Evaluation of genetic toxicity of 6-diazo-5-oxo-l-norleucine (DON).

DON (6-diazo-5-oxo-l-norleucine), a glutamine antagonist, was demonstrated to exhibit analgesic, antibacterial, antiviral and anticancer properties. The study was performed to characterize its in vitro and in vivo genetic toxicity potential. DON was tested in the bacterial reverse mutation assay (Ames test) using Salmonella typhimurium tester strains (TA98, TA100, TA1535 and TA1537) and Escherichia coli tester strain (WP2 uvrA) with and without S9 and also with reductive S9. In addition, DON was tested for the chromosome aberrations in Chinese hamster ovary (CHO) cells with or without S9 to evaluate the clastogenic potential. Furthermore, DON was also evaluated for its in vivo clastogenic activity by detecting micronuclei in polychromatic erythrocyte (PCE) cells in bone marrow collected from the male mice dosed intravenously with 500, 100, 10, 1 and 0.1 mg/kg at 24 and 48-h post-dose. The Ames mutagenicity assay showed no positive mutagenic responses. However, the in vitro chromosome aberration assay demonstrated dose dependent statistically positive increase in structural aberrations at 4 and 20-h exposure without S9 and also at 4-h exposure with S9. The in vivo micronucleus assay also revealed a statistically positive response for micronucleus formation at 500, 100 and 10 mg/kg at 24 and 48-h post-dose. Thus, DON appears to be negative in the Ames test but positive in the in vitro chromosome aberration assay and in the in vivo micronucleus assay. In conclusion, the results indicate DON is a genotoxic compound with a plausible epigenetic mechanism.

6-(4-Amino-2-butyl-imidazoquinolyl)-norleucine: Toll-like receptor 7 and 8 agonist amino acid for self-adjuvanting peptide vaccine.

Generally, small peptides by themselves are weak to induce antibody responses. Toll-like receptor (TLR) ligands are attractive candidates of vaccine adjuvants to improve their antigenicity. The covalent conjugation of TLR ligands with antigens to produce self-adjuvanting peptide vaccine is a promising approach. Based on the structure of TLR7/8 ligands, a series of synthetic amino acids 6-imidazoquinolyl-norleucines were synthesized, wherein an imidazoquinoline structure as the TLR7/8 agonistic pharmacophores was constructed on the ε-NH2 group of Lys. Of them, 6-(4-amino-2-butyl-imidazoquinolyl)-norleucine showed the most potent TLR7 and TLR8 agonistic activities with EC50 values of 8.55 and 106 µM, respectively. Subsequently, mice were immunized with the influenza A virus M2e antigen mixed with or covalently conjugated to the TLR7/8 agonist amino acid, which led to induction of M2e specific antibody productions in the absence of other adjuvant. We successfully developed a novel efficient tool for self-adjuvanting peptide vaccines targeting TLR7/8.

Leucine in food mediates some of the postprandial rise in plasma leptin concentrations.

In vitro, leptin secretion is regulated at the level of mRNA translation by the rapamycin-sensitive mammalian target of rapamycin (mTOR) and its agonist leucine (Leu). Studies were conducted on meal-trained rats to evaluate the potential physiological relevance of these in vitro findings and the role of Leu in affecting rises in plasma leptin observed after a meal. In the first study, we correlated changes in plasma insulin and Leu to mTOR-signaling pathway activation and plasma leptin at different times during meal feeding. Rapid rises in plasma insulin and Leu, along with mTOR signaling (phosphorylation of eIF4G, S6K1, rpS6, and 4E-BP1) in adipose tissue were observed during the 3-h meal and declined thereafter. Plasma leptin rose more slowly, peaking at 3 h, and was inhibited by rapamycin (0.75 mg/kg) pretreatment. In another experiment, oral Leu or norleucine was provided instead of a meal. Leu and norleucine stimulated a rise in plasma leptin; however, the magnitude was less than the response to a complete meal. In a third study, rats were provided a meal that lacked Leu, branched-chain amino acids, or all amino acids. Stimulation of leptin secretion was reduced approximately 40% in animals provided the Leu-deficient meal. Further reductions were not observed by removing the other amino acids. Thus Leu appears to regulate most of the effects of dietary amino acids on the postprandial rise in plasma leptin but is responsible only for part of the leptin response to meal feeding.

Branched-chain and other amino acids in tissues of rats fed leucine-limiting amino acid diets containing norleucine.

Amino acid concentrations were measured in plasma, brain, muscle and liver from rats fed leucine-limiting diets containing varying proportions of other indispensable amino acids (IAA), the branched-chain amino acids (BCAA) and norleucine, a BCAA analog known to compete with large neutral amino acids (LNAA) for transport into tissues. Leucine was low and other IAA were high when dietary IAA were 125% and leucine was 65% of requirements; higher leucine and lower IAA concentrations occurred when dietary IAA were 75% of requirements. Tissue leucine was high and isoleucine and valine were low in rats fed excess leucine. Norleucine induced dose-dependent reductions in BCAA, especially in brain and muscle in which isoleucine or valine were sometimes undetectable. Leucine was not depressed further when control values were low as in the rats fed 125% IAA. Norleucine frequently prevented the high BCAA found after feeding additional BCAA. Other LNAA tended to be low in the brain and muscle of rats fed norleucine. Lysine was high only in the tissues of rats fed 75% IAA and norleucine; this effect was prevented when added leucine was given. Brain tryptophan, but not always serotonin, was low in rats fed norleucine. The results show transport-related, selective and usually marked depletions of tissue BCAA in rats fed norleucine; this suggests norleucine may be an aid in the treatment of clinical conditions involving excesses of BCAA.

Dietary amino acid analogues and transport of lysine or valine across the blood-brain barrier in rats.

Studies were undertaken to determine if dietary disproportions of amino acids would alter flux into brain of the amino acid present in the diet in a growth-limiting concentration. Rats were adapted to a lysine-limiting diet before receiving a meal of this control diet, alone or with added lysine or homoarginine (a competitor for lysine transport) or both, before intravenous infusion of [14C]lysine. The brain-to-plasma radioactivity ratio was lower in rats fed extra lysine or homoarginine than in rats fed the control diet, whereas lysine flux and brain lysine concentration were high in rats fed extra lysine alone. Flux and concentration were lower in rats fed homoarginine + lysine than in rats fed extra lysine alone. Other rats were fed a valine-limiting diet containing added valine, norleucine (a competitor for valine transport) or both, before [14C]valine was infused. Valine flux and brain valine concentrations were higher in rats fed extra valine than in control rats, whereas flux was lower in the group fed norleucine alone. Valine flux was higher in rats fed norleucine + valine than in the rats fed norleucine alone. Our studies show that dietary disproportions of amino acids can alter the flux of specific amino acids across the blood-brain barrier.

Effects of 13-nle-motilin in man--inhibition of gastric evacuation and stimulation of pepsin secretion.

In 3 healthy male volunteers, graded doses of 13-norleucine-motilin (13-nle-motilin)--synthetic analogue of motilin and biologically equivalent to the natural polypeptide--were given intravenously on separate days. Gastric emptying, as determined by means of a radioisotope method, was slowed down dose-dependently by increasing doses of 13-nle-motilin. This was indicated by increasing "half-lives" and "starting indices" of the gastric emptying process, and by decreasing pyloric loss rates, respectively. The gastric pepsin output rose markedly from the basal level during intravenous infusion of 13-nle-motilin.