Evidence of amino acids in hydrolysates of compounds formed by ionizing radiations. II. Aqueous solutions of CH3CN and C2H5CN

It has been shown that the action of ionizing radiations on dilute, oxygen-free, aqueous solutions of acetonitrile and propionitrile leads to the formation of oligomers, which upon hydrolyses release amino acids. The presence of nine amino acids, the same as those found in irradiated aqueous cyanides, has been established. those amino acids with asymmetric carbon atoms separated by GC method, appeared to consist of nearly equal amounts of D and L isomers. Glycine is the most abundant amino acid in hydrolysates of acetonitrile, while alanine appears in the samples of propionitrile. A comparison of all amino acids, identified in hydrolysates of various cyanides and nitriles, suggests that it is the cyano group, and a free-radical initiated mechanism, that is primarily involved in these radiation-chemical changes of potential interest to prebiotic chemistry.     

Evidence of amino acids in hydrolysates of compounds formed by ionizing radiations

It has been shown that the action of ionizing radiations on dilute, oxygen-free, aqueous solutions of acetonitrile and propionitrile leads to the formation of oligomers, which upon hydrolyses release amino acids. The presence of nine amino acids, the same as those found in irradiated aqueous cyanides, has been established. Those amino acids with asymmetric carbon atoms separated by GC method, appeared to consist of nearly equal amounts of D and L isomers. Glycine is the most abundant amino acid in hydrolysates of acetonitrile, while alanine appears in the samples of propionitrile. A comparison of all amino acids, identified in hydrolysates of various cyanides and nitriles, suggests that it is the cyano group, and a free-radical initiated mechanism, that is primarily involved in these radiation-chemical changes of potential interest to prebiotic chemistry.     

Submerged culture conidial germination and conidiation of the bioherbicideColletotrichum truncatum are influenced by the amino acid composition of the medium

Summary Submerged culture experiments were conducted to determine the optimal nitrogen source for rapidly producing conidia of the bioherbicide,Colletotrichum truncatum. Germination ofC. truncatum conidial inocula in submerged culture occurred most rapidly (>95% in 6 h) in media provided with a complete complement of amino acids. When (NH₄)₂SO₄, urea, or individual amino acids were provided as the sole nitrogen source, conidial germination was less than 20% after 6 h incubation. Conidia production was delayed inC. truncatum cultures grown in media with urea or individual amino acids as nitrogen sources compared to cultures supplied with Casamino acids or complete synthetic amino acid nitrogen sources. The use of methionine, lysine, tryptophan, isoleucine, leucine or cysteine as a sole nitrogen source severely inhibitedC. truncatum conidia production. Media with synthetic amino acid mixtures less these inhibitory amino acids produced significantly higher conidia yields compared to media with amino acid mixtures containing these amino acids. When various amounts of each individual inhibitory amino acid were added to media which contained amino acid mixtures, cysteine and methionine were shown to be most effective in reducing conidiation. An optimal nitrogen source forC. truncatum conidiation in submerged culture should contain a complete mixture of amino acids with low levels of cysteine, methionine, leucine, isoleucine, lysine and tryptophan for rapid conidiation and optimal conidia yield.The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.     

Membrane transport in Schistosoma mansoni: transport of amino acids by adult males.

The mechanisms by which adult male Schistosoma mansoni transport amino acids have been investigated using radioactive amino acids during 2-min incubation times. The transport constants (K_t) for mediated uptake of glycine, proline, methionine, arginine, glutamate, and tryptophan were calculated to be 0.60-1.05, 1.67-1.98, 2.0, 0.10-0.35, 0.30-0.50, and 0.5-1.0 mM, respectively. Maximal velocities (V_max) were 5.5–7.5, 25, 6.4, 1.5-2.0, 2.5, and 3.0–6.0 μmoles absorbed/g worm protein/2 min, respectively. Cysteine is taken up solely by diffusion. Proline uptake is unique in that no significant diffusion component was found. The other amino acids studied were absorbed by diffusion as well as by specific transport systems. In the 2-min incubation periods employed glycine, proline, glutamate, and methionine were not significantly metabolized indicating that the uptake studies using these substrates reflect transport. Metabolism of the other amino acids used in these studies was not examined. The specificity of the transport systems was studied by testing the inhibitory effects of various amino acids on the uptake of each of the amino acids studied. The results suggest the presence of at least five transport systems. There is a highly specific transport locus for proline, and one for acidic amino acids. There are probably at least two transport systems, each of broad and overlapping specificity, for most of the neutral amino acids. Basic amino acids also appear to be taken up by complex transport systems, at least one of which overlaps with the neutral sites. The results are discussed with respect to the nutrition of the parasite and the host-parasite relationship.     

Physiological studies on Phymatotrichum omnivorum X. Influence of nitrogen sources on free and bound amino acid pools

The effect of nitrogen source on the free and bound amino acids of mycelium of Phymatotrichum omnivorum (Shear) Dugg was investigated. The largest free amino acid pool was present in the natural medium and the smallest in the synthetic medium. Phymatotrichum omnivorum was able to utilize different nitrogen sources with the best growth occurring with NH₄NO₃. The ratio of glycine to alanine and aspartic to glutamic was around 0.25 in the free amino acid pool and around 1 in the bound amino acid pool. The free pool of glutamic acid ranged from 5.6 % to 27.2 % depending upon the nitrogen source in the media. The free pool of alanine ranged from 35.7 % to 17.2 % in relation to the nitrogen source. Most other amino acid ratios did not vary significantly between the free amino acids and the bound amino acids.     

Role of Amino Acids and Vitamins in Nutrition of Mesophilic Methanococcus spp

In this study we found that autotrophic methanococci similar to Methanococcus maripaludis obtained up to 57% of their cellular carbon from exogenous amino acids. About 85% of the incorporation was into protein. Primarily nonpolar and basic amino acids and glycine were incorporated; only small amounts of acidic and some polar amino acids were taken up. An additional 10% of the incorporation was into the nucleic acid fraction. Because little ¹⁴CO₂ was formed from the ¹⁴C-amino acids, little metabolism of the amino acids occurred. Therefore the growth stimulation by amino acids was probably due to the sparing of anabolic energy requirements. Of the amino acids incorporated, only alanine was also a sole nitrogen source for these methanococci. In contrast, Methanococcus vannielii and “Methanococcus aeolicus” are autotrophic methanococci which did not incorporate amino acids and did not utilize alanine as a sole nitrogen source. Although glutamine served as a sole nitrogen source for the autotrophic methanococci and Methanococcus voltae, a heterotrophic methanococcus, growth was due to chemical deamination in the medium. M. voltae requires leucine and isoleucine for growth. However, these amino acids were not significant nitrogen sources, and alanine was not a sole nitrogen source for the growth of M. voltae. The branched-chain amino acids were not extensively metabolized by M. voltae. Pantoyl lactone and pantoic acid were readily incorporated by M. voltae. The intact vitamin pantothenate was neither stimulatory to growth nor incorporated. In conclusion, although amino acids and vitamins are nutritionally important to both autotrophic and heterotrophic methanococci, generally they are not subject to extensive catabolism.     

Hormonal regulation of the system a amino acid transport adaptive response mechanism in a kidney epithelial cell line (MDCK)

When mamalian cells are starved for amino acids, the activity of the A amino acid transport system increases, a phenomenon called adaptive regulation. We have examined the effects of those factors which support Madin-Darby canine kidney (MDCK) cell growth in a defined medium on the derepression of System A activity. Of the five factors which supported MDCK cell growth, insulin was found to be an absolute requirement for derepression. In contrast, PGE₁ was a negative controlling factor for the transport system. Growth of MDCK cells in the absence of PGE₁ resulted in elevated System A activity which derepressed poorly upon amino acid starvation. Kinetic analysis of α-(methylamino) isobutyric acid (mAIB) uptake as a function of substrate concentration showed that the elevated A activity observed when cells were grown in the absence of PGE₁ was kinetically similar to the activity induced by starvation for amino acids. Transport of mAIB by amino-acid-fed cells grown in the presence of PGE₁ was characterized by a linear Eadie-Hofstee graph and by a relatively low Vmax. Transport by cells starved for amino acids or by cells grown in the absence of PGE₁ was characterized by biphasic kinetics for mAIB transport and by elevated Vmax values. An influence of growth factors on the inactivation of derepressed A activity was also observed. In the presence of cycloheximide the rate of loss of A activity in amino-acid-starved cells was 1/4–1/2 that of amino-acid-fed cells. Insulin slowed inactivation in the absence of most amino acids in a protein-synthesis-independent manner, but insulin did not influence the more rapid inactivation observed in amino-acid-fed cells. These results indicate that the level of System A activity observed in response to regulation by amino acids represents a balance between carrier synthesis and inactivation, which can be positively or negatively influenced by growth factors.     

Enzymatic characterization of peptidic materials isolated from aqueous solutions of ammonium cyanide (pH 9) and hydrocyanic acid (pH 6) exposed to ionizing radiation

The enzymatic digestion of some radiolytically produced peptidic materials was examined. The substrates were compounds isolated from 0.1 molar solutions of NH₄CN (pH 9) and HCN (pH 6), after their exposure to rays from a⁶⁰Co source (15–20 Mrad doses). Commercial proteolytic enzymes pronase and aminopeptidase M were used. The examined materials were of composite nature and proteolytic action was systematically observed after their subsequent purification. In some fractions the effect was found to be positive with up to 30% of peptide bonds cleaved with respect to the amino acid content. These findings support our previous conclusions on the free radical induced formation of peptidic backbones without the intervention of amino acids. Some side effects were also noted which might be of interest in observations on enzymatic cleavage of other composite peptidic materials of abiotic origin.     

6 Lignin-protein complex in cell walls of Pinus elliottii: Amino acid constituents

Cell walls of Pinus elliottii callus contain ca 12 % protein. Klason lignin prepared from the walls contained 9 % protein and represented 4.5 % of the wall. The lignin fraction was increased to 22 % of the wall weight by reacting washed cell-wall tissue with coniferyl alcohol and H₂O₂, a reaction catalysed by peroxidase that remained bound to the wall. The augmented lignin preparation yielded 10 % protein. The acid hydrolysate of whole wall tissue included five amino acids at a concentration higher than hydroxyproline. The hydrolysates of both natural and augmented lignin preparations yielded distributions of amino acids in which the concentration of hydroxyproline was higher than that of all other amino acids. The results suggest that polymerizing lignin links covalently with cell-wall glycoprotein, and that the bonds may be formed preferentially with hydroxyproline.     

The radiolysis of aqueous acetonitrile: Compounds of interest to chemical evolution studies

Summary Oxygen-free aqueous solutions of CH₃CN (0.1 M, pH 6) were exposed to gamma rays from a⁶⁰Co source, the mixture of nonvolatile radiolytic products was fractionated and the fractions were analysed. Succinic, maleic, fumaric, malonic and pyruvic acids were identified. Glycol aldehyde, glucose and probably ribose were observed in the hydrolysate of fractionated material. It has been suggested that an oligomer is formed which has a fragment with the polyhydroxy structure and on hydrolysis releases the carbohydrates. Radiolytic products which release amino acids on hydrolysis were found in several fractions. The amino acid contents of the hydrolysates were up to about 2.8% of the fraction mass. The presence of several protein and nonprotein amino acids suggests that their origin should be in a peptidic structure, which is probably a fragment of an oligomer radiolytically produced. A direct analysis of the irradiated solution shows the presence of acetaldehyde, propionaldehyde, glyoxal and of biacetyl. Experimental findings are discussed and a free-radical mechanism is proposed to account for the chemical changes observed.     

The atmosphere of the primitive Earth and the prebiotic synthesis of amino acids

The atmosphere of the Earth at the time of its formation is now generally believed to have been reducing, an idea proposed by Oparin and extensively discussed by Urey. This atmosphere would have contained CH₄, N₂ with traces of NH₃, water and hydrogen. Only traces of NH₃ would have been present because of its solubility in water. UV light and electric discharges were the major sources of energy for amino acid synthesis, with electric discharges being the most efficient, although most other sources of energy also give amino acids.The first prebiotic electric discharge synthesis of amino acids showed that surprisingly high yields of amino acids were synthesized. Eleven amino acids were identified, four of which occur in proteins. Hydroxy acids, simple aliphatic acids and urea were also identified. These experiments have been repeated recently, and 33 amino acids were identified, ten of which occur in proteins, including all of the hydrophobic amino acids.Methionine can be synthesized by electric discharges if H₂S or CH₃SH is added to the reduced gases. The prebiotic synthesis of phenylalanine, tyrosine and trytophan involves pyrolysis reactions combined with plausible solution reactions.Eighteen amino acids have been identified in the Murchison meteorite, a type II carbonaceous chondrite, of which six occur in proteins. All of the amino acids found in the Murchison meteorite have been found among the electric discharge products. Furthermore, the ratios of amino acids in the meteorite show a close correspondence to the ratios from the electric discharge synthesis, indicating that the amino acids on the parent body of the carbonaceous chondrites were synthesized by electric discharges or by an analogous process.     

Aflatoxin Contamination Detected in Nutrient and Anti-Oxidant Rich Edible Stink Bug Stored in Recycled Grain Containers

Recently, there has been multi-agency promotion of entomophagy as an environmentally-friendly source of food for the ever increasing human population especially in the developing countries. However, food quality and safety concerns must first be addressed in this context. We addressed these concerns in the present study using the edible stink bug Encosternum delegorguei, which is widely consumed in southern Africa. We analysed for mycotoxins, and health beneficials including antioxidants, amino acids and essential fatty acids using liquid chromatography coupled to quadrupole time of flight mass spectrometry (LC-Qtof-MS) and coupled gas chromatography (GC)-MS. We also performed proximate analysis to determine nutritional components. We identified the human carcinogen mycotoxin (aflatoxin B₁) at low levels in edible stink bugs that were stored in traditonally woven wooden dung smeared baskets and gunny bags previously used to store cereals. However, it was absent in insects stored in clean zip lock bags. On the other hand, we identified 10 fatty acids, of which 7 are considered essential fatty acids for human nutrition and health; 4 flavonoids and 12 amino acids of which two are considered the most limiting amino acids in cereal based diets. The edible stink bug also contained high crude protein and fats but was a poor source of minerals, except for phosphorus which was found in relatively high levels. Our results show that the edible stink bug is a nutrient- and antioxidant-rich source of food and health benefits for human consumption. As such, use of better handling and storage methods can help eliminate contamination of the edible stink bug with the carcinogen aflatoxin and ensure its safety as human food.     

Influence of the nitrogen source and concentration on N fractions and free amino acid levels of grape vine explants

The influence of the source of inorganic nitrogen (KNO₃, (NH₄)₂SO₄ and NH₄NO₃) and its concentration (5, 10, 20 and 30 mM N) on total N incorporation, as well as on N distribution into different fractions (amminiacal, amino, amide and protein) and on free amino acid levels has been determined in grape vine explants cultured in vitro.Increasing concentrations of the nitrogen source resulted in increased total N content in tissues. This effect was small for KNO₃, higher for (NH₄)₂SO₄ and maximal for NH₄NO₃. In addition, nitrate promoted an increase in amino-N only, whereas ammonium increased both the ammoniacal-N and the amino-N fractions. Incorporation of N into amide-N and protein-N were not affected significantly by the N sources tested.The application of increasing quantities of N enhanced the accumulation of most free amino acids, especially arginine, alanine and proline, but to different extents, depending on both the N source and its concentration. The combination of ammonium and nitrate resulted in a higher accumulation of amino acids than that observed with either one of the two forms alone.     

Partitioning of acidic, basic and neutral amino acids into imidazolium-based ionic liquids

In this paper, partitioning behaviors of typical neutral (Alanine), acidic (Glutamic acid) and basic (Lysine) amino acids into imidazolium-based ionic liquids [C₄mim][PF₆], [C₆mim][PF₆], [C₈mim][PF₆], [C₆mim][BF₄] and [C₈mim][BF₄] as extracting solvents were examined. [C₆mim][BF₄] showed the best efficiency for partitioning of amino acids. The partition coefficients of amino acids in ionic liquids were found to depend strongly on pH of the aqueous solution, amino acid and ionic liquid chemical structures. Different chemical forms of amino acids in aqueous solutions were pH dependent, so the pH value of the aqueous phase was a determining factor for extraction of amino acids into ionic liquid phase. Both water content of ionic liquids and charge densities of their anionic and cationic parts were important factors for partitioning of cationic and anionic forms of amino acids into ionic liquid phase. Extracted amino acids were back extracted into phosphate buffer solutions adjusted on appropriate pH values. The results showed that ionic liquids could be used as suitable modifiers on the stationary phase of an HPLC column for efficient separation of acidic, basic, and neutral amino acids.     

Long distance translocation of sucrose, serine, leucine, lysine, and carbon dioxide assimilates: I. Soybean

To determine the selectivity of movement of amino acids from source leaves to sink tissues in soybeans (Glycine max [L.] Merr. `Wells'), ¹⁴C-labeled serine, leucine, or lysine was applied to an abraded spot on a fully expanded trifoliolate leaflet, and an immature sink leaf three nodes above was monitored with a GM tube for arrival of radioactivity. Comparisons were made with ¹⁴C-sucrose and ¹⁴CO₂ assimilates. Radioactivity was detected in the sink leaf for all compounds applied to the source leaflet. A heat girdle at the source leaf petiole essentially blocked movement of applied compounds, suggesting phloem transport. Transport velocities were similar (ranged from 0.75 to 1.06 cm/min), but mass transfer rates for sucrose were much higher than those for amino acids. Hence, the quantity of amino acids entering the phloem was much smaller than that of sucrose. Extraction of source, path, and sink tissues at the conclusion of the experiments revealed that 80 to 90% of the radioactivity remained in the source leaflet. Serine was partially metabolized in the transport path, whereas lysine and leucine were not. Although serine is found in greater quantities than leucine and lysine in the source leaf and path of soybeans, applied leucine and lysine were transported at comparable velocities and in only slightly lower quantities than was applied serine. Thus, no selective barrier against entry of these amino acids into the phloem exists.     

Electrochemical behaviors of amino acids at multiwall carbon nanotubes and Cu2O modified carbon paste electrode

A carbon paste electrode modified with multiwall carbon nanotubes and copper(I) oxide (MWCNT-Cu₂O CPME) was fabricated, and the electrochemical behaviors of 19 kinds of natural amino acids at this modified electrode were studied. The experimental results showed that the various kinds of amino acids without any derivatization displayed obvious oxidation current responses at the modified electrode. It was also found that the current response values of amino acids were dependent mainly on pH values of buffer solutions. The phenomenon could be explained by the fact that the amino acids suffered complexation or electrocatalytic oxidation processes under different pH values. Six kinds of amino acids (arginine, tryptophan, histidine, threonine, serine, and tyrosine), which performed high-oxidation current responses in alkaline buffers, were selected to be detected simultaneously by capillary zone electrophoresis coupled with amperometric detection (CZE-AD). These amino acids could be perfectly separated within 20 min, and their detection limits were as low as 10⁻⁷ or 10⁻⁸ mol L⁻¹ magnitude (signal/noise ratio = 3). The above results demonstrated that MWCNT-Cu₂O CPME could be successfully employed as an electrochemical sensor for amino acids with some advantages of convenient preparation, high sensitivity, and good repeatability.     

Protein content and amino acids composition of bee-pollens from major floral sources in Al-Ahsa,eastern Saudi Arabia

Protein content and amino acids composition of bee-pollens from major pollen floral sources in Al-Ahsa, Saudi Arabia were determined to investigate the nutritive value of pollen protein relative to requirements of honeybees and adult humans. The major pollen sources were alfalfa, date palm, rape, summer squash, and sunflower. Bee-pollens from alfalfa and date palm showed high content of crude protein and amino acid concentrations. Bee-pollen from sunflower had low content of those components. Eighteen amino acids were found in bee-pollens from the five major floral sources. The highest concentrations of individual amino acids valine, leucine, isoleucine, phenylalanine and proline were obtained from alfalfa bee-pollen; lysine, arginine, cysteine, tryptophan and tyrosine from date palm; methionine, histidine, glycine and alanine from summer squash; threonine, serine and glutamic acid from sunflower; and aspartic acid from rape bee-pollen. The amino acid composition obtained from sunflower bee-pollen showed the lowest concentrations of the essential amino acids: isoleucine, leucine, methionine, phenylalanine and valine. Apart from methionine, arginine and isoleucine, the essential amino acids of bee-pollen from alfalfa, date palm, summer squash and rape exceeded the honeybees’ requirements. Methionine was the limiting amino acid in bee-pollens from the five selected sources. Concentrations of essential amino acids in the tested bee-pollens were variable and significantly correlated to their botanical origin of pollen. Bee-pollens from alfalfa, date palm and summer squash was found to be rich source of protein and amino acids for bees and for humans.     

Chemiluminescence determination of hydroperoxides following radiolysis and photolysis of free amino acids

Hydroperoxides were determined in selected amino acids using three free radical generating systems by a sensitive (50 pmol limit of detection) and specific high performance liquid chromatography (HPLC)/chemiluminescence method. UVB and gamma radiation produced significant hydroperoxide formation, particularly in the aromatic amino acids tyrosine and tryptophan. Hydroperoxide yield was found to be dependent on both amino acid and irradiation source. Generation of hydrogen peroxide as a by-product of irradiation caused interference with chemiluminescence detection demonstrating the need for catalase addition. Hydroperoxides were not detectable following metal-catalysed H₂O₂ breakdown. We suggest that metal ions could interfere with the detection of hydroperoxides by causing preferential decomposition.     

Radiolysis,racemization, and the origin of optical activity

An investigation has been undertaken to determine whether ionizing radiation might engender racemization (radioracemization) of optically active amino acids, along with their well-known radiolysis. We have exposed a number of solid and dissolved optically active amino acids to the ionizing radiation from a 3000-Ci ⁶⁰Co γ-ray source for periods of time which would engender substantial, but not total radiolysis. γ-Ray doses which caused 55–68% radiolysis of solid amino acids typically engendered 2–5% racemization. Aqueous solutions of the sodium salts of amino acids which underwent 53–66% radiolysis typically showed 5–11% racemization. The corresponding hydrochloride salts in aqueous solution, however, underwent little or no racemization. In aqueous solution both percentage degradation and percentage racemization were approximately proportional to γ-ray dosage within the range employed (1–36 × 10⁶ rads). Mechanisms for the radioracemization of amino acids in the solid state and as dissolved sodium salts are proposed, and the absence of racemization for dissolved hydrochloride salts is rationalized. Implications of these observations with regard to the origin of optical activity by the Vester-Ulbricht β-decay mechanism are discussed, as are their implications regarding the use of diagenetic racemization rates of ancient amino acid samples as criteria for geochronological and geothermometric calculations.     

Tissue concentration changes of amino acids and biogenic amines in the central nervous system of mice with experimental autoimmune encephalomyelitis (EAE)

We have characterized the changes in tissue concentrations of amino acids and biogenic amines in the central nervous system (CNS) of mice with MOG_35-55-induced experimental autoimmune encephalomyelitis (EAE), an animal model commonly used to study multiple sclerosis (MS). High performance liquid chromatography was used to analyse tissue samples from five regions of the CNS at the onset, peak and chronic phase of MOG_35-55 EAE. Our analysis includes the evaluation of several newly examined amino acids including d-serine, and the inter-relations between the intraspinal concentration changes of different amino acids and biogenic amines during EAE. Our results confirm many of the findings from similar studies using different variants of the EAE model as well as those examining changes in amino acid and biogenic amine levels in the cerebrospinal fluid (CSF) of MS patients. However, several notable differences were observed between mice with MOG_35-55-induced EAE with findings from human studies and other EAE models. In addition, our analysis has identified strong correlations between different amino acids and biogenic amines that appear to change in two distinct groups during EAE. Group I analyte concentrations are increased at EAE onset and peak but then decrease in the chronic phase with a large degree of variability. Group II is composed of amino acids and biogenic amines that change in a progressive manner during EAE. The altered levels of these amino acids and biogenic amines in the disease may represent a critical pathway leading to neurodegenerative processes that are now recognized to occur in EAE and MS.