Growth Responses of Juvenile Red Drum Sciaenops ocellatus to Dietary Phenylalanine and Tyrosine Can Be Used to Calculate the Total Aromatic Amino Acid Requirement.

BACKGROUND: A current priority in aquaculture is the replacement of fish meal with alternative feedstuffs to ensure the industry's sustainability. However, most alternative protein sources are deficient in at least 1 indispensable amino acid (IAA). Therefore, there is a critical need to establish refined estimates of IAA requirements of fish. OBJECTIVES: The objectives of this study were to determine the total aromatic amino acid (TAAA) requirement (Phe + Tyr) and the Tyr replacement value for Phe in juvenile red drum Sciaenops ocellatus. METHODS: The TAAA requirement was obtained by feeding juvenile red drum diets that contained incremental amounts of Phe (0.54, 0.84, 1.14, 1.44, 1.74, and 2.04 g/100 g dry diet) and a fixed concentration of Tyr. Because of the TAAA requirement obtained, a second feeding trial was conducted to determine the maximum Tyr replacement value for Phe when a control diet was prepared with an 80%:20% Phe-to-Tyr ratio (Phe:Tyr; 1.60 g Phe/100 g dry diet and 0.41 g Tyr/100 g), and 5 experimental diets were prepared by decreasing the inclusion of Phe and increasing the inclusion of Tyr (Phe:Tyr of 70%:30%, 60%:40%, 50%:50%, 40%:60%, and 30%:70%). RESULTS: Weight gain, the feed efficiency ratio, and the protein efficiency ratio increased 354%, 133%, and 134%, respectively, relative to that of fish fed the basal diet as the Phe concentration increased to 1.44 g/100 g of dry diet; at higher concentrations of Phe, no significant differences were found between treatments. Analysis of the weight gain data with a broken-line model estimated the TAAA requirement of red drum to be 2.10 g/100 g dry diet (1.69 g Phe + 0.41 g Tyr). The maximum Tyr replacement value for Phe was estimated at 40%:60% Phe:Tyr, because only fish fed the diet with a 30%:70% Phe:Tyr ratio had a significant reduction (42%) in growth performance. CONCLUSION: The TAAA requirement for maximum growth of juvenile red drum was estimated to be 2.10 ± 0.08 g/100 g dry diet. Moreover, Tyr can account for up to 60% of the TAAA requirement of juvenile red drum.

Aromatic amino acid auxotrophs constructed by recombinant marker exchange in Methylophilus methylotrophus AS1 cells expressing the aroP-encoded transporter of Escherichia coli.

The isolation of auxotrophic mutants, which is a prerequisite for a substantial genetic analysis and metabolic engineering of obligate methylotrophs, remains a rather complicated task. We describe a novel method of constructing mutants of the bacterium Methylophilus methylotrophus AS1 that are auxotrophic for aromatic amino acids. The procedure begins with the Mu-driven integration of the Escherichia coli gene aroP, which encodes the common aromatic amino acid transporter, into the genome of M. methylotrophus. The resulting recombinant strain, with improved permeability to certain amino acids and their analogues, was used for mutagenesis. Mutagenesis was carried out by recombinant substitution of the target genes in the chromosome by linear DNA using the FLP-excisable marker flanked with cloned homologous arms longer than 1,000 bp. M. methylotrophus AS1 genes trpE, tyrA, pheA, and aroG were cloned in E. coli, sequenced, disrupted in vitro using a Kmr marker, and electroporated into an aroP carrier recipient strain. This approach led to the construction of a set of marker-less M. methylotrophus AS1 mutants auxotrophic for aromatic amino acids. Thus, introduction of foreign amino acid transporter genes appeared promising for the following isolation of desired auxotrophs on the basis of different methylotrophic bacteria.

Activation of caspases and cleavage of Bid are required for tyrosine and phenylalanine deficiency-induced apoptosis of human A375 melanoma cells.

Deprivation of tyrosine (Tyr) and phenylalanine (Phe) inhibits growth and induces programmed cell death (apoptosis) of human A375 melanoma cells. Herein, we found that activation of caspases and release of mitochondrial cytochrome c are required for this process. Culturing A375 cells in Tyr/Phe-free medium, containing 10% dialyzed fetal bovine serum, results in activation of caspase-3-like activity. This is accompanied by decreased cell viability and increased apoptosis. Tyr/Phe deprivation also stimulates proteolytic cleavage of the DNA repair enzyme, poly(ADP-ribose) polymerase (PARP). Western blot analysis showed that caspases 3, 7, 8, and 9 are activated by deprivation of Tyr/Phe. Tyr/Phe deprivation decreases mitochondrial membrane potential, induces cleavage of Bid, increases translocation of Bax from the cytosol to mitochondria, and results in release of cytochrome c from the mitochondria to the cytosol. Apoptosis due to Tyr/Phe deprivation is almost completely inhibited by the broad-spectrum cell-permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z.VAD.fmk). This inhibitor suppresses the cleavage of Bid, the release of cytochrome c from the mitochondria to the cytosol, and the cleavage of PARP. Decylubiquinone, a mitochondrial permeability transition pore inhibitor, does not suppress the activation of caspase 8 but suppresses release of cytochrome c, activation of caspase 9, and induction of apoptosis. These results indicate that activation of caspases, cleavage of Bid, and mitochondrial release of cytochrome c are required for apoptosis induced by Tyr/Phe deprivation.