Changes In Amino Acid Composition Research Articles

Environmentally Induced Changes in Amino Acid Composition in the Grain of Durum Wheat Grown under Different Water and Temperature Regimes in a Mediterranean Environment

Amino acid composition is an important feature in determining the nutritional value of wheat grain for human and animal diets. Environmental conditions are known to influence protein quantity as well as grain production and, in turn, amino acid composition. In this study, grain yield, protein content, and amino acid composition were determined in 10 durum wheat genotypes under three water and temperature regimes in a Mediterranean environment. The highest value for grain-protein content (15.7%) was found in the warmer and driest environment and the lowest (12.8%) in the irrigated environment. Although amino acid composition showed significant variation for all genotypes, with the exception of arginine and cysteine, major changes in amino acid composition were caused by environmental conditions and in particular by water availability and temperature during the grain-filling period, which significantly altered the duration of grain development. The amino acids with the highest percentage of variation between environments were tyrosine (26.4%), lysine (23.7%), methionine (20.3%), threonine (19.3%), and valine (15.6%), whereas phenylalanine (5.1%), glycine (6.4%), and aspartic acid (6.8%) showed the least variation between environments. Whereas the content of glutamine, phenylalanine, and proline increased with the decrease in grain-filling duration, the remaining amino acids tended to diminish, presumably because high temperature and drought favored the deposition of gliadins (proteins particularly rich in glutamine and proline), to the detriment of albumins and globulins (proteins especially rich in threonine, lysine, methionine, valine, and histidine). Despite the negative correlations found between the percentage of protein and its content in essential amino acids, the results indicate that reductions in lysine per unit of food were not very pronounced (0.32 to 0.29 g/100 g of flour) with increases of up 22.7% in grain-protein content, whereas threonine did not change and valine even slightly increased.

A Combinatorial Approach to the Discovery of Biocidal Six‐Residue Cyclic d,l‐α‐Peptides Against the Bacteria Methicillin‐Resistant Staphylococcus aureus (MRSA) and E. coli and the Biofouling Algae Ulva linza and Navicula perminuta

A combinatorial approach has been used to rapidly identify cyclic d,l-alpha-peptide hexamer sequences that exert biocidal activity towards both methicillin-resistant Staphylococcus aureus (MRSA) and E. coli bacteria, as well as the marine algae Ulva linza and Navicula perminuta. Evaluation of the effects against marine algae was facilitated by the development of a reliable, automated assay for toxicity, which should be of general utility for biofouling investigations. While the selective toxicity of cyclic D,L-alpha-peptides towards bacteria has been proven to be highly sensitive to minor changes in amino acid composition, this study demonstrates that this phenomenon extends to eukaryotic species as well, despite their significant structural differences. In performing toxicity assays on both prokaryotic and eukaryotic organisms in parallel, we have discovered examples of six-residue cyclic D,L-alpha-peptide sequences with either broad-spectrum or highly selective biocidal activities. Sequence [KWFFFH] (underlined amino acid abbreviations represent D-amino acid residues) was found to display 100-fold selectivity towards U. linza, demonstrating that the approach described herein may help lead to the development of new biofouling tools which are not generally toxic to all organisms, but rather specifically target microbial agents of interest.

The changes of fatty acid and amino acid compositions in sea bream ( Sparus aurata) during irradiation process

Aqua cultured fish (sea bream) were irradiated by Cobalt-60 at commercial irradiation facility at dose of 2.5 and 5 kGy at 2–4 °C. The proximate composition, fatty acid and amino acid composition changes of irradiated aqua cultured sea bream ( Sparus aurata) of Aegean Sea were investigated. Total saturated (28.01%) and total monounsaturated (28.42%) fatty acid contents of non-irradiated decreased content of 27.69–27.97% for 2.5 kGy irradiated groups and increased content of 28.33–28.56% for 5 kGy irradiated groups after irradiation process. Total polyunsaturated fatty acid content for irradiated samples was lower than that of non-irradiated samples. Aspartic acid, glutamic acid, serine, glycine, arginine, alanine, tyrosine, cystine, tryptophan, lysine and proline contents for 2.5 and 5 kGy irradiated sea bream are significantly different ( p<0.05).

A Combinatorial Approach to the Discovery of Biocidal Six-Residue Cyclic D, L-α-Peptides Against Bacteria MRSA and E. coli. and the Biofouling Algae Ulva linza and Navicula perminuta

A combinatorial approach has been used to rapidly identify cyclic D, L-α-peptide hexamer sequences that exert biocidal activity towards both MRSA and E. coli. bacteria, as well as the marine algae Ulva linza and Navicula perminuta. Evaluation of the effects against marine algae was facilitated by the development of a reliable, automated assay for marine toxicity that should be of general utility for biofouling investigations. While the selective toxicity of cyclic D, L-α-peptides towards bacteria has been proven to be highly sensitive to minor changes in amino acid composition, this study demonstrates that this phenomenon extends to eukaryotic species as well, despite their significant structural differences. In performing toxicity assays on both prokaryotic and eukaryotic organisms in parallel, we have discovered examples of 6-residue cyclic D, L-α-peptide sequences with either broad-spectrum or highly selective biocidal activities. Sequence [KWFFFH] was found to display 100-fold selectivity towards U. linza demonstrating that the approach described herein may help lead to the development of new biofouling tools that are not generally toxic to all organisms, but rather specifically target microbial agents of interest.

Side Chain-dependent Stacking Modulates Tau Filament Structure

The misfolding of proteins into highly ordered fibrils with similar physical properties is a hallmark of many degenerative diseases. Here, we use the microtubule associated protein tau as a model system to investigate the role of amino acid side chains in the formation of such fibrils. We identify a region (positions 272-289) in the tau protein that, in the fibrillar state, either forms part of a core of parallel, in-register, beta-strands, or remains unfolded. Single point mutations are sufficient to control this conformational switch with disease mutants G272V and DeltaK280 (found in familial forms of dementia) inducing a folded state. Through systematic mutagenesis we derive a propensity scale for individual amino acids to form fibrils with parallel, in-register, beta-strands. This scale should not only apply to tau fibrils but generally to all fibrils with same strand arrangement.

Comparison of aspartic acid racemization between mammoth and human dentinal tissues

Objective Comprehensive research has revealed the complexity and heterogeneity of amino acid racemization (AAR) in biological samples. Therefore, to date, inherent uncertainties in relative dating as well as in determination of age at death have highlighted the significance of further investigations to explore aspartic acid kinetics in biominerals. Here we present data on an archaeological sample obtained from a mammoth tusk, in comparison to a previously well-described biomineral, to modern human dentin. Design Since no experimental investigations were carried out on this topic earlier, we provide the first estimates on racemization kinetics and time dependent tendencies in mammoth dentin. We analysed our samples using a protocol, which has been adopted for tooth dentin investigation. We measured different racemization rates amongst mammoth dentin layers. Results and conclusions Significantly, an overall low racemization rate was detected compared to human dentin. Kinetic experiments were performed by heating the samples, and linear correlations were established between racemization rates and heating time. The results allow us to derive racemization rate constants and activation energy for both samples. Extrapolation for ambient temperature showed increased velocity in mammoth dentin, which can be explained by biodeterioration of ancient collagen and by diagenetic changes in amino acid composition influenced by environmental conditions.

Changes in amino acid composition of Ulva intestinalis (Chlorophyceae) following addition of ammonium or nitrate

M.W. Taylor, N.G. Barr, C.M. Grant and T.A.V. Rees. 2006. Changes in amino acid composition of Ulva intestinalis (Chlorophyceae) following addition of ammonium or nitrate. Phycologia 45: 270–276. DOI: 10.2216/05-15.1Nitrogen assimilation was investigated in the marine macroalga Ulva intestinalis. Following incubation of the alga in the presence of ammonium or nitrate, resulting changes in free amino acid content were determined. After 10 h, the largest changes by far occurred in the levels of glutamine and asparagine, which both increased more than 10-fold regardless of nitrogen source. Other amino acids increased slightly, but these two – together with their precursors glutamate and aspartate – comprised 83 and 76% of the total free protein amino acid-N pool upon addition of ammonium or nitrate, respectively. In subsequent time-course experiments, with ammonium as nitrogen source, glutamate initially decreased (with a concomitant increase in glutamine) before recovering to at least its original level. Asparagine levels began to increase after 1 h. Saturation of the glutamine and glutamate pools occurred after approximately 6 h, and coincided with the transition from the surge phase of ammonium uptake to the internally controlled uptake phase. This study represents the first investigation into how levels of specific amino acids change during ammonium assimilation in macroalgae, and as such extends our current knowledge of this poorly understood process.

Combined use of selective inhibitors and fluorogenic substrates to study the specificity of somatic wild‐type angiotensin‐converting enzyme

Somatic angiotensin-converting enzyme (ACE) contains two homologous domains, each bearing a functional active site. Studies on the selectivity of these ACE domains towards either substrates or inhibitors have mostly relied on the use of mutants or isolated domains of ACE. To determine directly the selectivity properties of each ACE domain, working with wild-type enzyme, we developed an approach based on the combined use of N-domain-selective and C-domain-selective ACE inhibitors and fluorogenic substrates. With this approach, marked differences in substrate selectivity were revealed between rat, mouse and human somatic ACE. In particular, the fluorogenic substrate Mca-Ala-Ser-Asp-Lys-DpaOH was shown to be a strict N-domain-selective substrate of mouse ACE, whereas with rat ACE it displayed marked C-domain selectivity. Similar differences in selectivity between these ACE species were also observed with a new fluorogenic substrate of ACE, Mca-Arg-Pro-Pro-Gly-Phe-Ser-Pro-DpaOH. In support of these results, changes in amino-acid composition in the binding site of these three ACE species were pinpointed. Together these data demonstrate that the substrate selectivity of the N-domain and C-domain depends on the ACE species. These results raise concerns about the interpretation of functional studies performed in animals using N-domain and C-domain substrate selectivity data derived only from human ACE.

P.156 Serologic profiles at the haemodialysis in Hospital constantine (Algeria)

The envelope 2 protein of hepatitis C virus (HCV) presents three hypervariable regions, named HVR1, HVR2 and HVR3, in which the presence of antigenic sites has been described. Genetic variability in these regions may reflect the generation of escape mutants as a consequence of the immune response. Therefore, these regions would tend to accumulate amino acid changes along the infection process, an effect that could be accelerated by antiviral treatments. In this study, we have analyzed the E1–E2 region of 23 HCV patients non-responders to antiviral treatment, 7 of which were infected with subtype 1a, 15 with subtype 1b, and 1 with a new HCV-1 subtype, before and after 6 and/or 12 months of peg-interferon + ribavirin treatment. We have sequenced about 100 clones from each sample, analyzing a total of 4906 sequences. A detailed analysis of the evolutionary forces acting along the genome region studied confirmed the existence of the three hypervariable regions, characterized by significant changes in amino acid composition between samples taken at different times from the same patient and a high number of sites evolving under positive selection. Moreover, for the recently described HVR3, our results suggest that its location could be restricted to residues 434–450, instead of the originally postulated 431–466.

Lack of immunological cross-reactivity between parasite-derived and recombinant forms of ES-62, a secreted protein of Acanthocheilonema viteae

The longevity of filarial nematodes is dependent on secreted immunomodulatory products. Previous investigation of one such product, ES-62, has suggested a critical role for post-translationally attached phosphorylcholine (PC) moieties. In order to further investigate this, ES-62 lacking PC was produced, using the Pichia pastoris recombinant gene expression system. Unlike parasite-derived ES-62, which is tetrameric the recombinant material was found to consist of a mixture of apparently stable tetramers, dimers and monomers. Nevertheless, the recombinant protein was considered to be an adequate PC-free ES-62 as it was recognized by existing antisera against the parasite-derived protein. However, subsequent to this, recognition of parasite-derived ES-62 by antibodies produced against the recombinant protein was found to be absent. In an attempt to explain this, recombinant ES-62 was subjected to structural analysis and was found to (i) contain 3 changes in amino acid composition; (ii) demonstrate significant alterations in glycosylation; (iii) show major differences in protein secondary structure. The effects of these alterations in relation to the observed change in immunogenicity were investigated and are discussed. The data presented clearly show that recognition by existing antibodies is insufficient proof that recombinant proteins can be used to mimic parasite-derived material in studies on nematode immunology and vaccination.

Ontogenic changes of amino acid composition in planktonic crustacean species

Changes in amino acid composition (AAC) during ontogeny of some planktonic crustacean species commonly found in fresh and brackish coastal waters were compared. For these comparisons two calanoid copepods (Eurytemora velox and Calanipeda aquae-dulcis), two cyclopoid copepods (Diacyclops bicuspidatus odessanus and Acanthocyclops robustus) and two Daphnia (Daphnia pulicaria and Daphnia magna) species were selected. A discriminant analysis was performed to determine whether there were significant differences between the AAC of the different stages of each species. Results show gradual changes in AAC during ontogeny of the copepod species. Calanoids showed the greatest differences in AAC between stages, followed by cyclopoids. Gradual changes in AAC were due to the increase in some amino acids such as alanine, valine, glutamic acid, glycine, arginine, proline and tyrosine from nauplii to adults. The latter showed a remarkable increase in all copepod species. In contrast, Daphnia species showed a relatively constant AAC during development, with only minor changes being detected, and not related with ontogeny. Differences in the physico-chemical variables of the lagoons do not seem to be the cause of copepod ontogenic changes in AAC. Data suggest that AAC differences found between stages of copepod species could indicate a gradual change in diet during the life cycle of these copepods.

Effect of age and temperature on amino acid composition and the content of different protein types of juvenile Atlantic cod (Gadus morhua) otoliths

The purpose of this study was to analyse the amino acid composition of otolith matrix protein, estimate the proportion of the water-soluble protein fraction, and analyse the effect of matrix composition on otolith visual appearance. Juvenile Atlantic cod (Gadus morhua) were reared under constant temperature and feeding conditions and sampled at the beginning and the end of the experiment. The amino acid composition was dominated by asparagine, glutamic acid, leucine, serine, and proline. A change in amino acid composition was observed with increasing temperature and time, caused by changing proportions of the water-soluble and -insoluble protein fractions. Feeding level had no effect. The relative content of water-soluble protein was linearly related to fish dry weight and temperature. Otolith opacity, defined as the percentage of incident light absorbed by an otolith section, did not differ significantly between experimental treatments. The soluble protein fraction had a positive, albeit insignificant, correlation with opacity. Using opacity and otolith volume, deposited total otolith protein content was estimated with an R2 of 0.91, where otolith volume alone explained 83% of the observed variation.

Metabolic economics and microbial proteome evolution

Abstract Natural selection is thought to act upon protein structures to optimize biochemical properties related to their specific cellular function. Selection pressures related to efficient synthesis, rather than proper function, of proteins may act globally on the amino acid composition of the proteome, but are less firmly established. A substantial fraction of bacterial energy budgets are devoted to biosynthesis of amino acids, the building blocks of proteins. The fueling reactions of central metabolism provide precursor metabolites for synthesis of amino acids. Thus, synthesis of an amino acid entails a dual cost; energy is lost by diverting chemical intermediates from fueling reactions and additional energy is required to convert precursor metabolites to amino acids. Selection to reduce the energetic costs predicts increases in the abundance of less energetically costly amino acids in highly expressed proteins. Using synonymous codon usage bias as a measure of translation rates, we show that amino acid composition in the proteomes of Escherichia coli and Bacillus subtilis reflects the action of natural selection to enhance metabolic efficiency. The primary structures of proteins may also reflect natural selection to enhance the rate and accuracy of their synthesis. Differences in cellular concentrations of tRNAs could lead to translation selection both within and among synonymous families. In yeast, usage of several amino acids show striking associations with gene expression. These changes in amino acid composition result in stronger correlations between amino acid usage and tRNA abundances in highly expressed genes than in less expressed loci. Proteome-wide patterns of amino acid composition in microbes appears to reflect natural selection to enhance the overall physiology of cells as well as the specific functions of proteins. Contact: akashi@psu.edu&amp;#x2028; http://www.bio.psu.edu/people/faculty/akashi/

Transcription factor genes with expression correlated to nitrate‐related root plasticity of Arabidopsis thaliana

ABSTRACTThe root response to changes in nitrogen availability was examined in Arabidopsis thaliana. Plants grown on vertically oriented Petri plates with nutrient media containing either 10 or 0.5 mm KNO3 supplemented with 5 mm glutamine were analysed for changes in root architecture, morphology and nitrogen status compared to plants grown on 0.5 mm KNO3. Fourteen‐day‐old 10 mm KNO3‐grown plants displayed a decrease in number, length and density of lateral roots, had the highest tissue NO3– content, but no change in amino acid composition. Lateral root development was arrested at a stage after the initiation of primordia but prior to emergence. In contrast, plants grown on 0.5 mm KNO3 supplemented with glutamine had a slightly higher number and density of lateral root primordia and lateral roots, lower root NO3– content, and higher tissue glutamine content. A macro‐array containing 126 putative A. thaliana transcriptional factor expressed sequence tags was used to screen for regulatory genes with expression correlated to the root plasticity response. Genes for a basic leucine zipper (bZIP) and a LIM transcription factor were identified and via Northern analysis it was confirmed that their expression is preferentially observed in the roots and correlated to the root response to nitrate availability.

The role of biominerals in the sinking flux and preservation of amino acids in the Southern Ocean along 170°W

Abstract In a study of the transport and transformation of particulate amino acids in the Southern Ocean, we found that silicate and calcium carbonate biominerals play an important role in the preservation of amino acids throughout the water column and surface sediments. Plankton, sinking particle and sediment samples were collected during the USJGOFS AESOPS transect across the Antarctic Polar Front (APF) along 170°W. Total hydrolyzable amino acids (THAA) made up of 17–27% of total organic carbon (Corg) in sinking particles and 6–23% of Corg in surface sediments. In addition to THAA, we measured amino acids bound in silicate (SiTHAA) and calcium carbonate (CaTHAA) biominerals. Although the fraction of biomineral bound to total amino acids in plankton was small, Amino acid composition has been used to develop a Degradation Index (sensu Dauwe and Middelburg, 1998). The unusual amino acid composition of Southern Ocean plankton, i.e., dominated by diatom cell walls, resulted in an apparent mismatch between the absolute value of the Degradation Index and the presumed extent of degradation. However, changes in amino acid composition that accompanied degradation were similar to those found in previous studies. Principal components analysis suggests that the greatest change in THAA composition occurred between the sediment surface floc layer and deeper sediments where particles had the longest residence time. Compositional changes observed in the water column suggested that degradation processes resulted in complete removal of amino acids, whereas changes in sediments were consistent both with selective degradation of plankton amino acids with depth and with the conversion of primarily phytoplankton biomass to that of bacterial biomass.

The essentiality of oxygen for life

Understanding the many roles that oxygen plays in biology has fascinated scientists for decades. One estimates that over 350 different types of enzymes currently exist that have been shaped during evolution to accommodate the need for biological systems to capture the energy associated with the conversion of oxygen (or its intermediate reduction products) to water. Further, a large number of enzymes have evolved to protect sensitive cellular components from deleterious intermediary oxygen-reduction products. In this presentation, we discuss a question surrounding the most prevalent of the oxygen reacting enzymes—the superfamily of monooxygenase hemeproteins called cytochrome P450. We ask, “Can we understand the changes that have occurred with time that have dictated modifications in the structure and functions of the P450s?” We use as an example a comparison of differences for one P450, CYP17A, a steroid hydroxylating enzyme located at a branch point of steroid hormone synthesis. We have compared by homologous modeling the structural location of amino acids altered during the evolution of CYP17A from closely related species of ungulates of the Order Artiodactyla. These studies indicate that amino acids located in loops on the surface of the P450 molecule are most frequently altered. Analysis of the codon sequences of the cDNAs indicate changes in amino acid composition may result from single nucleotide polymorphisms (SNPs).

Morphological parameters in varieties of silk fibers determined by small-angle X-ray scattering

The small-angle X-ray scattering intensity data recorded from silk fibers were compared with the simulated data obtained from a linear paracrystalline model. For this purpose, an exponential distribution function for the amorphous and crystalline phase lengths was used. There are significant changes in phase lengths because of amino acid compositional changes in different families of silk fibers. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 85: 2382–2388, 2002

Changes in amino acid composition in the tissues of African catfish (Clarias gariepinus) as a consequence of dietary L-carnitine supplements

A study was undertaken to examine the effect of different amounts of dietary lysine (13 and 21 g kg−1 diet), lipid (80 and 160 g kg−1 diet) and L-carnitine (0.2 and 1.0 g kg−1 diet) on growth performance, proximate composition and amino acid metabolism of the African catfish (Clarias gariepinus). Juvenile African catfish (23 ± 1.5 g/fish) were stocked into 70-L aquaria (16 aquaria, 28 fish/aquarium) connected to a recirculation system during a maximum period of 74 days. All groups were fed at a level of 24 g kg−0.8 day−1 in an experiment run at pair feeding. Animals receiving 1.0 g carnitine accumulated up to six times more carnitine in their tissues than animals receiving 0.2 g (P aspartic acid > glycine > alanine > arginine > serine > threonine in skeletal muscle tissue (P < 0.05). Total amino acid concentration in muscle and liver tissues (dry-matter basis) increased from 506 to 564 and from 138 to 166 mg g−1, respectively, when diets were offered with high L-carnitine, low lysine and low fat levels. These data suggest that dietary L-carnitine supplementation may increase fatty acid oxidation and possibly decrease amino acid combustion for energy.

Acetylcholinesterase of the California red scale Aonidiella aurantii Mask.: Catalysis, inhibition, and reactivation

The properties of the California red scale, Aonidiella aurantii, acetylcholinesterase (AChE) were studied using various substrates, inhibitors, and reactivators. The enzyme is inhibited by di( p-allyl- N-methylaminophenyl)pentane-3-one, is insensitive to tetraisopropyl pyrophosphoamidate, and displays the substrate inhibition phenomenon. The insect AChE deviates from its vertebrate counterparts by requiring a comparatively higher level of substrate to attain inhibition, by effectively hydrolyzing the larger homologous substrate propionyl thiocholine, by having a very high sensitivity to a bulkier inhibitor such as chlopyrifos-oxon, by having insensitivity to the peripheral anionic site (PAS) inhibitor fasciculin, and by being reactivated by pyridine-2-aldoxime methyl iodide but not by 1-(4-aminocarbonylpyridinium-1 ′-(2 ′-pyridiniumaldoxime)dimethyl dibromide. Those differences were discussed as being associated with probable changes of amino acid composition within the enzyme acyl pocket and the PAS. Based on the above comparison, the insect enzyme may be regarded as an intermediate between AChE and butyrylcholinesterase. Among the organophosphorus (OP) compounds, chlopyrifos-oxon and 7-(methylethoxyphosphinyloxy)-1-methyl quinolinium were the most powerful inhibitors, while paraoxon was two orders of magnitude less effective. Among the carbamates, carbaryl was similar to paraoxon in its inhibitory effects followed by aldicarb and pirimicarb. The alkaloid huperzine A is an extremely potent inhibitor of the diaspidid AChE (with a K i value in the subnanomolar range). The quaternary ammonium ligands propidium, edrophonium, and d-tubocurarine displayed high levels of inhibition. Toxogonin was the most powerful reactivator of OP-inhibited enzyme, while its nonoxime analog 1,1-bis(4-tertbutylpyridinium)dimethylether dichloride was without effect. Possible reasons for the enhanced inhibition of AChE in the presence of carbaryl and Toxogonin were discussed.

Cellular and extracellular production of carbohydrates and amino acids by the marine diatom Skeletonema costatum: diel variations and effects of N depletion

β-1,3-Glucan (chrysolaminaran) is the principal storage polysaccharide in diatoms (Bacillariophyceae), the major primary producers in the sea. The glucan generally contributes a substantial fraction of the algal biomass, but its level varies markedly in response to growth conditions. The scope of this work was to study the metabolism and function of the polysaccharide in marine diatoms. Axenic cultures of the marine planktonic diatom Skeletonema costatum (Grev.) Cleve were used in the experiments. Glucan metabolism was studied by growing the alga in batch culture, and measuring metabolite fluxes by chemical analyses as well as by 14C tracer technique using labeled bicarbonate. A photobioreactor was developed for strictly controlled growth of microalgae. Fine pH regulation was obtained by relay-activated titration with dilute acid (HCl) and base (NaOH). Irradiance and temperature were also carefully controlled. Batch cultures were grown with a 14:10 h light:dark cycle, and pH curves were recorded during different growth phases.A new method was developed for the combined determination of β-1,3-glucan and cell wall polysaccharides in diatoms, representing total cellular carbohydrate. The glucan is rapidly extracted by hot dilute H2SO4, and the cell wall polysaccharides are subsequently hydrolyzed by cold 80% H2SO4overnight. Each carbohydrate fraction is finally determined by the phenol-sulphuric acid method. This procedure is simple and rapid compared to previous methods, and applies well to laboratory cultures as well as natural phytoplankton populations dominated by diatoms.Synthesis and mobilization of β-1,3-glucan in N-limited S. costatum were studied by combined 14C tracer technique and chemical analyses. Radiolabeled bicarbonate was added to the cultures, and 14C incorporation in different metabolites was determined using biochemical fractionation. In a pulse phase, 14C label was mainly incorporated in the glucan fraction (85%) during photosynthesis under nitrogen limitation. Subsequently, a 14C chase was carried out by adding NH4+ and incubating the cells under different light conditions. Radiolabeled glucan decreased significantly (by 26% in the dark, and by 19% in low light) whereas radiolabeled amino acids, proteins and other polysaccharides increased significantly during NH4+ assimilation. Chemical analyses of β-1,3-glucan and cellular free amino acids supported the 14C measurements. Changes in amino acid composition strongly indicated that de novo biosynthesis took place, with a Gln/Glu ratio increasing from 0.4 to 10. This study provides new evidence of β-1,3-glucan supplying carbon skeletons for synthesis of amino acids and protein in diatoms. Mobilization of glucan yields glucose, which is further metabolized by the respiratory pathways to provide precursors as well as energy. The results from the 14C chase also indicated significant synthesis of other polysaccharides or possibly RNA from glucan.In a different study, dark carbon fixation in N-limited S. costatum was measured using 14C-bicarbonate. Addition of NH4+ resulted in 4-fold increase in carboxylation rate, and biochemical fractionation showed that mainly amino acids were radiolabeled. Chemical analyses confirmed that cellular free amino acids increased rapidly (with increasing Gln/Glu), and showed that cellular glucan decreased significantly (by 28%) during NH4+ assimilation. The results strongly indicate that β-carboxylation provides C4 precursors for amino acid synthesis, and β-1,3-glucan is likely to be the ultimate substrate for β-carboxylation. Moreover, a C/N uptake ratio of 0.33 indicated that β-carboxylation was related to protein synthesis.A detailed study was made of the production of carbohydrates and amino acids by S. costatum during different growth phases. During exponential growth under diel light conditions, the glucan level oscillated between 17% (end of scotophase) and 42% (end of photophase) of cellular organic carbon, and the corresponding protein/glucan ratio alternated between 2.3 and 0.7. Concurrently, the cellular free amino acid pool oscillated between 8% (end of scotophase) and 22% (end of photophase) of cellular organic nitrogen, and the corresponding Gln/Glu ratio alternated between 0.05 and 2. Depletion of nitrogen from the medium resulted in rapid accumulation of glucan, reaching 75-80% of cellular organic carbon, whereas the cellular nitrogenous components decreased significantly. Consequently, the protein/glucan ratio decreased to <0.1. This study indicates that β-1,3-glucan functions both as a short-term diurnal reserve and a long-term stockpile reserve.Field investigations by other workers suggest that glucan plays a very active role in the dynamics of natural diatom populations, and the protein/glucan ratio has been used as a sensitive parameter for nutrient status. The glucan dynamics may be involved in physiological control of buoyancy. Glucan accumulation by nutrient-deplete cells causes increased cellular density and sinking below the nutricline. Upon nutrient replenishment and mobilization of glucan, the cells rise toward the surface of the water column, thereby transporting deep nutrients to the euphotic zone. β-1,3-Glucan also seems to play an important role in the development of resting stages in diatoms.