Changes In Amino Acid Composition Research Articles

A Nutritional Evaluation on Whole Cottonseed Removed Germination Ability by Heat-treatment

National Institute of Animal Science, RDA, Suwon 441-706, KoreaABSTRACTIn Korea, wide spread use of whole cottonseed, which is primarily a GMO plant imported from foreign countries and being fed to animals as raw state, has aroused concern that it may disturb the existing ecology of the country unless dispersion of the seed is under proper control. The objective of this study was to elucidate the changes in various nutritive parameters due to heat treatment and to determine the effective condition for removing germination ability of whole cottonseed(WCS). Of the various temperatures applied(76, 78, 80, 85, 100°C/30 min) 85°C for 30 min was confirmed to be the lowest temperature treatment which resulted in a complete removal of the germination ability of WCS. Therefore, based on the determined temperature condition(85°C 30min) we tried to examine the changes of various nutritional parameters, including nutrient composition, in vitro digestibilities and ruminal protein degradabilities, comparing raw whole cotton seed(RWCS) and heated whole cotton seed (HWCS). Some changes in amino acid composition were observed with heat treatment of WCS, but these were regarded to originate from the variation in plant quality and seed morphology, which are usually affected by different environmental factors during the vegetation period. As for fatty acid composition, no significant differences were observed to occur during heat treatment. However, WCS heated at 85°C for 30 min in a circulating oven showed a significant decrease(p<0.05) of in situ rumen degradability in both dry matter(DM) and crude protein(CP), as compared to raw WCS. Overall results obtained in the study indicate that the heating condition used in this study, which was proven to be the most appropriate and economic to remove germination ability of WCS, may also improve the nutritional value of the ruminant with regard to reducing its protein degradability within the rumen.(Key words :Whole cottonseed, Germination ability, Heat treatment, Degradability)

太湖水体溶解性氨基酸的空间分布特征

Amino acids( AAs) are important bio-available components of organic matter( OM) and are the major forms of organic nitrogen( ON) in aquatic ecosystem. They also represent significant fractions of OM in recent coastal marine sediments,water column particles and dissolved organic matters. They are typically labile composed relative to bulk OM and ON,and account for a considerable portion of the particulate OC and ON recycled in both water column and sediments,and therefore are an important nutrient form. Owing to the different AAs monomers have different biogeochemical stability,AAs are often used as an indicator of organic matter sources and as an index of OM degradation potential. Changes in amino acid composition exhibit consistent trends and demonstrate the degradation of cellular organic matter as it sinks through water column and is incorporated into sediments. Due to the labile character of AAs,the percentage of OC and N represented by amino acid( AA-C / OC% and AA-N / ON%) and the percentage of total amino acids have proven to be useful degradation indicators. They are sensitive at different stages of OM alteration and appear uncompromised by source variation. Dissolved amino acids( DAAs) characteristics were investigated in water of Lake Taihu,a large,shallow and eutrophic freshwater lake in China. The northern lake is characterized by phytoplankton dominance,high water turbidity,and severe Microcystis spp. algal blooms. In contrast,eastern lake is characterized by submersed vegetation,clear water,and diverse communities of fishes and invertebrates. The main upstream land use types of south Taihu were classified by the mountain forests and farmland. DAAs concentrations in Taihu ranged from 0. 27 to 3. 95 μmol / L,with a mean value of 1.38 μmol / L. The large regional differences in concentrations and yields of DAAs suggested that DOM produced in situ is an important source of bio-reactive of organic matter in waters of Taihu. The spatial distributions of DAAs concentrations in Taihu were less variable than those previously reported from other aquatic environments. DAAs concentrations of north Taihu were significantly higher than south lake and east lake,with mean values of( 2. 59±0. 71) μmol / L in north Taihu,( 0. 48±0. 14) μmol / L in south Taihu,and( 0. 48 ± 0. 16) μmol / L in east Taihu,respectively( P 0. 001). Based on the relationship between amino acid yields and percent labile DOC,( 17. 65 ±17. 84) % of fresh DOC in north lake is labile,which is higher than in south lake or east lake. The results showed internal sources mainly from the decomposing of phytoplankton should be the main source of DAAs in Taihu,and DAAs was important fraction of DOM. DAAs compositions were dominated by Phenylalanine( PHE) and lysine( LYS) in north lake,while only LYS in south and east lake.Compositions and concentrations of DAAs can be possible used as an indicator,which could provide useful information about the nutritional status and bio-available DOM in Lake Taihu. But because of high spatial heterogeneity in Taihu,we need to establish DAAs-liable DOC linear model for Lake Taihu,by the means of the algal-dominated and macrophytedominated DOM degradation experiments.

The influence of flushes on some constituents of mushrooms (Agaricus bisporus) cultivated on different composts

The effect of two different composts: horse manure and broiler chicken manure and the influence of flushes during the growing cycle on some chemical components contained in the mushroom (&lt;i&gt;Agaricus bisporus&lt;/i&gt;) was studied. The strain Somycel 11 was analyzed. The following determinations were made: dry matter, total nitrogen and crude protein, nitrates, nitrites and amino acids composition. It has been noted that the crude protein from. combined flushes III to VI of mushrooms cultivated on horse manure was about 24% higher than in those grown on broiler chicken manure. Some changes in amino acids composition, especially lysine, histidine, arginine, aspartic acid, serine, methionine, glutamic acid and alanine were observed, depending on the size of the fruit-body, flushes and type of compost.

Thermal aggregation properties of whey protein glycated with various saccharides

Aggregation of whey proteins during thermal processing is a challenge to maintain the dispersibility, particularly at pH near isoelectric point and increased ionic strength. We report glycation of whey protein isolate (WPI) with lactose and maltodextrin enabled transparent dispersions after heating at 88 °C for 2 min, when tested at 7%w/v protein, pH 3.0–7.0 and 0–150 mM NaCl or CaCl2. Transparent dispersions corresponded to particles smaller than 14 nm based on atomic force microscopy. In comparison, WPI glycated with glucose behaved similarly to WPI control, forming turbid dispersions or gels at many conditions. Based on analytical ultracentrifugation, reducing saccharide with a smaller molecular weight was glycated at a larger number on each protein molecule, agreeing with changes in amino acid composition. Glycation lowered the isoelectric point and increased denaturation temperatures of WPI. The results suggest that repulsive steric interactions are the dominant mechanism controlling whey protein aggregation and thus transparency of dispersions.

Molecular Characterization of Cold Adaptation of Membrane Proteins in the Vibrionaceae Core-Genome

Cold-adaptation strategies have been studied in multiple psychrophilic organisms, especially for psychrophilic enzymes. Decreased enzyme activity caused by low temperatures as well as a higher viscosity of the aqueous environment require certain adaptations to the metabolic machinery of the cell. In addition to this, low temperature has deleterious effects on the lipid bilayer of bacterial membranes and therefore might also affect the embedded membrane proteins. Little is known about the adaptation of membrane proteins to stresses of the cold. In this study we investigate a set of 66 membrane proteins from the core genome of the bacterial family Vibrionaceae to identify general characteristics that discern psychrophilic and mesophilic membrane proteins. Bioinformatical and statistical methods were used to analyze the alignments of the three temperature groups mesophilic, intermediate and psychrophilic. Surprisingly, our results show little or no adaptation to low temperature for those parts of the proteins that are predicted to be inside the membrane. However, changes in amino acid composition and hydrophobicity are found for complete sequences and sequence parts outside the lipid bilayer. Among others, the results presented here indicate a preference for helix-breaking and destabilizing amino acids Ile, Asp and Thr and an avoidance of the helix-forming amino acid Ala in the amino acid composition of psychrophilic membrane proteins. Furthermore, we identified a lower overall hydrophobicity of psychrophilic membrane proteins in comparison to their mesophilic homologs. These results support the stability-flexibility hypothesis and link the cold-adaptation strategies of membrane proteins to those of loop regions of psychrophilic enzymes.

Post-secretion processing influences spider silk performance

Phenotypic variation facilitates adaptations to novel environments. Silk is an example of a highly variable biomaterial. The two-spidroin (MaSp) model suggests that spider major ampullate (MA) silk is composed of two proteins-MaSp1 predominately contains alanine and glycine and forms strength enhancing β-sheet crystals, while MaSp2 contains proline and forms elastic spirals. Nonetheless, mechanical properties can vary in spider silks without congruent amino acid compositional changes. We predicted that post-secretion processing causes variation in the mechanical performance of wild MA silk independent of protein composition or spinning speed across 10 species of spider. We used supercontraction to remove post-secretion effects and compared the mechanics of silk in this 'ground state' with wild native silks. Native silk mechanics varied less among species compared with 'ground state' silks. Variability in the mechanics of 'ground state' silks was associated with proline composition. However, variability in native silks did not. We attribute interspecific similarities in the mechanical properties of native silks, regardless of amino acid compositions, to glandular processes altering molecular alignment of the proteins prior to extrusion. Such post-secretion processing may enable MA silk to maintain functionality across environments, facilitating its function as a component of an insect-catching web.

Gly6 of kalata B1 is critical for the selective binding to phosphatidylethanolamine membranes

The membrane interaction of the cyclotide kalata B1, an all-d-analogue and a single alanine substituted analogue (G6A), was studied by surface plasmon resonance (SPR) and atomic force microscopy (AFM). Kalata B1 showed a strong binding selectivity for dimyristoyl-phosphatidylethanolamine (DMPE) compared to dimyristoyl-phoshatidylcholine (DMPC)-containing lipids. However, when the interaction was visualized by AFM the peptide interacted with DMPC and DMPE in a similar manner. There was no apparent change in membrane morphology with either lipid, suggesting that kalata B1 does not act via a carpet-like disruption mechanism. The d-analogue showed similar binding by SPR and the same strong selectivity for DMPE, indicating that the membrane-interaction and lipid selectivity are not stereo-specific. SPR studies of the G6A analogue revealed that it interacted in a similar way to kalata B1 on the DMPC containing lipids, but showed no increased response on the DMPE containing lipids observed for kalata B1 and d-kalata B1. These results indicate that the Gly6 residue directly influences membrane binding as it is located near a putative membrane interacting hydrophobic patch. Overall, the data suggest that very small changes in amino acid composition (with no change in conformation) can influence specific self-association in combination with membrane binding and mediate the activity of kalata B1.

Effect of Low Temperature on Growth and Ultra-Structure of Staphylococcus spp

The effect of temperature fluctuation is an important factor in bacterial growth especially for pathogens such as the staphylococci that have to remain viable during potentially harsh and prolonged transfer conditions between hosts. The aim of this study was to investigate the response of S. aureus, S. epidermidis, and S. lugdunensis when exposed to low temperature (4°C) for prolonged periods, and how this factor affected their subsequent growth, colony morphology, cellular ultra-structure, and amino acid composition in the non-cytoplasmic hydrolysate fraction. Clinical isolates were grown under optimal conditions and then subjected to 4°C conditions for a period of 8 wks. Cold-stressed and reference control samples were assessed under transmission electron microscopy (TEM) to identify potential ultra-structural changes. To determine changes in amino acid composition, cells were fractured to remove the lipid and cytoplasmic components and the remaining structural components were hydrolysed. Amino acid profiles for the hydrolysis fraction were then analysed for changes by using principal component analysis (PCA). Exposure of the three staphylococci to prolonged low temperature stress resulted in the formation of increasing proportions of small colony variant (SCV) phenotypes. TEM revealed that SCV cells had significantly thicker and more diffuse cell-walls than their corresponding WT samples for both S. aureus and S. epidermidis, but the changes were not significant for S. lugdunensis. Substantial species-specific alterations in the amino acid composition of the structural hydrolysate fraction were also observed in the cold-treated cells. The data indicated that the staphylococci responded over prolonged periods of cold-stress treatment by transforming into SCV populations. The observed ultra-structural and amino acid changes were proposed to represent response mechanisms for staphylococcal survival amidst hostile conditions, thus maintaining the viability of the species until favourable conditions arise again.

Planthopper “adaptation” to resistant rice varieties: Changes in amino acid composition over time

The brown planthopper, Nilaparvata lugens, shows considerable geographic and temporal variability in its response to varieties of cultivated rice. N. lugens has repeatedly “adapted” to resistant rice varieties; however, the physiological changes underlying planthopper adaptation are poorly understood. Endosymbionts within planthoppers, such as yeast-like endosymbionts (YLS) could play a role as they produce essential amino acids for planthoppers. We used a full factorial study to determine how natal rice variety, exposed rice variety, YLS presence, and the number of reared generations affected nymphal development, planthopper total nitrogen content, and planthopper hydrolyzed amino acid profiles. Nymphal development was strongly influenced by a four-way interaction between the exposed rice variety, natal rice variety, number of reared generations, and YLS presence. While symbiosis improved nymphal performance in the 8th generation, it appeared to be a drain on nymphs in the 11th generation, when the aposymbiotic nymphs actually showed higher performance than the symbiotic nymphs. This suggests that the symbiotic relationship may be acting beneficially in one generation while acting as a drain during another generation. Aposymbiotic planthoppers reared for 11 generations had a higher proportional concentration of rare amino acids than those reared for 8 generations, indicating that the planthopper itself appears to improve its ability to acquire rare amino acids. Therefore, the change in amino acid composition of planthoppers suggests an underlying change in protein expression or amino acid metabolism over time.

Hypertrophic cardiomyopathy: from mutation to functional analysis of defective protein

AimTo analyze the genesis of hypertrophic cardiomyopathy on a large cohort of patients from molecular genetics point of view and perform the functional analysis of the 3D molecular model of defective myosin-7 protein in silico.MethodsThe study enrolled 153 patients with diagnosed hypertrophic cardiomyopathy from different parts of the Czech Republic. DNA samples were analyzed for mutations in exons 21 and 22 of the MYH7 gene, which have been associated with high mutation clustering. The 3D model of human myosin-7 was built using the x-ray structure of nucleotide-free scallop myosin S1 as the structural template. We performed de novo structure prediction of mutant and wild type peptides spanning the 769-788 amino acids region of the myosin-7 protein.ResultsThe Arg870His and Asp778Val amino acid alterations were found in 2 unrelated patients with a severe form of hypertrophic cardiomyopathy. The Asp778Val variation was chosen for subsequent 3D molecular modeling in silico. The mutation of the Asp by Val not only changes the character of the interaction pattern with other amino acids or ions but Val, being a small hydrophobic amino acid, can also completely change the stability of the region.ConclusionMutation location in the MYH7 gene and changes in amino acid composition may have a crucial negative impact on the outcome of the disease in patients with hypertrophic cardiomyopathy. In addition, a mutation that changes the charge of the amino acid is more likely to affect protein function than a conservative mutation.

Differences in variability of hypervariable region 1 of hepatitis C virus (HCV) between acute and chronic stages of HCV infection.

Distinguishing between acute and chronic HCV infections is clinically important given that early treatment of infected patients leads to high rates of sustained virological response. Analysis of 2179 clonal sequences derived from hypervariable region 1 (HVR1) of the HCV genome in samples obtained from patients with acute (n = 49) and chronic (n = 102) HCV infection showed that intra-host HVR1 diversity was 1.8 times higher in patients with chronic than acute infection. Significant differences in frequencies of 5 amino acids (positions 5, 7, 12, 16 and 18) and the average genetic distances among intra-host HVR1 variants were found using analysis of molecular variance. Differences were also observed in the polarity, volume and hydrophobicity of 10 amino acids (at positions 1, 4, 5, 12, 14, 15, 16, 21, 22 and 29). Based on these properties, a classification model could be constructed, which permitted HVR1 variants from acute and chronic cases to be discriminated with an accuracy of 88%. Progression from acute to chronic stage of HCV infection is accompanied by characteristic changes in amino acid composition of HVR1. Identifying these changes may permit diagnosis of recent HCV infection.

Effect of water on the heat-yellowing of silk fabric and the changes in amino acid composition in the silk fibroin in the sealed tubes by heat-treatment

Effect of water on the heat-yellowing of silk fabric and the changes in amino acid composition in the silk fibroin in the sealed tubes by heat-treatment

Transcriptional profiling of an Fd-GOGAT1/GLU1 mutant in Arabidopsis thaliana reveals a multiple stress response and extensive reprogramming of the transcriptome.

BackgroundGlutamate plays a central position in the synthesis of a variety of organic molecules in plants and is synthesised from nitrate through a series of enzymatic reactions. Glutamate synthases catalyse the last step in this pathway and two types are present in plants: NADH- or ferredoxin-dependent. Here we report a genome wide microarray analysis of the transcriptional reprogramming that occurs in leaves and roots of the A. thaliana mutant glu1-2 knocked-down in the expression of Fd-GOGAT1 (GLU1; At5g04140), one of the two genes of A. thaliana encoding ferredoxin-dependent glutamate synthase.ResultsTranscriptional profiling of glu1-2 revealed extensive changes with the expression of more than 5500 genes significantly affected in leaves and nearly 700 in roots. Both genes involved in glutamate biosynthesis and transformation are affected, leading to changes in amino acid compositions as revealed by NMR metabolome analysis. An elevated glutamine level in the glu1-2 mutant was the most prominent of these changes. An unbiased analysis of the gene expression datasets allowed us to identify the pathways that constitute the secondary response of an FdGOGAT1/GLU1 knock-down. Among the most significantly affected pathways, photosynthesis, photorespiratory cycle and chlorophyll biosynthesis show an overall downregulation in glu1-2 leaves. This is in accordance with their slight chlorotic phenotype. Another characteristic of the glu1-2 transcriptional profile is the activation of multiple stress responses, mimicking cold, heat, drought and oxidative stress. The change in expression of genes involved in flavonoid biosynthesis is also revealed. The expression of a substantial number of genes encoding stress-related transcription factors, cytochrome P450 monooxygenases, glutathione S-transferases and UDP-glycosyltransferases is affected in the glu1-2 mutant. This may indicate an induction of the detoxification of secondary metabolites in the mutant.ConclusionsAnalysis of the glu1-2 transcriptome reveals extensive changes in gene expression profiles revealing the importance of Fd-GOGAT1, and indirectly the central role of glutamate, in plant development. Besides the effect on genes involved in glutamate synthesis and transformation, the glu1-2 mutant transcriptome was characterised by an extensive secondary response including the downregulation of photosynthesis-related pathways and the induction of genes and pathways involved in the plant response to a multitude of stresses.

Evolution from Primitive Life to Homo sapiens Based on Visible Genome Structures: The Amino Acid World

It is not too much to say that molecular biology, including genome research, has progressed based on the determination of nucleotide or amino acid sequences. However, these ap-proaches are limited to the analysis of relatively small numbers of the same genes among spe-cies. On the other hand, by graphical presenta-tion of the ratios of the numbers of amino acids present to the total numbers of amino acids presumed from the target gene(s) or genome or those of the numbers of nucleotides present to the total numbers of nucleotides calculated from the target gene(s) or genome, we can readily draw conclusions from extraordinarily huge data sets integrated by human intelli-gence. 1) Assuming polymerization of amino acids or nucleotides in a simulation analysis based on a random choice, proteins were formed by simple amino acid polymerization, while nucleotide polymerization to form nucleic acids encoding specific proteins needed certain specific control. These results proposed that protein formation chronologically preceded codon formation during the establishment of primitive life forms. In the prebiotic phase, amino acid composition was a dominant factor that determined protein characteristics; the “Amino Acid World”. 2) The genome is constructed homogeneou- sly from putative small units displaying similar codon usages and coding for similar amino acid compositions; the unit is a gene assembly en-coding 3,000 - 7,000 amino acid residues and this unit size is independent not only of genome size, but also of species. 3) In codon evolution, all nucleotide alterna-tions are correlated, not only in coding regions, but also in non-coding regions; the correlations can be expressed by linear formulas; y = ax + b, where “y” and “x” represent nucleotide con-tents, and “a” and “b” are constant. 4) The basic pattern of cellular amino acid compositions obtained from whole cell lysates is conserved from bacteria to Homo sapiens, and resembles that calculated from complete genomes. This basic pattern is characterized by a “star-shape” that changes slightly among species, and changes in amino acid composi-tion seem to reflect biological evolution. 5) Organisms can essentially be classified according to two codon patterns. Biological evolution due to nucleotide sub-stitutions can be expressed by simple linear formulas based on mathematical principles, while natural selection must affect species pre- servation after nucleotide alternations. There-fore, although Darwin’s natural selection is not directly involved in nucleotide alternations, it contributes obviously to the selection of nu-cleotide alternations. Thus, Darwin’s natural selection is doubtless an important factor in biological evolution.

Evaluation of the effect of CpG hypermutability on human codon substitution

Understanding the cause underlying the changes in amino acid composition of proteins is essential for understanding protein evolution and function. Accurate models of DNA and protein evolution are essential for studying molecular evolution. Although many models have been developed, most models assume that each site evolves independently and that substitutions are time reversible. In mammals and other organisms, CpG hypermutability is one of the major causes of nucleotide mutations because CpG dinucleotides are often methylated at C, and the methyl-C mutation spontaneously deaminates to yield T about 3 times more rapidly than other types of point mutations. In this study, we evaluate the effect of CpG hypermutability on codon substitution by comparing thousands of coding regions in the human and chimpanzee genomes and by inferring ancestral sequences by using mouse as the outgroup. We found that 14% of synonymous and nonsynonymous substitutions on human genes were caused by CpG hypermutability. Based on these results, we developed a model that incorporates CpG hypermutability as well as the transition/transversion ratio and changes in the chemical properties of amino acids.

Amino acid biogeochemistry and organic matter degradation state across the Pakistan margin oxygen minimum zone

To assess whether the oxygen minimum zone (OMZ) across the Pakistan Margin causes differences in the lability of sedimentary organic matter, sediments were collected in the core of the OMZ, in the upper and lower transition zones and below the OMZ. Sediment samples were analysed for total nitrogen (TN) and organic carbon (OC) contents, mineral surface area (SA), and total hydrolysable amino acids (THAA) and enzymatically hydrolysable amino acids (EHAA). OC contents and organic carbon per unit of mineral surface area (OC/SA) values were clearly elevated in the core and lower OMZ transition zone. These sediments also contained more labile sedimentary organic matter, as discerned by higher concentrations of THAA and the contribution of N in THAA to TN. A protein amino acid-based degradation index revealed that all sedimentary organic matter has undergone significant degradation, but sediments in the upper OMZ transition zone and below the OMZ are more degraded than inside the OMZ. Changes in amino acid composition during diagenesis are attributed to a combination of factors: (1) selective preservation in which amino acids in cell walls are better preserved than amino acids in cell plasma, (2) formation and accumulation of bacterially derived organic matter; there were relatively more living bacteria in the core of the OMZ and an accumulation of peptidoglycan-derived amino acids in degraded sediments in the upper OMZ transition zone and below the OMZ, and (3) bacterial transformation, as the molar percentages of bacterial transformation products β-alanine (Bala), γ-amino butyric acid (Gaba), and ornithine (Orn), increased with increasing degradation.

Changes in amino acid composition during wine vinegar production in a fully automatic pilot acetator

Changes in free amino acid, ammonium ion and urea contents in the culture medium were investigated during controlled acetification in the presence of acetic acid bacteria. The bacteria were found to use l-proline, l-leucine and ammonium ion as their primary sources of nitrogen; in fact, these three species accounted for 68.1% of their total nitrogen uptake. Concentration changes were very similar for all amino acids and consistent with changes in cell concentrations. Thus, cell concentration in the medium decreased through bacterial autolysis as the amino acid concentration increased. By contrast, when a cellular growth is produced the amino acid concentration was decreased. The concentration of acetic acid bacteria oscillated widely during the last fermentation stage. This can be ascribed to the bacteria using an apoptosis-like way in response to the stressing conditions produced by the high concentration of acetic acid present in the medium.

Decomposition process of organic matter derived from freshwater phytoplankton

We investigated the biodegradation process of freshwater phytoplanktonic organic matter using incubation experiments, with special reference to changes in three major biomolecules: neutral aldoses, amino acids and fatty acids. The concentration of neutral aldoses decreased drastically relative to amino acids and fatty acids during the early decomposition phase (days 0–7), owing largely to the rapid decomposition of storage carbohydrate. This resulted in a temporary decrease in the C/N ratio of organic matter. During the late phase (days 7–60), however, the rate of decrease in neutral aldoses slowed, while the considerable decrease in amino acids and fatty acids continued. The inconspicuous change in amino acid composition was probably due to the fact that no protein and/or peptide is composed of a limited species of amino acid. Although the compositional variety of organic matter among the phytoplankton was clearly observed at the start of decomposition, it became obscure in the course of 60 days. This indicates that while the organic composition of the labile fraction of phytoplanktonic organic matter varies depending on the phytoplankton groups, the refractory fraction has similar composition. The addition of bacterial organic matter is likely another reason for the similar composition of the remaining organic matter at day 60.

Cancer and nutrition: role of amino acids (AA) for the regulation of hepatocellular apoptosis

Since decades nutrition is known to play a central role in development of cardiac diseases, cancer and diabetes. However, the causative nutritional factors and their mode of action are not well understood. Accumulating evidence suggest that certain nutrients (including glucose and AA) may regulate cell signalling events/gene expression independently of hormones, possibly acting through specific nutrient sensor mechanisms [1]. Previous in vivo studies by ourselves on the regulation of rat hepatocyte proliferation and apoptosis revealed that the lack of glucose and/or AA blocks the initiation of DNA synthesis (G1/Stransition). Furthermore, feed restriction favoured apoptosis in rat liver. Here we report on the role of AA for apoptosis of human hepatoma cells (HCC-1.2). The following changes in AA composition – as compared to standard RPMI 1640 medium – exerted a pro-apoptotic action, without and with TGF-β1 treatment: (1) deprivation of branched chain AA, with Val > Leu > Ile when tested individually; (2) deprivation of Phe, Trp, Lys, Met, Thr, either as group or, with the exception of Met, when tested individually; (3) deprivation of His and Gln seemed less effective than the other individual AA tested; (4) occurrence of autophagy was demonstrated lightand electron-microscopically. In summary, this first series of experiments revealed HCC-1.2 cells sensitive to the AA deprivation, rendering this model suitable for studying pharmacological actions of AA.

Using evolutionary tools to refine the new hypervariable region 3 within the envelope 2 protein of hepatitis C virus

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.