Biochemical selection systems for mammalian cells: The essential amino acids

The essential amino acid requirement of cultured mammalian cells can be satisfied by 19 amino acid derivatives. This finding (a) confirms the results of animal nutritional studies and (b) identifies 19 essential amino acid derivatives and should permit the isolation of a new class of auxotrophic mutants. Five naturally occurring auxotrophic markers have been detected in this survey; namely, inability to utilize cystathionine and citrulline in Chinese hamster ovary (CHO) cells, inability to metabolize citrulline by HTC⁺ hepatoma cells, and confirmation of Eagle's observation that KB cells can utilize homocystine in place of methionine or cystine and d-cystine in place of l-cystine.This study was presented as an abstract at the 14th Annual Somatic Cell Genetics Conference, Houston, Texas, October 29-November 1, 1975.     

Antioxidant effects of sulfur-containing amino acids

Sulfur is an essential element for the entire biological kingdom because of its incorporation into amino acids, proteins and other biomolecules. Sulfur atoms are also important in the iron-containing flavoenzymes. Unlike humans, plants can use inorganic sulfur to synthesize sulfur-containing amino acids. Therefore, plants are an important source of sulfur for humans. Sulfur-containing compounds are found in all body cells and are indispensable for life. Some of sulfur-containing antioxidant compounds are, cysteine, methionine, taurine, glutathione, lipoic acid, mercaptopropionylglycine, N-acetylcysteine, and the three major organosulfur compounds of garlic oil, diallylsulfide, diallyldisulfide and diallyltrisulfide. In a comparison of the structure-function relationship among these sulfur-containing antioxidant compounds, dihydrolipoic acid (the reduced form of LA) is the most effective antioxidant. Dihydrolipoic acid contains two sulfhydryl groups and can undergo further oxidation reaction to form lipoic acid. The antioxidative activities of sulfur-containing compounds follow a general trend, the more highly reduced forms are stronger antioxidants and the number of sulfur atoms determine, at least in part, their modulatory activites on the glutathione related antioxidant enzymes. In this article, the antioxidant effects and the antioxidative activities, of sulfur-containing amino acids, are reviewed. In addition, the general antioxidant effects and the structure-function relationship of some sulfur-containing compounds are also reviewed.     

Mutational analysis of the Helicobacter pylori vacuolating toxin amino terminus: identification of amino acids essential for cellular vacuolation

The functional importance of the amino terminus of the Helicobacter pylori vacuolating cytotoxin (VacA) was investigated by analyzing the relative levels of vacuolation of HeLa cells transfected with plasmids encoding wild-type and mutant forms of the toxin. Notably, VacA's intracellular activity was found to be sensitive to small truncations and internal deletions at the toxin's amino terminus. Moreover, alanine-scanning mutagenesis revealed the first VacA point mutations (at proline 9 or glycine 14) that completely abolish the toxin's intracellular activity.     

Identification of amino acids essential for catalytic activity of pneumococcal neuraminidase A

We characterised pneumococcal neuraminidase A (NanA) by determining key amino acids required for the enzymatic activity of the protein. Single replacement of two residues, hypothesised to be important for the catalytic activity of neuraminidases, resulted in total loss of activity (E647 with Q or Y752 with F). The mutation of R663 to H caused substantial reduction in the catalytic ability of the enzyme. The inactive neuraminidases thus produced were protective immunogens against pneumococcal pneumonia in mice.     

L-histidine: effects on sensitivity of cat spinal neurones to amino acids.

L-Histidine enhanced the inhibitory actions of GABA, muscimol and beta-alanine rather than that of glycine on spinal neurones in pentobarbitone-anaesthetised cats. L-Histidine also enhanced the excitatory action of L-glutamate, D- and L-aspartate, L-homocysteate and especially that of quisqualate, whereas the actions of acetylcholine, kainate, N-methyl-D-aspartate and D-homocysteate were more commonly reduced. These actions of L-histidine are best ascribed to an effect on amino acid transport systems, probably of the low affinity type, which therefore appear to be partially responsible for the inactivation of exogenously administered amino acids.     

Treatment of carbamyl phosphate synthetase deficiency with keto analogues of essential amino acids.

Congenital carbamyl phosphate synthetase deficiency was diagnosed by liver biopsy in a 13-year-old girl, alpha-Keto analogues of essential amino acids have been shown to spare nitrogen by reducing urea formation; hence, they were given to this patient in the hope of reducing hyperammonemia and improving protein tolerance. After intravenous infusion of the keto analogues of valine, leucine, isoleucine, methionine and phenylalanine, the corresponding plasma amino acids, including alloisoleucine and tyrosine, rose sharply. Twenty-four hours later, fasting plasma ammonia had fallen from the preinfusion value of 0.050 to 0.028 mM. Protein intake was kept at 0.5 g per kilogram for two weeks. Addition of keto acids by mouth reduced plasma ammonia and alanine to normal or near normal levels. Seizures and episodes of vomiting and lethargy decreased in frequency. Urinary nitrogen decreased, suggesting that nitrogen balance improved. These data indicate that keto acids may be useful in the treatment of congenital hyperammonemia.     

Dietary self-selection of protein-unbalanced diets supplemented with three essential amino acids in Nile tilapia

Animals do not eat whatever food item they encounter, but choose different foods that best match their requirements. Fish exhibit such "nutritional wisdom" and adapt their feeding behaviour and food intake according to their needs and the nutritional properties of diets. In this paper, we tested the ability of Nile tilapia to select between diets with a balanced or unbalanced composition of essential amino acids. To this end, three different diets were prepared: a gelatine based diet (D(1)), a gelatine diet supplemented with three essential amino acids (EAA, l-tryptophane, l-methionine, l-threonine) (D(2)), and a diet containing only cellulose and the three crystalline EAA (D(3)). In addition, the putative role of both orosensorial factors (using pellets vs capsules) and social interactions (single vs groups of ten fish) was investigated. To this end, a total of 68 male tilapia of about 141±48 g (mean±S.D.) were challenged, individually or in groups, to select between D(1)vs D(2) using pellets dispensed by self-feeders (exp. 1). In another experiment (exp. 2), 11 individual fish were challenged to select encapsulated diets with non flavour or smell proprieties (D(1)vs D(2)), and in exp. 3 fish were challenged to self-supplementation in EAA (D(1)vs D(3)). The results showed the ability of tilapia to avoid the EAA-deficient diet, choosing 82.2% D(2) in the case of individual fish, and 80.8% D(2) in the case of fish groups. Dietary selection was not directly driven by the orosensorial characteristics of food, since tilapia sustained a higher preference for D(2) when fed with encapsulated diets. Finally, in exp. 3 tilapia self-supplemented the EAA deficiency by selecting a synchronised combination of D(1) and D(3) that matched their nutritional requirements. These findings highlighted the capacity of fish to make dietary selection based on the EAA content, which should be considered when discussing food intake regulation mechanisms, and diet formulation and supplementation with EAA.     

Modulation of antimicrobial effects of beta-lactams by amino acids in vitro

Glycine as well as 11 and 10, respectively, out of a total of 12 D-amino-acids tested increased the antimicrobial efficacy of imipenem (IMI) and of ampicillin (AMP) using the serosensitive strain E. coli ATCC 8739. D-proline was ineffective in assays with IMI as well as D-proline and D-leucine in assays with AMP. - In contrast, L-amino-acids behaved differently: In assays with IMI, 9 out of 13 isomers were ineffective whereas 3 were antagonistic (L-phenylalanine, L-serine, L-tryptophan). In combination with AMP, however, 10 L-amino acids had an antagonistic effect and 2 (L-leucine, L-methionine) were ineffective. L-alanine was an exception and showed a synergism with both antibiotics which was assumed to have been due to a racemase activity of cells. - Seroresistance of E. coli apparently reduced the synergistic effect of glycine and beta-lactams. - Glycine, alanine and tryptophan lost their typical synergistic or antagonistic effect with AMP when tested as di- or tri-amino-acid compounds. This was not the case with di-L-alanine - It is supposed that the synergistic effect of glycine or of D-amino-acids with beta-lactams can be explained mainly by an inhibition of carboxypeptidases.     

Random mutagenesis of Helicobacter pylori vacA to identify amino acids essential for vacuolating cytotoxic activity

VacA is a secreted toxin that plays a role in Helicobacter pylori colonization of the stomach and may contribute to the pathogenesis of peptic ulcer disease and gastric cancer. In this study, we analyzed a library of plasmids expressing randomly mutated forms of recombinant VacA and identified 10 mutant VacA proteins that lacked vacuolating cytotoxic activity when added to HeLa cells. The mutations included six single amino acid substitutions within an amino-terminal hydrophobic region and four substitutions outside the amino-terminal hydrophobic region. All 10 mutations mapped within the p33 domain of VacA. By introducing mutations into the H. pylori chromosomal vacA gene, we showed that secreted mutant toxins containing V21L, S25L, G121R, or S246L mutations bound to cells and were internalized but had defects in vacuolating activity. In planar lipid bilayer and membrane depolarization assays, VacA proteins containing V21L and S25L mutations were defective in formation of anion-selective membrane channels, whereas proteins containing G121R or S246L mutations retained channel-forming capacity. These are the first point mutations outside the amino-terminal hydrophobic region that are known to abrogate vacuolating toxin activity. In addition, these are the first examples of mutant VacA proteins that have defects in vacuolating activity despite exhibiting channel activities similar to those of wild-type VacA.     

86Rubidium release from cultured primary astrocytes: effects of excitatory and inhibitory amino acids

The effects of high K+, glutamate and its analogue, kainate, on K+ release were studied in primary astrocyte cultures prepared from newborn rat brains using 86Rb+ as a tracer for K+. An increase in 86Rb+ release was observed when the extracellular K+ concentration was elevated (10-40 mM). Glutamate and kainate stimulated the release in a dose-dependent manner, 100 microM concentrations being about as equally effective as high K+ (40 mM). Both compounds also caused an increase in the absorbance of the cyanine dye, 3,3'-diethylthiadicarbocyanine, indicating depolarization of the membrane. No significant Na+-dependent uptake of [3H]kainate occurred in the cells, thus excluding the possibility that depolarization was due to electrogenic uptake of amino acid into the cells. GABA and taurine significantly depressed the high K+- and glutamate-induced 86Rb+ release. Taurine itself caused a small increase in 86Rb+ release and the membrane was depolarized, judging from the increase in the absorbance of the cyanine dye, 3,3'-diethylthiadicarbocyanine. No effect of taurine was observed when the Cl- concentration was reduced in the experimental medium. The results suggest that cultured astrocytes respond by membrane depolarization to high external K+ and to glutamate and kainate. The degree of this depolarization can be modified by the inhibitory amino acids GABA, taurine and glycine, the effect of taurine probably being mediated by an increase in Cl- conductance across the cell membrane. The role of functional receptors for amino acid transmitters and the effects observed are discussed.     

The carboxyl 35 amino acids of SV40 Vp3 are essential for its nuclear accumulation

To identify the moiety responsible for nuclear localization of the SV40 structural protein Vp3 in its natural environment, a transfection vector containing the entire coding regions of Vp2, Vp3, and agnoprotein, and one-third of the coding region of Vp1, was constructed. Several mutations were introduced into the plasmid and the subcellular distribution of Vp3 or mutant Vp3 was examined following DEAE-dextran-mediated DNA transfection into TC7 cells. Our study shows that Vp3 is synthesized and is transported into the nucleus in the absence of Vp2, agnoprotein, and intact Vp1. However, in the absence of its carboxyl-terminal 35 amino acids, the truncated Vp3 is limited to a cytoplasmic and perinuclear accumulation. Thus, the carboxyl 35 amino acids of Vp3 are required for its nuclear localization and may contain a nuclear accumulation signal.