Effect of oxygen deficiency on nitrogen assimilation and amino acid metabolism of soybean root segments

Plants submitted to O₂ deficiency present a series of biochemical modifications, affecting overall root metabolism. Here, the effect of hypoxia on the metabolic fate of ¹⁵N derived from ¹⁵NO₃ ^?, ¹⁵NO₂ ^? and ¹⁵NH₄ ⁺ in isolated soybean root segments was followed by gas chromatography–mass spectrometry, to provide a detailed analysis of nitrogen assimilation and amino acid biosynthesis under hypoxia. O₂ deficiency decreased the uptake of the nitrogen sources from the solution, as ratified by the lower ¹⁵NO₃ ^? and ¹⁵NH₄ ⁺ enrichment in the root segments. Moreover, analysis of endogenous NO₂ ^? and ¹⁵NH₄ ⁺ levels suggested a slower metabolism of these ions under hypoxia. Accordingly, regardless of the nitrogen source, hypoxia reduced total ¹⁵N incorporation into amino acids. Analysis of ¹⁵N enrichment patterns and amino acid levels suggest a redirecting of amino acid metabolism to alanine and γ-aminobutyric acid synthesis under hypoxia and a differential sensitivity of individual amino acid pathways to this stress. Moreover, the role of glutamine synthetase in nitrogen assimilation both under normoxia and hypoxia was ratified. In comparison with ¹⁵NH₄ ⁺, ¹⁵NO₂ ^? assimilation into amino acids was more strongly affected by hypoxia and NO₂ ^? accumulated in root segments during this stress, indicating that nitrite reductase may be an additional limiting step. NO₂ ^? accumulation was associated with a higher nitric oxide emission. ¹⁵NO₃ ^? led to much lower ¹⁵N incorporation in both O₂ conditions, probably due to the limited nitrate reductase activity of the root segments. Overall, the present work shows that profound alterations of root nitrogen metabolism occur during hypoxic stress.     

Induction of experimental phenylketonuria-like conditions in chick embryo. Effect on amino acid concentration in brain,liver and plasma

Experimental hyperphenylalaninemia has been induced in chick embryos between 11–20 days of incubation by daily injection of α-methylphenylalanine and phenylalanine. Brain and liver weight decreased after 8 days of treatment. An increase of nearly 14-fold in the brain phenylalanine/tyrosine ratio was observed after 9 days of treatment. Similar results were obtained in liver, although the increase found in this case was smaller than in brain. Chronic hyperphenylalaninemia induced a clear rise in the levels of plasma and liver valine, leucine and isoleucine, while in brain these levels did not change significantly. Plasma and brain glycine content was also enhanced by this treatment. Brain tyrosine concentration was clearly decreased in these conditions, in contrast to the enhancement reported after this and other treatments in various animal species. Thus, the higher value of the brain phenylalanine/tyrosine ratio obtained by α-methylphenylalanine plus phenylalanine administration was due to both an increase in the phenylalanine and a decrease in the tyrosine levels, conditions that have been also found in human phenylketonurics. Therefore, the treatment here reported was an excellent method for imitating the conditions of phenylketonuria during the period of rapid myelination in the chick, one of the most dramatic in nervous system development.     

A sulfur amino acid deficiency changes the amino acid composition of body protein in piglets

Experiments carried out to determine the amino acid requirement in growing animals are often based on the premise that the amino acid composition of body protein is constant. However, there are indications that this assumption may not be correct. The objective of this study was to test the effect of feeding piglets a diet deficient or not in total sulfur amino acids (TSAA; Met + Cys) on nitrogen retention and amino acid composition of proteins in different body compartments. Six blocks of three pigs each were used in a combined comparative slaughter and nitrogen balance study. One piglet in each block was slaughtered at 42 days of age, whereas the other piglets received a diet deficient or not in TSAA for 19 days and were slaughtered thereafter. Two diets were formulated to provide either 0.20% Met and 0.45% TSAA (on a standardized ileal digestible basis) or 0.46% Met and 0.70% TSAA. Diets were offered approximately 25% below ad libitum intake. At slaughter, the whole animal was divided into carcass, blood, intestines, liver, and the combined head, tail, feet and other organs (HFTO), which were analyzed for nitrogen and amino acid contents. Samples of the longissimus muscle (LM) were analyzed for myosin heavy chain (MyHC) and actin contents. Nitrogen retention was 20% lower in piglets receiving the TSAA-deficient diet (P < 0.01). In these piglets, the nitrogen content in tissue gain was lower in the empty body, carcass, LM and blood (P < 0.05) or tended to be lower in HFTO (P < 0.10), but was not different in the intestines and liver. The Met content in retained protein was lower in the empty body, LM and blood (P < 0.05), and tended to be lower in the carcass (P < 0.10). The Cys content was lower in LM, but higher in blood of piglets receiving the TSAA-deficient diet (P < 0.05). Skeletal muscle appeared to be affected most by the TSAA deficiency. In LM, the Met content in retained protein was reduced by 12% and total Met retention by more than 60%. The MyHC and actin contents in LM were not affected by the TSAA content of the diet. These results show that a deficient TSAA supply affects the amino acid composition of different body proteins. This questions the use of a constant ideal amino acid profile to express dietary amino acid requirements, but also illustrates the plasticity of the animal to cope with nutritional challenges.     

Manipulation of amino acid composition in soybean seeds by the combination of deregulated tryptophan biosynthesis and storage protein deficiency

The ability of genetic manipulation to yield greatly increased concentrations of free amino acids (FAAs) in seeds of soybean was evaluated by introduction of a feedback-insensitive mutant enzyme of tryptophan (Trp) biosynthesis into two transformation-competent breeding lines deficient in major seed storage proteins. The storage protein-deficient lines exhibited increased accumulation of certain other seed proteins as well as of FAAs including arginine (Arg) and asparagine in mature seeds. Introduction of the gene for a feedback-insensitive mutant of an α subunit of rice anthranilate synthase (OASA1D) into the two high-FAA breeding lines by particle bombardment resulted in a >10-fold increase in the level of free Trp in mature seeds compared with that in nontransgenic seeds. The amount of free Trp in these transgenic seeds was similar to that in OASA1D transgenic seeds of the wild-type cultivar Jack. The composition of total amino acids in seeds of the high-FAA breeding lines remained largely unaffected by the expression of OASA1D with the exception of an increase in the total Trp content. Our results therefore indicate that the extra nitrogen resource originating from storage protein deficiency was used exclusively for the synthesis of inherent alternative nitrogen reservoirs such as free Arg and not for deregulated Trp biosynthesis conferred by OASA1D. The intrinsic null mutations responsible for storage protein deficiency and the OASA1D transgene affecting Trp content were thus successfully combined and showed additive effects on the amino acid composition of soybean seeds.     

Uncharged tRNA-phosphofructokinase interaction in amino acid deficiency

Summary When the tRNA of mammalian cells is incompletely charged due to amino acid deficiency or by analogs which cannot be activated, many metabolic events become limited. This rapid demise of cell function appears to be due to the inhibition of phosphofructokinase (PFK) by uncharged tRNA (FEBS Lett 302: 113 (1992)). Charged tRNA has been shown to be sequestered within the protein synthetic machinery, (Negrutskii, B. S. and Deutscher, M. P. (1992) Proc Natl Acad Sci USA 89: 3601) and would therefore be removed from an inhibitory role. Besides the direct demonstration that tRNA inhibits PFK in an assay regarded as indicative of its control mechanism, several reports in the literature support this model. These include 1) The rapid onset of inhibition of glycolysis and glucose uptake by intact cells upon amino acid deficiency and the similar lesion at the 43S ribosomal subunit on glucose or amino acid deprivation. 2) The recognition that unusually high concentrations of cAMP required to stimulate protein synthesis in energy depleted or gel filtered lysates correlates with its action on PFK as an analog of the positive effector, adenosine-5-monophosphate. 3) The often repeated observation that the product of PFK activity, fructose-1,6-diphosphate, is a stimulant of protein synthesis (see Jackson, R. J., et al. (1983) Eur J Biochem 131: 289). This diphosphate has been shown to be the proximate effector binding to eIF-2B, the guanine nucleotide exchange factor (Singh, L. P. Arror, A. R. and Wahba, A. J. (1994), FASEB J. 8: 279) which by releasing GDP bound to the inactive GDP: eIF-2 complex, permits the factor to initiate a new peptide chain. The above information supports the view that the block at the G1 restriction point in the cell cycle of normal cells brought about by amino acid deprivation is a result of inhibition of protein synthesis through the phosphofructokinase-uncharged tRNA mechanism. This is consistent with observations in the literature that tumor and transformed cells, which are more resistant to this block (Pardee, A. B., Proc Natl Acad Sci USA 71: 1286–1291 (1974)) have a higher phosphofructokinase activity or higher levels of fructose-1,6-diphosphate.Presented at the 4th International Congress on Amino Acids, Vienna, Austria, August 7–11, 1995. (Amino Acids (1995) 9: 23, Abstract).     

Effect of vitamin D deficiency on bone formation in the chick.

1. The process of diaphyseal bone formation can be investigated by studying the rate of incorporation of radioactive precursors, administered in vivo into bone fractions of increasing density. 2. In the 4-week-old vitamin D-treated chick most of the osteoid becomes calcified within 12h and almost all within 2 days. The low-density calcified phase that is formed is converted into a higher density form and within 7 days the greater proportion of the calcified tissue is in the higher density form. 3. In the vitamin D-deficient chick of similar age the rate of calcification of osteoid is decreased, as is the rate of conversion into the higher density phase with the resultant accumulation of the lower density calcified form. 4. The higher density phase probably corresponds to hydroxyapatite and the lower density one to the ACP-pase described by Termine & Posner [(1967) Calcif. Tissue Res. 1, 8--23]. 5. The disorder in the process of calcification seems to be unrelated to the alteration in blood Ca2+ and phosphate concentrations, but related to the presence or absence of cholecalciferol.     

Effect of dietary amino acid on the amino acid pool of Aedes aegypti

Ion-exchange chromatography analysis of whole body extracts of Aedes aegypti mosquitoes which had received amino acids in their diet revealed that generally there were changes in the titre of two or more amino acids. Cysteine produced the greatest number of changes and was toxic to the insect. Of the ten amino acids provided, none resulted in the significant change in the concentration of tyrosine following a blood meal as was observed in previous studies. Evidence is presented for the conversion of arginine to ornithine and for the synthesis of arginine from glutamic acid. The data presented tend to support the hypothesis of lysine synthesis from α-ketoglutarate and for the use of proline as an energy reserve in the insect.