Effect of natural amino acids and sugars on cyclase activities in infusoria Tetrahymena pyriformis and Dileptus anser

Natural amino acids and sugars in intracellular eukaryotes are known to regulate adenylyl cyclase (AC) and guanylyl cyclase (GC) systems that control the most important cell processes. The goal of the present work consisted in study of effects of natural amino acids and sugars and some of their derivatives on AC and GC activities of infusoria Tetrahymena pyriformis and Dileptus anser. Methionine, arginine, lysine, and tryptamine stimulated basic AC activity of T. pyriformis, whereas alanine, thyrosine, and cysteine decreased it. Methionine, glycine, alanine, thyrosine, arginine, and to the lesser degree tryptamine and histidine stimulated AC of D. anser. The GC activity of T. pyriformis are increased in the presence of tryptamine, tryptophane, histidine, arginine, and lysine, whereas glycine and aspartic acid, on the contrary, decreased it. Tryptamine, tryptophan, leucine, glutamic acid, serine, histidine, and alanine stimulated the GC activity of D. anser. Glucose, fructose, and sucrose stimulated the basal AC activity of both infusorians and GC of T. pyriformis, with glucose and sucrose increasing AC of T. pyriformis twice, while that of D. anser 4.5 times. Lactose stimulated AC and GC of T. pyriformis and was inefficient with respect to the D. anser cyclases, whereas mannose and galactose did not affect the enzyme activities in both infusorians. The study of the chemotactic response of infusorians to amino acids and sugars indicates that involved in realization of this response can be signaling pathways both dependent on and independent of cyclic nucleotides. Thus, it has been established for the first time that several amino acids and sugars affect functional activity of enzymes with cyclase activity of the infusorians T. pyriformis and D. anser. This confirms the hypothesis that at early stages of evolution the large spectrum of comparatively simple natural molecules has a hormone-like action.     

Molecular mechanisms of action of natural amino acids and serotonin on infusorian adenylyl cyclase and guanylyl cyclase

Earlier, it has been shown that some amino acids and their derivatives are able to regulate activities of adenylyl cyclase (AC) and guanylyl cyclase (GC) in free-living infusoria Dileptus anser and Tetrahymena pyriformis. The goal of this work consisted in studying the molecular mechanisms of action of methionine, tyrosine, alanine, and neurohormone serotonin on the activity of enzyme-cyclases and in identification of their specific receptors in D. anser and T. pyriformis. Methionine and serotonin significantly increased the basal AC activity in both infusoria; the effect of serotonin on AC in T. pyriformis took place with participation of the Ca²⁺-dependent form of AC and of the heterotrimeric G-proteins. The AC-stimulating effect of tyrosine and alanine was expressed weakly and was revealed only in D. anser. Serotonin in both infusoria and alanine in D. anser stimulated GC activity, whereas methionine and tyrosine did not affect GC. Methionine and serotonin were bound with a high affinity to the surface receptors of infusoria. The K_D for [methyl-³H]methionine binding to D. anser and T. pyriformis were equal to 7.5 and 35.6 nM, and for [³H]serotonin binding, they were 2.7 and 4.7 nM, respectively. Alanine and tyrosine were bound to infusoria with low affinity. Thus, in the infusoria D. anser and T. pyriformis, there are chemosignal systems regulated by amino acids and their derivatives, including enzymes with cyclase activity. These systems are suggested to be similar to the hormonal signal systems of the higher eukaryotes and to be their predecessors.     

Regulation by cyclic adenosine monophosphate of functional activity of the adenylyl cyclase system in the infusorian <Emphasis Type="Italic">Dileptus anser</Emphasis>

In some unicellular eukaryotes, cAMP performs functions not only of the secondary messenger, but also of hormone, the primary messenger. We have found that cAMP is bound to surface receptors of the free-living infusorian Dileptus anser and stimulates activity of the adenylyl cyclase signaling system (AC-system) including heterotrimeric G-proteins and the enzyme, adenylyl cyclase (AC). The binding of cAMP to receptor is performed with a high affinity (K _D = 27 nM) and is highly specific, as cGMP and adenosine do not produce a marked effect on it. The infusorian cAMP-receptors have been shown to be coupled to G-proteins, which is indicated by a decrease of their affinity to the ligand in the presence of GTP, stimulation of the GTP-binding of G-proteins with the cyclic nucleotide, and block of the cAMP regulatory effects with suramin, an inhibitor of heterotrimeric G-proteins. cAMP stimulates dose-dependently the AC activity, its effect remaining virtually unchanged in the presence of cGMP, AMP, GMP, and adenosine. N⁶,O^2′-dibutyryl-cAMP, a non-hydrolyzed cAMP analogue, only at comparatively high concentrations competes with cAMP for binding sites and decreases the cAMP stimulating effects on the AC activity and GTP binding. Thus, we have shown for the first time that the AC system of the infusorians D. anser is stimulated by the extracellular cAMP that in this case functions as the external signal regulates activity of extracellular cAMP-dependent effector systems.     

The study of molecular mechanisms of action of natural amino acids and serotonin on adenylyl and guanylyl cyclases of the ciliates

It has been previously shown that some amino acids and their derivatives are capable of regulating the activity of adenylyl cyclase (AC) and guanylate cyclase (GC) in free-living ciliates Dileptus anser and Tetrahymena. The aim of this work was to study the molecular mechanisms of action of methionine, tyrosine, alanine and neurohormone serotonin on the activity of enzymes-cyclases and the identification of their specific receptors in D. anser and T. pyriformis. Methionine and serotonin significantly increased the basal AC activity in both ciliates, and the AC effect of serotonin in T. pyriformis was carried out with the participation of Ca2+-dependent form of AC and heterotrimetic G proteins. AC stimulating effect of tyrosine and alanine was expressed weakly and only detected in D. anser. Serotonin is both ciliates and alanine in D. anser stimulated GC activity, whereas methionine and tyrosine had no effect on GC. Methionine and serotonin bind to surface receptors of the ciliates with high affinity. K(D) for [methyl-3H] methionine binding to D. anser and T. pyriformis were 7.5 and 35.6 nM, and for [3H] serotonin binding were 2.7 and 4.7 nM, respectively. Alanine and tyrosine bind to the ciliates with low affinity. Thus, ciliates D. anser and T. pyriformis have chemosignaling systems regulated by amino acids and their derivatives and including the enzymes with cyclase activity. There is an assumption that these systems are similar to hormonal signaling systems of higher eukaryotes and are their predecessors.     

Inhibition of functional activity of the adenylyl cyclase signaling system of the ciliate Dileptus anser by colchicine and vinblastine

Cytoskeleton plays a key role in the functioning of hormonal signaling systems in vertebrate animals. However, data on the effect of cytoskeletal components, in particular microtubules, on the functional activity of chemosignaling systems of unicellular organisms are currently lacking. The goal of this work consisted of studying the effects of microtubule-disrupting agents, colchicine and vinblastine, on the adenylyl cyclase system of free living infusoria Dileptus anser. The incubation of D. anser with colchicine and vinblastine (10^?5–10^?6 M) weakly affected the basal activity of adenylyl cyclase (AC), but led to a significant decrease in or complete block of AC stimulation with nonhormonal (GppNHp, sodium fluoride) and hormonal agents (adrenaline, serotonin, glucagon). The basal level of GTP binding in heterotrimeric G proteins decreased and there was observed inhibition of stimulation of G proteins by hormones. Colchicine and vinblastine have been shown to interrupt adrenalin-produced AC stimulation achieved through G_s-protein, but weakly affect its inhibiting AC effect caused by the G_i-protein. Thus, it has been established for the first time that, in unicellular organisms, i.e., infusoria D. anser, microtubules are involved in the regulation of the functional activity of the AC system and their action is realized at the level of G proteins, which is similar to Gs-proteins in vertebrate animals.     

Histone-antihistone interactions in the <Emphasis Type="Italic">Escherichia coli-Tetrahymena pyriformis</Emphasis> association

Using the Escherichia coli-Tetrahymena pyriformis model system, we revealed the involvement of bacterial antihistone activity and protozoan histones in interactions between pro-and eukaryotic microorganisms. Antihistone activity enhanced the viability of E. coli in association with T. pyriformis, according to our data on the dynamics of E. coli cell numbers. The strain with antihistone activity induced incomplete phagocytosis in the infusorians, resulting in cytological changes and ultrastructural alterations that indicated the retention of bacterial cells in phagosomes. Bacteria with antihistone activity located in the T. pyriformis cytoplasm influenced the eukaryotic nucleus. This was accompanied by in macronucleus decompactization and a decrease in the average histone content in the population of infusorians. The data obtained suggest that protozoan histone inactivation by bacteria is one of the mechanisms involved in prokaryote persistence in associations with eukaryotic microorganisms.     

Regulatory calcium effect on adenylyl cyclase functional activity in the infusorian <Emphasis Type="Italic">Dileptis anser</Emphasis>

Earlier we have shown that some non-hormonal activators of adenylyl cyclase (AC) and hormones of higher vertebrate animals are able to affect functional activity of the AC system in the infusorian Dileptus anser. In the present work, sensitivity of this infusorian AC to Ca²⁺ was studied and it was found that calcium cations at concentrations of 0.5–10 μM stimulated significantly the enzyme activity in D. anser partially purified membranes. An increase of Ca²⁺ concentrations to 100 μM and higher led to the complete block of their stimulatory effect. In the EDTA-treated membranes the enzyme activity was reduced markedly, but it was restored significantly by addition of Ca²⁺. Calmodulin antagonists—chlorpromazine, W-7, and W-5—caused a dose-dependent decrease of the enzyme activity stimulated by 5 μM Ca²⁺ with IC₅₀ values of 35, 137, and 174 M, respectively. The AC-stimulating effects of biogenic amines (serotonin and octopamine) were completely retained in the presence of 2.5 and 100 μM Ca²⁺, whereas effects of peptide hormones (relaxine and EGF) were hardly changed in the presence of 2.5 μM calcium ions, but were markedly inhibited by 100 μM Ca²⁺. In the EDTA-treated membranes, the AC effects of biogenic amines were reduced, while the effects of peptide hormones were not revealed. On addition of Ca²⁺, the AC effects of biogenic amines were completely restored, whereas the effects of peptide hormones were not detected or restored to a non-significant degree. Calmodulin antagonists slightly affected the AC effects of peptide hormones at concentrations efficient in the case of vertebrate AC, but decreased them markedly at higher concentrations. The AC effects of biogenic amines were little sensitive even to high antagonist concentrations. The obtained data show that targets of action of peptide hormones in the infusorian D. anser cell culture are the AC forms whose activity depends on calcium cations and possibly is regulated by Ca²⁺/calmodulin, whereas targets of action of biogenic amines are calcium-independent enzyme forms.     

Detection of amino acids in artificial nectars by two tropical ants,Leptothorax and Monomorium

Summary Many ants forage at extrafloral nectar on plants and provide the plant with some measure of protection from herbivory. These nectars contain sugars, amino acids and, often, other compounds. The role of amino acids in attracting ants to extrafloral nectars was studied by baiting with Karo-syrup-based solutions. Control (without amino acids) and experimental (with amino acids) solutions were placed in second growth forest in Trinidad, W.I. The number of ants visiting the solutions was counted at fiveminute intervals for 45 min. In tests of solutions with only one amino acid, both Leptothorax sp. and Monomorium sp. visited solutions with alanine, arginine, serine, cysteine, methionine or aspartic acid more frequently than sugaronly controls. Monomorium preferred control solutions to tyrosine solutions; Leptothorax preferred control solutions to histidine solutions. Leptothorax did not discriminate between control and tyrosine solutions; Monomorium did not discriminate between control and histidine solutions. However, in six of eight tests of combinations of amino acids, ants visited control solutions more frequently than experimental solutions. These results suggest that ants can act as selective agents, favoring plants with particular amino acids in their nectars.     

Studies on Amino Acid Contents of Processed Soybean

The influence of sugars on the heat destruction of the basic and sulphur containing amino acids of soybean products, soybean protein and also pure amino acids has been investigated.

Amino acids were estimated by microbiological assay procedure. Cystine was also determined chromatographically.

Lysine, arginine and histidine were destroyed in different ways behaved when soybean products, soybean protein or pure amino acids mixed with and without sugars were autoclaved; and the condition of added sugars tended to influence the way of destruction.

Cystine was the most heat-labile amino acid. But the destruction did not appear to be ascribed to added sugars except 50% ethyl alcohol-soluble sugar solution of defatted soybean flour. This finding was also substantiated by the chromatographic analysis.

Methionine, in soybean products and soybean protein or as pure amino acid with and without the addition of sugars, was not influenced by autoclaving.     

The chemical stimuli of human skin surface for the attachment response of Schistosoma mansoni cercariae

and 1986. The chemical stimuli of human skin surface for the attachment response of S mansoni cercariae. International Journal for Parasitology 16: 575–579. The stimuli for the S. mansoni attachment response were analysed by offering skin extracts and chemicals to cercariae through Millipore filters. The attachment stimulating components were contained in the hydrophilic skin extract. Whereas sugars had no stimulating activity, a mixture of electrolytes, lactate and urea had a weak, and amino acids a very strong, effect. The amino acids of the hydrophilic skin extract were qualitatively and quantitatively identified and their effectivity as pure chemicals compared with that of the skin extract. The acid amino acids and histidine and ornithine had a weak effect, whereas arginine alone stimulated as intensely as the whole hydrophilic skin extract. The specialization on arginine as the main attachment trigger could be interpreted as adaptation to the fact that creeping and penetrating cercariae secrete arginine with the contents of their post acetabular glands. Thus they might communicate their successful host identification to further cercariae.     

Antimutagenic Effect of Amino Acids on the Mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)

The antimutagenic activity of protein-constituting amino acids except histidine on the mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) was investigated in vitro using Salmonella typhinurium TA-100 as an indicator bacterium (Ames test), and concentrations (IC₅₀) of amino acids that inhibit 50% of the mutagenecity were measured. Cysteine was found to be most active and glycine, tryptophan, lysine, and arginine were strong antimutagenic amino acids. Other amino acids showed moderate or weak antimutagenic activities, depending on the amino acids. The results indicate that amino acids play a substantial role in chemoprevention of N-nitroso amine-induced mutagenicity.     

Amino acid utilization by the ruminal bacterium Synergistes jonesii strain 78-1

The ruminal bacterium Synergistes jonesii strain 78-1, which is able to degrade the pyridinediol toxin in the plant Leucaena leucephala, was studied for its ability to utilise amino acids. The organism used arginine, histidine and glycine from a complex mixture of amino acids, and both arginine and histidine supported growth in a semi-defined medium. The products of (U-¹⁴C)-arginine metabolism were CO₂ acetate, butyrate, citrulline and ornithine. The labelling pattern of end products from (U-¹⁴C)-histidine metabolism differed in that carbon also flowed into formate and propionate. Arginine was catabolised by the arginine deiminase pathway which was characterised by the presence of arginine deiminase, ornithine transcarbamylase and carbamate kinase. This is the first report of a rumen bacterium that uses arginine and histidine as major energy yielding substrates.     

Speculations on the evolution of the genetic code II

An evolutionary scheme is postulated in which a primitive code, involving only guanine and cytosine, would code for glycine (GG), alanine (GC), arginine (CG) and proline (CC). From each of these amino acids and their codons, there evolves a family of related amino acids as the code expands. The four families are: (1)alanine valine, leucine, isoleucine, phenylalanine, tyrosine, methionine and tryptophane; (2)proline, threonine and serine; (3)arginine, lysine, and histidine; (4)glycine, serine, cysteine, glutamic acid, glutamine, aspartic acid and asparagine. Except for the glycine relation to glutamic acid and aspartic acid, all amino acids are related by chemical similarities in their side chains. Glycine not having a side chain would permit a more complex set of substitutions.     

Regulation of Adenylyl Cyclase Signaling System in Cell Cultures of Infusoria Dileptus anser and Tetrahymena pyriformis by Peptides of Insulin Superfamily

Hormone-sensitive adenylyl cyclase signaling system (ACS) provides transduction of a wide spectrum of hormonal signals in cells of the higher eucaryotes. At the same time, ACS in the lower eucaryotes at present is practically not studied. We studied regulatory effects on ACS of the infusoria Dileptus anser and Tetrahymena pyriformis of peptide hormones of the higher eukaryotes—insulin, IGF-1, and relaxin, whose action on ACS of the higher eucaryotes was the subject of our earlier studies. The action of these hormones at concentrations of 10^–10–10^–8 M on the AC activity in infusoria had clearly stimulating character, the dose–effect curves being of a bell-shaped form with a maximum of the stimulating effect of the hormones at concentrations of 10^–9–10^–8 M. the shape of the curves and the value of the stimulating effect of the peptide hormones depended substantially on the level of the AC basal activity in homogenates of infusorian cell cultures. All the hormones (10^–8 M) stimulated GTP-binding activity of G-proteins. It was shown by the example of relaxin that its stimulating effect on GTP-binding in infusorian cells was dose-dependent and increased in the range of hormone concentrations from 10^–10 to 10^–8 M to reach its maximum at concentrations of 10^–8–10^–7 M. In the presence of suramin, an inhibitor of heterotrimeric G-proteins, the stimulating effects of the hormones on the GTP-binding and the AC activity decreased essentially or were absent completely. This indicates that the heterotrimeric G-proteins are ones of components of the signaling cascade that mediates regulatory effects of the hormones of the insulin group on the AC activity in infusorian cell cultures. Based on the obtained data, it is suggested that the basic molecular mechanisms of regulation of ACS by insulin and the related peptides that are similar to those found in the higher vertebrates already begin to be formed as early as at the level of the lower eucaryotes.     

Functional characteristics of calcium-sensitive adenylyl cyclase of the infusorian <Emphasis Type="Italic">Tetrahymena pyriformis</Emphasis>

The calcium-sensitive forms of adenylyl cyclases (AC) have been revealed in the majority of vertebrate and invertebrate animals, as well as in several representatives of unicellular organisms, including infusoria. We have found for the first time that the AC activity in the infusorian Tetrahymena pyriformis changes in the presence of calcium ions. Calcium ions at concentrations of 0.2–20 μM stimulated the activity of this enzyme, with the maximum of the stimulatory effect being observed at 2 μM Ca²⁺. At a concentration of 100 μM and higher, the calcium cations inhibited the AC activity. Antagonists of calmodulin W-5 and W-7 at concentrations of 20–100 μM decreased the stimulatory effect of 5 μM Ca²⁺, while at the higher concentrations inhibited it completely. Another calmodulin antagonist, chloropromazine, decreased the Ca²⁺-stimulated AC activity only at concentrations of 200–1000 μM. The stimulatory effect of serotonin, EGF, and cAMP on AC activity was enhanced in the presence of 5 μM Ca²⁺. The stimulatory effect of EGF, cAMP, and insulin on AC was decreased in the presence of 100 μM Ca²⁺, while the effect of cAMP was also observed in the presence of calmodulin antagonists (500 μM). At the same time, stimulatory effect of D-glucose did not change in the presence of Ca²⁺ and calmodulin antagonists. The obtained data indicate that, in the infusorian T. pyriformis, there are calcium-sensitive forms of AC that can be stimulated by EGF, cAMP, insulin, and serotonin.     

Nutritional studies of some members of mucorales IV. 1. Sugars,amino- and organic acids of the mycelium

Summary and Conclusions The sugars and amino acids of the fungal mycelium of six species of the order Mucorales were determined with the use of two-dimensional as well as circular paper chromatography. A critical survey of the above conclusions revealed that aspartic acid, glutamic acid, glycine and serine and -alanine were found in either free and bound form. Glutamine and arginine were absent in bound form. Threonine, histidine, isoleucine and tryptophane were absent in the ethanol soluble fraction. The presence and absence of certain amino acids in these species are good subsidiary characters for distinguishing one of them from the other.In the majority of the species three sugars viz., glucose, fructose and sucrose were found. InM. indica andC. bertholletiae sucrose was absent. Amongst the organic acid only tartaric and malic acids were detected in all the organisms.     

Effects of Nutrient Media on the Composition of Free Amino Acids in Saccharomyces cerevisiae

The quantitative and qualitative compositions of free amino acids of the yeast Saccharomyces cerevisiaeY-503 cultivated in different nutrient media were studied by liquid chromatography. The yeast grown in a medium containing geothermal water was shown to accumulate more amino acids. During lyophilization, the stabilization of the physiological activity of the yeast in this nutrient medium was observed. The increased biological value of dry yeast was shown to depend on the content of free amino acids, including essential amino acids: arginine, histidine, leucine, isoleucine, lysine, threonine, serine, and phenylalanine.     

The influence of peptides on the uptake of amino acids inFusobacterium; predicted interactions withPorphyromonas gingivalis

A study of the uptake of amino acids and its influence by a peptide source was carried out withFusobacterium varium as a convenient representative of the genus. Reference strains and a clinical isolate had similar amino acid uptake profiles, but most amino acids were incorporated at lower concentrations by the latter. In general, high levels of serine, asparagine, glutamate, cysteine, and arginine were incorporated by all species. Histidine, lysine, threonine, and aspartate were taken up at lower levels, whereas the nonpolar neutral amino acids such as alanine, valine, leucine, isoleucine, glycine, proline, phenylalanine, and methionine were poorly metabolized. Yeast extract, as a source of peptides, stimulated the uptake of several amino acids such as histidine and glutamate, whereas others such as methionine, threonine, and asparagine were repressed. The incorporation of some amino acids such as aspartate, ornithine, lysine, and arginine was unaffected by the presence of peptides. Equimolar nitrogen concentrations of amino acids or ammonia could not replace the peptide requirement, emphasizing the importance of peptides as an energy source. The limited capacity ofFusobacterium spp. to hydrolyze proteins increased approximately 30% in the presence of the proteolytic species,Porphyromonas gingivalis, and may represent one bacterial interaction in which peptides may become available toFusobacterium species in vivo.     

Ypq3p-dependent histidine uptake by the vacuolar membrane vesicles of Saccharomyces cerevisiae

The vacuolar membrane proteins Ypq1p, Ypq2p, and Ypq3p of Saccharomyces cerevisiae are known as the members of the PQ-loop protein family. We found that the ATP-dependent uptake activities of arginine and histidine by the vacuolar membrane vesicles were decreased by ypq2Δ and ypq3Δ mutations, respectively. YPQ1 and AVT1, which are involved in the vacuolar uptake of lysine/arginine and histidine, respectively, were deleted in addition to ypq2Δ and ypq3Δ. The vacuolar membrane vesicles isolated from the resulting quadruple deletion mutant ypq1Δypq2Δypq3Δavt1Δ completely lost the uptake activity of basic amino acids, and that of histidine, but not lysine and arginine, was evidently enhanced by overexpressing YPQ3 in the mutant. These results suggest that Ypq3p is specifically involved in the vacuolar uptake of histidine in S. cerevisiae. The cellular level of Ypq3p-HA³ was enhanced by depletion of histidine from culture medium, suggesting that it is regulated by the substrate.