Modeling and resistant alleles explain the selectivity of antimalarial compound 49c towards apicomplexan aspartyl proteases.

Toxoplasma gondii aspartyl protease 3 (TgASP3) phylogenetically clusters with Plasmodium falciparum Plasmepsins IX and X (PfPMIX, PfPMX). These proteases are essential for parasite survival, acting as key maturases for secreted proteins implicated in invasion and egress. A potent antimalarial peptidomimetic inhibitor (49c) originally developed against Plasmepsin II selectively targets TgASP3, PfPMIX, and PfPMX To unravel the molecular basis for the selectivity of 49c, we constructed homology models of PfPMIX, PfPMX, and TgASP3 that were first validated by identifying the determinants of microneme and rhoptry substrate recognition. The flap and flap-like structures of several reported Plasmepsins are highly flexible and critically modulate the access to the binding cavity. Molecular docking of 49c to TgASP3, PfPMIX, and PfPMX models predicted that the conserved phenylalanine residues in the flap, F344, F291, and F305, respectively, account for the sensitivity toward 49c. Concordantly, phenylalanine mutations in the flap of the three proteases increase twofold to 15-fold the IC50 values of 49c. Compellingly the selection of mutagenized T. gondii resistant strains to 49c reproducibly converted F344 to a cysteine residue.

Three-year audit of the hyperphenylalaninaemia/phenylketonuria spectrum in Victoria.

AIMS: To determine the prevalence, the types and severity of hyperphenylalaninaemia (including phenylketonuria (PKU)) in Victoria and to report on a new treatment modality of PKU. METHODS: We reviewed the medical records of all patients diagnosed with high blood phenylalanine levels by newborn screening between November 2001 and October 2004. RESULTS: We identified 17 newborn babies with high levels of blood phenylalanine (total samples: 190,835). Dihydrobiopterin reductase deficiency was excluded in all babies. Five babies had persistent phenylalanine levels of 200-300, and do not receive any dietary or pharmaceutical therapy. One baby was diagnosed as having pyruvoyl tetrahydro-pterin synthase deficiency. Following reports of tetrahydrobiopterin (BH(4))-responsive PKU, we have performed a BH(4) load (20 mg/kg, 6R-5,6,7,8-tetrahydro-L-biopetrin dehydrochloride; Schricks Laboratories, Jona, Switzerland) in 10 newborn babies with PKU (one baby with a phenylalanine level of 2600 micromol/L was started on diet without prior load). Three babies had a significant response to BH(4) (>35% decrease in phenylalanine level). Protein restriction (1.2 g/kg/day) and introduction of phenylalanine-free formula, in addition to BH(4) treatment, were necessary in one patient. The other patients maintain good metabolic control with BH(4) treatment only (at approximately 11 mg/kg/day) and an intake of 2-3 g protein per day. Of the nine babies who are on a full PKU diet, three have high phenylalanine tolerance (consistently >40 mg/kg/day). CONCLUSION: There is a spectrum of severity of hyperphenylalaninaemia in the population. The detection of BH(4)-responsive PKU patients offers them a less restrictive dietary regimen and an improved quality of life, and may enable near normal life-style in adolescence.

Phenylalanine ammonia-lyase modified with polyethylene glycol: potential therapeutic agent for phenylketonuria.

Phenylketonuria (PKU) is an autosomal recessive genetic disease caused by the defects in the phenylalanine hydroxylase (PAH) gene. Individuals homozygous for defective PAH alleles show elevated levels of systemic phenylalanine and should be under strict dietary control to reduce the risk of neuronal damage associated with high levels of plasma phenylalanine. Researchers predict that plant phenylalanine ammonia-lyase (PAL), which converts phenylalanine to nontoxic t-cinnamic acid, will be an effective therapeutic enzyme for the treatment of PKU. The problems of this potential enzyme therapy have been the low stability in the circulation and the antigenicity of the plant enzyme. Recombinant PAL originated from parsley (Petroselinum crispum) chemically conjugated with activated PEG2 [2,4-bis(O-methoxypolyethyleneglycol)-6-chloro-s-triazine] showed greatly enhanced stability in the circulation and was effective in reducing the plasma concentration of phenylalanine in the circulation of mice. PEG-PAL conjugate will be an effective therapeutic enzyme for the treatment of PKU.

Hydroxyl radical oxidation of cytochrome c by aerobic radiolysis.

The reaction of radiolytically generated *OH with cytochrome c was investigated by mass spectrometry. Tryptic digestion and characterization of the oxidized peptides by MALDI-TOF and ESI tandem mass spectrometry identified eight different amino acid residues with oxidized side chains with no cleavage of the protein detected. Solvent-accessible aromatic and methionine residues are the most susceptible to oxidation by *OH. These results support the careful use of *OH in characterizing protein surfaces. Dose-response studies identified the residues most prone to oxidation to be Phe-36, Phe-46, and Met-80. Hydroxylation of Phe-36 and Phe-46 should serve as indicators of the presence of *OH in the mitochondrial intermembrane space. Using solutions containing 50 at.% (18)O, our study also provides a novel method of determining the source of oxygen during *OH-mediated oxidation of proteins and contributes to identification of the modified residue type, with Phe>Tyr>Met in (18)O incorporation. During aerobic radiolysis, UV-vis spectroscopy indicates that ferrocytochrome c reaches a steady state concomitant with reduction of the heme.

Pegylated interferon-alpha2b treatment in melanoma patients: influence on amino acids, 5-hydroxyindolacetic acid and pteridine plasma concentrations.

Our objective was to study the influence of pegylated interferon-alpha2b (PEG-IFN-alpha) on the metabolism of amino acids and pteridines. We used an exploratory study into plasma concentrations of large neutral amino acids, 5-hydroxyindolacetic acid (5-HIAA), total biopterin (BIOP) and neopterin (NEOP) in 40 high-risk melanoma patients. Patients were randomized to treatment with PEG-IFN-alpha once a week in a dose of 6 microg/kg/week s.c. during 8 weeks, followed by a maintenance treatment of 3 microg/kg/week s.c. or to observation only. We found that treatment with PEG-IFN-alpha decreases tryptophan (TRP) concentrations in the first 3 months of treatment to a maximum of 25.3% compared to controls [95% confidence interval (CI): 14.9 to 34.4]. The TRP:LNAA ratio, an index for the availability of TRP to the central nervous system (CNS), decreases during 6 months with 18.8% (95% CI: 11.9 to 25.2). Concentrations of NEOP rose; however, concentrations of BIOP, the sum of tetrahydrobiopterin [BH4] and its oxidative products, did not decrease. The ratio of phenylalanine to tyrosine was increased with 11.7% (95% CI: 1.0 to 23.5) during 6 months. We conclude that, like conventional IFN-alpha, PEG-IFN-alpha lowers TRP concentrations and decreases the availability of TRP to the CNS. PEG-IFN-alpha has a similar influence on pteridine metabolism as standard IFN-alpha. If a lowered availability of TRP and a consequent decrease of serotonergic neurotransmission are indeed a mechanism underlying neuropsychiatric side-effects of IFN-alpha, patients on PEG-IFN-alpha are not at a lower risk of developing neuropsychiatric side-effects as patients on conventional IFN-alpha.

Amino acids in transmembrane domain two influence anesthetic enhancement of serotonin-3A receptor function.

Alcohols and volatile anesthetics affect the function of members of the nicotinic acetylcholine (nACh) superfamily of receptors. Studies on glycine and GABA(A) receptors implicate amino acid residues within transmembrane (TM) regions two and three of these receptors as critical for alcohol and anesthetic enhancement of receptor function. The serotonin-3 (5-HT(3)) receptor is a member of the nicotinic acetylcholine receptor superfamily, sharing sequence and structural homology with the other members. We tested the hypothesis that amino acids of the 5-HT(3) receptor homologous to those shown to affect alcohol and anesthetic potentiation in GABA(A) and glycine receptors also determine the effects of these compounds on the 5-HT(3) receptor. Six 5-HT(3A) mutant cDNAs were generated by site-directed mutagenesis of two amino acids, phenylalanine-269 (14') and lecucine-270 (15') in transmembrane domain two (TM2). When assayed electrophysiologically in Xenopus oocytes, wild-type 5-HT(3) receptors exhibit enhancement of function by enflurane, halothane, isoflurane, chloroform and ethanol, but not by decanol and propofol. Mutations in transmembrane domain two markedly affected alcohol and anesthetic enhancement of 5-HT(3) receptor function. Some mutations had differential effects on the abilities of the isomers enflurane and isoflurane to potentiate 5-HT(3) receptor function.

Tetrahydrobiopterin monotherapy for phenylketonuria patients with common mild mutations.

The effect of tetrahydrobiopterin (BH(4)) administration was studied in three infants with BH(4) responsive phenylalanine hydroxylase (PAH) deficiency by correlating different BH(4) doses with plasma phenylalanine levels under defined protein intake.

Tetrahydrobiopterin responsiveness in phenylketonuria differs between patients with the same genotype.

Recently, BH(4)-responsive phenylalanine hydroxylase (PAH) deficiency was reported in patients with specific mutations in the PAH gene, and it was suggested that BH(4) responsiveness may be determined by the respective genotypes. We now report on three patients with PAH deficiency and the same genotype but different responses to standardized BH(4) loading. Our results suggest that BH(4) responsiveness in PAH deficiency is at least partly independent from PAH genotype.

Alcohol-induced reductions in cardiac protein synthesis in vivo are not ameliorated by treatment with the dihydropyridine calcium channel blocker amlodipine.

BACKGROUND: Various studies have indicated that acute ethanol dosage perturbs cardiac function and/or structure with concomitant reductions in protein synthesis. Cellular calcium homeostasis is also perturbed, which may contribute to altered protein synthesis. This is supported by the observation that calcium channel blockers can prevent numerous features of alcohol-induced pathology. However, many of these studies have been carried out in vitro, employing supraphysiological levels of alcohol, or have failed to address whether their results obtained in isolated systems have direct relevance in vivo. The aim of the present investigation was to examine the response of cardiac protein synthesis in vivo due to a physiologically relevant dose of ethanol and determine whether a calcium channel antagonist could prevent these effects. METHODS: Changes in cardiac protein synthesis rates in vivo were assessed by measuring the fractional rates of protein synthesis (i.e., ks) using a "flooding dose" of [3H]phenylalanine. Rats were treated either acutely (10 mg/kg body weight, 3 hr) or chronically (10 mg/kg body weight/day, 30 days) with amlodipine, a dihydropyridine-type calcium channel blocker, before dosing with ethanol (75 mmol/kg body weight, 2.5 hr). RESULTS: Ethanol (75 mmol/kg body weight) inhibited cardiac protein synthesis after 1 hr. Similar responses were recorded at 2.5 and 6 hr after ethanol dosage. At 24 hr, ethanol decreased food intakes. However, a direct comparison between pair-fed controls and alcohol-dosed rats also showed a decrease in cardiac protein synthesis after 24 hr. Acute alcohol dosage reduced cardiac protein synthesis in mixed, myofibrillary, and sarcoplasmic protein fractions. Similar results were obtained when data were expressed relative to ribonucleic acid (i.e., kRNA). Neither acute nor chronic treatments with the calcium antagonist amlodipine ameliorated the deleterious actions of ethanol on protein synthesis. CONCLUSIONS: Ethanol may affect cardiac protein synthesis independently of altered calcium entry.

Troglitazone does not initiate hypertrophy but can sensitise cardiomyocytes to growth effects of serum.

Chronic administration of troglitazone might predispose to cardiac hypertrophy. The aims of the study were to determine if troglitazone could (i) initiate a trophic response directly in ventricular cardiomyocytes and (ii) modify responses to other trophic stimuli. After 24 h, troglitazone (10 nM-10 microM) (i) did not increase cellular protein mass and decreased incorporation of [14C]phenylalanine, a marker of protein synthesis, (ii) interacted with serum (10% v/v) and insulin-like growth factor-1 (10 nM) to produce small trophic responses, (iii) increased cellular protein mass but not protein synthesis with insulin (1 unit/ml). Troglitazone (1 microM) attenuated responses to phorbol-12-myristate-13-acetate (PMA) (100 nM), and noradrenaline (5 microM) and endothelin-1 (100 nM), which also activate protein kinase C. In summary, troglitazone does not initiate cardiomyocyte growth directly in vitro, and can inhibit protein kinase C-mediated growth mechanisms. However, the interaction of troglitazone with serum growth factors may contribute modestly to the development of hypertrophy. As troglitazone produced a moderate hypertrophic effect per se in re-differentiated cardiomyocytes, it may directly increase the severity of established hypertrophy.

An oral essential amino acid-carbohydrate supplement enhances muscle protein anabolism after resistance exercise.

This study was designed to determine the response of muscle protein to the bolus ingestion of a drink containing essential amino acids and carbohydrate after resistance exercise. Six subjects (3 men, 3 women) randomly consumed a treatment drink (6 g essential amino acids, 35 g sucrose) or a flavored placebo drink 1 h or 3 h after a bout of resistance exercise on two separate occasions. We used a three-compartment model for determination of leg muscle protein kinetics. The model involves the infusion of ring-(2)H(5)-phenylalanine, femoral arterial and venous blood sampling, and muscle biopsies. Phenylalanine net balance and muscle protein synthesis were significantly increased above the predrink and corresponding placebo value (P < 0.05) when the drink was taken 1 or 3 h after exercise but not when the placebo was ingested at 1 or 3 h. The response to the amino acid-carbohydrate drink produced similar anabolic responses at 1 and 3 h. Muscle protein breakdown did not change in response to the drink. We conclude that essential amino acids with carbohydrates stimulate muscle protein anabolism by increasing muscle protein synthesis when ingested 1 or 3 h after resistance exercise.

Gq/11-coupled receptors and protein synthesis in rat cardiomyocytes: role of Gi-proteins and protein kinase C-isozymes.

Our recent findings indicate that, in rat neonatal ventricular cardiomyocytes, endothelin-1 (ET-1) induces increases in the rate of protein synthesis in a partly pertussis toxin (PTX)-sensitive manner, and that angiotensin II-evoked increases in the rate of protein synthesis are brought about via local secretion of ET-1. The aim of this study was to find out: (1) whether noradrenaline (NA) and the thromboxane A2 (TXA2)-mimetic U 46619-induced increases in the rate of protein synthesis may be also partly PTX-sensitive and/or mediated by ET-1, and (2) whether the growth-promoting effects of NA and U 46619 as well as ET-1 might involve activation of the same set of protein kinase C (PKC) isozymes. For this purpose we first studied the effects of NA and U 46619 on inositol phosphate (IP)-formation (assessed as accumulation of total [3H]IPs in myo-[3H]inositol prelabelled cells) and on the rate of protein synthesis (assessed as [3H]phenylalanine incorporation) (1) in the presence and absence of the ET(A)-receptor antagonist BQ-123, and (2) in nontreated and PTX-pretreated cells. Second, we assessed the effects of the PKC-inhibitors bisindolylmaleimide I and Gö 6976 and of phorbol-12-myristate-13-acetate (PMA; 1 microM overnight)-pretreatment on U 46619-, NA- and ET-1-induced increases in the rate of protein synthesis. NA (0.01-10 microM) concentration-dependently increased IP-formation (maximum increase: 115-/+23% above basal, n=4) and [3H]phenylalanine incorporation (maximum increase: 40+/-3% above basal, n=20). Both responses were antagonized by the alpha1-adrenoceptor antagonist prazosin (1 microM), but were not significantly affected by BQ-123 (1 microM). U 46619 (0.01-100 microM) concentration-dependently increased IP-formation (maximum increase: 89+/-12% above basal, n=8) and [3H]phenylalanine incorporation (maximum increase: 33+/-4% above basal, n=16). Both responses were slightly but significantly antagonized by the TP-receptor antagonist SQ 29548 (1 microM), but were not affected by BQ-123 (1 microM). Pretreatment of the cardiomyocytes with 250 ng ml(-1) PTX overnight did not significantly affect NA- and U 46619-evoked increases in IP-formation and [3H]phenylalanine incorporation. The PKC-inhibitor bisindolylmaleimide I (5 microM) as well as pretreatment of the cells with PMA (1 microM) significantly reduced the effects of NA, U 46619 and ET- I on the rate of protein synthesis; in contrast, the PKC-inhibitor Gö 6976 (5 microM) was without any effects. We conclude that, in rat neonatal ventricular cardiomyocytes, stimulation of Gq/11-coupled receptors increases the rate of protein synthesis; this involves activation of the same PKC-isozymes (very likely PKC-delta and/or -epsilon). NA and U 46619 cause their growth-promoting effects in a PTX-insensitive manner; ET-1 is not involved in their effects.

Secretory, biosynthetic and cationic responses to 2-(N-PHENYL-INDOYL)IMIDAZOLE in rat pancreatic islets.

The secretory, biosynthetic and cationic effects of a novel insulinotropic agent with an imidazoline structure, 2-(N-phenyl-indoyl)imidazole hydrochloride (RX 871024) was investigated in rat pancreatic islets. In the 1.0-10-microM range, this agent augmented, in a concentration-related manner, the release of insulin from islets incubated at intermediate concentrations of d-glucose (4.0-7.0 mm), this enhancing action fading out at both lower a nd higher d-glucose levels. When the concentration of RX 871024 was raised to 1.0 mm, severe inhibition of glucose-stimulated insulin output was observed. The imidazole derivative (10 microM) failed to enhance glucose-stimulated biosynthetic activity in islets exposed to l-[4-3H]phenylalanine; a modest inhibition of the islet peptide tritiation was even recorded at 4.0 mm d-glucose. The positive insulinotropic action of RX 871024 (10 microM) coincided with a decrease in 45Ca net uptake, unchanged outflow of 86Rb and stimulation of 45Ca efflux from prelabelled islets, the latter effect being only partially suppressed in the absence of extracellular Ca2+. These findings suggest a multifactorial mode of action of RX 871024 in islet cells, with emphasis on both an apparent stimulation of Ca2+ influx and, independently of this effect, an intracellular redistribution of the divalent cation. The imidazole compound is proposed, therefore, to display suitable attributes to bypass site-specific defects of d-glucose metabolism in the B-cell of non-insulin-dependent diabetic patients.

Evidence for acute stimulation of fibrinogen production by glucagon in humans.

Fibrinogen, an acute-phase protein, and glucagon, a stress hormone, are often elevated in many conditions of physical and metabolic stress, including uncontrolled diabetes. However, the possible mechanisms for this association are poorly known. We have studied the acute effects of selective hyperglucagonemia (raised from -200 to -350 pg/ml for 3 h) on fibrinogen fractional secretion rate (FSR) in eight normal subjects during infusion of somatostatin and replacement doses of insulin, glucagon, and growth hormone. Fibrinogen FSR was evaluated by precursor-product relationships using either Phe (n = 8) or Leu (n = 2) tracers. Hyperglucagonemia did not change either plasma Phe or Tyr specific activity. After hyperglucagonemia, fibrinogen FSR increased by approximately 65% (from 12.9 +/- 3.6 to 21.5 +/- 6.1% per day, P < 0.025) using plasma Phe specific activity as the precursor pool. FSR increased by approximately 80% (from 16.6 +/- 4.8 to 29.4 +/- 8.8% per day, P < 0.025) if plasma Phe specific activity was corrected for the ketoisocaproate/Leu enrichment (or specific activity) ratio to obtain an approximate estimate of intrahepatic Phe specific activity. FSR increased by approximately 60% when using plasma Tyr specific activity as precursor pool (n = 8) (P < 0.05), as well as when using the Leu tracer precursor-product relationship (n = 2). In conclusion, selective hyperglucagonemia for approximately 3 h acutely stimulated fibrinogen FSR using a Phe tracer method. Thus, glucagon may be involved in the increase of fibrinogen concentration and FSR observed under stressed or pathologic conditions.

Activation of group III metabotropic glutamate receptors is neuroprotective in cortical cultures.

(RS)-alpha-Methyl-4-phosphonophenylglycine (MPPG) and (S)-alpha-methyl-3-carboxyphenylalanine (M3CPA), two novel preferential antagonists of group III metabotropic glutamate (mGlu) receptors, antagonized the neuroprotective activity of L-2-amino-4-phosphono-butanoate (L-AP4) or L-serine-O-phosphate in mice cultured cortical cells exposed to a toxic pulse of N-methyl-D-aspartate. In contrast, MPPG did not influence the neuroprotective activity of the selective group II mGlu receptor agonist, (2S,1'R,2'R,3'R)-2-(2,3-dicarboxy-cyclopropyl) glycine (DCG-IV). These results indicate that activation of group III mGu receptors exerts neuroprotective activity against excitotoxic neuronal death. At least one of the two major group III mGlu receptor subtypes, i.e. mGlu4 receptor, is expressed by cultured cortical neurons, as shown by immunocytochemical analysis with specific polyclonal antibodies.

The effect of hyperphenylalaninaemia on the muscarinic acetylcholine receptor in the HPH-5 mouse brain.

Previous studies on the effect of hyperphenylalaninaemia on the development of the muscarinic acetylcholine receptor in the cerebrum of the rat, using alpha-methylphenylalanine-induced hyperphenylalaninaemia, have shown a gradual and steady decrease in the number of binding sites for this neurotransmitter. The HPH-5 mouse, a phenylalanine hydroxylase mutant, can be hyperphenylalaninaemic without the use of a hydroxylase inhibitor. By employing quantitative autoradiography using [3H]quinuclinidylbenzilate to label muscarinic acetylcholine receptors, a refined analysis of this decrease in neurotransmitter binding sites can be made. The decrease was confirmed and is therefore due to the hyperphenylalaninaemia per se and not to the use of the inhibitor. Various areas of the brain reacted differently to hyperphenylalaninaemia, from no change (putamen) to a gradual decrease (external layer of the olfactory bulb, parietal, occipital and cingulate areas of the cerebral cortex, CA1 and CA3 layer of the hippocampus) to a decrease preceded by a transient increase (frontal area of the cerebral cortex, caudate nucleus). The extent of these changes depends on the duration of exposure to hyperphenylalaninaemia as well as on the degree of brain maturation, but can even be observed in the brain of the adult mouse on a hyperphenylalaninaemic regimen for 11 days. Since the hippocampus has been shown to be involved in the long-term storage of information, damage to this structure by hyperphenylalaninaemia may provide a clue to the global mental retardation observed in untreated PKU.

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

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

In vitro protein synthesis is affected by the herbicide 2,4-dichlorophenoxyacetic acid in Azospirillum brasilense.

The effects of 2,4-dichlorophenoxyacetic acid (2,4-D) on growth and protein, DNA and RNA synthesis of Azospirillum brasilense Cd were studied. At a concentration of 1 mM, 2,4-D inhibited cell growth, an effect that was reversed either by transferring bacteria to a control (2,4-D-free) medium or to a 2,4-D-treated medium supplemented with polyamines. The herbicide also affected in vitro protein synthesis, either when Azospirillum brasilense Cd's own cellular mRNA or an artificial mRNA was used. This effect was also reversed by the addition of polyamines to the 2,4-D-treated medium. Similar results were observed when DNA synthesis was studied in synchronous cultures. Taking into account the effects of this herbicide on animal cells (V.A. Rivarola and H.F. Balegno, Toxicology, 68 (1991) 109) we postulate that the mechanism of action of 2,4-D is similar on both procaryotic and eucaryotic cells, probably acting through the polyamine metabolism.

L-phenylalanine production by double auxotrophic mutants of Arthrobacter globiformis.

A number of tryptophan-plus-tyrosine double auxotrophs have been isolated from a glutamate producing Arthrobacter globiformis excreting L-phenylalanine by two-step mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. For the three potent mutants tested the medium of Alföldi was found to be the best. The optimum tryptophan, tyrosine and biotin concentrations for phenylalanine production of these mutants were 0.5 mmol/L, 0.1 mmol/L and 5 micrograms/L, respectively. At these levels strain TT-39 yielded 2.6 g phenylalanine per L of medium in flask culture with glucose (350 mmol/L) and NH4Cl (60 mmol/L).