Phenylalanine requirements using the direct amino acid oxidation technique, and the effects of dietary phenylalanine on food intake, gastric emptying, and macronutrient metabolism in adult cats.

Despite Phe being an indispensable amino acid for cats, the minimum Phe requirement for adult cats has not been empirically defined. The objective of study 1 was to determine the minimum Phe requirement, where Tyr is in excess, in adult cats using the direct amino acid oxidation (DAAO) technique. Four adult male cats were used in an 8 × 4 Latin rectangle design. Cats were adapted to a basal diet for 7 d, top dressed with Phe to meet 140% of the adequate intake (NRC, 2006. Nutrient requirements of dogs and cats. Washington, DC: Natl. Acad. Press). Cats were randomly assigned to one of eight experimental Phe diets (0.29%, 0.34%, 0.39%, 0.44%, 0.54%, 0.64%, 0.74%, and 0.84% Phe in the diet on a dry matter [DM] basis). Following 1 d of diet adaptation, individual DAAO studies were performed. During each DAAO study, cats were placed into individual indirect calorimetry chambers, and 75% of the cat's daily meal was divided into 13 equal meals supplied with a dose of L-[1-13C]-Phe. Oxidation of L-[1-13C]-Phe (F13CO2) during isotopic steady state was determined from the enrichment of 13CO2 in breath. Competing models were applied using the NLMIXED procedure in SAS to determine the effects of dietary Phe on 13CO2. The mean population minimum requirement for Phe was estimated at 0.32% DM and the upper 95% population confidence limit at 0.59% DM on an energy density of 4,200 kcal of metabolizable energy/kg DM calculated using the modified Atwater factors. In study 2, the effects of a bolus dose of Phe (44 mg kg-1 BW) on food intake, gastric emptying (GE), and macronutrient metabolism were assessed in a crossover design with 12 male cats. For food intake, cats were given Phe 15 min before 120% of their daily food was offered and food intake was measured. Treatment, day, and their interaction were evaluated using PROC GLIMMIX in SAS. Treatment did not affect any food intake parameters (P > 0.05). For GE and macronutrient metabolism, cats were placed into individual indirect calorimetry chambers, received the same bolus dose of Phe, and 15 min later received 13C-octanoic acid (5 mg kg-1 BW) on 50% of their daily food intake. Breath samples were collected to measure 13CO2. The effect of treatment was evaluated using PROC GLIMMIX in SAS. Treatment did not affect total GE (P > 0.05), but cats receiving Phe tended to delay time to peak enrichment (0.05 < P ≤ 0.10). Overall, Phe at a bolus dose of 44 mg kg-1 BW had no effect on food intake, GE, or macronutrient metabolism. Together, these results suggest that the bolus dose of Phe used may not be sufficient to elicit a GE response, but a study with a greater number of cats and greater food intake is warranted.

Two studies were conducted to evaluate 1) the minimum requirement for dietary Phe and 2) the effects of Phe on gastric emptying (GE) and food intake in adult cats. In study 1, the minimum Phe requirement was estimated using the direct amino acid oxidation (DAAO) technique. Four cats were used and received all diets in random order in a Latin rectangle design (0.29%, 0.34%, 0.39%, 0.44%, 0.54%, 0.64%, 0.74%, and 0.84% Phe in the diet on a dry matter [DM] basis). The minimum Phe requirement, in the presence of excess of Tyr, for adult cats was estimated to be 0.59% DM on an energy density of 4,200 kcal of metabolizable energy/kg DM calculated using the modified Atwater factors; higher than current recommendations set in place by the National Research Council and the American Association of Feed Control Officials. In study 2, we first validated the use of the 13C-octanoic acid breath test (13C-OABT) in cats. Then, the effects of an oral bolus of Phe on food intake, GE, and macronutrient metabolism were evaluated. Phe supplementation did not influence food intake, macronutrient metabolism, or total GE, but tended to delay the time to peak GE.

Anti-amyloidogenic amphipathic arginine-dehydrophenylalanine spheres capped selenium nanoparticles as potent therapeutic moieties for Alzheimer's disease.

Aggregation of both amyloid beta (Aß) peptide and hyperphosphorylated tau proteins is the major pathological hallmark of Alzheimer's disease (AD). Moieties that carry anti-amyloidogenic potency against both of the aggregating entities are considered to be promising drug candidatures for the disease. In the current work, we have synthesized amphipathic dipeptide vesicle-templated selenium nanoparticles (RΔF-SeNPs) as potential entities to combat AD. We have investigated and established their anti-amyloidogenic activity against different peptide-based amyloid models, such as the reductionist model based on the dipeptide phenylalanine-phenylalanine (FF) derived from Aß; a model based on the hexapeptide Ac-PHF6 (306VQIVYK311) derived from tau protein; and the full-length Aß42 polypeptide-based model. We also evaluated the neuroprotective characteristics of RΔF-SeNPs against FF, Ac-PHF6, and Aß42 fibril-induced toxicity in neuroblastoma, SH-SY5Y cells. RΔF-SeNPs further exhibited neuroprotective effects in streptozotocin (STZ) treated neuronal (N2a) cells carrying AD-like features. In addition, studies conducted in an intra-cerebroventricular STZ-instigated rat model of dementia revealed that RΔF-SeNP-treated animals showed improved cognitive activity and reduced Aß42 aggregate burden in brain tissues as compared with the STZ-treated group. Moreover, in vivo brain distribution studies conducted in animal models additionally demonstrated the brain-homing ability of RΔF-SeNPs. All together, these studies supported the potency of RΔF-SeNPs as efficient and propitious disease-modifying therapeutic agents for combating AD.

Green Tea Catechol (-)-Epigallocatechin Gallate (EGCG) Conjugated with Phenylalanine Shows Enhanced Autophagy Stimulating Activity in Human Aortic Endothelial Cells.

(-)-Epigallocatechin gallate (EGCG) is one of the autophagy stimulators that have been reported to protect vascular endothelial cells from oxidative stress-induced damage. In this study, we attempted potentiation of the autophagy-stimulating activity of EGCG in human aortic epithelial cells (HAECs) by using the EGCG-phenylalanine conjugate, E10. Autophagy-stimulating activity of E10 was evaluated by LC3-II measurement in the absence and presence of the lysosomal blocker chloroquine, CTYO-ID staining, and reporter assay using tandem fluorescence-tagged LC3. These experiments revealed significantly enhanced autophagic flux stimulation in HAECs by E10 compared with EGCG. Further elaboration of E10 showed that activation of AMPK through phosphorylation as the major mechanism of its autophagy stimulation. Like other autophagy stimulators, E10 protected HAECs from lipotoxicity as well as accompanying endothelial senescence. Finally, stimulation of autophagy by E10 was shown to protect HAECs from oxidative stress-induced apoptosis. These findings collectively suggest potential clinical implications of E10 for various cardiovascular complications through stimulation of autophagy.

Directed-evolution of translation system for efficient unnatural amino acids incorporation and generalizable synthetic auxotroph construction.

Site-specific incorporation of unnatural amino acids (UAAs) with similar incorporation efficiency to that of natural amino acids (NAAs) and low background activity is extremely valuable for efficient synthesis of proteins with diverse new chemical functions and design of various synthetic auxotrophs. However, such efficient translation systems remain largely unknown in the literature. Here, we describe engineered chimeric phenylalanine systems that dramatically increase the yield of proteins bearing UAAs, through systematic engineering of the aminoacyl-tRNA synthetase and its respective cognate tRNA. These engineered synthetase/tRNA pairs allow single-site and multi-site incorporation of UAAs with efficiencies similar to those of NAAs and high fidelity. In addition, using the evolved chimeric phenylalanine system, we construct a series of E. coli strains whose growth is strictly dependent on exogenously supplied of UAAs. We further show that synthetic auxotrophic cells can grow robustly in living mice when UAAs are supplemented.

Efficacy of Boron Neutron Capture Therapy in Primary Central Nervous System Lymphoma: In Vitro and In Vivo Evaluation.

BACKGROUND: Boron neutron capture therapy (BNCT) is a nuclear reaction-based tumor cell-selective particle irradiation method. High-dose methotrexate and whole-brain radiation therapy (WBRT) are the recommended treatments for primary central nervous system lymphoma (PCNSL). This tumor responds well to initial treatment but relapses even after successful treatment, and the prognosis is poor as there is no safe and effective treatment for relapse. In this study, we aimed to conduct basic research to explore the possibility of using BNCT as a treatment for PCNSL. METHODS: The boron concentration in human lymphoma cells was measured. Subsequently, neutron irradiation experiments on lymphoma cells were conducted. A mouse central nervous system (CNS) lymphoma model was created to evaluate the biodistribution of boron after the administration of borono-phenylalanine as a capture agent. In the neutron irradiation study of a mouse PCNSL model, the therapeutic effect of BNCT on PCNSL was evaluated in terms of survival. RESULTS: The boron uptake capability of human lymphoma cells was sufficiently high both in vitro and in vivo. In the neutron irradiation study, the BNCT group showed a higher cell killing effect and prolonged survival compared with the control group. CONCLUSIONS: A new therapeutic approach for PCNSL is urgently required, and BNCT may be a promising treatment for PCNSL. The results of this study, including those of neutron irradiation, suggest success in the conduct of future clinical trials to explore the possibility of BNCT as a new treatment option for PCNSL.

Phenylalanine Increases the Production of Antioxidant Phenolic Acids in Ginkgo biloba Cell Cultures.

The aims of this study were to evaluate the antioxidant properties, to investigate the content of major secondary metabolites in Ginkgo biloba cell cultures, and to determine the change in the production of phenolic acids by adding phenylalanine to the culture medium. Three in vitro methods, which depend on different mechanisms, were used for assessing the antioxidant activity of the extract: 1,1-diphenyl-2-picrylhydrazil (DPPH), reducing power and Fe2+ chelating activity assays. The extract showed moderate activity both in the DPPH and in the reducing power assays (IC50 = 1.966 ± 0.058 mg/mL; ASE/mL = 16.31 ± 1.20); instead, it was found to possess good chelating properties reaching approximately 70% activity at the highest tested dose. The total phenolic, total flavonoid, and condensed tannin content of G. biloba cell culture extract was spectrophotometrically determined. The phenolic acid content was investigated by RP-HPLC, and the major metabolites-protocatechuic and p-hydroxybenzoic acids-were isolated and investigated by 1H NMR. The results showed that phenylalanine added to G. biloba cell cultures at concentrations of 100, 150, and 200 mg/150 mL increased the production of phenolic acids. Cultures that were grown for 3 weeks and collected after 4 days of phenylalanine supplementation at high concentration showed maximal content of phenolic acids (73.76 mg/100 g DW).

Pitolisant, a wake-promoting agent devoid of psychostimulant properties: Preclinical comparison with amphetamine, modafinil, and solriamfetol.

Several therapeutic options are currently available to treat excessive daytime sleepiness (EDS) in patients suffering from narcolepsy or obstructive sleep apnea. However, there are no comparisons between the various wake-promoting agents in terms of mechanism of action, efficacy, or safety. The goal of this study was to compare amphetamine, modafinil, solriamfetol, and pitolisant at their known primary pharmacological targets, histamine H3 receptors (H3R), dopamine, norepinephrine, and serotonin transporters, and in various in vivo preclinical models in relation to neurochemistry, locomotion, behavioral sensitization, and food intake. Results confirmed that the primary pharmacological effect of amphetamine, modafinil, and solriamfetol was to increase central dopamine neurotransmission, in part by inhibiting its transporter. Furthermore, solriamfetol increased levels of extracellular dopamine in the nucleus accumbens, and decreased the 3,4-dihydroxyphenyl acetic acid (DOPAC)/DA ratio in the striatum, as reported for modafinil and amphetamine. All these compounds produced hyperlocomotion, behavioral sensitization, and hypophagia, which are common features of psychostimulants and of compounds with abuse potential. In contrast, pitolisant, a selective and potent H3R antagonist/inverse agonist that promotes wakefulness, had no effect on striatal dopamine, locomotion, or food intake. In addition, pitolisant, devoid of behavioral sensitization by itself, attenuated the hyperlocomotion induced by either modafinil or solriamfetol. Therefore, pitolisant presents biochemical, neurochemical, and behavioral profiles different from those of amphetamine and other psychostimulants such as modafinil or solriamfetol. In conclusion, pitolisant is a differentiated therapeutic option, when compared with psychostimulants, for the treatment of EDS, as this agent does not show any amphetamine-like properties within in vivo preclinical models.

L-carnitine protects DNA oxidative damage induced by phenylalanine and its keto acid derivatives in neural cells: a possible pathomechanism and adjuvant therapy for brain injury in phenylketonuria.

Although phenylalanine (Phe) is known to be neurotoxic in phenylketonuria (PKU), its exact pathogenetic mechanisms of brain damage are still poorly known. Furthermore, much less is known about the role of the Phe derivatives phenylacetic (PAA), phenyllactic (PLA) and phenylpyruvic (PPA) acids that also accumulate in this this disorder on PKU neuropathology. Previous in vitro and in vivo studies have shown that Phe elicits oxidative stress in brain of rodents and that this deleterious process also occurs in peripheral tissues of phenylketonuric patients. In the present study, we investigated whether Phe and its derivatives PAA, PLA and PPA separately or in combination could induce reactive oxygen species (ROS) formation and provoke DNA damage in C6 glial cells. We also tested the role of L-carnitine (L-car), which has been recently considered an antioxidant agent and easily cross the blood brain barrier on the alterations of C6 redox status provoked by Phe and its metabolites. We first observed that cell viability was not changed by Phe and its metabolites. Furthermore, Phe, PAA, PLA and PPA, at concentrations found in plasma of PKU patients, provoked marked DNA damage in the glial cells separately and when combined. Of note, these effects were totally prevented (Phe, PAA and PPA) or attenuated (PLA) by L-car pre-treatment. In addition, a potent ROS formation also induced by Phe and PAA, whereas only moderate increases of ROS were caused by PPA and PLA. Pre-treatment with L-car also prevented Phe- and PAA-induced ROS generation, but not that provoked by PLA and PPA. Thus, our data show that Phe and its major metabolites accumulated in PKU provoke extensive DNA damage in glial cells probably by ROS formation and that L-car may potentially represent an adjuvant therapeutic agent in PKU treatment.

L-phenylalanine attenuates high salt-induced hypertension in Dahl SS rats through activation of GCH1-BH4.

Amino acid metabolism plays an important role in controlling blood pressure by regulating the production of NO and ROS. The present study examined amino acid levels in the serum of Dahl SS rats and SS.13BN rats fed a low or high salt diet. We observed that 8 of 27 amino acids responded to a high salt diet in SS rats. Thus, we hypothesized that a defect in amino acids may contribute to the development of salt-induced hypertension. L-phenylalanine was used to treat SS rats with a low or high salt diet. The results demonstrated that L-phenylalanine supplementation significantly enhanced the serum nitrite levels and attenuated the high salt-induced hypertension in SS rats. Low levels of BH4 and nitrite and the impaired vascular response to acetylcholine were rescued by L-phenylalanine supplementation. Moreover, increased GTP cyclohydrolase (GCH1) mRNA, levels of BH4 and nitrite, and reduced superoxide production were observed in the kidneys of hypertensive SS rats with L-phenylalanine. The antihypertensive effects of L-phenylalanine might be mediated by enhancing BH4 biosynthesis and decreasing superoxide production from NO synthase, thereby protecting vascular and kidney function with reduced ROS and elevated NO levels. The present study demonstrated that L-phenylalanine supplementation restored vascular function, suggesting L-phenylalanine represented a potential target to attenuate high salt-sensitive hypertension through GCH1-BH4.

Multidisciplinary Approaches Identify Compounds that Bind to Human ACE2 or SARS-CoV-2 Spike Protein as Candidates to Block SARS-CoV-2-ACE2 Receptor Interactions.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently emerged virus that causes coronavirus infectious disease 2019 (COVID-19). SARS-CoV-2 spike protein, like SARS-CoV-1, uses the angiotensin converting enzyme 2 (ACE2) as a cellular receptor to initiate infection. Compounds that interfere with the SARS-CoV-2 spike protein receptor binding domain protein (RBD)-ACE2 receptor interaction may function as entry inhibitors. Here, we used a dual strategy of molecular docking and surface plasmon resonance (SPR) screening of compound libraries to identify those that bind to human ACE2 or the SARS-CoV-2 spike protein receptor binding domain (RBD). Molecular modeling screening interrogated 57,641 compounds and focused on the region of ACE2 that is engaged by RBD of the SARS-CoV-2 spike glycoprotein and vice versa. SPR screening used immobilized human ACE2 and SARS-CoV-2 Spike protein to evaluate the binding of these proteins to a library of 3,141 compounds. These combined screens identified compounds from these libraries that bind at KD (equilibrium dissociation constant) <3 µM affinity to their respective targets, 17 for ACE2 and 6 for SARS-CoV-2 RBD. Twelve ACE2 binders and six of the RBD binders compete with the RBD-ACE2 interaction in an SPR-based competition assay. These compounds included registered drugs and dyes used in biomedical applications. A Vero-E6 cell-based SARS-CoV-2 infection assay was used to evaluate infection blockade by candidate entry inhibitors. Three compounds demonstrated dose-dependent antiviral in vitro potency-Evans blue, sodium lifitegrast, and lumacaftor. This study has identified potential drugs for repurposing as SARS-CoV-2 entry inhibitors or as chemical scaffolds for drug development.IMPORTANCE SARS-CoV-2, the causative agent of COVID-19, has caused more than 60 million cases worldwide with almost 1.5 million deaths as of November 2020. Repurposing existing drugs is the most rapid path to clinical intervention for emerging diseases. Using an in silico screen of 57,641 compounds and a biophysical screen of 3,141 compounds, we identified 22 compounds that bound to either the angiotensin converting enzyme 2 (ACE2) and/or the SARS-CoV-2 spike protein receptor binding domain (SARS-CoV-2 spike protein RBD). Nine of these drugs were identified by both screening methods. Three of the identified compounds, Evans blue, sodium lifitegrast, and lumacaftor, were found to inhibit viral replication in a Vero-E6 cell-based SARS-CoV-2 infection assay and may have utility as repurposed therapeutics. All 22 identified compounds provide scaffolds for the development of new chemical entities for the treatment of COVID-19.

Application of self-assembly peptides targeting the mitochondria as a novel treatment for sorafenib-resistant hepatocellular carcinoma cells.

Currently, there is no appropriate treatment option for patients with sorafenib-resistant hepatocellular carcinoma (HCC). Meanwhile, pronounced anticancer activities of newly-developed mitochondria-accumulating self-assembly peptides (Mito-FF) have been demonstrated. This study intended to determine the anticancer effects of Mito-FF against sorafenib-resistant Huh7 (Huh7-R) cells. Compared to sorafenib, Mito-FF led to the generation of relatively higher amounts of mitochondrial reactive oxygen species (ROS) as well as the greater reduction in the expression of antioxidant enzymes (P < 0.05). Mito-FF was found to significantly promote cell apoptosis while inhibiting cell proliferation of Huh7-R cells. Mito-FF also reduces the expression of antioxidant enzymes while significantly increasing mitochondrial ROS in Huh7-R cells. The pro-apoptotic effect of Mito-FFs for Huh7-R cells is possibly caused by their up-regulation of mitochondrial ROS, which is caused by the destruction of the mitochondria of HCC cells.

Feedback control of two supplemental feeds during fed-batch culture on a platform process using inline Raman models for glucose and phenylalanine concentration.

The use of Raman models for glucose and phenylalanine concentrations to provide the signal for a control algorithm to continuously adjust the feed rate of two separate supplemental feeds during the fed-batch culture of a CHOK1SV GS-KO® cell line in a platform process was evaluated. Automated feed rate adjustment of the glucose feed using a Raman model for glucose concentration, maintained the glucose concentration within the desired target (average deviation ± 0.49 g/L). Automated feed rate adjustment of the nutrient feed using a Raman model for phenylalanine concentration, maintained phenylalanine concentrations within the target (average deviation ± 29.97 mg/L). The novel use of a Raman model for phenylalanine concentration, combined with a Raman model for glucose concentration, to maintain target glucose and phenylalanine concentrations through feed-rate adjustments, reduced the average cumulative glucose and nutrient feed additions (19% and 27% respectively) compared to manually adjusted cultures. Additionally, the proposed automation strategy led to lower osmolality during culture, maintained the nutrient environment more consistently, and achieved higher harvest product concentration (≈ 20% higher) compared to typical fed-batch process control for the cell line and platform process evaluated. Furthermore, the proposed feeding strategy yielded similar glycosylation and charge variant profiles compared to manually adjusted fed-batch process control. The ability to continuously adjust the feed rate addition of two separate feeds in this manner helps enable a shift away from the current daily offline sampling needed to control fed-batch mammalian cell culture during clinical and commercial manufacturing on platform processes.

Follow up long term preliminary studies on carcinogenic and toxic effects of ochratoxin A in rats and the putative protection of phenylalanine.

Carcinogenic effects of ochratoxin A (OTA) on liver, kidneys, intestine, lung and eyes of Wistar rats exposed to 10 ppm or 5 ppm OTA in the diet and additionally supplemented or not with phenylalanine (PHE) were examined during 24-months experimental period. OTA was seen to provoke strong degenerative changes and slight pericapillary oedema in most internal organs, e.g. kidneys, liver, intestine, spleen and brain. Six of total nine neoplasms were identified as malignant and three as benign. Five of total six malignant neoplasms and two of total three benign neoplasms were seen in male rats. The pathological finding in rats after two weeks feeding with OTA-contaminated feed was dominated by degenerative changes in various internal organs, which were weaker in the group additionally supplemented with PHE. The protective effect of PHE was evident with respect to OTA-induced decrease of serum glucose and serum protein, but this protection was not singnificant with respect to serum enzymes activity. The number of neoplasms in PHE-supplemented group exposed to 10 ppm OTA was similar to that in the group exposed to twice lower feed levels of OTA alone, suggesting about a possible protective effect of PHE. The rats would not be able to serve as experimental model for humans with regard to OTA-induced tumorigenesis, because the target organ of OTA-toxicity in humans and pigs is mainly the kidney as opposed to the significant damages and carcinogenic effects seen in various organs in rats exposed to OTA.

Antimicrobial secondary metabolites of an endolichenic Aspergillus niger isolated from lichen thallus of Parmotrema ravum.

A new 6-benzyl-γ-pyrone (1), named aspergyllone was isolated from the culture filtrates of an endolichenic fungus Aspergillus niger Tiegh, obtained from lichen thallus Parmotrema ravum (Krog & Swinscow) Serus, collected in India. 1 was isolated for the first time from an endolichenic fungus together with six other known metabolites identified as aurasperones A (2) and D (3), asperpyrone A (4), fonsecinone A (5), carbonarone A (6) and pyrophen (7). The compounds were tested against a panel of human, plant, food borne and fish pathogens. Aspergyllone showed strong selective antifungal activity against Candida parapsilosis (Ashford) Langeron & Talice, with an IC50 of 52 µg/mL. Aurasperone A and pyrophen showed moderate to strong antimicrobial activity inhibiting seven different test pathogens, being pyrophen active with IC50 ranging from 35 to 97 µg/mL.[Formula: see text].

Antidiabetic effect of free amino acids supplementation in human visceral adipocytes through adiponectin-dependent mechanism.

BACKGROUND & OBJECTIVES: : Amino acids are general nutrients having anti-diabetic property. The present study was undertaken to investigate the mechanism of anti-diabetic effects of amino acids in human visceral adipocyte cells in high glucose environment. METHODS: : Experiments were carried out in human visceral adipocytes. Adiponectin (APN) siRNAs were designed using Ambion tools. APN mRNA expression was quantified using real-time polymerase chain reaction, and protein level was studied using ELISA. AMP-activated kinase (AMPK) activity was measured and glucose uptake by 2-deoxyglucose uptake method. RESULTS: : Amino acids (proline and phenylalanine) exposure to adipocytes significantly (P <0.01) increased APN mRNA by 1.5-folds when compared to control whereas proline increased APN secretion by 10.6-folds (P <0.01), phenylalanine by 12.7-folds (P <0.001) and alanine by 6.3-folds (P <0.01). Free amino acid-induced AMPK activity and glucose uptake were decreased with the transient knockdown of APN. INTERPRETATION & CONCLUSIONS: : Antidiabetic effect of the tested amino acids was exhibited by increased glucose uptake through the AMPK pathway by an APN-dependent mechanism in human visceral adipocytes. This should be tested and confirmed in in vivo system. Newer treatment modalities with amino acids which can enhance glucose uptake and APN secretion can be developed as drug for treating both diabetes and obesity.

Effects of dietary leucine and phenylalanine on gastrointestinal development and small intestinal enzyme activities in milk-fed holstein dairy calves.

This study was investigated the effects of dietary supplementation of leucine and phenylalanine on the development of the gastrointestinal tract and the intestinal digestive enzyme activity in male Holstein dairy calves. Twenty calves with a body weight of 38 ± 3 kg at 1 day of age were randomly divided into four groups: a control group, a leucine group (1.435 g·l-1), a phenylalanine group (0.725 g·l-1), and a mixed amino acid group (1.435 g·l-1 leucine plus 0.725 g·l-1 phenylalanine). The supplementation of leucine decreased the short-circuit current (Isc) of the rumen and duodenum (P<0.01); phenylalanine did not show any influence on the Isc of rumen and duodenum (P>0.05), and also counteracted the Isc reduction caused by leucine. Leucine increased the trypsin activity at the 20% relative site of the small intestine (P<0.05). There was no difference in the activity of α-amylase and of lactase in the small intestinal chyme among four treatments (P>0.05). The trypsin activity in the anterior segment of the small intestine was higher than other segments, whereas the α-amylase activity in the posterior segment of the small intestine was higher than other segments. Leucine can reduce Isc of the rumen and duodenum, improve the development of the gastrointestinal tract, and enhance trypsin activity; phenylalanine could inhibit the effect of leucine in promoting intestinal development.

Amphiphilic bromelain-synthesized oligo-phenylalanine grafted with methoxypolyethylene glycol possessing stabilizing thermo-responsive emulsion properties.

A thermo-responsive amphiphile was developed from oligo-phenylalanine [oligo(Phe)]. The hydrophobic moiety of the amphiphile, oligo(Phe) was synthesized via reverse hydrolysis catalyzed by bromelain in dimethyl sulfoxide and dioxane solutions. The production of oligo(Phe) increased by 80.7% by screening suitable reaction conditions. The average degree of polymerization of oligo(Phe) was determined to be four by 1H NMR. By grafting with aldehyde-ended methoxypolyethylene glycol (mPEG), oligo(Phe) was converted to amphiphilic oligo(Phe)-mPEG. The surface tension of oligo(Phe)-mPEG solution increased with decreasing chain length of the mPEG moiety. Cytotoxicity studies showed oligo(Phe)-mPEGs are biocompatible. On varying temperature, a reversible phase transition of oligo(Phe)-mPEG solutions could be observed. N-octane-in-water emulsions and 0.5% beta-carotene containing squalene-in-water emulsions stabilized by oligo(Phe)-mPEGs occurred at 25 °C but de-emulsification took place at >40 °C. Emulsification could be restored once the separated mixture cooled and re-homogenized. The emulsification/de-emulsification cycling could be repeated many times. The time required for de-emulsification decreased with elevated temperature but increased with a reduced concentration of oligo(Phe)-mPEGs and a reduction in the chain length of the mPEG moiety.

Enhanced accumulation of harpagide and 8-O-acetyl-harpagide in Melittis melissophyllum L. agitated shoot cultures analyzed by UPLC-MS/MS.

Harpagide and its derivatives have valuable medicinal properties, such as anti-inflammatory, analgesic and potential antirheumatic effects. There is the demand for searching plant species containing these iridoids or developing biotechnological methods to obtain the compounds. The present study investigated the effects of methyl jasmonate (MeJa, 50 µM), ethephon (Eth, 50 µM) and L-phenylalanine (L-Phe, 2.4 g/L of medium), added to previously selected variant of Murashige and Skoog medium (supplemented with plant growth regulators: 6-benzylaminopurine 1.0 mg/L, α-naphthaleneacetic acid 0.5 mg/L, gibberellic acid 0.25 mg/L) on the accumulation of harpagide and 8-O-acetyl-harpagide in Melittis melissophyllum L. agitated shoot cultures. Plant material was harvested 2 and 8 days after the supplementation. Iridoids were quantitatively analyzed by the UPLC-MS/MS method in extracts from the biomass and the culture medium. It was found that all of the variants caused an increase in the accumulation of harpagide. In the biomass harvested after 2 days, the highest harpagide content of 247.3 mg/100 g DW was found for variant F (L-Phe and Eth), and the highest 8-O-acetyl-harpagide content of 138 mg/100 g DW for variant E (L-Phe and MeJa). After 8 days, in some variants, a portion of the metabolites was released into the culture medium. Considering the total amount of the compounds (in the biomass and medium), the highest accumulation of harpagide, amounting to 619 mg/100 g DW, was found in variant F, and the highest amount of 8-O-acetyl-harpagide, of 255.4 mg/100 g DW, was found in variant H (L-Phe, MeJa, Eth) when harvested on the 8th day. These amounts were, respectively, 24.7 and 4.8 times higher than in the control culture, and were, respectively, 15 and 6.7 times higher than in the leaves of the soil-grown plant. The total amount of the two iridoids was highest for variant F (0.78% DW) and variant H (0.68% DW) when harvested on the 8th day. The results indicate that the agitated shoot cultures of M. melissophyllum can be a rich source of harpagide and 8-O-acetyl-harpagide, having a potential practical application. To the best of our knowledge we present for the first time the results of the quantitative UPLC-MS/MS analysis of harpagide and 8-O-acetyl-harpagide in M. melissophyllum shoot cultures and the enhancement of their accumulation by means of medium supplementation with elicitors and precursor.

The effects of phenylalanine on exercise-induced fat oxidation: a preliminary, double-blind, placebo-controlled, crossover trial.

BACKGROUND: When combined with exercise, dietary amino acid (AA) supplementation is an effective method for accelerating fat mobilization. However, the effects of single AAs combined with exercise on fat oxidation remains unclear. We hypothesized that consumption of a specific amino acid, L- phenylalanine, may result in the secretion of glucagon, and when combined with exercise may promote fat oxidation. METHODS: Six healthy, active male volunteers were randomized in a crossover study to ingest either phenylalanine (3 g/dose) or placebo. Thirty minutes after ingestion each subject performed workload trials on a cycle ergometer for 1 h at 50% of maximal oxygen consumption. RESULTS: Oral intake of phenylalanine caused a significant increase in the concentrations of plasma glycerol and glucagon during exercise. The respiratory exchange ratio was also decreased significantly following ingestion of phenylalanine. CONCLUSION: These results suggested that pre-exercise supplementation of phenylalanine may stimulate whole body fat oxidation. No serious or study-related adverse events were observed. TRIAL REGISTRATION: UMIN000027502 Registered 26 May 2017. Restrospectively registered.

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

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.