Free amino acids in spider hemolymph

We examined the free amino acid composition of hemolymph from representatives of five spider families with an interest in knowing if the amino acid profile in the hemolymph of orb-web-building spiders reflects the high demands for small organic compounds in the sticky droplets of their webs. In nearly all analyses, on both orb and non-orb builders, glutamine was the most abundant free amino acid. Glycine, taurine, proline, histidine, and alanine also tended to be well-represented in orb and non-orb builders. While indications of taxon-specific differences in amino acid composition were observed, it was not apparent that two presumptive precursors (glutamine, taurine) of orb web sticky droplet compounds were uniquely enriched in araneids (orb builders). However, total amino acid concentrations were invariably highest in the araneids and especially so in overwintering juveniles, even as several of the essential amino acids declined during this winter diapause. Comparing the data from this study with those from earlier studies revealed a number of discrepancies. The possible origins of these differences are discussed.     

Uptake and utilization of lipids by Paramecium tetraurelia

Uptake rates of a variety of 14C-labeled fatty acids and complex lipids by Paramecium tetraurelia during 48 h of log-phase growth varied. Fatty acid uptake was maximal during lag phase of growth when phagosome (food vacuole) formation was minimal. Food vacuole formation was shown to be suppressed by the presence of exogenous lipids and by starvation. The rates of uptake of lipids were significantly greater than those of small organic compounds such as amino acids, cyclitols, fatty acid precursors and metabolic intermediates. Significant amounts of radioactivity from 14C-labeled fatty acids were metabolized to 14CO2. The uptake rates of different saturated, straight-chain fatty acids of even carbon numbers were different and were not correlated with chain length, results suggesting that the primary mechanism for uptake of these compounds was neither by bulk transport nor simple diffusion and that carrier-mediated processes could possibly be involved.     

Acid-labile amino acid precursors in the Murchison meteorite. 1: Chromatographic fractionation.

The amino acid content of a hot water extract of the Murchison meteorite can be increased by over 100 per cent by subjecting the extract to acid hydrolysis. The acid-labile compounds in the extract that account for this increase were fractionated by column chromatography on a cation exchange resin. Seventy mole per cent behaved as neutral or acidic compounds and were eluted from the column with an initial water wash. The remaining 30 mole per cent (basic precursors) were retained on the column and were eluted with the free amino acids by aqueous NH4OH. The acid-labile amino acid precursors in the water eluate could be retained and further fractionated on an anion exchange column, indicating that they are acidic compounds.     

Acid-labile amino acid precursors in the Murchison meteorite

The amino acid content of a hot water extract of the Murchison meteorite can be increased by over 100 per cent by subjecting the extract to acid hydrolysis. The acid-labile compounds in the extract that account for this increase were fractionated by column chromatography on a cation exchange resin. Sevently mole per cent behaved as neutral or acidic compounds and were eluted from the column with an initial water wash. The remaining 30 mole per cent (basic precursors) were retained on the column and were eluted with the free amino acids by aqueous NH₄OH. The acid-labile amino acid precursors in the water eluate could be retained and further fractionated on an anion exchange column, indicating that they are acidic compounds.Contribution number 101 from the Center for Meteorite Studies.     

Empirical evidence that soil carbon formation from plant inputs is positively related to microbial growth

Plant-carbon inputs to soils in the form of dissolved sugars, organic acids and amino acids fuel much of heterotrophic microbial activity belowground. Initial residence times of these compounds in the soil solution are on the order of hours, with microbial uptake a primary removal mechanism. Through microbial biosynthesis, the dissolved compounds become dominant precursors for formation of stable soil organic carbon. How the chemical class (e.g. sugar) of a dissolved compound influences stabilization in field soils is unknown and predictions from our understanding of microbial metabolism, turnover and identity are contradictory. We show that soil carbon formation, from chronic amendments of dissolved compounds to fertilized and unfertilized grasslands, is 2.4-times greater from a sugar than an amino acid. Formation rates are negatively correlated with respiration rates of the compounds, and positively correlated with their recovery in microbial biomass. These relationships suggest that the efficiency of microbial growth on a compound is positively related to formation rates of soil organic carbon. Fertilization does not alter these findings, but together nitrogen and phosphorus additions reduce soil carbon formation. Our results highlight the need to consider both nutrient enrichment and global-change induced shifts in the form of dissolved root inputs to soils to predict future soil carbon stocks and hence phenomena such as climate warming and food security to which these stock sizes are intimately tied.     

Role of Ferrocyanides in the Prebiotic Synthesis of α-Amino Acids

We investigated the synthesis of α-amino acids under possible prebiotic terrestrial conditions in the presence of dissolved iron (II) in a simulated prebiotic ocean. An aerosol-liquid cycle with a prebiotic atmosphere is shown to produce amino acids via Strecker synthesis with relatively high yields. However, in the presence of iron, the HCN was captured in the form of a ferrocyanide, partially inhibiting the formation of amino acids. We showed how HCN captured as Prussian Blue (or another complex compound) may, in turn, have served as the HCN source when exposed to UV radiation, allowing for the sustained production of amino acids in conjunction with the production of oxyhydroxides that precipitate as by-products. We conclude that ferrocyanides and related compounds may have played a significant role as intermediate products in the prebiotic formation of amino acids and oxyhydroxides, such as those that are found in iron-containing soils and that the aerosol cycle of the primitive ocean may have enhanced the yield of the amino acid production.     

Acid-labile amino acid precursors in the Murchison meteorite. II: A search for peptides and amino acyl amides.

The basic fraction of the acid-labile amino acid precursors found in hot water extracts of the Murchison meteorite was treated with ninhydrin under conditions that give destruction of free amino acids but allow nearly complete recovery to peptides and amino acylamides. After this treatment and a subsequent acid hydrolysis, the amount of amino acids found corresponded to less than 30 per cent of the precursor content of the basic fraction. Since only 30 percent of the total extract precursors are found in the basic fraction, these results indicate that such compounds, if present, account for no more than 9 mole per cent of the total acid-labile amino acid precursors of the meteorite hot water extract.     

Squalene-hopene cyclases

Hopanoids and sterols are members of a large group of cyclic triterpenoic compounds that have important functions in many prokaryotic and eukaryotic organisms. They are biochemically synthesized from linear precursors (squalene, 2,3-oxidosqualene) in only one enzymatic step that is catalyzed by squalene-hopene cyclase (SHC) or oxidosqualene cyclase (OSC). SHCs and OSCs are related in amino acid sequences and probably are derived from a common ancestor. The SHC reaction requires the formation of five ring structures, 13 covalent bonds, and nine stereo centers and therefore is one of the most complex one-step enzymatic reactions. We summarize the knowledge of the properties of triterpene cyclases and details of the reaction mechanism of Alicyclobacillus acidocaldarius SHC. Properties of other SHCs are included.     

Chemical Evolution and Meteorites: An Update

Carbonaceous chondrites are a primitive group of meteorites, which contain abundant organic material and provide a unique natural record of prebiotic chemical evolution. This material comprises a varied suite of soluble organic compounds that are similar, sometimes identical, to those found in the biosphere, such as amino acids, carboxylic acids, and sugar derivatives. Some amino acids of this suite also show L-enantiomeric excesses, and suggest the possibility they may have contributed to terrestrial homochirality by direct input of meteoritic material to the early Earth. This optical activity appears to be limited to the subgroup of alpha-methyl amino acids which, although not common in the extant biosphere, would have been well suited to provide the early earth with both enantiomeric excesses and means for their amplification by subsequent chemical evolution. We can also envision this exogenous delivery of carbonaceous material by meteorites and comets as having coincided with the endogenous formation of prebiotic precursors and influenced their evolution by complementary reactions or catalysis.     

Formation of heterocyclic amines using model systems

Initially, modeling was used to identify the mutagenic heterocyclic amines and their precursors. Major precursors have been shown to be single amino acids or amino acids together with creatine or creatinine. There is also evidence that Maillard reactions are involved since heating sugar and amino acids together with creatine or creatinine has been shown to produce several of the mutagenic heterocyclic amines, especially the aminoimidazoazaarenes (AIA compounds), e.g., IQ, MeIQ, MeIQx, DiMeIQx and PhIP. Due to a low yield in the model systems, the mechanisms behind the formation of the mutagenic heterocyclic amines are still unclear and need further substantiation. The fact that some AIA compounds are also produced in the absence of sugar casts some doubts on an obligatory participation of the Maillard reaction; alternative routes might exist. Further work using isotopically labeled precursors needs to be done and so far such work has only been performed for PhiP. The formation of mutagenic heterocyclic amines is dependent on time, temperature, pH, concentration of the precursors, type of amino acid, and the presence of certain divalent ions. Water may have an impact both as a temperature regulator and as a solvent medium for the reactants.     

Pyrolysis experiment of simulated exogenous complex organics synthesized from the gas mixtures of CO, NH3, and H2O by 3 MeV proton irradiation.

High molecular weight organic matter synthesized from mixtures of carbon monoxide, ammonia and water gases similar to those found in the interstellar medium were irradiated with a 3 MeV proton beam and analyzed by Curie point pyrolysis with detection by gas chromatograph and mass spectrometer (Pyr-GC-MS). A wide variety of organic compounds, not only a number of amide compounds, but also heterocyclic and polycyclic aromatic hydrocarbons (PAHs), were detected among the products of the pyrolysis. The present data shows that primary and primitive organic matter serving as precursors to bioorganic compounds such as amino acids, nucleic acid bases and sugar might have been formed in a gaseous mixture of similar composition to that of the interstellar dust environment.     

Ability of melanins to protect against the radiolysis of thymine and thymidine

Individuals with black skin rarely get skin cancer, and melanomas, tumors arising from pigmented cells, are generally resistant to radiation therapy. The role of melanin in these two phenomena has not been defined, but oxygen-radical species have been implicated in both effects. These studies were undertaken to determine the ability of various melanins to compete for ionizing radiation-produced radicals which destroy nucleic acid bases. The ability of Sigma eumelanin (S-eumelanin) to protect against the radiolysis of thymidine in buffered solutions was compared to the protective ability of seven amino acids, including melanin precursors; bovine serum albumin, as a model protein; ficoll, as a model polysaccharide; and DNA. Both proteins and polysaccharides are known to scavenge hydroxyl radicals in cells. The concentration of thymidine after exposure to gamma radiation was determined by High Performance Liquid Chromatography (HPLC) analysis after removal of insoluble melanin by acid precipitation. S-eumelanin was more effective at competing with thymidine for free radicals than bovine serum albumin, Ficoll, or DNA, but less effective than certain of the small molecules. Several of the above compounds were also examined for ability to protect against thymine radiolysis. In addition, melanins from other sources were compared to S-eumelanin. Of these, enzymatically synthesized phaeomelanin was the most effective. The results indicate that melanins can compete for base- and nucleoside-damaging free radicals more effectively than other cellular macromolecules. Of the small molecules, the phenolic compounds had the greatest scavenging ability. In vivo, melanins are found in melanosomes bound to protein. Therefore, the relevance of these findings to the photo- and radiobiology of melanins in vivo has yet to be determined.     

On the possible role of organic melanoidin polymers as matrices for prebiotic activity

Summary One of the major diagenetic pathways of organic matter in recent sediments involves the condensation of cellular constituents, particularly amino acids and sugars, into insoluble melanoidin-type polymers. These polymers consist mainly of humic and fulvic acids and make up the major part of the organic carbon reservoir in recent sediments. We suggest that a similar set of reactions between abiotically formed amino acids and sugars, and more generally between aldehydes and amines, occurred on a large scale in the prebiotic hydrosphere. The rapid formation of this insoluble polymeric material would have removed the bulk of the dissolved organic carbon from the primitive oceans and would thus have prevented the formation of an "organic soup".Melanoidin polymers have several properties which make them attractive hypothetical precursors of contemporary oxidation-reduction coenzymes: 1. they contain heterocyclic nitrogen compounds similar to the nitrogenous bases; 2. they contain a high concentration of stable free radicals; and 3. they tend to concentrate those heavy metals which play prominent roles in contemporary enzymic redox processes. The prebiotic formation of similar polymers could, therefore, have provided the starting point for a basic class of biochemical reactions.We suggest that the prebiotic scenario involved chemical and protoenzymic reactions at the sediment-ocean interface in relatively shallow waters and under conditions not much different from those of the recent environment.On leave from the Isotope Department, Weizmann Institute of Sciences, Rehovot, Israel. This is the address for reprint requests.On leave from the Department of Cell and Molecular Biology, San Francisco State University, San Francisco, California.On leave from the Department of Biophysics, University of Houston, Houston, Texas.     

Reduction of ultraviolet light-induced oxidative stress by amino acid-based iron chelators

The generation of free radicals by ultraviolet (UV) light accelerates skin aging, which is known as photoaging. Cutaneous iron catalyzes the generation of free radicals. We designed novel antioxidants that suppressed the iron-catalyzed free radical generation and the ensuing UV-induced damage by mimicking the binding site of iron sequestering proteins. These antioxidants, N-(2-hydroxybenzyl)amino acids, were prepared by condensation of amino acids such as glycine and L-serine with salicylaldehyde and followed by catalytic reduction. The compounds formed a 2:1 complex to iron ion. These amino acid derivatives inhibited the iron-induced hydroxyl radical generation (the Fenton reaction). The compounds also suppressed UV-induced lipid peroxidation in murine dermal fibroblast homogenates. In addition, N-(2-hydroxybenzyl)-L-serine showed protective activity against UV-induced cytotoxicity in murine dermal fibroblasts. Desferrioxamine, a strong iron sequestering compound, was effective in inhibiting the Fenton reaction and the lipid peroxidation, but it was ineffective in protecting against UV-induced cytotoxicity. The results suggest that UV-induced oxidative stress can be reduced by these amino acid derivatives.     

Synthesis of Photolabile Caged Amino Acids for Measuring Amino Acid Transporters

Photolabile precursors (caged compounds) of amino acids such as Ala, Leu, Lys, and Ser were prepared by some simple reactions. These compounds were designed for the rapid, photochemically initiated release of amino acids. These amino acid transporters were expressed in Xenopus oocyte by injecting mRNA prepared from rat kidney. The electrical response of each transporter was examined by applying the amino acids and caged compounds before and after photolysis. Photolysis of the caged amino acids increased the electrical response of the facilitated amino acid transporters expressed in the oocyte. Consequently, these synthesized caged amino acids would be applicable to kinetic investigations on the transporters when combined with a pulsed laser or xenon arc flash lamp.     

Evidence for the complex relationship between free amino acid and sugar concentrations and acrylamide‐forming potential in potato

Free amino acids and reducing sugars participate in the Maillard reaction during high‐temperature cooking and processing. This results not only in the formation of colour, aroma and flavour compounds, but also undesirable contaminants, including acrylamide, which forms when the amino acid that participates in the reaction is asparagine. In this study, tubers of 13 varieties of potato (Solanum tuberosum), which had been produced in a field trial in 2010 and sampled immediately after harvest or after storage for 6 months, were analysed to show the relationship between the concentrations of free asparagine, other free amino acids, sugars and acrylamide‐forming potential. The varieties comprised five that are normally used for crisping, seven that are used for French fry production and one that is used for boiling. Acrylamide formation was measured in heated flour, and correlated with glucose and fructose concentration. In French fry varieties, which contain higher concentrations of sugars, acrylamide formation also correlated with free asparagine concentration, demonstrating the complex relationship between precursor concentration and acrylamide‐forming potential in potato. Storage of the potatoes for 6 months at 9°C had a significant, variety‐dependent impact on sugar and amino acid concentrations and acrylamide‐forming potential.     

Protein synthesis by membrane-bound and free ribosomes of the developing rat cerebral cortex

1. Rates of RNA and protein synthesis were measured in rat cerebral-cortex slices, and compared with amino acid incorporation into protein by membrane-bound and free ribosomes from the same tissue, in the first 3 weeks of life. 2. A rapid age-dependent decline in the incorporation of labelled precursors into both RNA and protein was observed, which was more marked for amino acid incorporation into protein. 3. Although membrane-bound ribosomes comprise only a small fraction of total ribosomes, they were more active in incorporating amino acids into protein than were free ribosomes, especially immediately after birth. The decline in activity with age was more marked in the membrane-bound fraction than in free ribosomes. This loss of activity was largely independent of alterations in soluble factors or endogenous mRNA content and appeared to involve some alteration of the function of the ribosome itself, with relatively small alterations in the ratio of membrane-bound to free ribosomes. 4. Thyroidectomy, performed soon after birth, had no effect on the incorporation of radioactive precursors into RNA or protein by either slices or the cell-free preparations during the first 3-4 weeks of life.     

Amino acids and their derivatives as radioprotective agents

Summary Numerous amino acids and their analogs are capable of protecting biological systems from the toxic effects of ionizing radiation. These radioprotective agents can be classified into two broad groups, depending upon the presence or absence of a free or potentially free sulfhydryl group. The sulfhydryl-containing compounds have been studied extensively and are thought to exert their radioprotective effects by several mechanisms, including free radical scavenging and hydrogen atom donation. Several non-sulfhydryl-containing amino acids are also being investigated for their radioprotective effects. These agents are less well known than the familiar sulfhydryl compounds, but possess very interesting protective qualities. In short, the study of amino acids and their derivatives as radioprotective agents continues to contribute to an understanding of processes involved in radiation toxicity and to offer new compounds with potential application to situations of human exposure.     

Inhibitory effect of some acetyl esters and acetamides on glycation of the histone H1

Non-enzymatic glycosylation (glycation) is a spontaneous set of reactions between reducing sugars and free amino groups in proteins or other biomolecules leading to the formation of fluorescent and coloured compounds known as advanced glycation end products (AGEs). AGEs cause structural changes of key proteins in humans, and therefore they are related with a number of physiological processes and diseases such as aging, atherosclerosis, cataract, arthritis, Alzheimer's disease. Two main strategies have been employed to prevent the formation of AGEs: a) low carbohydrate diet and b) pharmacological intervention. The latter includes treatment with reactive compounds which might be either sugar competitors (type A), carbonyl traps (type B) or free radical trapping antioxidants (type C). Acetylsalicylic acid (ASA, aspirin) is a good example of sugar competitor capable of inhibiting glycation by acetylating epsilon-amino groups of lysine residues in proteins. Taking into consideration the inhibiting effect of ASA on glycation we designed to study the antiglycation activity of other acetyl group-containing compounds (acetamides and acetyl esters) using the lysine-rich protein histone H1 as a model. The glycation of the histone H1 was carried out by either fructose or a complex mixture of glycating agents obtained from E. coli and monitored by fluorescent spectroscopy, SDS-PAGE and measurement of the content of reactive carbonyl groups in the target protein. Our results showed that the inhibitory effect of phenyl acetate, acetanilide, 4-acetamidophenylacetic acid and isopropenyl acetate was comparable to that of ASA. Based on the obtained results we conclude that these compounds act as free radical scavengers protecting proteins from the damaging effect of reactive oxygen species produced during the formation of AGEs.     

Mechanisms for elongation in the biosynthesis of fatty acid components of epi-cuticular waxes

It is known that branched-chain amino acids can serve as precursors to iso- and anteiso-branched components of epi-cuticular waxes. Keto acid deamination products of Val, Leu and Ile are thought to serve as primers which are elongated by fatty acid synthase. However, the origin of elongation carbons has not been studied directly. Nor has the mechanism for formation of odd-carbon-length, straight- or branched-chain, cuticular ester fatty acids or free odd-carbon-length, straight fatty acid components of waxes been characterized. It is not known that α-oxidation of even-length precursors or elongation of odd-length primers is involved in these cases. Here, we present evidence which substantiates the expectation that elongation of branched as well as straight-chain precursors to wax ester acids occurs by fatty acid synthase catalyzed by addition of two carbon units via acetate. Also, we present evidence which indicates that odd-carbon-length acids can result from elongation of odd-carbon-length primers (at least branched), rather than even-length acids shortened by α-oxidation.