limit of detection on column (LOD) was 2.5 ng for antagonist G, M1-3 and G-P and the limit of quantitation (LOQ) was 20 ng/ml for G and 100 ng/ml for M1-3. Sample clean-up by solid-phase extraction using C2-bonded 40 microm silica particles (Bond Elut, 1 ml reservoirs) resulted in elimination of interference from plasma constituents. Within-day and between-day precision and accuracy over a broad range of concentrations (100 ng/ml-100 microg/ml) normally varied by < 10%, although at the highest concentrations of M1 and M2 studied (50 microg/ml), increased variability and reduced recovery were observed. The new assay will aid in the clinical development of antagonist G.

Transcriptional control of monolignol biosynthesis in Pinus taeda: factors affecting monolignol ratios and carbon allocation in phenylpropanoid metabolism

NASA Technical Reports Server (NTRS)

Anterola, Aldwin M.; Jeon, Jae-Heung; Davin, Laurence B.; Lewis, Norman G.

2002-01-01

Transcriptional profiling of the phenylpropanoid pathway in Pinus taeda cell suspension cultures was carried out using quantitative real time PCR analyses of all known genes involved in the biosynthesis of the two monolignols, p-coumaryl and coniferyl alcohols (lignin/lignan precursors). When the cells were transferred to a medium containing 8% sucrose and 20 mm potassium iodide, the monolignol/phenylpropanoid pathway was induced, and transcript levels for phenylalanine ammonia lyase, cinnamate 4-hydroxylase, p-coumarate 3-hydroxylase, 4-coumarate:CoA ligase, caffeoyl-CoA O-methyltransferase, cinnamoyl-CoA reductase, and cinnamyl alcohol dehydrogenase were coordinately up-regulated. Provision of increasing levels of exogenously supplied Phe to saturating levels (40 mm) to the induction medium resulted in further up-regulation of their transcript levels in the P. taeda cell cultures; this in turn was accompanied by considerable increases in both p-coumaryl and coniferyl alcohol formation and excretion. By contrast, transcript levels for both cinnamate 4-hydroxylase and p-coumarate 3-hydroxylase were only slightly up-regulated. These data, when considered together with metabolic profiling results and genetic manipulation of various plant species, reveal that carbon allocation to the pathway and its differential distribution into the two monolignols is controlled by Phe supply and differential modulation of cinnamate 4-hydroxylase and p-coumarate 3-hydroxylase activities, respectively. The coordinated up-regulation of phenylalanine ammonia lyase, 4-coumarate:CoA ligase, caffeoyl-CoA O-methyltransferase, cinnamoyl-CoA reductase and cinnamyl alcohol dehydrogenase in the presence of increasing concentrations of Phe also indicates that these steps are not truly rate-limiting, because they are modulated according to metabolic demand. Finally, the transcript profile of a putative acid/ester O-methyltransferase, proposed as an alternative catalyst for O-methylation leading to coniferyl alcohol, was not up-regulated under any of the conditions employed, suggesting that it is not, in fact, involved in monolignol biosynthesis.

Transcriptional control of monolignol biosynthesis in Pinus taeda: factors affecting monolignol ratios and carbon allocation in phenylpropanoid metabolism.

PubMed

Anterola, Aldwin M; Jeon, Jae-Heung; Davin, Laurence B; Lewis, Norman G

2002-05-24

Transcriptional profiling of the phenylpropanoid pathway in Pinus taeda cell suspension cultures was carried out using quantitative real time PCR analyses of all known genes involved in the biosynthesis of the two monolignols, p-coumaryl and coniferyl alcohols (lignin/lignan precursors). When the cells were transferred to a medium containing 8% sucrose and 20 mm potassium iodide, the monolignol/phenylpropanoid pathway was induced, and transcript levels for phenylalanine ammonia lyase, cinnamate 4-hydroxylase, p-coumarate 3-hydroxylase, 4-coumarate:CoA ligase, caffeoyl-CoA O-methyltransferase, cinnamoyl-CoA reductase, and cinnamyl alcohol dehydrogenase were coordinately up-regulated. Provision of increasing levels of exogenously supplied Phe to saturating levels (40 mm) to the induction medium resulted in further up-regulation of their transcript levels in the P. taeda cell cultures; this in turn was accompanied by considerable increases in both p-coumaryl and coniferyl alcohol formation and excretion. By contrast, transcript levels for both cinnamate 4-hydroxylase and p-coumarate 3-hydroxylase were only slightly up-regulated. These data, when considered together with metabolic profiling results and genetic manipulation of various plant species, reveal that carbon allocation to the pathway and its differential distribution into the two monolignols is controlled by Phe supply and differential modulation of cinnamate 4-hydroxylase and p-coumarate 3-hydroxylase activities, respectively. The coordinated up-regulation of phenylalanine ammonia lyase, 4-coumarate:CoA ligase, caffeoyl-CoA O-methyltransferase, cinnamoyl-CoA reductase and cinnamyl alcohol dehydrogenase in the presence of increasing concentrations of Phe also indicates that these steps are not truly rate-limiting, because they are modulated according to metabolic demand. Finally, the transcript profile of a putative acid/ester O-methyltransferase, proposed as an alternative catalyst for O-methylation leading to coniferyl alcohol, was not up-regulated under any of the conditions employed, suggesting that it is not, in fact, involved in monolignol biosynthesis.

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.

Severe epilepsy as the major symptom of new mutations in the mitochondrial tRNA(Phe) gene.

PubMed

Zsurka, G; Hampel, K G; Nelson, I; Jardel, C; Mirandola, S R; Sassen, R; Kornblum, C; Marcorelles, P; Lavoué, S; Lombès, A; Kunz, W S

2010-02-09

To present 2 families with maternally inherited severe epilepsy as the main symptom of mitochondrial disease due to point mutations at position 616 in the mitochondrial tRNA(Phe) (MT-TF) gene. Histologic stainings were performed on skeletal muscle slices from the 2 index patients. Oxidative phosphorylation activity was measured by oxygraphic and spectrophotometric methods. The patients' complete mitochondrial DNA (mtDNA) and the relevant mtDNA region in maternal relatives were sequenced. Muscle histology showed only decreased overall COX staining, while a combined respiratory chain defect, most severely affecting complex IV, was noted in both patients' skeletal muscle. Sequencing of the mtDNA revealed in both patients a mutation at position 616 in the MT-TF gene (T>C or T>G). These mutations disrupt a base pair in the anticodon stem at a highly conserved position. They were apparently homoplasmic in both patients, and had different heteroplasmy levels in the investigated maternal relatives. Deleterious mutations in the mitochondrial tRNA(Phe) may solely manifest with epilepsy when segregating to homoplasmy. They may be overlooked in the absence of lactate accumulation and typical mosaic mitochondrial defects in muscle.

Chromium (d-Phenylalanine)3 Alleviates High Fat-Induced Insulin Resistance and Lipid Abnormalities

PubMed Central

Kandadi, Machender Reddy; Unnikrishnan, MK; Warrier, Ajaya Kumar Sankara; Du, Min; Ren, Jun; Sreejayan, Nair

2010-01-01

High-fat diet has been implicated as a major cause of insulin resistance and dyslipidemia. The objective of this study was to evaluate the impact of dietary-supplementation of chromium (d-phenylalanine)3 [Cr(d-Phe)3] on -glucose and -insulin tolerance in high-fat diet fed mice. C57BL/6-mice were randomly assigned to orally receive vehicle or Cr(d-Phe)3 (45 μg of elemental chromium/kg/day) for 8-weeks. High-fat-fed mice exhibited impaired whole-body -glucose and- insulin tolerance and elevated serum triglyceride levels compared to normal chow-fed mice. Insulin-stimulated glucose up- take in the gastrocnemius muscles, assessed as 2-[3H-deoxyglucose] incorporation was markedly diminished in high-fat fed mice compared to control mice. Treatment with chromium reconciled the high-fat diet-induced alterations in carbohydrate and lipid metabolism. Treatment of cultured, differentiated myotubes with palmitic acid evoked insulin resistance as evidenced by lower levels of insulin-stimulated Akt-phosphorylation, elevated JNK-phosphorylation, (assessed by Western blotting), attenuation of phosphoinositol-3-kinase activity (determined in the insulin-receptor substrate-1-immunoprecipitates by measuring the extent of phosphorylation of phosphatidylinositol by γ-32P-ATP), and impairment in cellular glucose up-take, all of which were inhibited by Cr(d-Phe)3. These results suggest a beneficial effect of chromium-supplementation in insulin resistant conditions. It is likely that these effects of chromium may be mediated by augmenting downstream insulin signaling. PMID:21134603

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

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.

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

Bis(PheOH) maleic acid amide-fumaric acid amide photoizomerization induces microsphere-to-gel fiber morphological transition: the photoinduced gelation system.

PubMed

Frkanec, Leo; Jokić, Milan; Makarević, Janja; Wolsperger, Kristina; Zinić, Mladen

2002-08-21

The photoinduced gelation system based on 1 (non-gelling) to 2 (gelling) molecular photoisomerization in water results by microspheres (1) to gel fibers (2) transformation at the supramolecular level.

Inactivation of substance P and its C-terminal fragments in rat plasma and its inhibition by Captopril.

PubMed

Couture, R; Regoli, D

1981-06-01

The metabolic degradation of substance P(SP), some of its C-terminal fragments, and some analogues by rat plasma has been evaluated from the disappearance of the biological activities of these peptides on the guinea pig isolated ileum. The experiments were performed by dissolving each peptide in saline and by adding 20% (v/v) of rat plasma for incubation at 37 degrees C for various periods of time. It was found that SP and octapeptide 4-11 are inactivated quite rapidly and at approximately the same rate whereas SP-free acid, heptapeptide 5-11, hexapeptide 6-11, and [D-Trp8]-SP are inactivated more slowly. The replacement of Phe7 by D-Trp does not protect the undecapeptide SP from inactivation. The degradation of SP and of all the C-terminal fragments was completely blocked by Captopril at a concentration of 10 micrograms/mL of plasma. Under these conditions, Captopril also slightly reduced the rate of inactivation of bradykinin and of SP-free acid. These results were interpreted as indicative of the presence in rat plasma of an endopeptidase that hydrolyses a peptide bond in the C-terminal pentapeptide sequence of SP. This endopeptidase is completely inactivated by Captopril, which thus appears to be not as specific for the angiotensin-converting enzyme as it was thought to be.

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.

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.

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.

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.

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.

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.

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.

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

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.

Measurements of the strong-interaction widths of the kaonic 3He and 4He 2p levels

PubMed Central

Bazzi, M.; Beer, G.; Bombelli, L.; Bragadireanu, A.M.; Cargnelli, M.; Curceanu (Petrascu), C.; dʼUffizi, A.; Fiorini, C.; Frizzi, T.; Ghio, F.; Guaraldo, C.; Hayano, R.S.; Iliescu, M.; Ishiwatari, T.; Iwasaki, M.; Kienle, P.; Levi Sandri, P.; Longoni, A.; Marton, J.; Okada, S.; Pietreanu, D.; Ponta, T.; Rizzo, A.; Romero Vidal, A.; Sbardella, E.; Scordo, A.; Shi, H.; Sirghi, D.L.; Sirghi, F.; Tatsuno, H.; Tudorache, A.; Tudorache, V.; Vazquez Doce, O.; Wünschek, B.; Widmann, E.; Zmeskal, J.

2012-01-01

The kaonic 3He and 4He X-rays emitted in the 3d→2p transitions were measured in the SIDDHARTA experiment. The widths of the kaonic 3He and 4He 2p states were determined to be Γ2p(He3)=6±6(stat.)±7 (syst.) eV, and Γ2p(He4)=14±8 (stat.)±5 (syst.) eV, respectively. Both results are consistent with the theoretical predictions. The width of kaonic 4He is much smaller than the value of 55±34 eV determined by the experiments performed in the 70ʼs and 80ʼs, while the width of kaonic 3He was determined for the first time. PMID:22876000

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.

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

Dominant ELOVL1 mutation causes neurological disorder with ichthyotic keratoderma, spasticity, hypomyelination and dysmorphic features.

PubMed

Kutkowska-Kaźmierczak, Anna; Rydzanicz, Małgorzata; Chlebowski, Aleksander; Kłosowska-Kosicka, Kamila; Mika, Adriana; Gruchota, Jakub; Jurkiewicz, Elżbieta; Kowalewski, Cezary; Pollak, Agnieszka; Stradomska, Teresa Joanna; Kmieć, Tomasz; Jakubowski, Rafał; Gasperowicz, Piotr; Walczak, Anna; Śladowski, Dariusz; Jankowska-Steifer, Ewa; Korniszewski, Lech; Kosińska, Joanna; Obersztyn, Ewa; Nowak, Wieslaw; Śledziński, Tomasz; Dziembowski, Andrzej; Płoski, Rafał

2018-06-01

Ichthyosis and neurological involvement occur in relatively few known Mendelian disorders caused by mutations in genes relevant both for epidermis and neural function. To identify the cause of a similar phenotype of ichthyotic keratoderma, spasticity, mild hypomyelination (on MRI) and dysmorphic features (IKSHD) observed in two unrelated paediatric probands without family history of disease. Whole exome sequencing was performed in both patients. The functional effect of prioritised variant in ELOVL1 (very-long-chain fatty acids (VLCFAs) elongase) was analysed by VLCFA profiling by gas chromatography-mass spectrometry in stably transfected HEK2932 cells and in cultured patient's fibroblasts. Probands shared novel heterozygous ELOVL1 p.Ser165Phe mutation (de novo in one family, while in the other family, father could not be tested). In transfected cells p.Ser165Phe: (1) reduced levels of FAs C24:0-C28:0 and C26:1 with the most pronounced effect for C26:0 (P=7.8×10 -6  vs HEK293 cells with wild type (wt) construct, no difference vs naïve HEK293) and (2) increased levels of C20:0 and C22:0 (P=6.3×10 -7 , P=1.2×10 -5 , for C20:0 and C22:0, respectively, comparison vs HEK293 cells with wt construct; P=2.2×10 -7 , P=1.9×10 -4 , respectively, comparison vs naïve HEK293). In skin fibroblasts, there was decrease of C26:1 (P=0.014), C28:0 (P=0.001) and increase of C20:0 (P=0.033) in the patient versus controls. There was a strong correlation (r=0.92, P=0.008) between the FAs profile of patient's fibroblasts and that of p.Ser165Phe transfected HEK293 cells. Serum levels of C20:0-C26:0 FAs were normal, but the C24:0/C22:0 ratio was decreased. The ELOVL1 p.Ser165Phe mutation is a likely cause of IKSHD. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

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.

A fluorescently labeled undecapeptide derived from a protein in royal jelly of the honeybee-royalisin-for specific detection of oxidized low-density lipoprotein.

PubMed

Sato, Akira; Unuma, Hiroto; Yamazaki, Yoji; Ebina, Keiichi

2018-06-01

The probes for detection of oxidized low-density lipoprotein (ox-LDL) in plasma and in atherosclerotic plaques are expected to facilitate the diagnosis, prevention, and treatment of atherosclerosis. Recently, we have reported that a heptapeptide (Lys-Trp-Tyr-Lys-Asp-Gly-Asp, KP6) coupled through the ε-amino group of N-terminal Lys to fluorescein isothiocyanate (FITC), (FITC)KP6, can be useful as a fluorescent probe for specific detection of ox-LDL. In the present study, to develop a novel fluorescent peptide for specific detection of ox-LDL, we investigated the interaction (with ox-LDL) of an undecapeptide corresponding to positions 41 to 51 of a potent antimicrobial protein (royalisin, which consists of 51 residues; from royal jelly of honeybees), conjugated at the N-terminus to FITC in the presence of 6-amino-n-caproic acid (AC) linker, (FITC-AC)-royalisin P11, which contains both sequences, Phe-Lys-Asp and Asp-Lys-Tyr, similar to Tyr-Lys-Asp in (FITC)KP6. The (FITC-AC)-royalisin P11 bound with high specificity to ox-LDL in a dose-dependent manner, through the binding to major lipid components in ox-LDL (lysophosphatidylcholine and oxidized phosphatidylcholine). In contrast, a (FITC-AC)-shuffled royalisin P11 peptide, in which sequences Phe-Lys-Asp and Asp-Lys-Tyr were modified to Lys-Phe-Asp and Asp-Tyr-Lys, respectively, hardly bound to LDL and ox-LDL. These findings strongly suggest that (FITC-AC)-royalisin P11 may be an effective fluorescent probe for specific detection of ox-LDL and that royalisin from the royal jelly of honeybees may play a role in the treatment of atherosclerosis through the specific binding of the region at positions 41 to 51 to ox-LDL. Copyright © 2018 European Peptide Society and John Wiley & Sons, Ltd.

New stable isotope method to measure protein digestibility and response to pancreatic enzyme intake in cystic fibrosis.

PubMed

Engelen, M P K J; Com, G; Anderson, P J; Deutz, N E P

2014-12-01

Adequate protein intake and digestion are necessary to prevent muscle wasting in cystic fibrosis (CF). Accurate and easy-to-use methodology to quantify protein maldigestion is lacking in CF. To measure protein digestibility and the response to pancreatic enzyme intake in CF by using a new stable isotope methodology. In 19 CF and 8 healthy subjects, protein digestibility was quantified during continuous (sip) feeding for 6 h by adding (15)N-labeled spirulina protein and L-[ring-(2)H5]phenylalanine (PHE) to the nutrition and measuring plasma ratio [(15)N]PHE to [(2)H5]PHE. Pancreatic enzymes were ingested after 2 h in CF and the response in protein digestibility was assessed. To exclude difference in mucosal function, postabsorptive whole-body citrulline (CIT) production rate was measured by L-[5-(13)C-5,5-(2)H2]-CIT pulse and blood samples were taken to analyze tracer-tracee ratios. Protein digestibility was severely reduced in the CF group (47% of healthy subjects; P < 0.001). Intake of pancreatic enzymes induced a slow increase in protein digestibility in CF until 90% of values obtained by healthy subjects. Maximal digestibility was reached at 100 min and maintained for 80 min. Stratification into CF children (n = 10) and adults showed comparable values for protein digestibility and similar kinetic responses to pancreatic enzyme intake. Whole-body citrulline production was elevated in CF indicating preserved mucosal function. Protein digestibility is severely compromised in patients with CF as measured by this novel and easy-to-use stable isotope approach. Pancreatic enzymes are able to normalize protein digestibility in CF, albeit with a severe delay. Registration ClinicalTrials.gov = NCT01494909. Copyright © 2013 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

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.

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.

Absolute parameters for AI Phoenicis using WASP photometry

NASA Astrophysics Data System (ADS)

Kirkby-Kent, J. A.; Maxted, P. F. L.; Serenelli, A. M.; Turner, O. D.; Evans, D. F.; Anderson, D. R.; Hellier, C.; West, R. G.

2016-06-01

Context. AI Phe is a double-lined, detached eclipsing binary, in which a K-type sub-giant star totally eclipses its main-sequence companion every 24.6 days. This configuration makes AI Phe ideal for testing stellar evolutionary models. Difficulties in obtaining a complete lightcurve mean the precision of existing radii measurements could be improved. Aims: Our aim is to improve the precision of the radius measurements for the stars in AI Phe using high-precision photometry from the Wide Angle Search for Planets (WASP), and use these improved radius measurements together with estimates of the masses, temperatures and composition of the stars to place constraints on the mixing length, helium abundance and age of the system. Methods: A best-fit ebop model is used to obtain lightcurve parameters, with their standard errors calculated using a prayer-bead algorithm. These were combined with previously published spectroscopic orbit results, to obtain masses and radii. A Bayesian method is used to estimate the age of the system for model grids with different mixing lengths and helium abundances. Results: The radii are found to be R1 = 1.835 ± 0.014 R⊙, R2 = 2.912 ± 0.014 R⊙ and the masses M1 = 1.1973 ± 0.0037 M⊙, M2 = 1.2473 ± 0.0039 M⊙. From the best-fit stellar models we infer a mixing length of 1.78, a helium abundance of YAI = 0.26 +0.02-0.01 and an age of 4.39 ± 0.32 Gyr. Times of primary minimum show the period of AI Phe is not constant. Currently, there are insufficient data to determine the cause of this variation. Conclusions: Improved precision in the masses and radii have improved the age estimate, and allowed the mixing length and helium abundance to be constrained. The eccentricity is now the largest source of uncertainty in calculating the masses. Further work is needed to characterise the orbit of AI Phe. Obtaining more binaries with parameters measured to a similar level of precision would allow us to test for relationships between helium abundance and mixing length.

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.

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

Molecular Analysis of Asymptomatic Bacteriuria Escherichia coli Strain VR50 Reveals Adaptation to the Urinary Tract by Gene Acquisition

DOE PAGES

Beatson, Scott A.; Ben Zakour, Nouri L.; Totsika, Makrina; ...

2015-05-01

Urinary tract infections (UTIs) are among the most common infectious diseases of humans, with Escherichia coli for >80% of all cases. One extreme of UTI is asymptomatic bacteriuria (ABU), which occurs as an asymptomatic carrier state that resembles commensalism. Here, to understand the evolution and molecular mechanisms that underpin ABU, the genome of the ABU E. coli strain VR50 was sequenced. Analysis of the complete genome indicated that it most resembles E. coli K-12, with the addition of a 94-kb genomic island (GI-VR50-pheV), eight prophages, and multiple plasmids. GI-VR50- pheV has a mosaic structure and contains genes encoding a numbermore » of UTI-associated virulence factors, namely, Afa (afimbrial adhesin), two autotransporter proteins (Ag43 and Sat), and aerobactin. We demonstrated that the presence of this island in VR50 confers its ability to colonize the murine bladder, as a VR50 mutant with GI-VR50- pheV deleted was attenuated in a mouse model of UTI in vivo. We established that Afa is the island-encoded factor responsible for this phenotype using two independent deletion (Afa operon and AfaE adhesin) mutants. E. coli VR50 afa and VR50 afaE displayed significantly decreased ability to adhere to human bladder epithelial cells. In the mouse model of UTI, VR50 afa and VR50 afaE displayed reduced bladder colonization compared to wild-type VR50, similar to the colonization level of the GI-VR50- pheV mutant. In conlusion, our study suggests that E. coli VR50 is a commensal-like strain that has acquired fitness factors that facilitate colonization of the human bladder.« less

Molecular Analysis of Asymptomatic Bacteriuria Escherichia coli Strain VR50 Reveals Adaptation to the Urinary Tract by Gene Acquisition

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

Beatson, Scott A.; Ben Zakour, Nouri L.; Totsika, Makrina

Urinary tract infections (UTIs) are among the most common infectious diseases of humans, with Escherichia coli for >80% of all cases. One extreme of UTI is asymptomatic bacteriuria (ABU), which occurs as an asymptomatic carrier state that resembles commensalism. Here, to understand the evolution and molecular mechanisms that underpin ABU, the genome of the ABU E. coli strain VR50 was sequenced. Analysis of the complete genome indicated that it most resembles E. coli K-12, with the addition of a 94-kb genomic island (GI-VR50-pheV), eight prophages, and multiple plasmids. GI-VR50- pheV has a mosaic structure and contains genes encoding a numbermore » of UTI-associated virulence factors, namely, Afa (afimbrial adhesin), two autotransporter proteins (Ag43 and Sat), and aerobactin. We demonstrated that the presence of this island in VR50 confers its ability to colonize the murine bladder, as a VR50 mutant with GI-VR50- pheV deleted was attenuated in a mouse model of UTI in vivo. We established that Afa is the island-encoded factor responsible for this phenotype using two independent deletion (Afa operon and AfaE adhesin) mutants. E. coli VR50 afa and VR50 afaE displayed significantly decreased ability to adhere to human bladder epithelial cells. In the mouse model of UTI, VR50 afa and VR50 afaE displayed reduced bladder colonization compared to wild-type VR50, similar to the colonization level of the GI-VR50- pheV mutant. In conlusion, our study suggests that E. coli VR50 is a commensal-like strain that has acquired fitness factors that facilitate colonization of the human bladder.« less

Molecular analysis of asymptomatic bacteriuria Escherichia coli strain VR50 reveals adaptation to the urinary tract by gene acquisition.

PubMed

Beatson, Scott A; Ben Zakour, Nouri L; Totsika, Makrina; Forde, Brian M; Watts, Rebecca E; Mabbett, Amanda N; Szubert, Jan M; Sarkar, Sohinee; Phan, Minh-Duy; Peters, Kate M; Petty, Nicola K; Alikhan, Nabil-Fareed; Sullivan, Mitchell J; Gawthorne, Jayde A; Stanton-Cook, Mitchell; Nhu, Nguyen Thi Khanh; Chong, Teik Min; Yin, Wai-Fong; Chan, Kok-Gan; Hancock, Viktoria; Ussery, David W; Ulett, Glen C; Schembri, Mark A

2015-05-01

Urinary tract infections (UTIs) are among the most common infectious diseases of humans, with Escherichia coli responsible for >80% of all cases. One extreme of UTI is asymptomatic bacteriuria (ABU), which occurs as an asymptomatic carrier state that resembles commensalism. To understand the evolution and molecular mechanisms that underpin ABU, the genome of the ABU E. coli strain VR50 was sequenced. Analysis of the complete genome indicated that it most resembles E. coli K-12, with the addition of a 94-kb genomic island (GI-VR50-pheV), eight prophages, and multiple plasmids. GI-VR50-pheV has a mosaic structure and contains genes encoding a number of UTI-associated virulence factors, namely, Afa (afimbrial adhesin), two autotransporter proteins (Ag43 and Sat), and aerobactin. We demonstrated that the presence of this island in VR50 confers its ability to colonize the murine bladder, as a VR50 mutant with GI-VR50-pheV deleted was attenuated in a mouse model of UTI in vivo. We established that Afa is the island-encoded factor responsible for this phenotype using two independent deletion (Afa operon and AfaE adhesin) mutants. E. coli VR50afa and VR50afaE displayed significantly decreased ability to adhere to human bladder epithelial cells. In the mouse model of UTI, VR50afa and VR50afaE displayed reduced bladder colonization compared to wild-type VR50, similar to the colonization level of the GI-VR50-pheV mutant. Our study suggests that E. coli VR50 is a commensal-like strain that has acquired fitness factors that facilitate colonization of the human bladder. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

High terbinafine resistance in Trichophyton interdigitale isolates in Delhi, India harbouring mutations in the squalene epoxidase gene.

PubMed

Singh, Ashutosh; Masih, Aradhana; Khurana, Ananta; Singh, Pradeep Kumar; Gupta, Meenakshi; Hagen, Ferry; Meis, Jacques F; Chowdhary, Anuradha

2018-03-25

In the last few years, infections caused by dermatophytes along with a concomitant increase in the number of difficult to treat cases have increasingly been recognised, indicating that dermatophytosis remains a challenging public health problem. The majority of infections are caused by Trichophyton rubrum and Trichophyton mentagrophytes complex. Terbinafine, an allylamine antifungal used orally and topically is considered to be a first-line drug in the therapy of dermatophyte infections. Terbinafine resistance has been predominately attributed to point mutations in the squalene epoxidase (SQLE) target gene a key enzyme in the ergosterol biosynthetic pathway leading to single amino acid substitutions. Here, we report the largest series of 20 terbinafine-resistant Trichophyton interdigitale isolates obtained predominately from cases of tinea corporis/cruris in three hospitals in Delhi, India exhibiting elevated MICs (4 to ≥32 μg/mL) to terbinafine and all harbouring single-point mutations Leu393Phe or Phe397Leu in the SQLE gene. In 12 (60%) T. interdigitale isolates, the Phe397Leu substitution was observed, whereas in the remaining 8 (40%) isolates the substitution Leu393Phe was reported for the first time in T. interdigitale. Furthermore, 10 susceptible T. interdigitale isolates (0.125-2 μg/mL) had a wild-type genotype. Remarkably, considerably high terbinafine resistance rate of 32% was observed among 63 T. interdigitale isolates identified by sequencing of the internal transcribed spacer region. This high level of terbinafine resistance of Indian dermatophyte isolates is worrisome warranting antifungal susceptibility testing and mutation analysis for monitoring this emerging resistance. © 2018 Blackwell Verlag GmbH.

Molecular Analysis of Asymptomatic Bacteriuria Escherichia coli Strain VR50 Reveals Adaptation to the Urinary Tract by Gene Acquisition

PubMed Central

Ben Zakour, Nouri L.; Totsika, Makrina; Forde, Brian M.; Watts, Rebecca E.; Mabbett, Amanda N.; Szubert, Jan M.; Sarkar, Sohinee; Phan, Minh-Duy; Peters, Kate M.; Petty, Nicola K.; Alikhan, Nabil-Fareed; Sullivan, Mitchell J.; Gawthorne, Jayde A.; Stanton-Cook, Mitchell; Nhu, Nguyen Thi Khanh; Chong, Teik Min; Yin, Wai-Fong; Chan, Kok-Gan; Hancock, Viktoria; Ussery, David W.; Ulett, Glen C.

2015-01-01

Urinary tract infections (UTIs) are among the most common infectious diseases of humans, with Escherichia coli responsible for >80% of all cases. One extreme of UTI is asymptomatic bacteriuria (ABU), which occurs as an asymptomatic carrier state that resembles commensalism. To understand the evolution and molecular mechanisms that underpin ABU, the genome of the ABU E. coli strain VR50 was sequenced. Analysis of the complete genome indicated that it most resembles E. coli K-12, with the addition of a 94-kb genomic island (GI-VR50-pheV), eight prophages, and multiple plasmids. GI-VR50-pheV has a mosaic structure and contains genes encoding a number of UTI-associated virulence factors, namely, Afa (afimbrial adhesin), two autotransporter proteins (Ag43 and Sat), and aerobactin. We demonstrated that the presence of this island in VR50 confers its ability to colonize the murine bladder, as a VR50 mutant with GI-VR50-pheV deleted was attenuated in a mouse model of UTI in vivo. We established that Afa is the island-encoded factor responsible for this phenotype using two independent deletion (Afa operon and AfaE adhesin) mutants. E. coli VR50afa and VR50afaE displayed significantly decreased ability to adhere to human bladder epithelial cells. In the mouse model of UTI, VR50afa and VR50afaE displayed reduced bladder colonization compared to wild-type VR50, similar to the colonization level of the GI-VR50-pheV mutant. Our study suggests that E. coli VR50 is a commensal-like strain that has acquired fitness factors that facilitate colonization of the human bladder. PMID:25667270

The polycyclic aromatic hydrocarbons benzo[a]pyrene and phenanthrene inhibit intestinal lipase activity in rainbow trout (Oncorhynchus mykiss).

PubMed

de Gelder, Stefan; Sæle, Øystein; de Veen, Bas T H; Vos, Joëlle; Flik, Gert; Berntssen, Marc H G; Klaren, Peter H M

2017-08-01

Elevated levels of polycyclic aromatic hydrocarbons (PAHs) are detected in aquafeeds where fish oils are (partially) replaced by vegetable oils. The highly lipophilic PAHs solubilize readily in oil droplets and micelles in the intestinal lumen that can affect enzymatic lipid digestion by altering lipase activity. We therefore investigated the effect of two PAHs, benzo[a]pyrene (BaP) and phenanthrene (PHE), on bile salt-activated lipase (BAL) activity in desalted luminal extracts of the proximal intestine of rainbow trout (Oncorhynchus mykiss) using the triacylglycerides rapeseed oil and fish oil as substrates. The hydrolysis of rapeseed oil and fish oil measured at a calculated substrate concentration of 2.2mM, increased linearly up to 30min at 15°C. Substrate dependency under initial velocity conditions was described by simple Michaelis-Menten kinetics with a K m value of 1.2mM for rapeseed and fish oil. Rapeseed oil hydrolysis was inhibited by 1nM BaP and 10nM PHE. The hydrolysis of fish oil was only inhibited by 10μM BaP. The in vitro lipase activity data were corroborated by TLC/HPLC analysis of the reaction products, showing that in the presence of BaP and PHE, 46-80% less free fatty acids (FFA) were hydrolysed from rapeseed and fish oil triacylglycerides. The presence of low concentrations of BaP and PHE decreased rapeseed oil hydrolysis by BAL whereas fish oil hydrolysis was not affected. The replacement of fish oil by rapeseed oil in aquafeeds introduces PAHs that could affect lipid digestion. Copyright © 2017 Elsevier Inc. All rights reserved.

Molecular mechanisms of protein-cholesterol interactions in plasma membranes: Functional distinction between topological (tilted) and consensus (CARC/CRAC) domains.

PubMed

Fantini, Jacques; Di Scala, Coralie; Baier, Carlos J; Barrantes, Francisco J

2016-09-01

The molecular mechanisms that control the multiple possible modes of protein association with membrane cholesterol are remarkably convergent. These mechanisms, which include hydrogen bonding, CH-π stacking and dispersion forces, are used by a wide variety of extracellular proteins (e.g. microbial or amyloid) and membrane receptors. Virus fusion peptides penetrate the membrane of host cells with a tilted orientation that is compatible with a transient interaction with cholesterol; this tilted orientation is also characteristic of the process of insertion of amyloid proteins that subsequently form oligomeric pores in the plasma membrane of brain cells. Membrane receptors that are associated with cholesterol generally display linear consensus binding motifs (CARC and CRAC) characterized by a triad of basic (Lys/Arg), aromatic (Tyr/phe) and aliphatic (Leu/Val) amino acid residues. In some cases, the presence of both CARC and CRAC within the same membrane-spanning domain allows the simultaneous binding of two cholesterol molecules, one in each membrane leaflet. In this review the molecular basis and the functional significance of the different modes of protein-cholesterol interactions in plasma membranes are discussed. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Characterization of a plasma membrane-associated prenylcysteine-directed alpha carboxyl methyltransferase in human neutrophils.

PubMed

Pillinger, M H; Volker, C; Stock, J B; Weissmann, G; Philips, M R

1994-01-14

Signal transduction in human neutrophils requires prenylcysteine-directed carboxyl methylation of ras-related low molecular weight GTP-binding proteins. We now report the subcellular localization and characterization of a neutrophil prenylcysteine alpha carboxyl methyltransferase. The highest carboxyl methyltransferase activity copurified with biotinylated neutrophil surface membranes, supporting a plasma membrane localization of the enzyme. Neutrophil nuclear fractions contained little or no methyltransferase activity. Methyltransferase activity was detergent-sensitive but could be reconstituted by removal of detergent in the presence of phosphatidyl choline and an anionic phospholipid. N-Acetyl-S-trans,trans-farnesyl-L-cysteine (AFC) and N-acetyl-S-all-trans-geranylgeranyl-L-cysteine (AGGC) were effective substrates for neutrophil prenylcysteine-directed methyltransferase; Vmax values for AFC and AGGC (16.4 and 22.1 pmol of methylated/mg protein/min, respectively) are among the highest yet reported. Although both GTP gamma S and the chemoattractant fMet-Leu-Phe stimulated methylation of ras-related proteins, neither affected methylation of AFC. These data suggest that neutrophil plasma membranes contain a phospholipid-dependent, prenylcysteine-directed carboxyl methyltransferase of relatively high specific activity that modifies ras-related protein substrates in the GTP-bound, activated state.

The Diagnostic Utility of Computer-Assisted Auscultation for the Early Detection of Cardiac Murmurs of Structural Origin in the Periodic Health Evaluation

PubMed Central

Viviers, Pierre L.; Kirby, Jo-Anne H.; Viljoen, Jeandré T.; Derman, Wayne

2017-01-01

Background: Identification of the nature of cardiac murmurs during the periodic health evaluation (PHE) of athletes is challenging due to the difficulty in distinguishing between murmurs of physiological or structural origin. Previously, computer-assisted auscultation (CAA) has shown promise to support appropriate referrals in the nonathlete pediatric population. Hypothesis: CAA has the ability to accurately detect cardiac murmurs of structural origin during a PHE in collegiate athletes. Study Design: Cross-sectional, descriptive study. Level of Evidence: Level 3. Methods: A total of 131 collegiate athletes (104 men, 28 women; mean age, 20 ± 2 years) completed a sports physician (SP)–driven PHE consisting of a cardiac history questionnaire and a physical examination. An independent CAA assessment was performed by a technician who was blinded to the SP findings. Athletes with suspected structural murmurs or other clinical reasons for concern were referred to a cardiologist for confirmatory echocardiography (EC). Results: Twenty-five athletes were referred for further investigation (17 murmurs, 6 abnormal electrocardiographs, 1 displaced apex, and 1 possible case of Marfan syndrome). EC confirmed 3 structural and 22 physiological murmurs. The SP flagged 5 individuals with possible underlying structural pathology; 2 of these murmurs were confirmed as structural in nature. Fourteen murmurs were referred by CAA; 3 of these were confirmed as structural in origin by EC. One such murmur was not detected by the SP, however, and detected by CAA. The sensitivity of CAA was 100% compared with 66.7% shown by the SP, while specificity was 50% and 66.7%, respectively. Conclusion: CAA shows potential to be a feasible adjunct for improving the identification of structural murmurs in the athlete population. Over-referral by CAA for EC requires further investigation and possible refinements to the current algorithm. Further studies are needed to determine the true sensitivity, specificity, and cost efficacy of the device among the athletic population. Clinical Relevance: CAA may be a useful cardiac screening adjunct during the PHE of athletes, particularly as it may guide appropriate referral of suspected structural murmurs for further investigation. PMID:28661830

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.

Chromium (D-phenylalanine)3 alleviates high fat-induced insulin resistance and lipid abnormalities.

PubMed

Kandadi, Machender Reddy; Unnikrishnan, M K; Warrier, Ajaya Kumar Sankara; Du, Min; Ren, Jun; Sreejayan, Nair

2011-01-01

High-fat diet has been implicated as a major cause of insulin resistance and dyslipidemia. The objective of this study was to evaluate the impact of dietary-supplementation of chromium (D-phenylalanine)(3) [Cr(D-Phe)(3)] on glucose and insulin tolerance in high-fat diet fed mice. C57BL/6-mice were randomly assigned to orally receive vehicle or Cr(D-Phe)(3) (45 μg of elemental chromium/kg/day) for 8-weeks. High-fat-fed mice exhibited impaired whole-body-glucose and -insulin tolerance and elevated serum triglyceride levels compared to normal chow-fed mice. Insulin-stimulated glucose up-take in the gastrocnemius muscles, assessed as 2-[(3)H-deoxyglucose] incorporation was markedly diminished in high-fat fed mice compared to control mice. Treatment with chromium reconciled the high-fat diet-induced alterations in carbohydrate and lipid metabolism. Treatment of cultured, differentiated myotubes with palmitic acid evoked insulin resistance as evidenced by lower levels of insulin-stimulated Akt-phosphorylation, elevated JNK-phosphorylation, (assessed by Western blotting), attenuation of phosphoinositol-3-kinase activity (determined in the insulin-receptor substrate-1-immunoprecipitates by measuring the extent of phosphorylation of phosphatidylinositol by γ-(32)P-ATP), and impairment in cellular glucose up-take, all of which were inhibited by Cr(d-Phe)(3). These results suggest a beneficial effect of chromium-supplementation in insulin resistant conditions. It is likely that these effects of chromium may be mediated by augmenting downstream insulin signaling. Copyright © 2010 Elsevier Inc. All rights reserved.

Three novel variants (p.Glu178Lys, p.Val245Met, p.Ser250Phe) of the phenylalanine hydroxylase (PAH) gene impair protein expression and function in vitro.

PubMed

Zong, Yanan; Liu, Ning; Ma, Shanshan; Bai, Ying; Guan, Fangxia; Kong, Xiangdong

2018-08-20

Phenylketonuria (PKU) is the most common inherited metabolic disease, an autosomal recessive disorder affecting >10,000 newborns each year globally. It can be caused by over 1000 different naturally occurring mutations in the phenylalanine hydroxylase (PAH) gene. We analyzed three novel naturally occurring PAH gene variants: p.Glu178Lys (c.532G>A), p.Val245Met (c.733G>A) and p.Ser250Phe (c.749C>T). The mutant effect on the PAH enzyme structure and function was predicted by bioinformatics software. Vectors expressing the corresponding PAH variants were generated for expression in E. coli and in HEK293T cells. The RNA expression of the three PAH variants was measured by quantitative reverse transcription polymerase chain reaction (RT-qPCR). The mutant PAH protein levels were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot and enzyme-linked immunosorbent assay (ELISA). All three variants were predicted to be pathogenic by bioinformatics analysis. The transcription of the three PAH variants was similar to the wild type PAH gene in HEK293T cells. In contrast, the levels of mutant PAH proteins decreased significantly compared to the wild type control, in both E. coli and HEK293T cells. Our results indicate that the three novel PAH gene variants (p.Glu178Lys, p.Val245Met, p.Ser250Phe) impair PAH protein expression and function in prokaryotic and eukaryotic cells. Copyright © 2018. Published by Elsevier B.V.

Mutations of human NARS2, encoding the mitochondrial asparaginyl-tRNA synthetase, cause nonsyndromic deafness and Leigh syndrome.

PubMed

Simon, Mariella; Richard, Elodie M; Wang, Xinjian; Shahzad, Mohsin; Huang, Vincent H; Qaiser, Tanveer A; Potluri, Prasanth; Mahl, Sarah E; Davila, Antonio; Nazli, Sabiha; Hancock, Saege; Yu, Margret; Gargus, Jay; Chang, Richard; Al-Sheqaih, Nada; Newman, William G; Abdenur, Jose; Starr, Arnold; Hegde, Rashmi; Dorn, Thomas; Busch, Anke; Park, Eddie; Wu, Jie; Schwenzer, Hagen; Flierl, Adrian; Florentz, Catherine; Sissler, Marie; Khan, Shaheen N; Li, Ronghua; Guan, Min-Xin; Friedman, Thomas B; Wu, Doris K; Procaccio, Vincent; Riazuddin, Sheikh; Wallace, Douglas C; Ahmed, Zubair M; Huang, Taosheng; Riazuddin, Saima

2015-03-01

Here we demonstrate association of variants in the mitochondrial asparaginyl-tRNA synthetase NARS2 with human hearing loss and Leigh syndrome. A homozygous missense mutation ([c.637G>T; p.Val213Phe]) is the underlying cause of nonsyndromic hearing loss (DFNB94) and compound heterozygous mutations ([c.969T>A; p.Tyr323*] + [c.1142A>G; p.Asn381Ser]) result in mitochondrial respiratory chain deficiency and Leigh syndrome, which is a neurodegenerative disease characterized by symmetric, bilateral lesions in the basal ganglia, thalamus, and brain stem. The severity of the genetic lesions and their effects on NARS2 protein structure cosegregate with the phenotype. A hypothetical truncated NARS2 protein, secondary to the Leigh syndrome mutation p.Tyr323* is not detectable and p.Asn381Ser further decreases NARS2 protein levels in patient fibroblasts. p.Asn381Ser also disrupts dimerization of NARS2, while the hearing loss p.Val213Phe variant has no effect on NARS2 oligomerization. Additionally we demonstrate decreased steady-state levels of mt-tRNAAsn in fibroblasts from the Leigh syndrome patients. In these cells we show that a decrease in oxygen consumption rates (OCR) and electron transport chain (ETC) activity can be rescued by overexpression of wild type NARS2. However, overexpression of the hearing loss associated p.Val213Phe mutant protein in these fibroblasts cannot complement the OCR and ETC defects. Our findings establish lesions in NARS2 as a new cause for nonsyndromic hearing loss and Leigh syndrome.

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.

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.

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.

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.

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

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.

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.

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.

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)

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

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.

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.

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.

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.

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.

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.

Designing medical foods for inherited metabolic disorders: why intact protein is superior to amino acids.

PubMed

Ney, Denise Marie; Etzel, Mark Raymond

2017-04-01

Phenylketonuria and tyrosinemia are inherited metabolic disorders characterized by high blood levels of phenylalanine (Phe) or tyrosine (Tyr), due to mutations in genes affecting Phe and Tyr metabolism, respectively. The primary management is a lifelong diet restricted in protein from natural foods in combination with medical foods comprised mixtures of synthetic amino acids. Compliance is often poor after childhood leading to neuropsychological sequela. Glycomacropeptide, an intact 64 amino acid glycophosphopeptide isolated from cheese whey, provides a new paradigm for the management of phenylketonuria and tyrosinemia because glycomacropeptide contains no Phe and Tyr in its pure form, and is also a prebiotic. Medical foods made from glycomacropeptide have been used successfully for the management of phenylketonuria and tyrosinemia. Preclinical and clinical studies demonstrate that intact protein from glycomacropeptide provides a more acceptable and physiologic source of defined protein compared to amino acids in medical foods. For example, harmful gut bacteria were reduced, beneficial short chain fatty acids increased, renal workload decreased, protein utilization increased, and bone fragility decreased using intact protein versus amino acids. Advances in biotechnology will propel the transition from synthetic amino acids to intact proteins for the management of inherited metabolic disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

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.

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.

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.

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.

Effects of feeding reduced crude protein diets on growth performance, nitrogen excretion, and plasma uric acid concentration of broiler chicks during the starter period.

PubMed

Kriseldi, R; Tillman, P B; Jiang, Z; Dozier, W A

2018-05-01

An experiment (2 trials) was conducted to determine the effects of feeding reduced crude protein (CP) diets to Ross × Ross 708 male broilers while maintaining adequate essential amino acid (AA) concentrations on growth performance, nitrogen excretion, and plasma uric acid (UA) concentration during the starter period. In trial 1, 11 dietary treatments were fed from 1 to 18 d of age containing 1.20% digestible Lys. Diet 1 (23.2% CP) was formulated with DL-Met, L-Lys, and L-Thr to contain 1.70 total Gly + Ser to digestible Lys ratio whereas diets 2 (23.4% CP) to 11 were formulated with additional Gly to contain 1.90 total Gly + Ser to digestible Lys ratio. Free AA were added sequentially in the order of limitation (L-Val, L-Ile, L-Arg, L-Trp, L-His, L-Phe, and L-Leu) from diets 3 to 10 to decrease CP content from 22.6 to 18.8%, respectively. In diet 11, L-Gln was added to increase the CP content to 23.4%. Feed conversion of broilers fed diet 2 was lower (P < 0.05) than those consuming diets 6 to 11 from 1 to 17 d of age. Nitrogen excretion (mg/b/d) decreased (P < 0.001) by 14.1% when broilers were fed diet 4 compared with birds fed diet 2 from 15 to 16 d of age. Broilers fed diet 4 had lower (P = 0.011) plasma UA concentration than birds fed diet 2 at 18 d of age. In trial 2, 8 dietary treatments containing 1.25% digestible Lys and 1.70 total Gly + Ser to digestible Lys ratio were fed from 1 to 21 d of age. Diet 1 (24.0% CP) was supplemented with DL-Met, L-Lys, and L-Thr. Free AA (L-Val, Gly, L-Ile, L-Arg, L-Trp, L-His, and L-Phe) were sequentially supplemented in the order of limitation to decrease CP content in diets 2 to 8 from 23.8 to 20.3%. Broilers fed diet 1 had higher (P < 0.05) body weight gain and lower (P < 0.05) feed conversion when compared with diet 7 or 8. Plasma UA concentration of broiler provided diets 4 to 8 was lower (P < 0.05) compared with diet 1 at 21 d of age. Placing a minimum on dietary CP percentage may not be necessary when proper AA ratios are implemented in diet formulation.

Specific neurokinin receptors mediate plasma extravasation in the rat knee joint.

PubMed Central

Lam, F. Y.; Ferrell, W. R.

1991-01-01

1 Plasma extravasation in the rat knee joint was induced by intra-articular injection of neurokinins and specific neurokinin receptor agonists. 2 Pronounced plasma extravasation was produced by substance P (SP, 4-185 microM) and to a lesser extent by neurokinin-B (NKB, 83-413 microM), whereas neurokinin-A (NKA, 88-440 microM) and calcitonin gene-related peptide (CGRP, 26-130 microM) had no significant effect. 3 The specific neurokinin1 receptor agonist [Sar9, Met(O2)11]-substance P (NK1 agonist) in doses of 0.4-70 microM appeared to be more potent than SP in eliciting plasma extravasation. The neurokinin2 receptor agonist [Nle10]-neurokinin A4-10 (NK2 agonist) was not effective at 70 microM but produced a small and significant effect at 330 microM, whereas the neurokinin3 receptor agonist [MePhe7]-neurokinin B (NK3 agonist) was without effect at 40 microM or 400 microM. 4 Injections of SP or NKA into the synovial cavity of the rat knee were equally effective in producing marked plasma extravasation in remote sites such as the forelimb and hindlimb paws. 5 Co-administration experiments showed that the effects of SP were synergistic with NKA or the NK1 receptor agonist, but not with CGRP or the NK2 receptor agonist. 6 The rank order of potency was NK1 agonist greater than or equal to SP greater than NKB greater than NK2 agonist suggesting that NK1 receptors mediate plasma extravasation in the rat knee joint. PMID:1715229

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.

Intracellular pH in mammalian stages of Trypanosoma cruzi is K+-dependent and regulated by H+-ATPases.

PubMed

Van Der Heyden, N; Docampo, R

2000-02-05

Regulation of intracellular pH (pHi) was investigated in Trypanosoma cruzi amastigotes and trypomastigotes using 2',7'-bis-(carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF). pHi was determined to be 7.33 +/- 0.08 and 7.35 +/- 0.07 in amastigotes and trypomastigotes, respectively, and there were no significant differences in the regulation of pH, between the two stages. Steady-state pHi, recovery of pHi from acidification, and H+-efflux were all decreased markedly by the H+-ATPase inhibitors N,N'-dicyclohexylcarbodi-imide (DCCD), diethylstilbestrol (DES) and N-ethylmaleimide (NEM) supporting a significant role for a plasma membrane H+-ATPase in the regulation of pHi. pHi was maintained at neutrality over a range of external pH (pHe) from 5-8 in parasites suspended in a buffer containing Na+ and K+ (standard buffer) but was acidified at low pHe in the absence of these cations (choline buffer). The pHi of trypomastigotes decreased significantly when they transformed into amastigotes. The rate of recovery of pHi by acidified parasites was similar in Na+-free buffer and standard buffer but was slower in the absence of K+ (K+-free or choline buffer) and parasites suspended in choline buffer were acidic by 0.25 pH units as compared with controls. Ba2+ and Cs+ decreased the pHi of parasites suspended in standard but not choline buffer suggesting the presence of an inward directed K+ channel. The pHi of amastigotes and trypomastigotes suspended in Cl(-)-free buffer was decreased by 0.13 and 0.2 pH units, respectively, supporting the presence of a chloride conductive channel. No evidence of pH regulation via a Na+/H+ or Cl-/HCO3- exchanger was found. These results are consistent with the presence of a plasma membrane H+-ATPase that regulates pHi and is supported by K+ and Cl- channels.

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.

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.

Anabolic resistance assessed by oral stable isotope ingestion following bed rest in young and older adult volunteers: Relationships with changes in muscle mass.

PubMed

Biolo, Gianni; Pišot, Rado; Mazzucco, Sara; Di Girolamo, Filippo Giorgio; Situlin, Roberta; Lazzer, Stefano; Grassi, Bruno; Reggiani, Carlo; Passaro, Angelina; Rittweger, Joern; Gasparini, Mladen; Šimunič, Boštjan; Narici, Marco

2017-10-01

Aging and experimental bed rest are associated with muscle atrophy and resistance to post-prandial stimulation of protein synthesis or anabolic resistance (AR). We have used in young and older adult volunteers, during short-term bed rest, a quick and non-invasive method, based on a single oral bolus of the stable isotope L[ring- 2 H 5 ]phenylalanine (D 5 Phe), to determine post-prandial AR, defined as ratio between irreversible hydroxylation and incorporation into body protein of ingested phenylalanine. We compared in older (O, 59 ± 1 y) and young (Y, 23 ± 1 y) healthy male volunteers the effects of two-week bed rest on post-prandial protein kinetics, assessed during absorption of a standard ready-to-use oral nutritional supplement, through stable-labeled isotope amino acid D 5 Phe, diluted in water, given as single oral load. The metabolic fate of D 5 Phe is either utilization for protein synthesis or irreversible hydroxylation to L[ring- 2 H 4 ]tyrosine (D 4 Tyr). AR was defined as ratio between the areas under the curves of D 4 Tyr-to-D 5 Phe plasma concentrations over 6 h meal absorption. To determine the relationships between AR and muscle changes following bed rest, quadriceps muscle volume (QMV) was determined by magnetic resonance imaging (MRI). At baseline, in pooled Y and O subjects, values of AR were inversely correlated with QMV (R = -0.75; p < 0.03). Following 2-weeks of inactivity, there were significant bed rest effects on AR (p < 0.01) and QMV (p < 0.03), as well as significant bed rest × group interaction for AR (p < 0.03; +9.2% in Y; +21.9% in O) and QMV (p < 0.05; -5.7% in Y; -%7.3 in O). In pooled subjects, the percentage delta changes in AR and QMV, induced by bed rest, were inversely correlated (R = -0.57; p < 0.05). Bed rest-induced AR is much greater in the older than in younger adults. We have developed a new, simple, non-invasive method for the assessment of AR. The results indicate that this metabolic abnormality is a key mechanism for sarcopenia of aging and inactivity. Copyright © 2016 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

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

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.

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.

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

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

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

"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-.

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.

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

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.

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.

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%.

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

NASA Astrophysics Data System (ADS)

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

2013-05-01

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

The effect of perinatal anoxia on amino acid metabolism in the developing brain. Part II: The effect of perinatal anoxia on the free amino acid patterns in CSF of infants and children.

PubMed

Kaneko, K

1985-01-01

To clarify the effects of perinatal anoxia on the subsequent amino acid metabolism in the brain of children, free amino acid levels in the cerebrospinal fluid (CSF) were determined in 15 children diagnosed as having cerebral palsy and/or mental retardation with perinatal anoxia, and 58 control children without anoxia, aged from 4 days to 12 yrs. There was no significant difference in total amino acid levels between anoxic children and the controls. In the controls, the Gln level in CSF was high, Arg, Asp and Glu levels in CSF were almost the same during infancy and childhood, and the levels of Orn, Lys, His, Tau, Thr, Ser, Asn, Gly, Ala, Val, Met, Ile, Leu, Tyr and Phe in CSF decreased with age until pre-school age. In the newborns and infants among the anoxic children, the levels of most free amino acids in CSF were relatively high compared with those of the controls and, except Glu and Gln, decreased with age during infancy. The Orn, His, Gly, Tyr and Phe levels in CSF of anoxic children were lower than those of the controls in older infants. These results suggest that perinatal anoxia affected free amino acid patterns in CSF of newborns and infants and that the subsequent disturbance of amino acid metabolism in their brains remained after birth.

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.

Identification of BACE2 as an avid ß-amyloid-degrading protease

PubMed Central

2012-01-01

Background Proteases that degrade the amyloid ß-protein (Aß) have emerged as key players in the etiology and potential treatment of Alzheimer’s disease (AD), but it is unlikely that all such proteases have been identified. To discover new Aß-degrading proteases (AßDPs), we conducted an unbiased, genome-scale, functional cDNA screen designed to identify proteases capable of lowering net Aß levels produced by cells, which were subsequently characterized for Aß-degrading activity using an array of downstream assays. Results The top hit emerging from the screen was ß-site amyloid precursor protein-cleaving enzyme 2 (BACE2), a rather unexpected finding given the well-established role of its close homolog, BACE1, in the production of Aß. BACE2 is known to be capable of lowering Aß levels via non-amyloidogenic processing of APP. However, in vitro, BACE2 was also found to be a particularly avid AßDP, with a catalytic efficiency exceeding all known AßDPs except insulin-degrading enzyme (IDE). BACE1 was also found to degrade Aß, albeit ~150-fold less efficiently than BACE2. Aß is cleaved by BACE2 at three peptide bonds—Phe19-Phe20, Phe20-Ala21, and Leu34-Met35—with the latter cleavage site being the initial and principal one. BACE2 overexpression in cultured cells was found to lower net Aß levels to a greater extent than multiple, well-established AßDPs, including neprilysin (NEP) and endothelin-converting enzyme-1 (ECE1), while showing comparable effectiveness to IDE. Conclusions This study identifies a new functional role for BACE2 as a potent AßDP. Based on its high catalytic efficiency, its ability to degrade Aß intracellularly, and other characteristics, BACE2 represents a particulary strong therapeutic candidate for the treatment or prevention of AD. PMID:22986058

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

PubMed

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

2017-01-01

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

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.

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.

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.

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

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.

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.

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.

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.

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.

Interaction of hemoglobin Grey Lynn (Vientiane) with a non-deletional α(+)-thalassemia in an adult Thai proband.

PubMed

Singha, Kritsada; Fucharoen, Goonnapa; Fucharoen, Supan

2014-01-01

Hemoglobin (Hb) Grey Lynn is a Hb variant caused by a substitution of Phe for Leu at position 91 of α1-globin chain, originally described in individual of unknown ethnic background. This article addresses the interaction of Hb Grey Lynn with a non-deletional α(+)-thalassemia found in Thailand, a hitherto un-described condition. The proband was adult Thai woman referred for investigation of mild anemia with Hb 90 g/L. Hb analyses using low pressure liquid chromatography raised a suspicion of abnormal Hb presence, which was failed to demonstrate by cellulose acetate electrophoresis and capillary electrophoresis. DNA sequencing identified a CTT (Leu) to TTT (Phe) mutation at codon 91 corresponding to the Hb Grey Lynn (Vientiane) [α91(FG3)Leu>Phe (α1) on α1-globin gene and a C deletion between codons 36 and 37 on α2-globin gene causing α(+)-thalassemia. As compared to those observed in a compound heterozygote for Hb Grey Lynn / α(0)-thalassemia reported previously, higher MCV (81.7 fL) and MCH (26.3 pg) values with a lower level of Hb Grey Lynn (19.7%) were observed in the proband. The normochromic normocytic anemia observed could be due to the interaction of Hb Grey Lynn with α(+)-thalassemia. The two mutations could be identified using PCR-RFLP and allele-specific PCR assays developed.

Impact of bacterial activity on turnover of insoluble hydrophobic substrates (phenanthrene and pyrene)-Model simulations for prediction of bioremediation success.

PubMed

Rein, Arno; Adam, Iris K U; Miltner, Anja; Brumme, Katja; Kästner, Matthias; Trapp, Stefan

2016-04-05

Many attempts for bioremediation of polycyclic aromatic hydrocarbon (PAH) contaminated sites failed in the past, but the reasons for this failure are not well understood. Here we apply and improve a model for integrated assessment of mass transfer, biodegradation and residual concentrations for predicting the success of remediation actions. First, we provide growth parameters for Mycobacterium rutilum and Mycobacterium pallens growing on phenanthrene (PHE) or pyrene (PYR) degraded the PAH completely at all investigated concentrations. Maximum metabolic rates vmax and growth rates μ were similar for the substrates PHE and PYR and for both strains. The investigated Mycobacterium species were not superior in PHE degradation to strains investigated earlier with this method. Real-world degradation scenario simulations including diffusive flux to the microbial cells indicate: that (i) bioaugmentation only has a small, short-lived effect; (ii) Increasing sorption shifts the remaining PAH to the adsorbed/sequestered PAH pool; (iii) mobilizing by solvents or surfactants resulted in a significant decrease of the sequestered PAH, and (iv) co-metabolization e.g. by compost addition can contribute significantly to the reduction of PAH, because active biomass is maintained at a high level by the compost. The model therefore is a valuable contribution to the assessment of potential remediation action at PAH-polluted sites. Copyright © 2015 Elsevier B.V. All rights reserved.

Detection of tripeptidyl peptidase I activity in living cells by fluorogenic substrates.

PubMed

Steinfeld, Robert; Fuhrmann, Jens C; Gärtner, Jutta

2006-09-01

Tripeptidyl peptidase I (TPP-I) is a lysosomal peptidase with unclear physiological function. TPP-I deficiency is associated with late-infantile neuronal ceroid lipofuscinosis (NCL), a fatal neurodegenerative disease of childhood that is characterized by loss of neurons and photoreceptor cells. We have developed two novel fluorogenic substrates, [Ala-Ala-Phe]2-rhodamine 110 and [Arg-Nle-Nle]2-rhodamine 110, that are cleaved by TPP-I in living cells. Fluorescence of liberated rhodamine 110 was detected by flow cytometry and was dependent on the level of TPP-I expression. Rhodamine-related fluorescence could be suppressed by preincubation with a specific inhibitor of TPP-I. When investigated by fluorescent confocal microscopy, rhodamine signals colocalized with lysosomal markers. Thus, cleavage of these rhodamide-derived substrates is a marker for mature enzymatically active TPP-I. In addition, TPP-I-induced cleavage of [Ala-Ala-Phe]2-rhodamine 110 could be visualized in primary neurons. We conclude that [Ala-Ala-Phe]2-rhodamine 110 and [Arg-Nle-Nle]2-rhodamine 110 are specific substrates for determining TPP-I activity and intracellular localization in living cells. Further, these substrates could be a valuable tool for studying the neuronal pathology underlying classical late-infantile NCL. This article contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

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.

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.

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.

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.

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.

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.

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.

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).

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.

Embryonal Control of Yellow Seed Coat Locus ECY1 Is Related to Alanine and Phenylalanine Metabolism in the Seed Embryo of Brassica napus

PubMed Central

Wang, Fulin; He, Jiewang; Shi, Jianghua; Zheng, Tao; Xu, Fei; Wu, Guanting; Liu, Renhu; Liu, Shengyi

2016-01-01

Seed coat color is determined by the type of pigment deposited in the seed coat cells. It is related to important agronomic traits of seeds such as seed dormancy, longevity, oil content, protein content and fiber content. In Brassica napus, inheritance of seed coat color is related to maternal effects and pollen effects (xenia effects). In this research we isolated a mutation of yellow seeded B. napus controlled by a single Mendelian locus, which is named Embryonal Control of Yellow seed coat 1 (Ecy1). Microscopy of transverse sections of the mature seed show that pigment is deposited only in the outer layer of the seed coat. Using Illumina Hisequation 2000 sequencing technology, a total of 12 GB clean data, 116× coverage of coding sequences of B. napus, was achieved from seeds 26 d after pollination (DAP). It was assembled into 172,238 independent transcripts, and 55,637 unigenes. A total of 139 orthologous genes of Arabidopsis transparent testa (TT) genes were mapped in silico to 19 chromosomes of B. napus. Only 49 of the TT orthologous genes are transcribed in seeds. However transcription of all orthologs was independent of embryonal control of seed coat color. Only 55 genes were found to be differentially expressed between brown seeds and the yellow mutant. Of these 55, 50 were upregulated and five were downregulated in yellow seeds as compared to their brown counterparts. By KEGG classification, 14 metabolic pathways were significantly enriched. Of these, five pathways: phenylpropanoid biosynthesis, cyanoamino acid metabolism, plant hormone signal transduction, metabolic pathways, and biosynthesis of secondary metabolites, were related with seed coat pigmentation. Free amino acid quantification showed that Ala and Phe were present at higher levels in the embryos of yellow seeds as compared to those of brown seeds. This increase was not observed in the seed coat. Moreover, the excess amount of free Ala was exactly twice that of Phe in the embryo. The pigment substrate chalcone is synthesized from two molecules of Ala and one molecule of Phe. The correlation between accumulation of Ala and Phe, and disappearance of pigment in the yellow seeded mutant, suggests that embryonal control of seed coat color is related with Phe and Ala metabolism in the embryo of B. napus. PMID:26896439

Plasma pharmacokinetics, whole-body distribution, metabolism, and radiation dosimetry of 68Ga bombesin antagonist BAY 86-7548 in healthy men.

PubMed

Roivainen, Anne; Kähkönen, Esa; Luoto, Pauliina; Borkowski, Sandra; Hofmann, Birte; Jambor, Ivan; Lehtiö, Kaisa; Rantala, Tuija; Rottmann, Antje; Sipilä, Henri; Sparks, Rick; Suilamo, Sami; Tolvanen, Tuula; Valencia, Ray; Minn, Heikki

2013-06-01

This first-in-human study investigated the safety, tolerability, metabolism, pharmacokinetics, biodistribution, and radiation dosimetry of (68)Ga-bombesin antagonist (68)Ga-DOTA-4-amino-1-carboxymethylpiperidine-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 (BAY 86-7548). Five healthy men underwent dynamic whole-body PET/CT after an intravenous injection of BAY 86-7548 (138 ± 5 MBq). Besides total radioactivity, plasma samples were analyzed by radio-high-performance liquid chromatography for metabolism of the tracer. Dosimetry was calculated using the OLINDA/EXM software. Three radioactive plasma metabolites were detected. The proportion of unchanged BAY 86-7548 decreased from 92% ± 9% at 1 min after injection to 19% ± 2% at 65 min. The organs with the highest absorbed doses were the urinary bladder wall (0.62 mSv/MBq) and the pancreas (0.51 mSv/MBq). The mean effective dose was 0.051 mSv/MBq. BAY 86-7548 was well tolerated by all subjects. Intravenously injected BAY 86-7548 is safe, and rapid metabolism is demonstrated. A 150-MBq injection of BAY 86-7548 results in an effective dose of 7.7 mSv, which could be reduced to 5.7 mSv with frequent bladder voids.

/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.

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

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.

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.

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

Computational simulations assessment of mutations impact on streptokinase (SK) from a group G streptococci with enhanced activity - insights into the functional roles of structural dynamics flexibility of SK and stabilization of SK-μplasmin catalytic complex.

PubMed

Kazemi, Faegheh; Arab, Seyed Shahriar; Mohajel, Nasir; Keramati, Malihe; Niknam, Niloofar; Aslani, Mohammad Mehdi; Roohvand, Farzin

2018-05-28

Streptokinase (SK), a plasminogen activator (PA) that converts inactive plasminogen (Pg) to plasmin (Pm), is a protein secreted by groups A, C, and G streptococci (GAS, GCS, and GGS, respectively), with high sequence divergence and functional heterogeneity. While roles of some residual changes in altered SK functionality are shown, the underlying structural mechanisms are less known. Herein, using computational approaches, we analyzed the conformational basis for the increased activity of SK from a GGS (SKG132) isolate with four natural residual substitutions (Ile33Phe, Arg45Gln, Asn228Lys, Phe287Ile) compared to the standard GCS (SKC). Using the crystal structure of SK.Pm catalytic complex as main template SKC.μPm catalytic complex was modeled through homology modeling process and validated by several online validation servers. Subsequently, SKG132.μPm structure was constructed by altering the corresponding residual substitutions. Results of three independent MD simulations showed increased RMSF values for SKG132.μPm, indicating the enhanced structural flexibility compared to SKC.μPm, specially in 170 and 250 loops and three regions: R1 (149-161), R2 (182-215) and R3 (224-229). In parallel, the average number of Hydrogen bonds in 170 loop, R2 and R3 (especially for Asn228Lys) of SKG132 compared to that of the SKC was decreased. Accordingly, residue interaction networks (RINs) analyses indicated that Asn228Lys might induce more level of structural flexibility by generation of free Lys256, while Phe287Ile and Ile33Phe enhanced the stabilization of the SKG132.μPm catalytic complex. These results denoted the potential role of the optimal dynamic state and stabilized catalytic complex for increased PA potencies of SK as a thrombolytic drug.

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).

Assembly and stoichiometry of the core structure of the bacterial flagellar type III export gate complex.

PubMed

Fukumura, Takuma; Makino, Fumiaki; Dietsche, Tobias; Kinoshita, Miki; Kato, Takayuki; Wagner, Samuel; Namba, Keiichi; Imada, Katsumi; Minamino, Tohru

2017-08-01

The bacterial flagellar type III export apparatus, which is required for flagellar assembly beyond the cell membranes, consists of a transmembrane export gate complex and a cytoplasmic ATPase complex. FlhA, FlhB, FliP, FliQ, and FliR form the gate complex inside the basal body MS ring, although FliO is required for efficient export gate formation in Salmonella enterica. However, it remains unknown how they form the gate complex. Here we report that FliP forms a homohexameric ring with a diameter of 10 nm. Alanine substitutions of conserved Phe-137, Phe-150, and Glu-178 residues in the periplasmic domain of FliP (FliPP) inhibited FliP6 ring formation, suppressing flagellar protein export. FliO formed a 5-nm ring structure with 3 clamp-like structures that bind to the FliP6 ring. The crystal structure of FliPP derived from Thermotoga maritia, and structure-based photo-crosslinking experiments revealed that Phe-150 and Ser-156 of FliPP are involved in the FliP-FliP interactions and that Phe-150, Arg-152, Ser-156, and Pro-158 are responsible for the FliP-FliO interactions. Overexpression of FliP restored motility of a ∆fliO mutant to the wild-type level, suggesting that the FliP6 ring is a functional unit in the export gate complex and that FliO is not part of the final gate structure. Copurification assays revealed that FlhA, FlhB, FliQ, and FliR are associated with the FliO/FliP complex. We propose that the assembly of the export gate complex begins with FliP6 ring formation with the help of the FliO scaffold, followed by FliQ, FliR, and FlhB and finally FlhA during MS ring formation.

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.

Assembly and stoichiometry of the core structure of the bacterial flagellar type III export gate complex

PubMed Central

Fukumura, Takuma; Makino, Fumiaki; Dietsche, Tobias; Kinoshita, Miki; Kato, Takayuki; Wagner, Samuel; Namba, Keiichi; Imada, Katsumi

2017-01-01

The bacterial flagellar type III export apparatus, which is required for flagellar assembly beyond the cell membranes, consists of a transmembrane export gate complex and a cytoplasmic ATPase complex. FlhA, FlhB, FliP, FliQ, and FliR form the gate complex inside the basal body MS ring, although FliO is required for efficient export gate formation in Salmonella enterica. However, it remains unknown how they form the gate complex. Here we report that FliP forms a homohexameric ring with a diameter of 10 nm. Alanine substitutions of conserved Phe-137, Phe-150, and Glu-178 residues in the periplasmic domain of FliP (FliPP) inhibited FliP6 ring formation, suppressing flagellar protein export. FliO formed a 5-nm ring structure with 3 clamp-like structures that bind to the FliP6 ring. The crystal structure of FliPP derived from Thermotoga maritia, and structure-based photo-crosslinking experiments revealed that Phe-150 and Ser-156 of FliPP are involved in the FliP–FliP interactions and that Phe-150, Arg-152, Ser-156, and Pro-158 are responsible for the FliP–FliO interactions. Overexpression of FliP restored motility of a ∆fliO mutant to the wild-type level, suggesting that the FliP6 ring is a functional unit in the export gate complex and that FliO is not part of the final gate structure. Copurification assays revealed that FlhA, FlhB, FliQ, and FliR are associated with the FliO/FliP complex. We propose that the assembly of the export gate complex begins with FliP6 ring formation with the help of the FliO scaffold, followed by FliQ, FliR, and FlhB and finally FlhA during MS ring formation. PMID:28771466

Multidimensional free energy surface of unfolding of HP-36: Microscopic origin of ruggedness

NASA Astrophysics Data System (ADS)

Ghosh, Rikhia; Roy, Susmita; Bagchi, Biman

2014-10-01

The protein folding funnel paradigm suggests that folding and unfolding proceed as directed diffusion in a multidimensional free energy surface where a multitude of pathways can be traversed during the protein's sojourn from initial to final state. However, finding even a single pathway, with the detail chronicling of intermediates, is an arduous task. In this work we explore the free energy surface of unfolding pathway through umbrella sampling, for a small globular α-helical protein chicken-villin headpiece (HP-36) when the melting of secondary structures is induced by adding DMSO in aqueous solution. We find that the unfolding proceeds through the initial separation or melting of aggregated hydrophobic core that comprises of three phenylalanine residues (Phe7, Phe11, and Phe18). This separation is accompanied by simultaneous melting of the second helix. Unfolding is found to be a multistage process involving crossing of three consecutive minima and two barriers at the initial stage. At a molecular level, Phe18 is observed to reorient itself towards other hydrophobic grooves to stabilize the intermediate states. We identify the configuration of the intermediates and correlate the intermediates with those obtained in our previous works. We also give an estimate of the barriers for different transition states and observe the softening of the barriers with increasing DMSO concentration. We show that higher concentration of DMSO tunes the unfolding pathway by destabilizing the third minimum and stabilizing the second one, indicating the development of a solvent modified, less rugged pathway. The prime outcome of this work is the demonstration that mixed solvents can profoundly transform the nature of the energy landscape and induce unfolding via a modified route. A successful application of Kramer's rate equation correlating the free energy simulation results shows faster rate of unfolding with increasing DMSO concentration. This work perhaps presents the first systematic theoretical study of the effect of a chemical denaturant on the microscopic free energy surface and rates of unfolding of HP-36.

Bradykinin-mediated diseases.

PubMed

Kaplan, Allen P

2014-01-01

Diseases which have been demonstrated to be caused by increased plasma levels of bradykinin all have angioedema as the common major clinical manifestation. Angioedema due to therapy with angiotensin-converting enzyme (ACE) inhibitors is caused by suppressed bradykinin degradation so that it accumulates. This occurs because ACE metabolizes bradykinin by removal of Phe-Arg from the C-terminus, which inactivates it. By contrast, angioedema due to C1 inhibitor deficiency (either hereditary types I and II, or acquired) is caused by bradykinin overproduction. C1 inhibitor inhibits factor XIIa, kallikrein and activity associated with the prekallikrein-HK (high-molecular-weight kininogen) complex. In its absence, uncontrolled activation of the plasma bradykinin cascade is seen once there has been an initiating stimulus. C4 levels are low in all types of C1 inhibitor deficiency due to the instability of C1 (C1r, in particular) such that some activated C1 always circulates and depletes C4. In the hereditary disorder, formation of factor XIIf (factor XII fragment) during attacks of swelling causes C4 levels to drop toward zero, and C2 levels decline. A kinin-like molecule, once thought to be a cleavage product derived from C2 that contributes to the increased vascular permeability seen in hereditary angioedema (HAE), is now thought to be an artifact, i.e. no such molecule is demonstrable. The acquired C1 inhibitor deficiency is associated with clonal disorders of B cell hyperreactivity, including lymphoma and monoclonal gammopathy. Most cases have an IgG autoantibody to C1 inhibitor which inactivates it so that the presentation is strikingly similar to type I HAE. New therapies for types I and II HAE include C1 inhibitor replacement therapy, ecallantide, a kallikrein antagonist, and icatibant, a B2 receptor antagonist. A newly described type III HAE has normal C1 inhibitor, although it is thought to be mediated by bradykinin, as is an antihistamine-resistant subpopulation of patients with 'idiopathic' angioedema. The mechanism(s) for the formation of bradykinin in these disorders is unknown. © 2014 S. Karger AG, Basel.

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

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.

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.

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.

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.

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

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

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.

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.

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.

Selective albumin-binding surfaces modified with a thrombin-inhibiting peptide.

PubMed

Freitas, Sidónio C; Maia, Sílvia; Figueiredo, Ana C; Gomes, Paula; Pereira, Pedro J B; Barbosa, Mário A; Martins, M Cristina L

2014-03-01

Blood-contacting medical devices have been associated with severe clinical complications, such as thrombus formation, triggered by the activation of the coagulation cascade due to the adsorption of certain plasma proteins on the surface of biomaterials. Hence, the coating of such surfaces with antithrombotic agents has been used to increase biomaterial haemocompatibility. Biomaterial-induced clotting may also be decreased by albumin adsorption from blood plasma in a selective and reversible way, since this protein is not involved in the coagulation cascade. In this context, this paper reports that the immobilization of the thrombin inhibitor D-Phe-Pro-D-Arg-D-Thr-CONH2 (fPrt) onto nanostructured surfaces induces selective and reversible adsorption of albumin, delaying the clotting time when compared to peptide-free surfaces. fPrt, synthesized with two glycine residues attached to the N-terminus (GGfPrt), was covalently immobilized onto self-assembled monolayers (SAMs) having different ratios of carboxylate-hexa(ethylene glycol)- and tri(ethylene glycol)-terminated thiols (EG6-COOH/EG3) that were specifically designed to control GGfPrt orientation, exposure and density at the molecular level. In solution, GGfPrt was able to inactivate the enzymatic activity of thrombin and to delay plasma clotting time in a concentration-dependent way. After surface immobilization, and independently of its concentration, GGfPrt lost its selectivity to thrombin and its capacity to inhibit thrombin enzymatic activity against the chromogenic substrate n-p-tosyl-Gly-Pro-Arg-p-nitroanilide. Nevertheless, surfaces with low concentrations of GGfPrt could delay the capacity of adsorbed thrombin to cleave fibrinogen. In contrast, GGfPrt immobilized in high concentrations was found to induce the procoagulant activity of the adsorbed thrombin. However, all surfaces containing GGfPrt have a plasma clotting time similar to the negative control (empty polystyrene wells), showing resistance to coagulation, which is explained by its capacity to adsorb albumin in a selective and reversible way. This work opens new perspectives to the improvement of the haemocompatibility of blood-contacting medical devices. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Changing Metabolic Signatures of Amino Acids and Lipids During the Prediabetic Period in a Pig Model With Impaired Incretin Function and Reduced β-Cell Mass

PubMed Central

Renner, Simone; Römisch-Margl, Werner; Prehn, Cornelia; Krebs, Stefan; Adamski, Jerzy; Göke, Burkhard; Blum, Helmut; Suhre, Karsten; Roscher, Adelbert A.; Wolf, Eckhard

2012-01-01

Diabetes is generally diagnosed too late. Therefore, biomarkers indicating early stages of β-cell dysfunction and mass reduction would facilitate timely counteraction. Transgenic pigs expressing a dominant-negative glucose-dependent insulinotropic polypeptide receptor (GIPRdn) reveal progressive deterioration of glucose control and reduction of β-cell mass, providing a unique opportunity to study metabolic changes during the prediabetic period. Plasma samples from intravenous glucose tolerance tests of 2.5- and 5-month-old GIPRdn transgenic and control animals were analyzed for 163 metabolites by targeted mass spectrometry. Analysis of variance revealed that 26 of 163 parameters were influenced by the interaction Genotype × Age (P ≤ 0.0001) and thus are potential markers for progression within the prediabetic state. Among them, the concentrations of seven amino acids (Phe, Orn, Val, xLeu, His, Arg, and Tyr) were increased in 2.5-month-old but decreased in 5-month-old GIPRdn transgenic pigs versus controls. Furthermore, specific sphingomyelins, diacylglycerols, and ether phospholipids were decreased in plasma of 5-month-old GIPRdn transgenic pigs. Alterations in plasma metabolite concentrations were associated with liver transcriptome changes in relevant pathways. The concentrations of a number of plasma amino acids and lipids correlated significantly with β-cell mass of 5-month-old pigs. These metabolites represent candidate biomarkers of early phases of β-cell dysfunction and mass reduction. PMID:22492530

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.

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

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.

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.

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.

Metabolic syndrome in children and adolescents with phenylketonuria.

PubMed

Kanufre, Viviane C; Soares, Rosângelis D L; Alves, Michelle Rosa A; Aguiar, Marcos J B; Starling, Ana Lúcia P; Norton, Rocksane C

2015-01-01

This study aimed to identify markers of metabolic syndrome (MS) in patients with phenylketonuria (PKU). This was a cross-sectional study consisting of 58 PKU patients (ages of 4-15 years): 29 patients with excess weight, and 29 with normal weight. The biochemical variables assessed were phenylalanine (phe), total cholesterol, HDL-c, triglycerides, glucose, and basal insulin. The patients had Homeostasis Model Assessment (HOMA) and waist circumference assessed. No inter-group difference was found for phe. Overweight patients had higher levels of triglycerides, basal insulin, and HOMA, but lower concentrations of HDL-cholesterol, when compared to the eutrophic patients. Total cholesterol/HDL-c was significantly higher in the overweight group. A positive correlation between basal insulin level and HOMA with waist circumference was found only in the overweight group. The results of this study suggest that patients with PKU and excess weight are potentially vulnerable to the development of metabolic syndrome. Therefore, it is necessary to conduct clinical and laboratory monitoring, aiming to prevent metabolic changes, as well as excessive weight gain and its consequences, particularly cardiovascular risk. Copyright © 2014 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

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

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.

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...

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

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

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

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

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.

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.

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

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

Construction of PAH-degrading mixed microbial consortia by induced selection in soil.

PubMed

Zafra, German; Absalón, Ángel E; Anducho-Reyes, Miguel Ángel; Fernandez, Francisco J; Cortés-Espinosa, Diana V

2017-04-01

Bioremediation of polycyclic aromatic hydrocarbons (PAHs)-contaminated soils through the biostimulation and bioaugmentation processes can be a strategy for the clean-up of oil spills and environmental accidents. In this work, an induced microbial selection method using PAH-polluted soils was successfully used to construct two microbial consortia exhibiting high degradation levels of low and high molecular weight PAHs. Six fungal and seven bacterial native strains were used to construct mixed consortia with the ability to tolerate high amounts of phenanthrene (Phe), pyrene (Pyr) and benzo(a)pyrene (BaP) and utilize these compounds as a sole carbon source. In addition, we used two engineered PAH-degrading fungal strains producing heterologous ligninolytic enzymes. After a previous selection using microbial antagonism tests, the selection was performed in microcosm systems and monitored using PCR-DGGE, CO 2 evolution and PAH quantitation. The resulting consortia (i.e., C1 and C2) were able to degrade up to 92% of Phe, 64% of Pyr and 65% of BaP out of 1000 mg kg -1 of a mixture of Phe, Pyr and BaP (1:1:1) after a two-week incubation. The results indicate that constructed microbial consortia have high potential for soil bioremediation by bioaugmentation and biostimulation and may be effective for the treatment of sites polluted with PAHs due to their elevated tolerance to aromatic compounds, their capacity to utilize them as energy source. Copyright © 2016 Elsevier Ltd. All rights reserved.

Highly potent antagonists of luteinizing hormone-releasing hormone free of edematogenic effects.

PubMed

Bajusz, S; Kovacs, M; Gazdag, M; Bokser, L; Karashima, T; Csernus, V J; Janaky, T; Guoth, J; Schally, A V

1988-03-01

To eliminate the undesirable edematogenic effect of the luteinizing hormone-releasing hormone (LH-RH) antagonists containing basic D amino acids at position 6, exemplified by [Ac-D-Phe(pCl)1,2,D-Trp3,D-Arg6,D-Ala10]LH-RH [Phe(pCl) indicates 4-chlorophenylalanine], analogs with D-ureidoalkyl amino acids such as D-citrulline (D-Cit) or D-homocitrulline (D-Hci) at position 6 were synthesized and tested in several systems in vitro and in vivo. HPLC analysis revealed that the overall hydrophobicity of the D-Cit/D-Hci6 analogs was similar to that of the basic D-Arg6 antagonists. In vitro, most of the analogs completely inhibited LH-RH-mediated luteinizing hormone release in perfused rat pituitary cell systems at an antagonist to LH-RH molar ratio of 5:1. In vivo, the most active peptides, [Ac-D-Nal(2)1,D-Phe(pCl)2,D-Trp3,D-Cit6,D-Ala10]LH-RH [Nal(2) indicates 3-(2-naphthyl)alanine] and its D-Hci6 analog, caused 100% inhibition of ovulation in cycling rats in doses of 3 micrograms and suppressed the luteinizing hormone level in ovariectomized female rats for 47 hr when administered at doses of 25 micrograms. Characteristically, these peptides did not exert any edematogenic effects even at 1.5 mg/kg. These properties of the D-Cit/D-Hci6 antagonists may make them useful clinically.

Highly potent antagonists of luteinizing hormone-releasing hormone free of edematogenic effects.

PubMed Central

Bajusz, S; Kovacs, M; Gazdag, M; Bokser, L; Karashima, T; Csernus, V J; Janaky, T; Guoth, J; Schally, A V

1988-01-01

To eliminate the undesirable edematogenic effect of the luteinizing hormone-releasing hormone (LH-RH) antagonists containing basic D amino acids at position 6, exemplified by [Ac-D-Phe(pCl)1,2,D-Trp3,D-Arg6,D-Ala10]LH-RH [Phe(pCl) indicates 4-chlorophenylalanine], analogs with D-ureidoalkyl amino acids such as D-citrulline (D-Cit) or D-homocitrulline (D-Hci) at position 6 were synthesized and tested in several systems in vitro and in vivo. HPLC analysis revealed that the overall hydrophobicity of the D-Cit/D-Hci6 analogs was similar to that of the basic D-Arg6 antagonists. In vitro, most of the analogs completely inhibited LH-RH-mediated luteinizing hormone release in perfused rat pituitary cell systems at an antagonist to LH-RH molar ratio of 5:1. In vivo, the most active peptides, [Ac-D-Nal(2)1,D-Phe(pCl)2,D-Trp3,D-Cit6,D-Ala10]LH-RH [Nal(2) indicates 3-(2-naphthyl)alanine] and its D-Hci6 analog, caused 100% inhibition of ovulation in cycling rats in doses of 3 micrograms and suppressed the luteinizing hormone level in ovariectomized female rats for 47 hr when administered at doses of 25 micrograms. Characteristically, these peptides did not exert any edematogenic effects even at 1.5 mg/kg. These properties of the D-Cit/D-Hci6 antagonists may make them useful clinically. PMID:3278323

The Dehydratase ADT3 Affects ROS Homeostasis and Cotyledon Development1[OPEN

PubMed Central

Para, Alessia; Muhammad, DurreShahwar; Naldrett, Michael J.; Warpeha, Katherine M.

2016-01-01

During the transition from seed to seedling, emerging embryos strategically balance available resources between building up defenses against environmental threats and initiating the developmental program that promotes the switch to autotrophy. We present evidence of a critical role for the phenylalanine (Phe) biosynthetic activity of AROGENATE DEHYDRATASE3 (ADT3) in coordinating reactive oxygen species (ROS) homeostasis and cotyledon development in etiolated Arabidopsis (Arabidopsis thaliana) seedlings. We show that ADT3 is expressed in the cotyledon and shoot apical meristem, mainly in the cytosol, and that the epidermis of adt3 cotyledons contains higher levels of ROS. Genome-wide proteomics of the adt3 mutant revealed a general down-regulation of plastidic proteins and ROS-scavenging enzymes, corroborating the hypothesis that the ADT3 supply of Phe is required to control ROS concentration and distribution to protect cellular components. In addition, loss of ADT3 disrupts cotyledon epidermal patterning by affecting the number and expansion of pavement cells and stomata cell fate specification; we also observed severe alterations in mesophyll cells, which lack oil bodies and normal plastids. Interestingly, up-regulation of the pathway leading to cuticle production is accompanied by an abnormal cuticle structure and/or deposition in the adt3 mutant. Such impairment results in an increase in cell permeability and provides a link to understand the cell defects in the adt3 cotyledon epidermis. We suggest an additional role of Phe in supplying nutrients to the young seedling. PMID:27540109

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...

In Vitro and in Vivo Analysis of the Binding of the C Terminus of the HDL Receptor Scavenger Receptor Class B, Type I (SR-BI), to the PDZ1 Domain of Its Adaptor Protein PDZK1*

PubMed Central

Kocher, Olivier; Birrane, Gabriel; Tsukamoto, Kosuke; Fenske, Sara; Yesilaltay, Ayce; Pal, Rinku; Daniels, Kathleen; Ladias, John A. A.; Krieger, Monty

2010-01-01

The PDZ1 domain of the four PDZ domain-containing protein PDZK1 has been reported to bind the C terminus of the HDL receptor scavenger receptor class B, type I (SR-BI), and to control hepatic SR-BI expression and function. We generated wild-type (WT) and mutant murine PDZ1 domains, the mutants bearing single amino acid substitutions in their carboxylate binding loop (Lys14-Xaa4-Asn19-Tyr-Gly-Phe-Phe-Leu24), and measured their binding affinity for a 7-residue peptide corresponding to the C terminus of SR-BI (503VLQEAKL509). The Y20A and G21Y substitutions abrogated all binding activity. Surprisingly, binding affinities (Kd) of the K14A and F22A mutants were 3.2 and 4.0 μm, respectively, similar to 2.6 μm measured for the WT PDZ1. To understand these findings, we determined the high resolution structure of WT PDZ1 bound to a 5-residue sequence from the C-terminal SR-BI (505QEAKL509) using x-ray crystallography. In addition, we incorporated the K14A and Y20A substitutions into full-length PDZK1 liver-specific transgenes and expressed them in WT and PDZK1 knock-out mice. In WT mice, the transgenes did not alter endogenous hepatic SR-BI protein expression (intracellular distribution or amount) or lipoprotein metabolism (total plasma cholesterol, lipoprotein size distribution). In PDZK1 knock-out mice, as expected, the K14A mutant behaved like wild-type PDZK1 and completely corrected their hepatic SR-BI and plasma lipoprotein abnormalities. Unexpectedly, the 10–20-fold overexpressed Y20A mutant also substantially, but not completely, corrected these abnormalities. The results suggest that there may be an additional site(s) within PDZK1 that bind(s) SR-BI and mediate(s) productive SR-BI-PDZK1 interaction previously attributed exclusively to the canonical binding of the C-terminal SR-BI to PDZ1. PMID:20739281

In vitro and in vivo Analysis of the Binding of the C Terminus of the HDL Receptor Scavenger Receptor Class B type I (SR-BI) to the PDZ1 Domain of its Cytoplasmic Adaptor Protein PDZK1

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

O Kocher; G Birrane; K Tsukamoto

2011-12-31

The PDZ1 domain of the four PDZ domain-containing protein PDZK1 has been reported to bind the C terminus of the HDL receptor scavenger receptor class B, type I (SR-BI), and to control hepatic SR-BI expression and function. We generated wild-type (WT) and mutant murine PDZ1 domains, the mutants bearing single amino acid substitutions in their carboxylate binding loop (Lys(14)-Xaa(4)-Asn(19)-Tyr-Gly-Phe-Phe-Leu(24)), and measured their binding affinity for a 7-residue peptide corresponding to the C terminus of SR-BI ((503)VLQEAKL(509)). The Y20A and G21Y substitutions abrogated all binding activity. Surprisingly, binding affinities (K(d)) of the K14A and F22A mutants were 3.2 and 4.0 ?M,more » respectively, similar to 2.6 ?M measured for the WT PDZ1. To understand these findings, we determined the high resolution structure of WT PDZ1 bound to a 5-residue sequence from the C-terminal SR-BI ((505)QEAKL(509)) using x-ray crystallography. In addition, we incorporated the K14A and Y20A substitutions into full-length PDZK1 liver-specific transgenes and expressed them in WT and PDZK1 knock-out mice. In WT mice, the transgenes did not alter endogenous hepatic SR-BI protein expression (intracellular distribution or amount) or lipoprotein metabolism (total plasma cholesterol, lipoprotein size distribution). In PDZK1 knock-out mice, as expected, the K14A mutant behaved like wild-type PDZK1 and completely corrected their hepatic SR-BI and plasma lipoprotein abnormalities. Unexpectedly, the 10-20-fold overexpressed Y20A mutant also substantially, but not completely, corrected these abnormalities. The results suggest that there may be an additional site(s) within PDZK1 that bind(s) SR-BI and mediate(s) productive SR-BI-PDZK1 interaction previously attributed exclusively to the canonical binding of the C-terminal SR-BI to PDZ1.« less

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.

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.

Identification and Functional Characterization of the Glycogen Synthesis Related Gene Glycogenin in Pacific Oysters (Crassostrea gigas).

PubMed

Li, Busu; Meng, Jie; Li, Li; Liu, Sheng; Wang, Ting; Zhang, Guofan

2017-09-06

High glycogen levels in the Pacific oyster (Crassostrea gigas) contribute to its flavor, quality, and hardiness. Glycogenin (CgGN) is the priming glucosyltransferase that initiates glycogen biosynthesis. We characterized the full sequence and function of C. gigas CgGN. Three CgGN isoforms (CgGN-α, β, and γ) containing alternative exon regions were isolated. CgGN expression varied seasonally in the adductor muscle and gonadal area and was the highest in the adductor muscle. Autoglycosylation of CgGN can interact with glycogen synthase (CgGS) to complete glycogen synthesis. Subcellular localization analysis showed that CgGN isoforms and CgGS were located in the cytoplasm. Additionally, a site-directed mutagenesis experiment revealed that the Tyr200Phe and Tyr202Phe mutations could affect CgGN autoglycosylation. This is the first study of glycogenin function in marine bivalves. These findings will improve our understanding of glycogen synthesis and accumulation mechanisms in mollusks. The data are potentially useful for breeding high-glycogen oysters.

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…

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.

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.

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.

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

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.

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

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

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.

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