Phylogenetic mixture models for proteins

Standard protein substitution models use a single amino acid replacement rate matrix that summarizes the biological, chemical and physical properties of amino acids. However, site evolution is highly heterogeneous and depends on many factors: genetic code; solvent exposure; secondary and tertiary structure; protein function; etc. These impact the substitution pattern and, in most cases, a single replacement matrix is not enough to represent all the complexity of the evolutionary processes. This paper explores in maximum-likelihood framework phylogenetic mixture models that combine several amino acid replacement matrices to better fit protein evolution.We learn these mixture models from a large alignment database extracted from HSSP, and test the performance using independent alignments from TREEBASE.We compare unsupervised learning approaches, where the site categories are unknown, to supervised ones, where in estimations we use the known category of each site, based on its exposure or its secondary structure. All our models are combined with gamma-distributed rates across sites. Results show that highly significant likelihood gains are obtained when using mixture models compared with the best available single replacement matrices. Mixtures of matrices also improve over mixtures of profiles in the manner of the CAT model. The unsupervised approach tends to be better than the supervised one, but it appears difficult to implement and highly sensitive to the starting values of the parameters, meaning that the supervised approach is still of interest for initialization and model comparison. Using an unsupervised model involving three matrices, the average AIC gain per site with TREEBASE test alignments is 0.31, 0.49 and 0.61 compared with LG (named after Le & Gascuel 2008 Mol. Biol. Evol. 25, 1307-1320), WAG and JTT, respectively. This three-matrix model is significantly better than LG for 34 alignments (among 57), and significantly worse for 1 alignment only. Moreover, tree topologies inferred with our mixture models frequently differ from those obtained with single matrices, indicating that using these mixtures impacts not only the likelihood value but also the output tree. All our models and a PhyML implementation are available from http://atgc.lirmm.fr/mixtures.     

Influence of amino acid supply on ribosomes and protein synthesis of perfused rat liver

1. The livers of rats were perfused in situ with medium containing mixtures of amino acids in multiples of their concentration in normal rat plasma. The incorporation of labelled amino acid into protein of the liver and of the perfusing medium increased with increasing amino acid concentration. During 60min. perfusions, labelling of liver protein reached a plateau, and labelling of medium protein was inhibited when the initial concentration of the amino acid mixture was more than ten times the normal plasma value. 2. Examination of polysome profiles derived from livers perfused without amino acids in the medium showed that the number of large aggregates was decreased and the number of small aggregates, particularly monomers and dimers, was increased with time of perfusion. The addition of amino acids to the perfusion medium reversed this polysome shift to an extent that was dependent on the initial concentration of amino acids. Polysome profiles derived from livers perfused for 60min. with ten times the normal plasma concentration of amino acids were essentially the same as the polysome profiles of normal non-perfused livers. 3. The ability of ribosome preparations from perfused livers to incorporate amino acids into protein in vitro decreased with increasing time of perfusion when no amino acids were added to the medium, but increased as the concentration of amino acids in the perfusion medium was increased. 4. The ability of cell sap from perfused livers to support protein synthesis in vitro was not influenced by the amino acid concentration of the perfusion medium. 5. Livers were perfused for 60min. with medium containing amino acid mixtures at ten times the normal plasma concentration but deficient in one amino acid. Maximal incorporation of labelled amino acid into liver protein, the stability of the polysome profile and the ability of ribosome preparations to incorporate amino acids into protein were found to depend on the presence of 11 amino acids: arginine, asparagine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan and valine. A mixture of these 11 amino acids, at ten times their normal plasma concentration, stimulated the incorporation of labelled amino acid into liver protein, stabilized the polysome profile and increased the ability of ribosome preparations to incorporate amino acids into protein to the same extent as the complete mixture. 6. It is concluded that the availability of certain amino acids plays an important role in the control of protein synthesis, possibly by stimulating the ability of ribosomes to become, and to remain, attached to messenger RNA.     

Electro-olfactogram and multiunit olfactory receptor responses to complex mixtures of amino acids in the channel catfish, Ictalurus punctatus

In vivo electrophysiological recordings from populations of olfactory receptor neurons in the channel catfish, Ictalurus punctatus, clearly showed that both electro-olfactogram and integrated neural responses of olfactory receptor cells to complex mixtures consisting of up to 10 different amino acids were predictable with knowledge of (a) the responses to the individual components in the mixture and (b) the relative independence of the respective receptor sites for the component stimuli. All amino acid stimuli used to form the various mixtures were initially adjusted in concentration to provide approximately equal response magnitudes. Olfactory receptor responses to both multimixtures and binary mixtures were recorded. Multimixtures were formed by mixing equal aliquots of 3-10 different amino acids. Binary mixtures were formed by mixing equal aliquots of two equally stimulatory solutions. Solution 1 contained either one to nine different neutral amino acids with long side-chains (LCNs) or one to five different neutral amino acids with short side-chains (SCNs). Solution 2, comprising the binary mixture, consisted of only a single stimulus, either a LCN, SCN, basic, or acidic amino acid. The increasing magnitude of the olfactory receptor responses to mixtures consisting of an increasing number of neutral amino acids indicated that multiple receptor site types with highly overlapping specificities exist to these compounds. For both binary mixtures and multimixtures composed of neutral and basic or neutral and acidic amino acids, the receptor responses were significantly enhanced compared with those mixtures consisting of an equal number of only neutral amino acids. These results demonstrate that receptor sites for the basic and acidic amino acids, respectively, are highly independent of those for the neutral amino acids, and suggest that a mechanism for synergism is the simultaneous activation of relatively independent receptor sites by the components in the mixture. In contrast, there was no evidence for the occurrence of mixture suppression.     

Peptide libraries: Determination of relative reaction rates of protected amino acids in competitive couplings

In the solid phase preparation of synthetic peptide libraries, equimolarity of the resultant peptides in the mixture simplifies the identification of active compounds. Two primary methods for the preparation of combinatorial peptide mixtures are currently used. In the first method, the starting resin is divided into equal aliquots, individual amino acids are coupled to each aliquot, and the resin is then recombined. This process is repeated for each position. However, due to the physical process, each resin bead contains only one peptide sequence. Statistically, for mixtures of longer sequences, an ever-increasing amount of resin is necessary to ensure complete representation of each peptide in the library. Thus, each peptide will be represented in the library if a sufficient number of resin beads are used. In addition, the concentration of each peptide in the library depends on both the number of mixture positions in the library and the amount of resin used. In the second method, mixtures of amino acids are coupled simultaneously at each addition step. The proportion of each amino acid in the reaction mixture is varied inversely to its reaction rate such that, ideally, an equimolar mixture of each peptide is synthesized. An advantage of this method over the previous method is that each peptide is ensured to be represented in the library, although not necessarily in equimolar amounts. It is known that not only do the coupling rates of each amino acid vary, but the coupling rates of individual amino acids also change when coupled to different amino acid resins. Consequently, in order to obtain equimolar peptide mixtures through the use of mixtures of protected amino acids, the ratio of reaction rates of one amino acid over another must be constant irrespective of the resin-bound amino acid. If this premise is true, this method of synthesis offers a significant advantage over the previous method since, theoretically, equimolar peptide libraries could be synthesized. The influence of the resin-bound amino acid on the relative reaction rates of incoming amino acids was investigated in the current study. © 1994 John Wiley & Sons, Inc.     

Transport of threonine and tryptophan by rat brain slices: relation to other amino acids at concentrations found in plasma

Threonine content of brain decreases in young rats fed a threonine-limiting, low protein diet containing a supplement of small neutral amino acids (serine, glycine and alanine), which are competitors of threonine transport in other systems (Tews et al., 1977). Threonine transport by brain slices was inhibited more by a complex amino acid mixture resembling plasma from rats fed the small neutral amino acid supplement than by mixtures resembling plasma from control rats or from rats fed a supplement of large neutral amino acids. Greater inhibition was seen with mixtures containing only the small neutral amino acids than with mixtures containing only large neutral amino acids. On an equimolar basis, serine and alanine were the most inhibitory; large neutrals were moderately so; and glycine and lysine were without effect. Threonine transport was also strongly inhibited by α-amino-n-butyric acid and homoserine, less so by α-aminoisobutyric acid, and not at all by GABA. The complex amino acid mixtures strongly inhibited α-aminoisobutyric acid transport by brain or liver slices but, in contrast to effects in brain, the extent of the inhibition in liver was not much affected by altering the composition of the mixture. Tryptophan accumulation by brain slices was effectively inhibited by other large neutral amino acids in physiologically occurring concentrations. Threonine, or a mixture of serine, glycine and alanine only slightly inhibited tryptophan uptake; basic amino acids were without effect and histidine stimulated tryptophan transport slightly. These results support the conclusion that a diet-induced decrease in the concentration in brain of a specific amino acid may be related to increased inhibition of its transport into brain by increases in the concentrations of transport-related, plasma amino acids.     

Olfactory discrimination of complex mixtures of amino acids by the black bullhead Ameiurus melas

On the basis of previous findings of behavioural discrimination of amino acids and on the knowledge of electrophysiology of the catfish (genera Ictalurus and Ameiurus) olfactory organs, behavioural experiments that investigated olfactory discrimination of amino acid mixtures were carried out on the black bullhead Ameiurus melas. Repeated presentations of food‐rewarded mixtures released increased swimming activity measured by counting the number of turns >90° within 90 s of stimulus addition. Non‐rewarded amino acids and their mixtures released little swimming activity, indicating that A. melas discriminated between the conditioned and the non‐conditioned stimuli. Two questions of mixture discrimination were addressed: (1) Are A. melas able to detect components within simple and complex amino acid mixtures? (2) What are the smallest differences between two complex mixtures that A. melas can detect? Three and 13 component mixtures tested were composed primarily of equipotent amino acids [determined by equal electroolfactogram (EOG) amplitude] that contained L‐Cys at ×100 the equipotent concentration. Ameiurus melas initially perceived the ternary amino acid mixture as its more stimulatory component alone [i.e. cysteine (Cys)], whereas the conditioned 13 component mixture containing the more stimulatory L‐Cys was perceived immediately as different from L‐Cys alone. The results indicate that components of ternary mixtures are detectable by A. melas but not those of more complex mixtures. To test for the smallest detectable differences in composition between similar multimixtures, all mixture components were equipotent. Initially, A. melas were unable to discriminate the mixtures of six amino acids from the conditioned mixtures of seven amino acids, whereas they discriminated immediately the mixtures of four and five amino acids from the conditioned mixture. Experience with dissimilar mixtures enabled the A. melas to start discriminating the seven‐component conditioned mixture from its six‐component counterparts. After fewer than five training trials, A. melas discriminated the mixtures of nine and 10 amino acids from a conditioned mixture of 12 equipotent amino acids; however, irrespective of the number of training trials, A. melas were unable to discriminate the 12 component mixture from its 11 component counterparts.     

A statistical framework for the interpretation of mtDNA mixtures: forensic and medical applications

Background

Mitochondrial DNA (mtDNA) variation is commonly analyzed in a wide range of different biomedical applications. Cases where more than one individual contribute to a stain genotyped from some biological material give rise to a mixture. Most forensic mixture cases are analyzed using autosomal markers. In rape cases, Y-chromosome markers typically add useful information. However, there are important cases where autosomal and Y-chromosome markers fail to provide useful profiles. In some instances, usually involving small amounts or degraded DNA, mtDNA may be the only useful genetic evidence available. Mitochondrial DNA mixtures also arise in studies dealing with the role of mtDNA variation in tumorigenesis. Such mixtures may be generated by the tumor, but they could also originate in vitro due to inadvertent contamination or a sample mix-up.

Methods/Principal Findings

We present the statistical methods needed for mixture interpretation and emphasize the modifications required for the more well-known methods based on conventional markers to generalize to mtDNA mixtures. Two scenarios are considered. Firstly, only categorical mtDNA data is assumed available, that is, the variants contributing to the mixture. Secondly, quantitative data (peak heights or areas) on the allelic variants are also accessible. In cases where quantitative information is available in addition to allele designation, it is possible to extract more precise information by using regression models. More precisely, using quantitative information may lead to a unique solution in cases where the qualitative approach points to several possibilities. Importantly, these methods also apply to clinical cases where contamination is a potential alternative explanation for the data.

Conclusions/Significance

We argue that clinical and forensic scientists should give greater consideration to mtDNA for mixture interpretation. The results and examples show that the analysis of mtDNA mixtures contributes substantially to forensic casework and may also clarify erroneous claims made in clinical genetics regarding tumorigenesis.     

Preparation of 3-Methyl-3-(substituted phenyl)-pyruvic Acid

The relationships between dietary levels of the essential amino acids and hepatic polysome profiles of rats were investigated with special attention to the amino acid requirement pattern for the maximum rat growth as determined by other investigators. The basal diet contained a 7% essential amino acid mixture and a 3% non-essential amino acid mixture, with appropriate amounts of other nutrients. Rats were fed test diet for 5 hours and then the polysome profile was determined. The amounts of essential amino acids needed for maximum aggregation of polysome were low for methionine-cystine, leucine and tryptophan as compared with requirements for maximum growth. But in other essential amino acids, the amounts were in almost the same range as those reported for maximum growth by others. The differences between the amino acid requirement patterns for maximum aggregation of hepatic ribosomes and for maximum growth of rats might be due to a difference in amino acid requirements of the liver and whole body. Therefore, the hepatic polysome profile might be used to measure the effect of amino acid supplementation on dietary proteins. The requirement pattern of essential amino acids in other organs may be studied by polysome profile determination.     

TSSub: eukaryotic protein subcellular localization by extracting features from profiles

This paper introduces a new subcellular localization system (TSSub) for eukaryotic proteins. This system extracts features from both profiles and amino acid sequences. Four different features are extracted from profiles by four probabilistic neural network (PNN) classifiers, respectively (the amino acid composition from whole profiles; the amino acid composition from the N-terminus of profiles; the dipeptide composition from whole profiles and the amino acid composition from fragments of profiles). In addition, a support vector machine (SVM) classifier is added to implement the residue-couple feature extracted from amino acid sequences. The results from the five classifiers are fused by an additional SVM classifier. The overall accuracies of this TSSub reach 93.0 and 77.4% on Reinhardt and Hubbard's eukaryotic protein dataset and Huang and Li's eukaryotic protein dataset, respectively. The comparison with existing methods results shows TSSub provides better prediction performance than existing methods. AVAILABILITY: The web server is available from http://166.111.24.5/webtools/TSSub/index.html.     

The effect of feeding with a tryptophan-free amino acid mixture on rat liver magnesium ion-activated deoxyribonucleic acid-dependent ribonucleic acid polymerase

1. The Widnell & Tata (1966) assay method for Mg(2+)-activated DNA-dependent RNA polymerase was used for initial-velocity determinations of rat liver nuclear RNA polymerase. One unit (U) of RNA polymerase was defined as that amount of enzyme required for 1 mmol of [(3)H]GMP incorporation/min at 37 degrees C. 2. Colony fed rats were found to have a mean RNA polymerase activity of 65.9muU/mg of DNA and 18h-starved rats had a mean activity of 53.2muU/mg of DNA. Longer periods of starvation did not significantly decrease RNA polymerase activity further. 3. Rats that had been starved for 18h were used for all feeding experiments. Complete and tryptophan-deficient amino acid mixtures were given by stomach tube and the animals were killed 15-120min later. The response of RNA polymerase to the feeding with the complete amino acid mixture was rapid and almost linear over the first hour of feeding, resulting in a doubling of activity. The activity was still elevated above the starvation value at 120min after feeding. The tryptophan-deficient amino acid mixture produced a much less vigorous response about 45min after the feeding, and the activity had returned to the starvation value by 120min after the feeding. 4. The response of RNA polymerase to the feeding with the complete amino acid mixture was shown to occur within a period of less than 5min to about 10min after the feeding. 5. Pretreatment of the animals with puromycin or cycloheximide was found to abolish the 15min RNA polymerase response to the feeding with the complete amino acid mixture, but the activity of the controls was unaffected. 6. The characteristics of the RNA polymerase from 18h-starved animals and animals fed with the complete or incomplete amino acid mixtures for 1h were examined. The effects of Mg(2+) ions, pH, actinomycin D and nucleoside triphosphate omissions were determined. The [Mg(2+)]- and pH-activity profiles of the RNA polymerase from the animal fed with the complete mixture appeared to differ from those of the enzyme from the other groups, but this difference is probably not significant. 7. [5-(3)H]Orotic acid incorporation by rat liver nuclei in vivo was shown to be affected by the amino acid mixtures in a similar manner to the RNA polymerase. 8. The tryptophan concentrations of plasma and liver were determined up to 120 min after feeding with the amino acid mixtures. Feeding with the complete mixture produced a rapid increase in free tryptophan concentrations in both plasma and liver, but feeding with the incomplete mixture did not alter the plasma concentration. The liver tryptophan concentration increased at about 45min after feeding with the tryptophan-deficient diet. 9. There was a good correlation between the liver tryptophan concentration and RNA polymerase activity in all groups of animals. 10. It was concluded that the rat liver nucleus responded to an increase in amino acid supply by increased synthesis of RNA as a result of synthesis of RNA polymerase de novo. The correlation of tryptophan concentration and RNA polymerase activity appears to reflect the general amino acid concentration required to support hepatic protein synthesis and to produce new RNA polymerase. This new polymerase appears to differ from the basal RNA polymerase by its rapid synthesis and destruction, which may be a means of regulating RNA synthesis by the amino acid concentration in the liver.     

Study on optimization of moisture retention for golf green rootzone soil mixtures

Most golf course green have been constructed with pure sand or sand-based rootzone mixes. As we know, high sand content provides rapid drainage despite sand’s inefficiency in retaining moisture. However, drainage capability and water retention are both essential elements to the golf course green, and the addition of peat could increase the soil moisture retention, therefore, the research on the drainage capability and water retention of the sandy golf green has become more and more important these years. In this study, extreme vertex design was applied which is one of the mixture experiment designs widely used in mixture experiments, the study investigated the effects of the thirteen different rootzone soil mixtures using middle-coarse, fine sand, very fine sand plus silt and clay as well as peat as the materials under three kinds of golf green profile (1-layer profile, 2-layer profile, 3-layer profile) conditions on the water retention of green rootzone. Through the qualitative, quantitative and optimization analysis of water retention capability of the sandy golf green, evidence a basis for choice of green profiles and rootzone matrix could be provided. And the significantly correlative regression model was established between the moisture retention and components of rootzone soil mixture. In addition, the order of factor contribution ratio, effect of single and double factor and optimization of the model were analyzed in detail. The results were as follows: both green profile and soil mixture, which had interaction of each other, had significant effects on soil moisture retention. Additional attributes include high porosity and greater water holding capacity than sand, and the higher content of peat, fine sand plus silt and clay, the better water retention. The mixtures had much higher water content in 1-layer profile than that in the other two profiles. There was significantly higher water content in 2-layer profile for pure sand mixtures (A–E) and low peat mixture (F) than that in 3-layer profile, while there was no higher water content for other 7 high peat mixtures (G–M) (>5%) in 2-layer than that in 3-layer profiles. The significance of key factors in rootzone soil mixture on moisture retention were: very fine sand plus silt and clay > peat > middle-coarse > fine sand. According to the moisture retention 15–25% specification of USGA (United States Golf Association), the optimal soil mixture in 1-layer profile was: middle-coarse 71.4–73.5%; fine sand 17.8–21.5%; very fine sand plus silt and clay 6.8–8.4%; peat 0–1%. The optimal soil mixture in 2-layer profile was: middle-coarse 65.0–73.4%; fine sand 17.8–20.5%; very fine sand plus silt and clay 7.5–8.9%; peat 0.2–6.3%. The optimal soil mixture in 3-layer profile was: middle-coarse 62.3– 73.9%; fine sand 17.7–21.4%; very fine sand plus silt and clay 7.3–10.7%; peat 0–6.3%. These optimal recipes took through the limitation of previous research, which were practically important to golf green soil selection and profile design. Thus, both proportion and interaction should be considered when we choose the soil mixture.     

A novel amino acid supplementation strategy based on a stoichiometric model to enhance human IL-2 (interleukin-2) expression in high-cell-density Escherichia coli cultures

A novel amino acid supplementation strategy was developed for enhancing the production of IL-2 (interleukin-2; as a model protein) by recombinant Escherichia coli BL21 (pET21a-hil2) in fed-batch high-cell-density cultures. The amino acids most needed and their amounts were determined using a stoichiometric model, and full factorial design experiments were conducted to determine the effects of single amino acids and amino acid mixtures on production. One of the most effective amino acid mixtures was found to be leucine, aspartic acid and glycine. This amino acid mixture was utilized for the production of IL-2 in batch and fed-batch fermentations. The amount of IL-2 produced increased from 403 to 722 mg/l and from 5.15 × 103 to 8.08 × 103 mg/l in batch and fed-batch cultures respectively. The results also revealed that the above amino acid mixture specifically increases IL-2 concentration in the cells.     

The effect of feeding with a tryptophan-free amino acid mixture on rat-liver polysomes and ribosomal ribonucleic acid

1. Starving rats were given complete and tryptophan-deficient amino acid mixtures by stomach tube and were killed from 1 to 7hr. later. The polysome profile in the livers of rats fed with the tryptophan-deficient mixture showed a shift in distribution such that the large aggregates were decreased and the small aggregates were increased, particularly dimers. This polysome shift was reversed when the complete amino acid mixture was given by stomach tube 2hr. after administering the tryptophan-free amino acid mixture. 2. After removal of liver polysomes by centrifugation, some smaller ribosomal aggregates (oligosomes) remaining in suspension were harvested by prolonged centrifugation of the supernatant fluid. A large increase in the dimer population of this fraction was observed in the rats receiving the incomplete mixture. 3. When the polysome and oligosome fractions were incubated with cell sap, an energy-generating system and labelled amino acids dl-[1-(14)C]leucine and l-[Me-(14)C]tryptophan were incorporated into the cell fractions in the ratio 4.5:1. Preparations of polysomes and oligosomes from rats fed with the tryptophan-free amino acid mixture showed a decreased amino acid-incorporating activity compared with particulate preparations made from rats fed with the complete mixture. 4. The yield of free ribosomes prepared from the unfractionated liver microsomes by treatment with iso-octane was 40-50% greater in rats fed with the amino acid mixture deficient in tryptophan. 5. A post-microsomal fraction was prepared from cell sap and was shown to consist of ribosomal sub-units. When the animals were fed with the tryptophan-deficient mixture, there was an increase in content of this post-microsomal fraction and in the ratio 30s RNA/19s RNA. Rats were also given [5-(3)H]orotic acid at the time of feeding with the amino acids. Lack of tryptophan in the mixture caused a decrease in the specific activity of both RNA fractions which affected the 30s RNA more extensively than the 19s RNA. 6. These changes in the distribution and quantity of the cellular components engaged in protein synthesis are discussed in relation to RNA metabolism and amino acid-incorporating activity of the liver cell and their response to feeding with the tryptophan-free amino acid mixture.     

Studies on Amino Acid Contents of Processed Soybean

Effect of heating on the nutritive value of defatted soybean flour has been investigated by animal experiments. Loss due to heat degradation was evaluated in two ways. In the first method, the amino acids lost during overheating were supplemented by cystine and mixture of lysine, arginine, tryptophan, and serine at dietary levels of 1.6% nitrogen, and cystine and mixture of those amino acids plus histidine at dietary levels of 3.2% nitrogen. The other procedure adopted was the absorbent test used with amino acid mixtures based on the pattern of amino acids released by pancreatic hydrolysis of unheated, properly heated, and overheated defatted soybean flour at 6 and 120 hr hydrolysis.

At 1.6% dietary nitrogen level, the nutritive value of overheated soybean flour increased by supplementation with cystine and amino acid mixture, but at the 3.2% nitrogen level only cystine was effective. Supplementation of lost amino acids to overheated flour did not restore the nutritive value to that of the properly heated flour. Based on the amino acids released by pancreatic hydrolysis of unheated, properly heated, and overheated soybean flour after 6 and 120 hr reaction, amino acid mixtures were prepared and tested for their nutritive value. While the nutritive value of amino acid mixture prepared based on the pattern of amino acid liberated by 6 hr digestion of unheated, properly heated, and overheated flour did not show similar trend to that of 3 kinds of flour itself, the nutritive value of the amino acid mixture prepared after the data obtained by 120 hr digestion agreed well with the trend of unheated or heated soybean flour.

The nutritive value was also measured by the nitrogen balance of test animals.     

Lipid Mixtures Containing a Very High Proportion of Saturated Fatty Acids Only Modestly Impair Insulin Signaling in Cultured Muscle Cells

In vitro examinations of the effect of saturated fatty acids on skeletal muscle insulin action often use only one or two different fatty acid species, which does not resemble the human plasma fatty acid profile. We compared graded concentrations (0.1-0.8mM) of 3 different lipid mixtures: 1) a physiologic fatty acid mixture (NORM; 40% saturated fatty acids), 2) a physiologic mixture high in saturated fatty acids (HSFA; 60% saturated fatty acids), and 3) 100% palmitate (PALM) on insulin signaling and fatty acid partitioning into triacylglycerol (TAG) and diacylglycerol (DAG) in cultured muscle cells. As expected, PALM readily impaired insulin-stimulated pAkt^Thr308/Akt and markedly increased intracellular DAG content. In contrast, the fatty acid mixtures only modestly impaired insulin-stimulated pAkt^Thr308M/Akt, and we found no differences between NORM and HSFA. Importantly, NORM and HSFA did not increase DAG content, but instead dose-dependently increased TAG accumulation. Therefore, the robust impairment in insulin signaling found with palmitate exposure was attenuated with physiologic mixtures of fatty acids, even with a very high proportion of saturated fatty acids. This may be explained in part by selective partitioning of fatty acids into neutral lipid (i.e., TAG) when muscle cells were exposed to physiologic lipid mixtures.     

Which Amino Acids Should Be Used in Prebiotic Chemistry Studies?

The adsorption of amino acids on minerals and their condensation under conditions that resemble those of prebiotic earth is a well studied subject. However, which amino acids should be used in these experiments is still an open question. The main goal of this review is to attempt to answer this question. There were two sources of amino acids for the prebiotic earth: (1) exogenous -- meaning that the amino acids were synthesized outside the earth and delivered to our planet by interplanetary dust particles (IDPs), meteorites, comets, etc. and (2) endogenous -- meaning that they were synthesized on earth in atmospheric mixtures, hydrothermal vents, etc. For prebiotic chemistry studies, the use of a mixture of amino acids from both endogenous and exogenous sources is suggested. The exogenous contribution of amino acids to this mixture is very different from the average composition of proteins, and contains several non-protein amino acids. On the other hand, the mixture of amino acids from endogenous sources is seems to more closely resemble the amino acid composition of terrestrial proteins.     

Improvement of pulmonary surfactant activity by introducing D-amino acids into highly hydrophobic amphiphilic α-peptide Hel 13-5

The high costs of artificial pulmonary surfactants, ranging in hundreds per kilogram of body weight, used for treating the respiratory distress syndrome (RDS) premature babies have limited their applications. We have extensively studied soy lecithins and higher alcohols as lipid alternatives to expensive phospholipids such as DPPC and PG. As a substitute for the proteins, we have synthesized the peptide Hel 13-5D3 by introducing D-amino acids into a highly lipid-soluble, basic amphiphilic peptide, Hel 13-5, composed of 18 amino acid residues. Analysis of the surfactant activities of lipid-amphiphilic artificial peptide mixtures using lung-irrigated rat models revealed that a mixture (Murosurf SLPD3) of dehydrogenated soy lecithin, fractionated soy lecithin, palmitic acid (PA), and peptide Hel 13-5D3 (40:40:17.5:2.5, by weight) superior pulmonary surfactant activity than a commercially available pulmonary surfactant (beractant, Surfacten®). Experiments using ovalbumin-sensitized model animals revealed that the lipid-amphiphilic artificial peptide mixtures provided significant control over an increase in the pulmonary resistance induced by premature allergy reaction and reduced the number of acidocytes and neutrophils in lung-irrigated solution. The newly developed low-cost pulmonary surfactant system may be used for treatment of a wide variety of respiratory diseases.     

Amino acid and codon usage profiles: adaptive changes in the frequency of amino acids and codons

In the work presented, the changes in codon and amino acid contents have been studied as a function of environmental conditions by comparing pairs of homologs in a group of extremophilic/non-extremophilic genomes. Our results obtained based on such analysis highlights a number of notable observations: (i) the overall preference of amino acid usages in the proteins of a given organism is significantly affected by major environmental factors. The changes in amino acid preferences (amino acid usage profiles) in an extremophile compared to its non-extremophile relative recurs in the organisms of similar extreme habitats. (ii) On the other hand, changes in codon usage preferences in these extremophilic/non-extremophilic pairs, lack such persistency not only in different genome-pairs but also in the individual genes of a specific pair. (iii) We have noted a correlation between cellular function and codon usage profiles of the genes in the studied pairs. (iv) Based on this correlation, we could obtain a decent prediction of cellular functions solely based on codon usage profile data. (v) Comparisons made between two sets of randomly generated genomes suggest that different patterns of codon usage changes in genes of different functional categories result in a partial resistance towards the changes in the concentration of a given amino acid. This buffering capacity might explain the observed differences in codon usage trends in genes of different functions. In the end, we suggest codon usage and amino acid profiles as powerful tools that can be utilized to improve function predictions and genome-environment mappings.     

Bayesian analysis of amino acid substitution models

Models of amino acid substitution present challenges beyond those often faced with the analysis of DNA sequences. The alignments of amino acid sequences are often small, whereas the number of parameters to be estimated is potentially large when compared with the number of free parameters for nucleotide substitution models. Most approaches to the analysis of amino acid alignments have focused on the use of fixed amino acid models in which all of the potentially free parameters are fixed to values estimated from a large number of sequences. Often, these fixed amino acid models are specific to a gene or taxonomic group (e.g. the Mtmam model, which has parameters that are specific to mammalian mitochondrial gene sequences). Although the fixed amino acid models succeed in reducing the number of free parameters to be estimated--indeed, they reduce the number of free parameters from approximately 200 to 0--it is possible that none of the currently available fixed amino acid models is appropriate for a specific alignment. Here, we present four approaches to the analysis of amino acid sequences. First, we explore the use of a general time reversible model of amino acid substitution using a Dirichlet prior probability distribution on the 190 exchangeability parameters. Second, we then explore the behaviour of prior probability distributions that are'centred' on the rates specified by the fixed amino acid model. Third, we consider a mixture of fixed amino acid models. Finally, we consider constraints on the exchangeability parameters as partitions,similar to how nucleotide substitution models are specified, and place a Dirichlet process prior model on all the possible partitioning schemes.