Acyl homoserine lactone changes the abundance of proteins and the levels of organic acids associated with stationary phase in Salmonella Enteritidis.

PubMed

de Almeida, Felipe Alves; Pimentel-Filho, Natan de Jesus; Carrijo, Lanna Clícia; Bento, Cláudia Braga Pereira; Baracat-Pereira, Maria Cristina; Pinto, Uelinton Manoel; de Oliveira, Leandro Licursi; Vanetti, Maria Cristina Dantas

2017-01-01

Quorum sensing (QS) is cell-cell communication mechanism mediated by signaling molecules known as autoinducers (AIs) that lead to differential gene expression. Salmonella is unable to synthesize the AI-1 acyl homoserine lactone (AHL), but is able to recognize AHLs produced by other microorganisms through SdiA protein. Our study aimed to evaluate the influence of AI-1 on the abundance of proteins and the levels of organic acids of Salmonella Enteritidis. The presence of N-dodecyl-homoserine lactone (C12-HSL) did not interfere on the growth or the total amount of extracted proteins of Salmonella. However, the abundance of the proteins PheT, HtpG, PtsI, Adi, TalB, PmgI (or GpmI), Eno, and PykF enhanced while the abundance of the proteins RplB, RplE, RpsB, Tsf, OmpA, OmpC, OmpD, and GapA decreased when Salmonella Enteritidis was anaerobically cultivated in the presence of C12-HSL. Additionally, the bacterium produced less succinic, lactic, and acetic acids in the presence of C12-HSL. However, the concentration of extracellular formic acid reached 20.46 mM after 24 h and was not detected when the growth was in the absence of AI-1. Considering the cultivation period for protein extraction, their abundance, process and function, as well as the levels of organic acids, we observed in cells cultivated in presence of C12-HSL a correlation with what is described in the literature as entry into the stationary phase of growth, mainly related to nitrogen and amino acid starvation and acid stress. Further studies are needed in order to determine the specific role of the differentially abundant proteins and extracellular organic acids secreted by Salmonella in the presence of quorum sensing signaling molecules. Copyright © 2016 Elsevier Ltd. All rights reserved.

Estimation of endogenous protein and amino acid ileal losses in weaned piglets by regression analysis using diets with graded levels of casein

PubMed Central

2013-01-01

Background Many studies have investigated endogenous loss of proteins and amino acids (AAs) at the ileal level in growing pigs. However, only a few studies have researched this subject in piglets. Knowledge regarding AA ileal digestibility in piglets would be helpful during the formulation of diets for weaning piglets, rather than just using coefficients obtained in growing pigs. Therefore, in this study, we sought to estimate endogenous protein and AA ileal losses in piglets. Furthermore, apparent and true ileal digestibility (AID and TID) of protein and AAs from casein were measured. Results The average flow of protein was 20.8 g/kg of dry matter intake (DMI). Basal protein loss, as estimated by regression, was 16.9 g/kg DMI. Glutamic acid, arginine, and aspartic acid (2.2, 1.4, and 1.2 g/kg DMI, respectively) were the AAs for which greater losses were seen. The AID of protein and AAs increased as the protein level in the diet increased. A higher increment in AID was observed between diets with 80 and160 g CP/kg of feed; this finding was mainly attributable to increases in glycine and arginine (46.1% and 18%, respectively). The TID of protein was 97.8, and the TID of AAs varied from 93.9 for histidine to 100.2 for phenylalanine. Conclusions The basal endogenous protein loss in piglets was 16.9 g/kg DMI. Endogenous protein was rich in glutamic acid, aspartic acid, and arginine, which represented 32.7% of endogenous protein loss in weaning piglets. The TID of casein was high and varied from 93.0 for histidine to 100.2 for phenylalanine. PMID:24053636

Plasma Levels of Fatty Acid-Binding Protein 4, Retinol-Binding Protein 4, High-Molecular-Weight Adiponectin, and Cardiovascular Mortality Among Men With Type 2 Diabetes: A 22-Year Prospective Study.

PubMed

Liu, Gang; Ding, Ming; Chiuve, Stephanie E; Rimm, Eric B; Franks, Paul W; Meigs, James B; Hu, Frank B; Sun, Qi

2016-11-01

To examine select adipokines, including fatty acid-binding protein 4, retinol-binding protein 4, and high-molecular-weight (HMW) adiponectin in relation to cardiovascular disease (CVD) mortality among patients with type 2 diabetes mellitus. Plasma levels of fatty acid-binding protein 4, retinol-binding protein 4, and HMW adiponectin were measured in 950 men with type 2 diabetes mellitus in the Health Professionals Follow-up Study. After an average of 22 years of follow-up (1993-2015), 580 deaths occurred, of whom 220 died of CVD. After multivariate adjustment for covariates, higher levels of fatty acid-binding protein 4 were significantly associated with a higher CVD mortality: comparing extreme tertiles, the hazard ratio and 95% confidence interval of CVD mortality was 1.78 (1.22-2.59; P trend=0.001). A positive association was also observed for HMW adiponectin: the hazard ratio (95% confidence interval) was 2.07 (1.42-3.06; P trend=0.0002), comparing extreme tertiles, whereas higher retinol-binding protein 4 levels were nonsignificantly associated with a decreased CVD mortality with an hazard ratio (95% confidence interval) of 0.73 (0.50-1.07; P trend=0.09). A Mendelian randomization analysis suggested that the causal relationships of HMW adiponectin and retinol-binding protein 4 would be directionally opposite to those observed based on the biomarkers, although none of the Mendelian randomization associations achieved statistical significance. These data suggest that higher levels of fatty acid-binding protein 4 and HMW adiponectin are associated with elevated CVD mortality among men with type 2 diabetes mellitus. Biological mechanisms underlying these observations deserve elucidation, but the associations of HMW adiponectin may partially reflect altered adipose tissue functionality among patients with type 2 diabetes mellitus. © 2016 American Heart Association, Inc.

A Jasmonate ZIM-Domain Protein NaJAZd Regulates Floral Jasmonic Acid Levels and Counteracts Flower Abscission in Nicotiana attenuata Plants

PubMed Central

Oh, Youngjoo; Baldwin, Ian T.; Galis, Ivan

2013-01-01

Jasmonic acid is an important regulator of plant growth, development and defense. The jasmonate-ZIM domain (JAZ) proteins are key regulators in jasmonate signaling ubiquitously present in flowering plants but their functional annotation remains largely incomplete. Recently, we identified 12 putative JAZ proteins in native tobacco, Nicotiana attenuata, and initiated systematic functional characterization of these proteins by reverse genetic approaches. In this report, Nicotiana attenuata plants silenced in the expression of NaJAZd (irJAZd) by RNA interference were used to characterize NaJAZd function. Although NaJAZd transcripts were strongly and transiently up-regulated in the rosette leaves by simulated herbivory treatment, we did not observe strong defense-related phenotypes, such as altered herbivore performance or the constitutive accumulation of defense-related secondary metabolites in irJAZd plants compared to wild type plants, both in the glasshouse and the native habitat of Nicotiana attenuata in the Great Basin Desert, Utah, USA. Interestingly, irJAZd plants produced fewer seed capsules than did wild type plants as a result of increased flower abscission in later stages of flower development. The early- and mid-developmental stages of irJAZd flowers had reduced levels of jasmonic acid and jasmonoyl-L-isoleucine, while fully open flowers had normal levels, but these were impaired in NaMYB305 transcript accumulations. Previously, NaMYB305-silenced plants were shown to have strong flower abscission phenotypes and contained lower NECTARIN 1 transcript levels, phenotypes which are copied in irJAZd plants. We propose that the NaJAZd protein is required to counteract flower abscission, possibly by regulating jasmonic acid and jasmonoyl-L-isoleucine levels and/or expression of NaMYB305 gene in Nicotiana attenuata flowers. This novel insight into the function of JAZ proteins in flower and seed development highlights the diversity of functions played by jasmonates

Cinnamic acid and its derivatives inhibit fructose-mediated protein glycation.

PubMed

Adisakwattana, Sirichai; Sompong, Weerachat; Meeprom, Aramsri; Ngamukote, Sathaporn; Yibchok-Anun, Sirintorn

2012-01-01

Cinnamic acid and its derivatives have shown a variety of pharmacologic properties. However, little is known about the antiglycation properties of cinnamic acid and its derivatives. The present study sought to characterize the protein glycation inhibitory activity of cinnamic acid and its derivatives in a bovine serum albumin (BSA)/fructose system. The results demonstrated that cinnamic acid and its derivatives significantly inhibited the formation of advanced glycation end products (AGEs) by approximately 11.96-63.36% at a concentration of 1 mM. The strongest inhibitory activity against the formation of AGEs was shown by cinnamic acid. Furthermore, cinnamic acid and its derivatives reduced the level of fructosamine, the formation of N(ɛ)-(carboxymethyl) lysine (CML), and the level of amyloid cross β-structure. Cinnamic acid and its derivatives also prevented oxidative protein damages, including effects on protein carbonyl formation and thiol oxidation of BSA. Our findings may lead to the possibility of using cinnamic acid and its derivatives for preventing AGE-mediated diabetic complications.

Cinnamic Acid and Its Derivatives Inhibit Fructose-Mediated Protein Glycation

PubMed Central

Adisakwattana, Sirichai; Sompong, Weerachat; Meeprom, Aramsri; Ngamukote, Sathaporn; Yibchok-anun, Sirintorn

2012-01-01

Cinnamic acid and its derivatives have shown a variety of pharmacologic properties. However, little is known about the antiglycation properties of cinnamic acid and its derivatives. The present study sought to characterize the protein glycation inhibitory activity of cinnamic acid and its derivatives in a bovine serum albumin (BSA)/fructose system. The results demonstrated that cinnamic acid and its derivatives significantly inhibited the formation of advanced glycation end products (AGEs) by approximately 11.96–63.36% at a concentration of 1 mM. The strongest inhibitory activity against the formation of AGEs was shown by cinnamic acid. Furthermore, cinnamic acid and its derivatives reduced the level of fructosamine, the formation of Nɛ-(carboxymethyl) lysine (CML), and the level of amyloid cross β-structure. Cinnamic acid and its derivatives also prevented oxidative protein damages, including effects on protein carbonyl formation and thiol oxidation of BSA. Our findings may lead to the possibility of using cinnamic acid and its derivatives for preventing AGE-mediated diabetic complications. PMID:22408423

Impact of antinutritional factors in food proteins on the digestibility of protein and the bioavailability of amino acids and on protein quality.

PubMed

Sarwar Gilani, G; Wu Xiao, Chao; Cockell, Kevin A

2012-08-01

Dietary antinutritional factors have been reported to adversely affect the digestibility of protein, bioavailability of amino acids and protein quality of foods. Published data on these negative effects of major dietary antinutritional factors are summarized in this manuscript. Digestibility and the quality of mixed diets in developing countries are considerably lower than of those in developed regions. For example, the digestibility of protein in traditional diets from developing countries such as India, Guatemala and Brazil is considerably lower compared to that of protein in typical North American diets (54-78 versus 88-94 %). Poor digestibility of protein in the diets of developing countries, which are based on less refined cereals and grain legumes as major sources of protein, is due to the presence of less digestible protein fractions, high levels of insoluble fibre, and/or high concentrations of antinutritional factors present endogenously or formed during processing. Examples of naturally occurring antinutritional factors include glucosinolates in mustard and canola protein products, trypsin inhibitors and haemagglutinins in legumes, tannins in legumes and cereals, gossypol in cottonseed protein products, and uricogenic nucleobases in yeast protein products. Heat/alkaline treatments of protein products may yield Maillard reaction compounds, oxidized forms of sulphur amino acids, D-amino acids and lysinoalanine (LAL, an unnatural nephrotoxic amino acid derivative). Among common food and feed protein products, soyabeans are the most concentrated source of trypsin inhibitors. The presence of high levels of dietary trypsin inhibitors from soyabeans, kidney beans or other grain legumes have been reported to cause substantial reductions in protein and amino acid digestibility (up to 50 %) and protein quality (up to 100 %) in rats and/or pigs. Similarly, the presence of high levels of tannins in sorghum and other cereals, fababean and other grain legumes can cause

Proteomic Investigation of Protein Profile Changes and Amino Acid Residue Level Modification in Cooked Lamb Meat: The Effect of Boiling.

PubMed

Yu, Tzer-Yang; Morton, James D; Clerens, Stefan; Dyer, Jolon M

2015-10-21

Hydrothermal treatment (heating in water) is a common method of general food processing and preparation. For red-meat-based foods, boiling is common; however, how the molecular level effects of this treatment correlate to the overall food properties is not yet well-understood. The effects of differing boiling times on lamb meat and the resultant cooking water were here examined through proteomic evaluation. The longer boiling time was found to result in increased protein aggregation involving particularly proteins such as glyceraldehyde-3-phosphate dehydrogenase, as well as truncation in proteins such as in α-actinin-2. Heat-induced protein backbone cleavage was observed adjacent to aspartic acid and asparagine residues. Side-chain modifications of amino acid residues resulting from the heating, including oxidation of phenylalanine and formation of carboxyethyllysine, were characterized in the cooked samples. Actin and myoglobin bands from the cooked meat per se remained visible on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, even after significant cooking time. These proteins were also found to be the major source of observed heat-induced modifications. This study provides new insights into molecular-level modifications occurring in lamb meat proteins during boiling and a protein chemistry basis for better understanding the effect of this common treatment on the nutritional and functional properties of red-meat-based foods.

Advances in protein-amino acid nutrition of poultry.

PubMed

Baker, David H

2009-05-01

The ideal protein concept has allowed progress in defining requirements as well as the limiting order of amino acids in corn, soybean meal, and a corn-soybean meal mixture for growth of young chicks. Recent evidence suggests that glycine (or serine) is a key limiting amino acid in reduced protein [23% crude protein (CP) reduced to 16% CP] corn-soybean meal diets for broiler chicks. Research with sulfur amino acids has revealed that small excesses of cysteine are growth depressing in chicks fed methionine-deficient diets. Moreover, high ratios of cysteine:methionine impair utilization of the hydroxy analog of methionine, but not of methionine itself. A high level of dietary L: -cysteine (2.5% or higher) is lethal for young chicks, but a similar level of DL: -methionine, L: -cystine or N-acetyl-L: -cysteine causes no mortality. A supplemental dietary level of 3.0% L: -cysteine (7x requirement) causes acute metabolic acidosis that is characterized by a striking increase in plasma sulfate and decrease in plasma bicarbonate. S-Methylmethionine, an analog of S-adenosylmethionine, has been shown to have choline-sparing activity, but it only spares methionine when diets are deficient in choline and(or) betaine. Creatine, or its precursor guanidinoacetic acid, can spare dietary arginine in chicks.

Lower glutamic acid decarboxylase 65-kDa isoform messenger RNA and protein levels in the prefrontal cortex in schizoaffective disorder but not schizophrenia.

PubMed

Glausier, Jill R; Kimoto, Sohei; Fish, Kenneth N; Lewis, David A

2015-01-15

Altered gamma-aminobutyric acid (GABA) signaling in the prefrontal cortex (PFC) has been associated with cognitive dysfunction in patients with schizophrenia and schizoaffective disorder. Levels of the GABA-synthesizing enzyme glutamic acid decarboxylase 67-kDa isoform (GAD67) in the PFC have been consistently reported to be lower in patients with these disorders, but the status of the second GABA-synthesizing enzyme, glutamic acid decarboxylase 65-kDa isoform (GAD65), remains unclear. GAD65 messenger RNA (mRNA) levels were quantified in PFC area 9 by quantitative polymerase chain reaction from 62 subjects with schizophrenia or schizoaffective disorder and 62 matched healthy comparison subjects. In a subset of subject pairs, GAD65 relative protein levels were quantified by confocal immunofluorescence microscopy. Mean GAD65 mRNA levels were 13.6% lower in subjects with schizoaffective disorder but did not differ in subjects with schizophrenia relative to their matched healthy comparison subjects. In the subjects with schizoaffective disorder, mean GAD65 protein levels were 19.4% lower and were correlated with GAD65 mRNA levels. Lower GAD65 mRNA and protein levels within subjects with schizoaffective disorder were not attributable to factors commonly comorbid with the diagnosis. In concert with previous studies, these findings suggest that schizoaffective disorder is associated with lower levels of both GAD65 and GAD67 mRNA and protein in the PFC, whereas subjects with schizophrenia have lower mean levels of only GAD67 mRNA and protein. Because cognitive function is generally better preserved in patients with schizoaffective disorder relative to patients with schizophrenia, these findings may support an interpretation that GAD65 downregulation provides a homeostatic response complementary to GAD67 downregulation that serves to reduce inhibition in the face of lower PFC network activity. Copyright © 2015 Society of Biological Psychiatry. Published by Elsevier Inc

Human Protein and Amino Acid Requirements.

PubMed

Hoffer, L John

2016-05-01

Human protein and amino acid nutrition encompasses a wide, complex, frequently misunderstood, and often contentious area of clinical research and practice. This tutorial explains the basic biochemical and physiologic principles that underlie our current understanding of protein and amino acid nutrition. The following topics are discussed: (1) the identity, measurement, and essentiality of nutritional proteins; (2) the definition and determination of minimum requirements; (3) nutrition adaptation; (4) obligatory nitrogen excretion and the minimum protein requirement; (5) minimum versus optimum protein intakes; (6) metabolic responses to surfeit and deficient protein intakes; (7) body composition and protein requirements; (8) labile protein; (9) N balance; (10) the principles of protein and amino acid turnover, including an analysis of the controversial indicator amino acid oxidation technique; (11) general guidelines for evaluating protein turnover articles; (12) amino acid turnover versus clearance; (13) the protein content of hydrated amino acid solutions; (14) protein requirements in special situations, including protein-catabolic critical illness; (15) amino acid supplements and additives, including monosodium glutamate and glutamine; and (16) a perspective on the future of protein and amino acid nutrition research. In addition to providing practical information, this tutorial aims to demonstrate the importance of rigorous physiologic reasoning, stimulate intellectual curiosity, and encourage fresh ideas in this dynamic area of human nutrition. In general, references are provided only for topics that are not well covered in modern textbooks. © 2016 American Society for Parenteral and Enteral Nutrition.

Exploiting Amino Acid Composition for Predicting Protein-Protein Interactions

PubMed Central

Roy, Sushmita; Martinez, Diego; Platero, Harriett; Lane, Terran; Werner-Washburne, Margaret

2009-01-01

Background Computational prediction of protein interactions typically use protein domains as classifier features because they capture conserved information of interaction surfaces. However, approaches relying on domains as features cannot be applied to proteins without any domain information. In this paper, we explore the contribution of pure amino acid composition (AAC) for protein interaction prediction. This simple feature, which is based on normalized counts of single or pairs of amino acids, is applicable to proteins from any sequenced organism and can be used to compensate for the lack of domain information. Results AAC performed at par with protein interaction prediction based on domains on three yeast protein interaction datasets. Similar behavior was obtained using different classifiers, indicating that our results are a function of features and not of classifiers. In addition to yeast datasets, AAC performed comparably on worm and fly datasets. Prediction of interactions for the entire yeast proteome identified a large number of novel interactions, the majority of which co-localized or participated in the same processes. Our high confidence interaction network included both well-studied and uncharacterized proteins. Proteins with known function were involved in actin assembly and cell budding. Uncharacterized proteins interacted with proteins involved in reproduction and cell budding, thus providing putative biological roles for the uncharacterized proteins. Conclusion AAC is a simple, yet powerful feature for predicting protein interactions, and can be used alone or in conjunction with protein domains to predict new and validate existing interactions. More importantly, AAC alone performs at par with existing, but more complex, features indicating the presence of sequence-level information that is predictive of interaction, but which is not necessarily restricted to domains. PMID:19936254

[Chromosomal proteins: histones and acid proteins].

PubMed

Salvini, M; Gabrielli, F

1976-01-01

Experimental data about the chemistry and the biology of chromosomal proteins are reviewed. Paragraphs include: aminoacid sequential data and post-translational covalent modications of histones, histone chemical differences in different tissues of the same species and in homologous organs of different species, histone synthesis subcellular localization and its association with DNA synthesis, histone synthesis transcriptional and translational control, histone synthesis during meiosis, oogenesis and early embryogenesis. The possible role of histones as controllers of gene expression is discussed and a model of primary structure of chromatine is proposed. The "acidic proteins" data concern the high tissue eterogenity of these proteins and their role in the steroid-hormon-controlled gene expression. The possible role of acidic proteins as general controllers of gene expression in eucariotic cells is discussed.

A Dominant Conformational Role for Amino Acid Diversity in Minimalist Protein-Protein Interfaces

PubMed Central

Gilbreth, Ryan N.; Esaki, Kaori; Koide, Akiko; Sidhu, Sachdev S.; Koide, Shohei

2008-01-01

Recent studies have shown that highly simplified interaction surfaces consisting of combinations of just two amino acids, Tyr and Ser, exhibit high affinity and specificity. The high functional levels of such minimalist interfaces might thus indicate small contributions of greater amino acid diversity seen in natural interfaces. Toward addressing this issue, we have produced a pair of binding proteins built on the fibronectin type III scaffold, termed “monobodies”. One monobody contains the Tyr/Ser binary-code interface (termed YS) and the other contains an expanded amino acid diversity interface (YSX), but both bind to an identical target, maltose binding protein (MBP). The YSX monobody bound with higher affinity, a slower off rate and a more favorable enthalpic contribution than the YS monobody. High-resolution x-ray crystal structures revealed that both proteins bound to an essentially identical epitope, providing a unique opportunity to directly investigate the role of amino acid diversity in a protein interaction interface. Surprisingly, Tyr still dominates the YSX paratope and the additional amino acid types are primarily used to conformationally optimize contacts made by tyrosines. Scanning mutagenesis showed that while all contacting Tyr side-chains are essential in the YS monobody, the YSX interface was more tolerant to mutations. These results suggest that the conformational, not chemical, diversity of additional types of amino acids provided higher functionality and evolutionary robustness, supporting the dominant role of Tyr and the importance of conformational diversity in forming protein interaction interfaces. PMID:18602117

Detection of non-protein amino acids in the presence of protein amino acids. II.

NASA Technical Reports Server (NTRS)

Shapshak, P.; Okaji, M.

1972-01-01

Studies conducted with the JEOL 5AH amino acid analyzer are described. This instrument makes possible the programming of the chromatographic process. Data are presented showing the separations of seventeen non-protein amino acids in the presence of eighteen protein amino acids. It is pointed out that distinct separations could be obtained in the case of a number of chemically similar compounds, such as ornithine and lysine, N-amidino alanine and arginine, and iminodiacetic acid and S-carboxymethyl cysteine and aspartic acid.

Phthalic acid chemical probes synthesized for protein-protein interaction analysis.

PubMed

Liang, Shih-Shin; Liao, Wei-Ting; Kuo, Chao-Jen; Chou, Chi-Hsien; Wu, Chin-Jen; Wang, Hui-Min

2013-06-24

Plasticizers are additives that are used to increase the flexibility of plastic during manufacturing. However, in injection molding processes, plasticizers cannot be generated with monomers because they can peel off from the plastics into the surrounding environment, water, or food, or become attached to skin. Among the various plasticizers that are used, 1,2-benzenedicarboxylic acid (phthalic acid) is a typical precursor to generate phthalates. In addition, phthalic acid is a metabolite of diethylhexyl phthalate (DEHP). According to Gene_Ontology gene/protein database, phthalates can cause genital diseases, cardiotoxicity, hepatotoxicity, nephrotoxicity, etc. In this study, a silanized linker (3-aminopropyl triethoxyslane, APTES) was deposited on silicon dioxides (SiO2) particles and phthalate chemical probes were manufactured from phthalic acid and APTES-SiO2. These probes could be used for detecting proteins that targeted phthalic acid and for protein-protein interactions. The phthalic acid chemical probes we produced were incubated with epithelioid cell lysates of normal rat kidney (NRK-52E cells) to detect the interactions between phthalic acid and NRK-52E extracted proteins. These chemical probes interacted with a number of chaperones such as protein disulfide-isomerase A6, heat shock proteins, and Serpin H1. Ingenuity Pathways Analysis (IPA) software showed that these chemical probes were a practical technique for protein-protein interaction analysis.

Effects of antinutritional factors on protein digestibility and amino acid availability in foods.

PubMed

Gilani, G Sarwar; Cockell, Kevin A; Sepehr, Estatira

2005-01-01

Digestibility of protein in traditional diets from developing countries such as India, Guatemala, and Brazil is considerably lower compared to that of protein in typical North American diets (54-78 versus 88-94%). The presence of less digestible protein fractions, high levels of insoluble fiber, and high concentrations of antinutritional factors in the diets of developing countries, which are based on less refined cereals and grain legumes as major sources of protein, are responsible for poor digestibility of protein. The effects of the presence of some of the important antinutritional factors on protein and amino digestibilities of food and feed products are reviewed in this chapter. Food and feed products may contain a number of antinutritional factors that may adversely affect protein digestibility and amino acid availability. Antinutritional factors may occur naturally, such as glucosinolates in mustard and rapeseed protein products, trypsin inhibitors and hemagglutinins in legumes, tannins in legumes and cereals, phytates in cereals and oilseeds, and gossypol in cottonseed protein products. Antinutritional factors may also be formed during heat/alkaline processing of protein products, yielding Maillard compounds, oxidized forms of sulfur amino acids, D-amino acids, and lysinoalanine (LAL, an unnatural amino acid derivative). The presence of high levels of dietary trypsin inhibitors from soybeans, kidney beans, or other grain legumes can cause substantial reductions in protein and amino acid digestibilities (up to 50%) in rats and pigs. Similarly, the presence of high levels of tannins in cereals, such as sorghum, and grain legumes, such as fababean (Vicia faba L.), can result in significantly reduced protein and amino acid digestibilities (up to 23%) in rats, poultry, and pigs. Studies involving phytase supplementation of production rations for swine or poultry have provided indirect evidence that normally encountered levels of phytates in cereals and legumes

Interactive Effects of Indigestible Carbohydrates, Protein Type, and Protein Level on Biomarkers of Large Intestine Health in Rats

PubMed Central

Taciak, Marcin; Barszcz, Marcin; Tuśnio, Anna; Pastuszewska, Barbara

2015-01-01

The effects of indigestible carbohydrates, protein type, and protein level on large intestine health were examined in rats. For 21 days, 12 groups of six 12-week-old male Wistar rats were fed diets with casein (CAS), or potato protein concentrate (PPC), providing 14% (lower protein level; LP), or 20% (higher protein level; HP) protein, and containing cellulose, resistant potato starch, or pectin. Fermentation end-products, pH, and β-glucuronidase levels in cecal digesta, and ammonia levels in colonic digesta were determined. Cecal digesta, tissue weights, cecal and colon morphology, and colonocyte DNA damage were also analyzed. Digesta pH was lower, whereas relative mass of cecal tissue and digesta were higher in rats fed pectin diets than in those fed cellulose. Cecal parameters were greater in rats fed PPC and HP diets than in those fed CAS and LP diets, respectively. Short-chain fatty acid (SCFA) concentrations were unaffected by protein or carbohydrate type. Total SCFA, acetic acid, and propionic acid concentrations were greater in rats fed LP diets than in those fed HP. Cecal pool of isobutyric and isovaleric acids was greater in rats fed PPC than in those fed CAS diets. PPC diets decreased phenol concentration and increased ammonia concentration in cecal and colonic digesta, respectively. Cecal crypt depth was greater in rats fed PPC and HP diets, and was unaffected by carbohydrates; whereas colonic crypt depth was greater in rats fed cellulose. Myenteron thickness in the cecum was unaffected by nutrition, but was greater in the colon of rats fed cellulose. Colonocyte DNA damage was greater in rats fed LP diets than in those fed HP diets, and was unaffected by carbohydrate or protein type. It was found that nutritional factors decreasing cecal digesta weight contribute to greater phenol production, increased DNA damage, and reduced ammonia concentration in the colon. PMID:26536028

Fatty acid transfer between multilamellar liposomes and fatty acid-binding proteins.

PubMed

Brecher, P; Saouaf, R; Sugarman, J M; Eisenberg, D; LaRosa, K

1984-11-10

A simple experimental system was developed for studying the movement of long-chain fatty acids between multilamellar liposomes and soluble proteins capable of binding fatty acids. Oleic acid was incorporated into multilamellar liposomes containing cholesterol and egg yolk lecithin and incubated with albumin or hepatic fatty acid-binding protein. It was found that the fatty acid transferred from the liposomes to either protein rapidly and selectively under conditions where phospholipid and cholesterol transfer did not occur. More than 50% of the fatty acid contained within liposomes could become protein bound, suggesting that the fatty acid moved readily between and across phospholipid bilayers. Transfer was reduced at low pH, and this reduction appeared to result from decreased dissociation of the protonated fatty acid from the bilayer. Liposomes made with dimyristoyl or dipalmitoyl lecithin and containing 1 mol per cent palmitic acid were used to show the effect of temperature on fatty acid transfer. Transfer to either protein did not occur at temperatures where the liposomes were in a gel state but occurred rapidly at temperatures at or above the transition temperatures of the phospholipid used.

Combined effects of dietary arginine, leucine and protein levels on fatty acid composition and gene expression in the muscle and subcutaneous adipose tissue of crossbred pigs.

PubMed

Madeira, Marta S; Pires, Virgínia M R; Alfaia, Cristina M; Luxton, Richard; Doran, Olena; Bessa, Rui J B; Prates, José A M

2014-05-01

The cumulative effects of dietary arginine, leucine and protein levels on fat content, fatty acid composition and mRNA levels of genes controlling lipid metabolism in pig longissimus lumborum muscle and subcutaneous adipose tissue (SAT) were investigated. The experiment was performed on fifty-four intact male pigs (Duroc × Pietrain × Large White × Landrace crossbred), with a live weight ranging from 59 to 92 kg. The pigs were randomly assigned to one of six experimental treatments (n 9). The treatments followed a 2 × 3 factorial arrangement, with two levels of arginine supplementation (0 v. 1 %) and three levels of a basal diet (normal protein diet, NPD; reduced protein diet, RPD; reduced protein diet to achieve 2 % of leucine, RPDL). The results showed that dietary arginine supplementation did not affect the intramuscular fat (IMF) content and back fat thickness, but increased the total fat in SAT. This effect was associated with an increase in fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD) mRNA levels in SAT, which suggests that arginine might be involved in the differential regulation of some key lipogenic genes in pig muscle and SAT. The increase in IMF content under the RPD, with or without leucine supplementation, was accompanied by increased FASN and SCD mRNA levels. Arginine supplementation did not influence the percentage of main fatty acids, while the RPD had a significant effect on fatty acid composition in both tissues. Leucine supplementation of RPD did not change IMF, total fat of SAT and back fat thickness, but increased 16 : 0 and 18 : 1cis-9 and decreased 18 : 2n-6 in muscle.

Dietary fatty acids and membrane protein function.

PubMed

Murphy, M G

1990-02-01

In recent years, there has been growing public awareness of the potential health benefits of dietary fatty acids, and of the distinction between the effects of the omega6 and omega3 polyunsaturated fatty acids that are concentrated in vegetable and fish oils, respectively. A part of the biologic effectiveness of the two families of polyunsaturated fatty acids resides in their relative roles as precursors of the eicosanoids. However, we are also beginning to appreciate that as the major components of the hydrophobic core of the membrane bilayer, they can interact with and directly influence the functioning of select integral membrane proteins. Among the most important of these are the enzymes, receptors, and ion channels that are situated in the plasma membrane of the cell, since they carry out the communication and homeostatic processes that are necessary for normal cell function. This review examines current information regarding the effects of diet-induced changes in plasma membrane fatty acid composition on several specific enzymes (adenylate cyclase, 5'-nucleotidase, Na(+)/K(+)-ATPase) and cell-surface receptors (opiate, adrenergic, insulin). Dietary manipulation studies have demonstrated a sensitivity of each to a fatty acid environment that is variably dependent on the nature of the fatty acid(s) and/or source of the membrane. The molecular mechanisms appear to involve fatty acid-dependent effects on protein conformation, on the "fluidity" and/or thickness of the membrane, or on protein synthesis. Together, the results of these studies reinforce the concept that dietary fats have the potential to regulate physiologic function and to further our understanding of how this occurs at a membrane level.

Protein and amino acid nutrition

USDA-ARS?s Scientific Manuscript database

Dairy cow protein and amino acid nutrition have a significant role in sustainable dairying. Protein, amino acids, and nitrogen are inextricably linked through effects in the rumen, metabolism of the cow, and environmental nutrient management. Feeding systems have been making progress toward emphasiz...

Decrease in level of APG-2, a member of the heat shock protein 110 family, in murine brain following systemic administration of kainic acid.

PubMed

Ogita, K; Takagi, R; Oyama, N; Okuda, H; Ito, F; Okui, M; Shimizu, N; Yoneda, Y

2001-09-01

APG-2 belongs to the heat shock protein 110 family. Although kainic acid (KA)-induced seizures are known to elicit expression of inducible heat shock protein 70 (HSP70) in the brain, no investigation has been carried out on the APG-2 level after excitatory amino acid-induced seizures. By means of an immunoblot assay, we determined the levels of HSP70 and APG-2 in discrete brain structures of mice after a single intraperitoneal injection of KA or N-methyl-D-aspartic acid (NMDA). APG-2 level was significantly decreased in frontal cortex, hippocampus, and striatum three days after the administration of KA, while HSP70 level was increased in these regions following the administration. In any of these regions, APG-2 levels were returned to the control levels 10 days after the administration. However, no significant changes were observed in levels of both HSP70 and APG-2 in hypothalamus, midbrain, medulla-pons, and cerebellum of the mice. By contrast, NMDA administration did not significantly affect both levels in any of the regions examined. These findings indicate that the transient decrease in APG-2 expression is one of the intracellular events elicited by signals peculiar to KA, but not by those peculiar to NMDA, in telencephalon of murine brain.

Overexpression of heart-type fatty acid binding protein enhances fatty acid-induced podocyte injury.

PubMed

Gao, Qing; Sarkar, Alhossain; Chen, Yizhi; Xu, Bo; Zhu, Xiaojuan; Yuan, Yang; Guan, Tianjun

2018-02-01

Deregulated lipid metabolism is a characteristic of metabolic diseases including type 2 diabetes and obesity, and likely contributes to podocyte injury and end-stage kidney disease. Heart-type fatty acid binding protein (H-FABP) was reported to be associated with lipid metabolism. The present study investigated whether H-FABP contributes to podocyte homeostasis. Podocytes were transfected by lentiviral vector to construct a cell line which stably overexpressed H-FABP. Small interfering RNA capable of effectively silencing H-FABP was introduced into podocytes to construct a cell line with H-FABP knockdown. Certain groups were treated with palmitic acid (PA) and the fat metabolism, as well as inflammatory and oxidative stress markers were measured. PA accelerated lipid metabolism derangement, inflammatory reaction and oxidative stress in podocytes. Overexpression of H-FABP enhanced the PA-induced disequilibrium in podocytes. The mRNA and protein expression levels of acyl-coenzyme A oxidase 3 and monocyte chemotactic protein 1, and the protein expression levels of 8-hydroxy-2'-deoxyguanosine and 4-hydroxynonenal were upregulated in the H-FABP overexpression group, while the mRNA and protein expression of peroxisome proliferator activated receptor α was downregulated. Knockdown of H-FABP inhibited the PA-induced injury and lipid metabolism derangement, as well as the inflammatory reaction and oxidative stress in podocytes. These results indicated that overexpression of H-FABP enhances fatty acid-induced podocyte injury, while H-FABP inhibition may represent a potential therapeutic strategy for the prevention of lipid metabolism-associated podocyte injury.

Carbonyl-based blue autofluorescence of proteins and amino acids

PubMed Central

Niyangoda, Chamani; Miti, Tatiana; Breydo, Leonid; Uversky, Vladimir

2017-01-01

Intrinsic protein fluorescence is inextricably linked to the near-UV autofluorescence of aromatic amino acids. Here we show that a novel deep-blue autofluorescence (dbAF), previously thought to emerge as a result of protein aggregation, is present at the level of monomeric proteins and even poly- and single amino acids. Just as its aggregation-related counterpart, this autofluorescence does not depend on aromatic residues, can be excited at the long wavelength edge of the UV and emits in the deep blue. Differences in dbAF excitation and emission peaks and intensities from proteins and single amino acids upon changes in solution conditions suggest dbAF’s sensitivity to both the chemical identity and solution environment of amino acids. Autofluorescence comparable to dbAF is emitted by carbonyl-containing organic solvents, but not those lacking the carbonyl group. This implicates the carbonyl double bonds as the likely source for the autofluorescence in all these compounds. Using beta-lactoglobulin and proline, we have measured the molar extinction coefficients and quantum yields for dbAF in the monomeric state. To establish its potential utility in monitoring protein biophysics, we show that dbAF emission undergoes a red-shift comparable in magnitude to tryptophan upon thermal denaturation of lysozyme, and that it is sensitive to quenching by acrylamide. Carbonyl dbAF therefore provides a previously neglected intrinsic optical probe for investigating the structure and dynamics of amino acids, proteins and, by extension, DNA and RNA. PMID:28542206

Structural and functional interaction of fatty acids with human liver fatty acid-binding protein (L-FABP) T94A variant.

PubMed

Huang, Huan; McIntosh, Avery L; Martin, Gregory G; Landrock, Kerstin K; Landrock, Danilo; Gupta, Shipra; Atshaves, Barbara P; Kier, Ann B; Schroeder, Friedhelm

2014-05-01

The human liver fatty acid-binding protein (L-FABP) T94A variant, the most common in the FABP family, has been associated with elevated liver triglyceride levels. How this amino acid substitution elicits these effects is not known. This issue was addressed using human recombinant wild-type (WT) and T94A variant L-FABP proteins as well as cultured primary human hepatocytes expressing the respective proteins (genotyped as TT, TC and CC). The T94A substitution did not alter or only slightly altered L-FABP binding affinities for saturated, monounsaturated or polyunsaturated long chain fatty acids, nor did it change the affinity for intermediates of triglyceride synthesis. Nevertheless, the T94A substitution markedly altered the secondary structural response of L-FABP induced by binding long chain fatty acids or intermediates of triglyceride synthesis. Finally, the T94A substitution markedly decreased the levels of induction of peroxisome proliferator-activated receptor α-regulated proteins such as L-FABP, fatty acid transport protein 5 and peroxisome proliferator-activated receptor α itself meditated by the polyunsaturated fatty acids eicosapentaenoic acid and docosahexaenoic acid in cultured primary human hepatocytes. Thus, although the T94A substitution did not alter the affinity of human L-FABP for long chain fatty acids, it significantly altered human L-FABP structure and stability, as well as the conformational and functional response to these ligands. © 2014 FEBS.

An amino acid depleted cell-free protein synthesis system for the incorporation of non-canonical amino acid analogs into proteins.

PubMed

Singh-Blom, Amrita; Hughes, Randall A; Ellington, Andrew D

2014-05-20

Residue-specific incorporation of non-canonical amino acids into proteins is usually performed in vivo using amino acid auxotrophic strains and replacing the natural amino acid with an unnatural amino acid analog. Herein, we present an efficient amino acid depleted cell-free protein synthesis system that can be used to study residue-specific replacement of a natural amino acid by an unnatural amino acid analog. This system combines a simple methodology and high protein expression titers with a high-efficiency analog substitution into a target protein. To demonstrate the productivity and efficacy of a cell-free synthesis system for residue-specific incorporation of unnatural amino acids in vitro, we use this system to show that 5-fluorotryptophan and 6-fluorotryptophan substituted streptavidin retain the ability to bind biotin despite protein-wide replacement of a natural amino acid for the amino acid analog. We envisage this amino acid depleted cell-free synthesis system being an economical and convenient format for the high-throughput screening of a myriad of amino acid analogs with a variety of protein targets for the study and functional characterization of proteins substituted with unnatural amino acids when compared to the currently employed in vivo methodologies. Copyright © 2014 Elsevier B.V. All rights reserved.

Relationships among the serum omega fatty acid levels, serum C-reactive protein levels and arterial stiffness/wave reflection in Japanese men.

PubMed

Tomiyama, Hirofumi; Matsumoto, Chisa; Odaira, Mari; Yamada, Jiko; Yoshida, Masanobu; Shiina, Kazuki; Nagata, Mikio; Yamashina, Akira

2011-08-01

We examined the relationship among the serum omega-3 and omega-6 fatty acid (O3FA and O6FA) levels, serum C-reactive protein (CRP) levels, and arterial stiffness/wave reflection (AS/WR) in healthy Japanese men. In 2206 Japanese healthy men, parameters related to the AS/WR (i.e., brachial-ankle pulse wave velocity and radial arterial pulse wave analysis) were measured. No significant inverse relationships were observed between the serum O3FA levels and the AS/WR-related parameters. Adjusted values of the AS/WR-related parameters and serum CRP levels were higher in the subjects with serum O6FA levels in the highest tertile than in those with serum O6FA levels in the lowest tertile. In healthy Japanese men with known high dietary intakes of O3FAs, the serum O3FA levels may not reflect the pathophysiological abnormalities related to AS/WR. Increased serum O6FA levels appeared to be independently associated with the unfavorable conditions related to AS/WR and inflammation. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

High Protein Intake Does Not Prevent Low Plasma Levels of Conditionally Essential Amino Acids in Very Preterm Infants Receiving Parenteral Nutrition.

PubMed

Morgan, Colin; Burgess, Laura

2017-03-01

We have shown that increasing protein intake using a standardized, concentrated, added macronutrients parenteral (SCAMP) nutrition regimen improves head growth in very preterm infants (VPIs) compared with a control parenteral nutrition (PN) regimen. VPIs are at risk of conditionally essential amino acid (CEAA) deficiencies because of current neonatal PN amino acid (AA) formulations. We hypothesized that the SCAMP regimen would prevent low plasma levels of CEAAs. To compare the plasma AA profiles at approximately day 9 of life in VPIs receiving SCAMP vs a control PN regimen. VPIs (<29 weeks' gestation) were randomized to receive SCAMP (30% more PN AA) or a control regimen. Data were collected to measure parenteral and enteral protein, energy, and individual AA intake and the first plasma AA profile. Plasma profiles of the 20 individual protogenic AA levels were measured using ion exchange chromatography. Plasma AA profiles were obtained at median (interquartile range [IQR]) age of 9 (8-10) days in both SCAMP (n = 59) and control (n = 67) groups after randomizing 150 VPIs. Median (IQR) plasma levels of individual essential AAs were higher than the reference population mean (RPM) in both groups, especially for threonine. SCAMP infants had higher plasma levels of essential AAs than did the controls. Median (IQR) plasma levels of glutamine, arginine, and cysteine (CEAAs) were lower than the RPM in both groups. Plasma AA levels in PN-dependent VPIs indicate there is an imbalance in essential and CEAA provision in neonatal PN AA formulations that is not improved by increasing protein intake.

Statistical potential-based amino acid similarity matrices for aligning distantly related protein sequences.

PubMed

Tan, Yen Hock; Huang, He; Kihara, Daisuke

2006-08-15

Aligning distantly related protein sequences is a long-standing problem in bioinformatics, and a key for successful protein structure prediction. Its importance is increasing recently in the context of structural genomics projects because more and more experimentally solved structures are available as templates for protein structure modeling. Toward this end, recent structure prediction methods employ profile-profile alignments, and various ways of aligning two profiles have been developed. More fundamentally, a better amino acid similarity matrix can improve a profile itself; thereby resulting in more accurate profile-profile alignments. Here we have developed novel amino acid similarity matrices from knowledge-based amino acid contact potentials. Contact potentials are used because the contact propensity to the other amino acids would be one of the most conserved features of each position of a protein structure. The derived amino acid similarity matrices are tested on benchmark alignments at three different levels, namely, the family, the superfamily, and the fold level. Compared to BLOSUM45 and the other existing matrices, the contact potential-based matrices perform comparably in the family level alignments, but clearly outperform in the fold level alignments. The contact potential-based matrices perform even better when suboptimal alignments are considered. Comparing the matrices themselves with each other revealed that the contact potential-based matrices are very different from BLOSUM45 and the other matrices, indicating that they are located in a different basin in the amino acid similarity matrix space.

The peritumoural adipose tissue microenvironment and cancer. The roles of fatty acid binding protein 4 and fatty acid binding protein 5.

PubMed

Guaita-Esteruelas, S; Gumà, J; Masana, L; Borràs, J

2018-02-15

The adipose tissue microenvironment plays a key role in tumour initiation and progression because it provides fatty acids and adipokines to tumour cells. The fatty acid-binding protein (FABP) family is a group of small proteins that act as intracellular fatty acid transporters. Adipose-derived FABPs include FABP4 and FABP5. Both have an important role in lipid-related metabolic processes and overexpressed in many cancers, such as breast, prostate, colorectal and ovarian. Moreover, their expression in peritumoural adipose tissue is deregulated, and their circulating levels are upregulated in some tumours. In this review, we discuss the role of the peritumoural adipose tissue and the related adipokines FABP4 and FABP5 in cancer initiation and progression and the possible pathways implicated in these processes. Copyright © 2017 Elsevier B.V. All rights reserved.

Signaling Pathways Related to Protein Synthesis and Amino Acid Concentration in Pig Skeletal Muscles Depend on the Dietary Protein Level, Genotype and Developmental Stages

PubMed Central

Liu, Yingying; Li, Fengna; Kong, Xiangfeng; Tan, Bie; Li, Yinghui; Duan, Yehui; Blachier, François; Hu, Chien-An A.; Yin, Yulong

2015-01-01

Muscle growth is regulated by the homeostatic balance of the biosynthesis and degradation of muscle proteins. To elucidate the molecular interactions among diet, pig genotype, and physiological stage, we examined the effect of dietary protein concentration, pig genotype, and physiological stages on amino acid (AA) pools, protein deposition, and related signaling pathways in different types of skeletal muscles. The study used 48 Landrace pigs and 48 pure-bred Bama mini-pigs assigned to each of 2 dietary treatments: lower/GB (Chinese conventional diet)- or higher/NRC (National Research Council)-protein diet. Diets were fed from 5 weeks of age to respective market weights of each genotype. Samples of biceps femoris muscle (BFM, type I) and longissimus dorsi muscle (LDM, type II) were collected at nursery, growing, and finishing phases according to the physiological stage of each genotype, to determine the AA concentrations, mRNA levels for growth-related genes in muscles, and protein abundances of mechanistic target of rapamycin (mTOR) signaling pathway. Our data showed that the concentrations of most AAs in LDM and BFM of pigs increased (P<0.05) gradually with increasing age. Bama mini-pigs had generally higher (P<0.05) muscle concentrations of flavor-related AA, including Met, Phe, Tyr, Pro, and Ser, compared with Landrace pigs. The mRNA levels for myogenic determining factor, myogenin, myocyte-specific enhancer binding factor 2 A, and myostatin of Bama mini-pigs were higher (P<0.05) than those of Landrace pigs, while total and phosphorylated protein levels for protein kinase B, mTOR, and p70 ribosomal protein S6 kinases (p70S6K), and ratios of p-mTOR/mTOR, p-AKT/AKT, and p-p70S6K/p70S6K were lower (P<0.05). There was a significant pig genotype-dependent effect of dietary protein on the levels for mTOR and p70S6K. When compared with the higher protein-NRC diet, the lower protein-GB diet increased (P<0.05) the levels for mTOR and p70S6K in Bama mini-pigs, but

Fed levels of amino acids are required for the somatotropin-induced increase in muscle protein synthesis

USDA-ARS?s Scientific Manuscript database

Chronic somatotropin (pST) treatment in pigs increases muscle protein synthesis and circulating insulin, a known promoter of protein synthesis. Previously, we showed that the pST-mediated rise in insulin could not account for the pST-induced increase in muscle protein synthesis when amino acids were...

The interaction of albumin and fatty-acid-binding protein with membranes: oleic acid dissociation.

PubMed

Catalá, A

1984-10-01

Bovine serum albumin or fatty-acid-binding protein rapidly lose oleic acid when incubated in the presence of dimyristoyl lecithin liposomes. The phenomenon is dependent on vesicle concentration and no measurable quantities of protein are found associated with liposomes. Upon gel filtration on Sepharose CL-2B of incubated mixtures of microsomes containing [1-14C] oleic acid and albumin or fatty-acid-binding protein, association of fatty acid with the soluble proteins could be demonstrated. Both albumin and fatty-acid-binding protein stimulated the transfer of oleic acid from rat liver microsomes to egg lecithin liposomes. These results indicate that albumin is more effective in the binding of oleic acid than fatty-acid-binding protein, which allows a selective oleic acid dissociation during its interaction with membranes.

Overexpression of heart-type fatty acid binding protein enhances fatty acid-induced podocyte injury

PubMed Central

Gao, Qing; Sarkar, Alhossain; Chen, Yizhi; Xu, Bo; Zhu, Xiaojuan; Yuan, Yang; Guan, Tianjun

2018-01-01

Deregulated lipid metabolism is a characteristic of metabolic diseases including type 2 diabetes and obesity, and likely contributes to podocyte injury and end-stage kidney disease. Heart-type fatty acid binding protein (H-FABP) was reported to be associated with lipid metabolism. The present study investigated whether H-FABP contributes to podocyte homeostasis. Podocytes were transfected by lentiviral vector to construct a cell line which stably overexpressed H-FABP. Small interfering RNA capable of effectively silencing H-FABP was introduced into podocytes to construct a cell line with H-FABP knockdown. Certain groups were treated with palmitic acid (PA) and the fat metabolism, as well as inflammatory and oxidative stress markers were measured. PA accelerated lipid metabolism derangement, inflammatory reaction and oxidative stress in podocytes. Overexpression of H-FABP enhanced the PA-induced disequilibrium in podocytes. The mRNA and protein expression levels of acyl-coenzyme A oxidase 3 and monocyte chemotactic protein 1, and the protein expression levels of 8-hydroxy-2′-deoxyguanosine and 4-hydroxynonenal were upregulated in the H-FABP overexpression group, while the mRNA and protein expression of peroxisome proliferator activated receptor α was downregulated. Knockdown of H-FABP inhibited the PA-induced injury and lipid metabolism derangement, as well as the inflammatory reaction and oxidative stress in podocytes. These results indicated that overexpression of H-FABP enhances fatty acid-induced podocyte injury, while H-FABP inhibition may represent a potential therapeutic strategy for the prevention of lipid metabolism-associated podocyte injury. PMID:29434805

Amino acids augment muscle protein synthesis in neonatal pigs during acute endotoxemia by stimulating mTOR-dependent translation initiation.

PubMed

Orellana, Renán A; Jeyapalan, Asumthia; Escobar, Jeffery; Frank, Jason W; Nguyen, Hanh V; Suryawan, Agus; Davis, Teresa A

2007-11-01

In skeletal muscle of adults, sepsis reduces protein synthesis by depressing translation initiation and induces resistance to branched-chain amino acid stimulation. Normal neonates maintain a high basal muscle protein synthesis rate that is sensitive to amino acid stimulation. In the present study, we determined the effect of amino acids on protein synthesis in skeletal muscle and other tissues in septic neonates. Overnight-fasted neonatal pigs were infused with endotoxin (LPS, 0 and 10 microg.kg(-1).h(-1)), whereas glucose and insulin were maintained at fasting levels; amino acids were clamped at fasting or fed levels. In the presence of fasting insulin and amino acids, LPS reduced protein synthesis in longissimus dorsi (LD) and gastrocnemius muscles and increased protein synthesis in the diaphragm, but had no effect in masseter and heart muscles. Increasing amino acids to fed levels accelerated muscle protein synthesis in LD, gastrocnemius, masseter, and diaphragm. LPS stimulated protein synthesis in liver, lung, spleen, pancreas, and kidney in fasted animals. Raising amino acids to fed levels increased protein synthesis in liver of controls, but not LPS-treated animals. The increase in muscle protein synthesis in response to amino acids was associated with increased mTOR, 4E-BP1, and S6K1 phosphorylation and eIF4G-eIF4E association in control and LPS-infused animals. These findings suggest that amino acids stimulate skeletal muscle protein synthesis during acute endotoxemia via mTOR-dependent ribosomal assembly despite reduced basal protein synthesis rates in neonatal pigs. However, provision of amino acids does not further enhance the LPS-induced increase in liver protein synthesis.

Markedly Lower Glutamic Acid Decarboxylase 67 Protein Levels in a Subset of Boutons in Schizophrenia.

PubMed

Rocco, Brad R; Lewis, David A; Fish, Kenneth N

2016-06-15

Convergent findings indicate that cortical gamma-aminobutyric acid (GABA)ergic circuitry is altered in schizophrenia. Postmortem studies have consistently found lower levels of glutamic acid decarboxylase 67 (GAD67) messenger RNA (mRNA) in the prefrontal cortex (PFC) of subjects with schizophrenia. At the cellular level, the density of GABA neurons with detectable levels of GAD67 mRNA is ~30% lower across cortical layers. Knowing how this transcript deficit translates to GAD67 protein levels in axonal boutons is important for understanding the impact it might have on GABA synthesis. In addition, because reductions in GAD67 expression before, but not after, the maturation of GABAergic boutons results in a lower density of GABAergic boutons in mouse cortical cultures, knowing if GABAergic bouton density is altered in schizophrenia would provide insight into the timing of the GAD67 deficit. PFC tissue sections from 20 matched pairs of schizophrenia and comparison subjects were immunolabeled for the vesicular GABA transporter (vGAT) and GAD67. vGAT+ bouton density did not differ between subject groups, consistent with findings that vGAT mRNA levels are unaltered in the illness and confirming that the number of cortical GABAergic boutons is not lower in schizophrenia. In contrast, in schizophrenia subjects, the proportion of vGAT+ boutons with detectable GAD67 levels (vGAT+/GAD67+ boutons) was 16% lower and mean GAD67 levels were 14% lower in the remaining vGAT+/GAD67+ boutons. Our findings suggest that GABA production is markedly reduced in a subset of boutons in the PFC of schizophrenia subjects and that this reduction likely occurs after the maturation of GABAergic boutons. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Crude protein and essential amino acid requirements in chicks during the first week posthatch.

PubMed

Sklan, D; Noy, Y

2003-05-01

1. This study examined optimal lysine and sulphur amino acid supply in the first week posthatch in broilers and the relationship between essential amino acids and dietary crude protein during the first week posthatch on performance at 7 d and through marketing. 2. The optimal supply during the 7 d posthatch using a 230 g/kg crude protein diet for sulphur amino acids was 9.1 and for lysine was 10.3-10.8 g/kg with maximal body weight (BW) or feed efficiency as the criteria. 3. Feeding diets with crude protein content ranging from 200 to 260 g/kg with either constant amounts of essential amino acids at different crude protein levels or constant ratios of essential amino acids to crude protein resulted in enhanced performance at 7 but not at 4 d with high protein intake and proportionally increased essential amino acids. 4. Performance on diets with crude protein ranging from 160 to 280 g/kg, with constant ratios of essential amino acid to crude protein, was much enhanced with the high crude protein diets at 7 d. All chicks were transferred to standard diets after 7 d and the BW advantage due to the balanced amino acid-high crude protein diet remained through marketing. 5. Thus increasing essential amino acids in a constant ratio to crude protein enhanced performance during the 7 d posthatch.

Brain–blood amino acid correlates following protein restriction in murine maple syrup urine disease

PubMed Central

2014-01-01

Background Conventional therapy for patients with maple syrup urine disease (MSUD) entails restriction of protein intake to maintain acceptable levels of the branched chain amino acid, leucine (LEU), monitored in blood. However, no data exists on the correlation between brain and blood LEU with protein restriction, and whether correction in blood is reflected in brain. Methods To address this question, we fed intermediate MSUD mice diets of 19% (standard) and 6% protein, with collection of sera (SE), striata (STR), cerebellum (CE) and cortex (CTX) for quantitative amino acid analyses. Results LEU and valine (VAL) levels in all brain regions improved on average 28% when shifting from 19% to 6% protein, whereas the same improvements in SE were on average 60%. Isoleucine (ILE) in brain regions did not improve, while the SE level improved 24% with low-protein consumption. Blood-branched chain amino acids (LEU, ILE, and VAL in sera (SE)) were 362-434 μM, consistent with human values considered within control. Nonetheless, numerous amino acids in brain regions remained abnormal despite protein restriction, including glutamine (GLN), aspartate (ASP), glutamate (GLU), gamma-aminobutyric acid (GABA), asparagine (ASN), citrulline (CIT) and serine (SER). To assess the specificity of these anomalies, we piloted preliminary studies in hyperphenylalaninemic mice, modeling another large neutral aminoacidopathy. Employing an identical dietary regimen, we found remarkably consistent abnormalities in GLN, ASP, and GLU. Conclusions Our results suggest that blood amino acid analysis may be a poor surrogate for assessing the outcomes of protein restriction in the large neutral amino acidopathies, and further indicate that chronic neurotransmitter disruptions (GLU, GABA, ASP) may contribute to long-term neurocognitive dysfunction in these disorders. PMID:24886632

Brain-blood amino acid correlates following protein restriction in murine maple syrup urine disease.

PubMed

Vogel, Kara R; Arning, Erland; Wasek, Brandi L; McPherson, Sterling; Bottiglieri, Teodoro; Gibson, K Michael

2014-05-08

Conventional therapy for patients with maple syrup urine disease (MSUD) entails restriction of protein intake to maintain acceptable levels of the branched chain amino acid, leucine (LEU), monitored in blood. However, no data exists on the correlation between brain and blood LEU with protein restriction, and whether correction in blood is reflected in brain. To address this question, we fed intermediate MSUD mice diets of 19% (standard) and 6% protein, with collection of sera (SE), striata (STR), cerebellum (CE) and cortex (CTX) for quantitative amino acid analyses. LEU and valine (VAL) levels in all brain regions improved on average 28% when shifting from 19% to 6% protein, whereas the same improvements in SE were on average 60%. Isoleucine (ILE) in brain regions did not improve, while the SE level improved 24% with low-protein consumption. Blood-branched chain amino acids (LEU, ILE, and VAL in sera (SE)) were 362-434 μM, consistent with human values considered within control. Nonetheless, numerous amino acids in brain regions remained abnormal despite protein restriction, including glutamine (GLN), aspartate (ASP), glutamate (GLU), gamma-aminobutyric acid (GABA), asparagine (ASN), citrulline (CIT) and serine (SER). To assess the specificity of these anomalies, we piloted preliminary studies in hyperphenylalaninemic mice, modeling another large neutral aminoacidopathy. Employing an identical dietary regimen, we found remarkably consistent abnormalities in GLN, ASP, and GLU. Our results suggest that blood amino acid analysis may be a poor surrogate for assessing the outcomes of protein restriction in the large neutral amino acidopathies, and further indicate that chronic neurotransmitter disruptions (GLU, GABA, ASP) may contribute to long-term neurocognitive dysfunction in these disorders.

Differential regulation of protein synthesis by amino acids and insulin in peripheral and visceral tissues of neonatal pigs

PubMed Central

Suryawan, Agus; O’Connor, Pamela M. J.; Bush, Jill A.; Nguyen, Hanh V.

2009-01-01

The high efficiency of protein deposition during the neonatal period is driven by high rates of protein synthesis, which are maximally stimulated after feeding. In the current study, we examined the individual roles of amino acids and insulin in the regulation of protein synthesis in peripheral and visceral tissues of the neonate by performing pancreatic glucose–amino acid clamps in overnight-fasted 7-day-old pigs. We infused pigs (n = 8–12/group) with insulin at 0, 10, 22, and 110 ng kg−0.66 min−1 to achieve ~0, 2, 6 and 30 μU ml−1 insulin so as to simulate below fasting, fasting, intermediate, and fed insulin levels, respectively. At each insulin dose, amino acids were maintained at the fasting or fed level. In conjunction with the highest insulin dose, amino acids were also allowed to fall below the fasting level. Tissue protein synthesis was measured using a flooding dose of L-[4-3H] phenylalanine. Both insulin and amino acids increased fractional rates of protein synthesis in longissimus dorsi, gastrocnemius, masseter, and diaphragm muscles. Insulin, but not amino acids, increased protein synthesis in the skin. Amino acids, but not insulin, increased protein synthesis in the liver, pancreas, spleen, and lung and tended to increase protein synthesis in the jejunum and kidney. Neither insulin nor amino acids altered protein synthesis in the stomach. The results suggest that the stimulation of protein synthesis by feeding in most tissues of the neonate is regulated by the post-prandial rise in amino acids. However, the feeding-induced stimulation of protein synthesis in skeletal muscles is independently mediated by insulin as well as amino acids. PMID:18683020

Role of Protein and Amino Acids in Infant and Young Child Nutrition: Protein and Amino Acid Needs and Relationship with Child Growth.

PubMed

Uauy, Ricardo; Kurpad, Anura; Tano-Debrah, Kwaku; Otoo, Gloria E; Aaron, Grant A; Toride, Yasuhiko; Ghosh, Shibani

2015-01-01

Over a third of all deaths of children under the age of five are linked to undernutrition. At a 90% coverage level, a core group of ten interventions inclusive of infant and young child nutrition could save one million lives of children under 5 y of age (15% of all deaths) (Lancet 2013). The infant and young child nutrition package alone could save over 220,000 lives in children under 5 y of age. High quality proteins (e.g. milk) in complementary, supplementary and rehabilitation food products have been found to be effective for good growth. Individual amino acids such as lysine and arginine have been found to be factors linked to growth hormone release in young children via the somatotropic axis and high intakes are inversely associated with fat mass index in pre-pubertal lean girls. Protein intake in early life is positively associated with height and weight at 10 y of age. This paper will focus on examining the role of protein and amino acids in infant and young child nutrition by examining protein and amino acid needs in early life and the subsequent relationship with stunting.

Circulating intestinal fatty acid-binding protein (I-FABP) levels in acute decompensated heart failure.

PubMed

Kitai, Takeshi; Kim, Yong-Hyun; Kiefer, Kathryn; Morales, Rommel; Borowski, Allen G; Grodin, Justin L; Tang, W H Wilson

2017-06-01

Venous congestion has become increasingly recognized as a potential contributor to end-organ dysfunction in heart failure. Elevated I-FABP, which is excreted specifically from damaged intestinal epithelial cells, has been found in patients with abdominal hypertension and intestinal ischemia. We hypothesize that elevated intestinal fatty acid-binding protein (I-FABP) levels would identify patients with more advanced heart failure who have venous and intestinal congestion. Baseline serum I-FABP levels were measured in 69 acute decompensated heart failure (ADHF) patients admitted to the intensive care unit for invasive hemodynamic monitoring and tailored medical therapy. Comprehensive echocardiography examinations were performed in all study patients, and clinical outcomes (death, cardiac transplant or left ventricular assist device placement) were assessed. The median circulating I-FABP level was 853pg/ml (interquartile range: 533 to 1448pg/ml). Age, gender, race, and baseline comorbidities were comparable between patients with low and high I-FABP levels. Although there were no significant correlations between I-FABP levels and invasively-measured hemodynamic parameters nor echocardiographic parameters, patients with higher I-FABP levels (≥853g/ml) had significantly worse clinical outcomes compared to those with lower I-FABP levels (<853pg/ml, P=0.025). Circulating I-FABP levels had no association with invasively-measured hemodynamic parameters, but were associated with adverse clinical outcomes in patients with ADHF with systolic dysfunction. Copyright © 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Oleic acid transfer from microsomes to egg lecithin liposomes: participation of fatty acid binding protein.

PubMed

Catalá, A; Avanzati, B

1983-11-01

Oleic acid transfer from microsomes or mitochondria to egg lecithin liposomes was stimulated by fatty acid binding protein. By gel filtration, it could be demonstrated that this protein incorporates oleic acid into liposomes. Fatty acid binding protein transfer activity was higher using microsomes rather than mitochondria, which suggests a selective interaction with different kinds of membranes. Transfer of oleic acid by this soluble protein is greater than that of stearic acid. The results indicate that fatty acid binding protein may participate in the intracellular transport of fatty acids.

Amino Acid Flux from Metabolic Network Benefits Protein Translation: the Role of Resource Availability.

PubMed

Hu, Xiao-Pan; Yang, Yi; Ma, Bin-Guang

2015-06-09

Protein translation is a central step in gene expression and affected by many factors such as codon usage bias, mRNA folding energy and tRNA abundance. Despite intensive previous studies, how metabolic amino acid supply correlates with protein translation efficiency remains unknown. In this work, we estimated the amino acid flux from metabolic network for each protein in Escherichia coli and Saccharomyces cerevisiae by using Flux Balance Analysis. Integrated with the mRNA expression level, protein abundance and ribosome profiling data, we provided a detailed description of the role of amino acid supply in protein translation. Our results showed that amino acid supply positively correlates with translation efficiency and ribosome density. Moreover, with the rank-based regression model, we found that metabolic amino acid supply facilitates ribosome utilization. Based on the fact that the ribosome density change of well-amino-acid-supplied genes is smaller than poorly-amino-acid-supply genes under amino acid starvation, we reached the conclusion that amino acid supply may buffer ribosome density change against amino acid starvation and benefit maintaining a relatively stable translation environment. Our work provided new insights into the connection between metabolic amino acid supply and protein translation process by revealing a new regulation strategy that is dependent on resource availability.

A nine-country study of the protein content and amino acid composition of mature human milk

PubMed Central

Feng, Ping; Gao, Ming; Burgher, Anita; Zhou, Tian Hui; Pramuk, Kathryn

2016-01-01

Background Numerous studies have evaluated protein and amino acid levels in human milk. However, research in this area has been limited by small sample sizes and study populations with little ethnic or racial diversity. Objective Evaluate the protein and amino acid composition of mature (≥30 days) human milk samples collected from a large, multinational study using highly standardized methods for sample collection, storage, and analysis. Design Using a single, centralized laboratory, human milk samples from 220 women (30–188 days postpartum) from nine countries were analyzed for amino acid composition using Waters AccQ-Tag high-performance liquid chromatography and total nitrogen content using the LECO FP-528 nitrogen analyzer. Total protein was calculated as total nitrogen×6.25. True protein, which includes protein, free amino acids, and peptides, was calculated from the total amino acids. Results Mean total protein from individual countries (standard deviation [SD]) ranged from 1,133 (125.5) to 1,366 (341.4) mg/dL; the mean across all countries (SD) was 1,192 (200.9) mg/dL. Total protein, true protein, and amino acid composition were not significantly different across countries except Chile, which had higher total and true protein. Amino acid profiles (percent of total amino acids) did not differ across countries. Total and true protein concentrations and 16 of 18 amino acid concentrations declined with the stage of lactation. Conclusions Total protein, true protein, and individual amino acid concentrations in human milk steadily decline from 30 to 151 days of lactation, and are significantly higher in the second month of lactation compared with the following 4 months. There is a high level of consistency in the protein content and amino acid composition of human milk across geographic locations. The size and diversity of the study population and highly standardized procedures for the collection, storage, and analysis of human milk support the validity and

Combined protein- and nucleic acid-level effects of rs1143679 (R77H), a lupus-predisposing variant within ITGAM.

PubMed

Maiti, Amit K; Kim-Howard, Xana; Motghare, Prasenjeet; Pradhan, Vandana; Chua, Kek Heng; Sun, Celi; Arango-Guerrero, María Teresa; Ghosh, Kanjaksha; Niewold, Timothy B; Harley, John B; Anaya, Juan-Manual; Looger, Loren L; Nath, Swapan K

2014-08-01

Integrin alpha M (ITGAM; CD11b) is a component of the macrophage-1 antigen complex, which mediates leukocyte adhesion, migration and phagocytosis as part of the immune system. We previously identified a missense polymorphism, rs1143679 (R77H), strongly associated with systemic lupus erythematosus (SLE). However, the molecular mechanisms of this variant are incompletely understood. A meta-analysis of published and novel data on 28 439 individuals with European, African, Hispanic and Asian ancestries reinforces genetic association between rs1143679 and SLE [Pmeta = 3.60 × 10(-90), odds ratio (OR) = 1.76]. Since rs1143679 is in the most active region of chromatin regulation and transcription factor binding in ITGAM, we quantitated ITGAM RNA and surface protein levels in monocytes from patients with each rs1143679 genotype. We observed that transcript levels significantly decreased for the risk allele ('A') relative to the non-risk allele ('G'), in a dose-dependent fashion: ('AA' < 'AG' < 'GG'). CD11b protein levels in patients' monocytes were directly correlated with RNA levels. Strikingly, heterozygous individuals express much lower (average 10- to 15-fold reduction) amounts of the 'A' transcript than 'G' transcript. We found that the non-risk sequence surrounding rs1143679 exhibits transcriptional enhancer activity in vivo and binds to Ku70/80, NFKB1 and EBF1 in vitro, functions that are significantly reduced with the risk allele. Mutant CD11b protein shows significantly reduced binding to fibrinogen and vitronectin, relative to non-risk, both in purified protein and in cellular models. This two-pronged contribution (nucleic acid- and protein-level) of the rs1143679 risk allele to decreasing ITGAM activity provides insight into the molecular mechanisms of its potent association with SLE. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

PLASMA PROTEIN PRODUCTION INFLUENCED BY AMINO ACID MIXTURES AND LACK OF ESSENTIAL AMINO ACIDS

PubMed Central

Madden, S. C.; Anderson, F. W.; Donovan, J. C.; Whipple, G. H.

1945-01-01

When blood plasma proteins are depleted by bleeding with return of red cells suspended in saline (plasmapheresis) it is possible to bring dogs to a steady state of hypoproteinemia and a constant level of plasma protein production if the diet nitrogen intake is controlled and limited. Such dogs are outwardly normal but have a lowered resistance to infection and intoxication and probably to vitamin deficiency. When the diet nitrogen is provided by certain mixtures of the ten growth essential amino acids plus glycine, given intravenously at a rapid rate, plasma protein production is good. The same mixture absorbed subcutaneously at a slower rate may be slightly better utilized. Fed orally the same mixture is better utilized and associated with a lower urinary nitrogen excretion. An ample amino acid mixture for the daily intake of a 10 kilo dog may contain in grams dl-threonine 1.4, dl-valine 3, dl-leucine 3, dl-isoleucine 2, l(+)-lysine·HCl·H2O 2.2, dl-tryptophane 0.3, dl-phenylalanine 2, dl-methionine 1.2, l(+)-histidine·HCl·H2O 1, l(+)-arginine·HCl 1, and glycine 2. Half this quantity is inadequate and not improved by addition of a mixture of alanine, serine, norleucine, proline, hydroxyproline, and tyrosine totalling 1.4 gm. Aspartic acid appears to induce vomiting when added to a mixture of amino acids. The same response has been reported for glutamic acid (8). Omission from the intake of leucine or of leucine and isoleucine results in negative nitrogen balance and rapid weight loss but plasma protein production may be temporarily maintained. It is possible that leucine may be captured from red blood cell destruction. Tryptophane deficiency causes an abrupt decline in plasma protein production. No decline occurred during 2 weeks of histidine deficiency but the urinary nitrogen increased to negative balance. Plasma protein production may be impaired during conditions of dietary deficiency not related to the protein or amino acid intake. Skin lesions and liver

Regulation of intestinal protein metabolism by amino acids.

PubMed

Bertrand, Julien; Goichon, Alexis; Déchelotte, Pierre; Coëffier, Moïse

2013-09-01

Gut homeostasis plays a major role in health and may be regulated by quantitative and qualitative food intake. In the intestinal mucosa, an intense renewal of proteins occurs, at approximately 50% per day in humans. In some pathophysiological conditions, protein turnover is altered and may contribute to intestinal or systemic diseases. Amino acids are key effectors of gut protein turnover, both as constituents of proteins and as regulatory molecules limiting intestinal injury and maintaining intestinal functions. Many studies have focused on two amino acids: glutamine, known as the preferential substrate of rapidly dividing cells, and arginine, another conditionally essential amino acid. The effects of glutamine and arginine on protein synthesis appear to be model and condition dependent, as are the involved signaling pathways. The regulation of gut protein degradation by amino acids has been minimally documented until now. This review will examine recent data, helping to better understand how amino acids regulate intestinal protein metabolism, and will explore perspectives for future studies.

Hypochlorous and peracetic acid induced oxidation of dairy proteins.

PubMed

Kerkaert, Barbara; Mestdagh, Frédéric; Cucu, Tatiana; Aedo, Philip Roger; Ling, Shen Yan; De Meulenaer, Bruno

2011-02-09

Hypochlorous and peracetic acids, both known disinfectants in the food industry, were compared for their oxidative capacity toward dairy proteins. Whey proteins and caseins were oxidized under well controlled conditions at pH 8 as a function of the sanitizing concentration. Different markers for protein oxidation were monitored. The results established that the protein carbonyl content was a rather unspecific marker for protein oxidation, which did not allow one to differentiate the oxidant used especially at the lower concentrations. Cysteine, tryptophan, and methionine were proven to be the most vulnerable amino acids for degradation upon hypochlorous and peracetic acid treatment, while tyrosine was only prone to degradation in the presence of hypochlorous acid. Hypochlorous acid induced oxidation gave rise to protein aggregation, while during peracetic acid induced oxidation, no high molecular weight aggregates were observed. Protein aggregation upon hypochlorous acid oxidation could primarily be linked to tryptophan and tyrosine degradation.

Serum levels of albumin, triglycerides, total protein and glucose in rats are altered after oral treatment with low doses of 13-cis-retinoic acid or all-trans-retinoic acid.

PubMed

Cisneros, F J; Gough, B J; Patton, R E; Ferguson, S A

2005-01-01

Currently used to treat severe acne, 13-cis-retinoic acid (13-cis-RA) is under investigation for its anticancer effects as is the isomer, all-trans-retinoic acid (all-trans-RA). Here, the effects of oral 13-cis-RA or all-trans-RA treatment on serum chemistry, leptin and adiponectin levels were evaluated. Adult Sprague-Dawley rats were gavaged once daily for 7 consecutive days with 13-cis-RA (7.5 or 15 mg kg(-1)), all-trans-RA (10 or 15 mg kg(-1)) (n=24/sex/dose), or soy oil (n=16/sex) and blood was sampled 30-480 min after the last gavage. The body weight was unaffected; however, the liver/body weight ratios were increased by both doses of all-trans-RA. Sex differences were noted for levels of cholesterol, creatine, triglycerides, albumin, alanine aminotransferase and total protein. Both doses of all-trans-RA reduced albumin levels to approximately 90% of the control and total protein levels to approximately 93% of the control while substantially elevating triglyceride levels to approximately 66%-99% above the control. Additionally, triglyceride levels of the 15 mg kg(-1) 13-cis RA group were approximately 62% higher than the controls and total protein levels were approximately 5% less. Glucose levels were affected by sex and RA treatment in that males treated with 15 mg kg(-1) of 13-cis-RA or 10 mg kg(-1) all-trans-RA had lower (13%-19%) levels than the same-sex controls; however, females were not similarly affected. Neither 13-cis-RA nor all-trans-RA treatment had significant effects on the levels of blood urea nitrogen, aspartate amino transferase, leptin or adiponectin. On a mg kg(-1) basis, all-trans-RA was more potent than 13-cis-RA. These results replicate previous findings of RA-induced increased triglyceride levels. Additionally, several new findings indicate there may be sex-specific effects of RA treatment. Finally, neither treatment appeared to alter the typical diurnal cycles of these endpoints. Copyright (c) 2005 John Wiley & Sons, Ltd.

A simple atomic-level hydrophobicity scale reveals protein interfacial structure.

PubMed

Kapcha, Lauren H; Rossky, Peter J

2014-01-23

Many amino acid residue hydrophobicity scales have been created in an effort to better understand and rapidly characterize water-protein interactions based only on protein structure and sequence. There is surprisingly low consistency in the ranking of residue hydrophobicity between scales, and their ability to provide insightful characterization varies substantially across subject proteins. All current scales characterize hydrophobicity based on entire amino acid residue units. We introduce a simple binary but atomic-level hydrophobicity scale that allows for the classification of polar and non-polar moieties within single residues, including backbone atoms. This simple scale is first shown to capture the anticipated hydrophobic character for those whole residues that align in classification among most scales. Examination of a set of protein binding interfaces establishes good agreement between residue-based and atomic-level descriptions of hydrophobicity for five residues, while the remaining residues produce discrepancies. We then show that the atomistic scale properly classifies the hydrophobicity of functionally important regions where residue-based scales fail. To illustrate the utility of the new approach, we show that the atomic-level scale rationalizes the hydration of two hydrophobic pockets and the presence of a void in a third pocket within a single protein and that it appropriately classifies all of the functionally important hydrophilic sites within two otherwise hydrophobic pores. We suggest that an atomic level of detail is, in general, necessary for the reliable depiction of hydrophobicity for all protein surfaces. The present formulation can be implemented simply in a manner no more complex than current residue-based approaches. © 2013.

Effect of Dietary Protein Level on the Expression of Proteins in the Gastrointestinal Tract of Young Pigs.

PubMed

Ma, Xianyong; Tian, Zhimei; Deng, Dun; Cui, Yiyan; Qiu, Yueqin

2018-05-02

The objective of this research is to investigate the effect of protein level on proteins expression in the gastrointestinal tract of young pigs. Eighteen piglets (Duroc × Landrace × Yorkshire) were weaned at 28 days of age and randomly assigned to three diets with 20%, 17%, and 14% CP level, and four essential amino acids, Lys, Met, Thr, and Trp, in three diets met the requirements of weaned piglets. The experimental period lasted 45 days. Compared with the control (20% CP level), the average daily feed intake, the average daily gain, and gain feed ratio of the 17% CP group did not decrease ( P > 0.05), but those of 14% CP group decreased ( P < 0.05). The proteomics profiles result of three tissues (gastric antrum, duodenum, and jejunum) showed that, compared with the control, the immune system, protein digestion and absorption, lipid or carbon digestion and absorption, etc. were up-regulated in 17% CP group, while most of them were down-regulated in 14% CP group. Amino acids metabolism of gastric, pancreatic secretion of duodenum or steroid hormone biosynthesis of jejunum was down-regulated in the 17% CP group, but the lipid metabolism was up-regulated in the 14% CP group. Six proteins were selected for identification by Western-blot, and their changes had the same trend as the proteomics results. The protein level decreased from 20% to 17%, the growth performance was not affected, while the nutrient digestion and absorption or the immune function were improved, which implied that 17% protein level maybe benefit for nutrients absorption of pigs.

Structures and functions of proteins and nucleic acids in protein biosynthesis

NASA Astrophysics Data System (ADS)

Miyazawa, Tatsuo; Yokoyama, Shigeyuki

Infrared and Raman spectroscopy is useful for studying helical conformations of polypeptides, which are determined by molecular structure parameters. Nuclear magnetic resonance spectroscopy, as well as X-ray analysis, is now established to be important for conformation studies of proteins and nucleic acids in solution. This article is mainly concerned with the conformational aspect and function regulation in protein biosynthesis. The strict recognition of transfer ribonucleic acid (tRNA) by aminoacyl-tRNA synthetase (ARS) is achieved by multi-step mutual adaptation. The conformations of ARS-bound amino acids have been elucidated by transferred nuclear Overhauser effect analysis. Aminoacyl-tRNA takes the 3‧-isomeric form in the polypeptide chain elongation cycle. The regulation of codon recognition by post-transcriptional modification is achieved by conversion of the conformational characteristic of the anticodon of tRNA. The cytidine → lysidine modification of the anticodon of minor isoleucine tRNA concurrently converts the amino acid specificity and the codon specificity. As novel protein engineering, a basic strategy has been established for in vivo biosynthesis of proteins that are substituted with unnatural amino acids (alloproteins).

Effect of a keto acid-amino acid supplement on the metabolism and renal elimination of branched-chain amino acids in patients with chronic renal insufficiency on a low protein diet.

PubMed

Teplan, V; Schück, O; Horácková, M; Skibová, J; Holecek, M

2000-10-27

The aim of our study was to evaluate the effect of a low-protein diet supplemented with keto acids-amino acids on renal function and urinary excretion of branched-chain amino acids (BCAA) in patients with chronic renal insufficiency (CRI). In a prospective investigation 28 patients with CRI (16 male, 12 female, aged 28-66 yrs, CCr 18.6 +/- 10.2 ml/min) on a low-protein diet (0.6 g of protein /kg BW/day and energy intake 140 kJ/kg BW/day) for a period of one month were included. Subsequently, this low protein diet was supplemented with keto acids-amino acids at a dose of 0.1 g/kg BW/day orally for a period of 3 months. Examinations performed at baseline and at the end of the follow-up period revealed significant increase in the serum levels of BCAA leucine (p < 0.02), isoleucine (p < 0.03), and valine (p < 0.02) while their renal fractional excretion declined (p < 0.02, p < 0.01 resp.). Keto acid-amino acid administration had no effect on renal function and on the clearance of inulin, para-aminohippuric acid. Endogenous creatinine and urea clearance remained unaltered. A significant correlation between fractional excretion of sodium and leucine (p < 0.05) and a hyperbolic relationship between inulin clearance and fractional excretion of BCAA (p < 0.01) were seen. Moreover, a significant decrease in proteinuria (p < 0.02), plasma urea concentration and renal urea excretion and a rise in albumin level (p < 0.03) were noted. We conclude that in patients with CRI on a low protein diet the supplementation of keto acids-amino acids does not affect renal hemodynamics, but is associated--despite increases in plasma concentrations--with a reduction of renal amino acid and protein excretion suggesting induction of alterations in the tubular transport mechanisms.

Gender difference in plasma fatty-acid-binding protein 4 levels in patients with chronic obstructive pulmonary disease

PubMed Central

Zhang, Xue; Li, Diandian; Wang, Hao; Pang, Caishuang; Wu, Yanqiu; Wen, Fuqiang

2016-01-01

COPD (chronic obstructive pulmonary disease) is characterized by airway inflammation and increases the likelihood of the development of atherosclerosis. Recent studies have indicated that FABP4 (fatty-acid-binding protein 4), an intracellular lipid chaperone of low molecular mass, plays an important role in the regulation of inflammation and atherosclerosis. We carried out a preliminary clinical study aiming at investigating the relationships between circulating FABP4 levels in patients with COPD and inflammation and lung function. We enrolled 50 COPD patients and 39 healthy controls in the study. Lung function tests were performed in all subjects. Plasma levels of FABP4 and adiponectin, TNFα (tumour necrosis factor α) and CRP (C-reactive protein) were measured. The correlations between FABP4 and lung function, adipokine (adiponectin), inflammatory factors and BMI (body mass index) were analysed. Compared with both males with COPD and healthy females, plasma FABP4 levels in females with COPD were significantly increased. Adiponectin and CRP levels were significantly higher in patients with COPD. Furthermore, we found that FABP4 levels were inversely correlated with FEV1% predicted (FEV1 is forced expiratory volume in 1 s) and positively correlated with adiponectin and TNFα in COPD patients. In addition, a positive correlation between plasma FABP4 and CRP was found in females with COPD. However, FABP4 levels were not correlated with BMI. Our results underline a gender difference in FABP4 secretion in stable COPD patients. Further studies are warranted to clarify the exact role of FABP4 in the pathogenesis of COPD. PMID:26823558

Effect of Maillard browning reaction on protein utilization and plasma amino acid response by rainbow trout (Salmo gairdneri).

PubMed

Plakas, S M; Lee, T C; Wolke, R E; Meade, T L

1985-12-01

The effect of the Maillard browning reaction in the diet of rainbow trout (Salmo gairdneri) on growth and amino acid availability was investigated. Chemical and enzymatic hydrolysis methods were applied for the detection of the losses of amino acids in a model protein browning system. Arginine and lysine exhibited the greatest losses in the mixture of fish protein isolate and glucose stored for 40 d at 37 degrees C. The apparent digestibility and absorption of individual amino acids, particularly lysine, was lower in trout fed browned protein than in those fed the control protein. Plasma lysine levels were significantly depressed, while the plasma levels of glucose and most other amino acids were elevated in relation to the loss in nutritive value of dietary protein after browning. The early Maillard reaction derivative of lysine, epsilon-deoxy-fructosyl-lysine, was recovered from browned protein (by using the in vitro enzymatic hydrolysis procedure) and from the plasma of trout fed browned protein. Analysis of plasma free amino acids provided an indication of lysine bioavailability and identified lysine as the first-limiting amino acid in the diets containing browned protein.

Heart-type fatty acid binding protein levels in elderly diabetics without known cardiovascular disease.

PubMed

Beysel, Selvihan; Kizilgul, Muhammed; Ozbek, Mustafa; Caliskan, Mustafa; Kan, Seyfullah; Apaydin, Mahmut; Ozcelik, Ozgur; Cakal, Erman

2017-01-01

Cardiovascular disease (CVD) is reported to be higher in elderly diabetics. Serum heart-type fatty acid binding protein (H-FABP) is a serum marker of myocardial ischemia. We aimed to investigate the association between serum H-FABP level and conventional cardiovascular risk factors, inflammatory markers and subclinical atherosclerosis in elderly diabetics without overt CVD. A total of 50 elderly diabetic patients without overt CVD and 30 age-, sex- and body mass index (BMI)-matched healthy controls were enrolled. Anthropometric and biochemical parameters, serum H-FABP, high-sensitivity C-reactive protein (hs-CRP), fibrinogen and carotid intima-media thickness (CIMT) were measured. Logistic regression analyses (adjustments for age, sex, hypertension, smoking, diabetes, BMI, blood pressure, lipid, blood glucose, hemoglobin A1c, hs-CRP and fibrinogen) were performed to evaluate the association between H-FABP and cardiovascular risk factors and atherosclerosis indices. Serum fibrinogen (421.50±85.52 mg/dL vs 319.17±30.77 mg/dL, p =0.023), CIMT (0.70±0.12 mm vs 0.59±0.06 mm, p <0.001) and hs-CRP (5.72±4.50 mg/dL vs 1.60±0.72 mg/dL, p <0.001) were significantly higher in diabetic patients than controls. The mean serum H-FABP level did not differ between groups (1571.79±604.60 ng/mL vs 1500.25±463.35 ng/mL, p =0.905). H-FABP was positively correlated with fibrinogen ( r 2 =0.473, p <0.001), hs-CRP ( r 2 =0.323, p =0.003) and CIMT ( r 2 =0.467, p <0.001). After full adjustments, the serum H-FABP level was independently associated with an increase in the fibrinogen level (odds ratio [OR] =4.21, 95% confidence level [CI] =1.49-11.90). Serum H-FABP was similar in the elderly diabetic patients without known CVD when compared with the nondiabetic control group. H-FABP does not possess a high diagnostic value as a cardiovascular marker when used alone; however, it may add supplementary information in patients with a high fibrinogen level.

Glutamic Acid as Enhancer of Protein Synthesis Kinetics in Hepatocytes from Old Rats.

PubMed

Brodsky, V Y; Malchenko, L A; Butorina, N N; Lazarev Konchenko, D S; Zvezdina, N D; Dubovaya, T K

2017-08-01

Dense cultures of hepatocytes from old rats (~2 years old, body weight 530-610 g) are different from similar cultures of hepatocytes from young rats by the low amplitude of protein synthesis rhythm. Addition of glutamic acid (0.2, 0.4, or 0.6 mg/ml) into the culture medium with hepatocytes of old rats resulted in increase in the oscillation amplitudes of the protein synthesis rhythm to the level of young rats. A similar action of glutamic acid on the protein synthesis kinetics was observed in vivo after feeding old rats with glutamic acid. Inhibition of metabotropic receptors of glutamic acid with α-methyl-4-carboxyphenylglycine (0.01 mg/ml) abolished the effect of glutamic acid. The amplitude of oscillation of the protein synthesis rhythm in a cell population characterizes synchronization of individual oscillations caused by direct cell-cell communications. Hence, glutamic acid, acting as a receptor-dependent transmitter, enhanced direct cell-cell communications of hepatocytes that were decreased with aging. As differentiated from other known membrane signaling factors (gangliosides, norepinephrine, serotonin, dopamine), glutamic acid can penetrate into the brain and thus influence the communications and protein synthesis kinetics that are disturbed with aging not only in hepatocytes, but also in neurons.

Interactions between Therapeutic Proteins and Acrylic Acid Leachable.

PubMed

Liu, Dengfeng; Nashed-Samuel, Yasser; Bondarenko, Pavel V; Brems, David N; Ren, Da

2012-01-01

Leachables are chemical compounds that migrate from manufacturing equipment, primary containers and closure systems, and packaging components into biopharmaceutical and pharmaceutical products. Acrylic acid (at concentration around 5 μg/mL) was detected as leachable in syringes from one of the potential vendors (X syringes). In order to evaluate the potential impact of acrylic acid on therapeutic proteins, an IgG 2 molecule was filled into a sterilized X syringe and then incubated at 45 °C for 45 days in a pH 5 acetate buffer. We discovered that acrylic acid can interact with proteins at three different sites: (1) the lysine side chain, (2) the N-terminus, and (3) the histidine side chain, by the Michael reaction. In this report, the direct interactions between acrylic acid leachable and a biopharmaceutical product were demonstrated and the reaction mechanism was proposed. Even thought a small amount (from 0.02% to 0.3%) of protein was found to be modified by acrylic acid, the modified protein can potentially be harmful due to the toxicity of acrylic acid. After being modified by acrylic acid, the properties of the therapeutic protein may change due to charge and hydrophobicity variations. Acrylic acid was detected to migrate from syringes (Vendor X) into a therapeutic protein solution (at a concentration around 5 μg/mL). In this study, we discovered that acrylic acid can modify proteins at three different sites: (1) the lysine side chain, 2) the N-terminus, and 3) the histidine side chain, by the Michael reaction. In this report, the direct interactions between acrylic acid leachable and a biopharmaceutical product were demonstrated and the reaction mechanism was proposed.

Cloning and characteristics of fish glial fibrillary acidic protein: implications for optic nerve regeneration.

PubMed

Cohen, I; Shani, Y; Schwartz, M

1993-08-15

Mammalian central nervous system neurons do not regenerate after axonal injury, unlike their counterparts in fish and amphibians. After axonal injury, glial cells in mammals do not support regrowth of axons, while in fish they support the regeneration process. Controversy exists as to whether or not the intact fish optic nerve expresses glial fibrillary acidic protein, a well-known marker for mature astrocytes, and thus whether its astrocytes differ in this respect from those of the brain and spinal cord, as well as from optic nerve astrocytes of other species. In an attempt to resolve this question we cloned fish glial fibrillary acidic protein. Two different complementary DNA clones were isolated from a carp brain complementary DNA library, each encoding a different form of glial fibrillary acidic protein apparently originating from different genes. Monospecific polyclonal antibodies were raised against a peptide synthesized according to the predicted amino acid sequence, and used to identify and localize the fish glial fibrillary acidic protein. Two glial fibrillary acidic proteins (of 49 kDa and 51 kDa) were identified by the antibodies in all tested fish central nervous system tissues. The antibodies were then used to examine glial fibrillary acidic protein immunoreactivity in sections taken from uninjured and injured optic nerves of goldfish. Injury was followed by an elevation in glial fibrillary acidic protein immunoreactivity along the whole length of the nerve, except at the site of the injury, where--as in the case of vimentin--no immunoreactivity was detectable. However, in contrast to vimentin-positive glial cells, which repopulate the site of the injury soon after the optic nerve is injured, glial fibrillary acidic protein-positive glial cells remained outside the injury site for as long as 6 weeks after the injury. Despite the injury-induced changes in glial fibrillary acidic protein immunoreactivity, no change was observed in the level of transcript

Real-time Measurements of Amino Acid and Protein Hydroperoxides Using Coumarin Boronic Acid*

PubMed Central

Michalski, Radoslaw; Zielonka, Jacek; Gapys, Ewa; Marcinek, Andrzej; Joseph, Joy; Kalyanaraman, Balaraman

2014-01-01

Hydroperoxides of amino acid and amino acid residues (tyrosine, cysteine, tryptophan, and histidine) in proteins are formed during oxidative modification induced by reactive oxygen species. Amino acid hydroperoxides are unstable intermediates that can further propagate oxidative damage in proteins. The existing assays (oxidation of ferrous cation and iodometric assays) cannot be used in real-time measurements. In this study, we show that the profluorescent coumarin boronic acid (CBA) probe reacts with amino acid and protein hydroperoxides to form the corresponding fluorescent product, 7-hydroxycoumarin. 7-Hydroxycoumarin formation was catalase-independent. Based on this observation, we have developed a fluorometric, real-time assay that is adapted to a multiwell plate format. This is the first report showing real-time monitoring of amino acid and protein hydroperoxides using the CBA-based assay. This approach was used to detect protein hydroperoxides in cell lysates obtained from macrophages exposed to visible light and photosensitizer (rose bengal). We also measured the rate constants for the reaction between amino acid hydroperoxides (tyrosyl, tryptophan, and histidine hydroperoxides) and CBA, and these values (7–23 m−1 s−1) were significantly higher than that measured for H2O2 (1.5 m−1 s−1). Using the CBA-based competition kinetics approach, the rate constants for amino acid hydroperoxides with ebselen, a glutathione peroxidase mimic, were also determined, and the values were within the range of 1.1–1.5 × 103 m−1 s−1. Both ebselen and boronates may be used as small molecule scavengers of amino acid and protein hydroperoxides. Here we also show formation of tryptophan hydroperoxide from tryptophan exposed to co-generated fluxes of nitric oxide and superoxide. This observation reveals a new mechanism for amino acid and protein hydroperoxide formation in biological systems. PMID:24928516

STRUCTURAL AND FUNCTIONAL INTERACTION OF FATTY ACIDS WITH HUMAN LIVER FATTY ACID BINDING PROTEIN (L-FABP) T94A VARIANT

PubMed Central

Huang, Huan; McIntosh, Avery L.; Martin, Gregory G.; Landrock, Kerstin K.; Landrock, Danilo; Gupta, Shipra; Atshaves, Barbara P.; Kier, Ann B.; Schroeder, Friedhelm

2014-01-01

The human liver fatty acid binding protein (L-FABP) T94A variant, the most common in the FABP family, has been associated with elevated liver triglyceride (TG) levels. How this amino acid substitution elicits these effects is not known. This issue was addressed with human recombinant wild-type (WT, T94T) and T94A variant L-FABP proteins as well as cultured primary human hepatocytes expressing the respective proteins (genotyped as TT, TC, and CC). T94A substitution did not or only slightly alter L-FABP binding affinities for saturated, monounsaturated, or polyunsaturated long chain fatty acids (LCFA), nor did it change the affinity for intermediates in TG synthesis. Nevertheless, T94A substitution markedly altered the secondary structural response of L-FABP induced by binding LCFA or intermediates of TG synthesis. Finally, T94A substitution markedly diminished polyunsaturated fatty acid, eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), induction of peroxisome proliferator-activated receptor alpha (PPARα) - regulated proteins such as L-FABP, fatty acid transport protein 5 (FATP5), and PPARα itself in cultured primary human hepatocytes. Thus, while T94A substitution did not alter the affinity of human L-FABP for LCFAs, it significantly altered human L-FABP structure and stability as well as conformational and functional response to these ligands. PMID:24628888

Effects of Whey Protein Hydrolysate Ingestion on Postprandial Aminoacidemia Compared with a Free Amino Acid Mixture in Young Men

PubMed Central

Nakayama, Kyosuke; Sanbongi, Chiaki; Ikegami, Shuji

2018-01-01

To stimulate muscle protein synthesis, it is important to increase the plasma levels of essential amino acids (EAA), especially leucine, by ingesting proteins. Protein hydrolysate ingestion can induce postprandial hyperaminoacidemia; however, it is unclear whether protein hydrolysate is associated with higher levels of aminoacidemia compared with a free amino acid mixture when both are ingested orally. We assessed the effects of whey protein hydrolysate (WPH) ingestion on postprandial aminoacidemia, especially plasma leucine levels, compared to ingestion of a free amino acid mixture. This study was an open-label, randomized, 4 × 4 Latin square design. After 12–15 h of fasting, 11 healthy young men ingested the WPH (3.3, 5.0, or 7.5 g of protein) or the EAA mixture (2.5 g). Blood samples were collected before ingestion and at time points from 10 to 120 min after ingestion, and amino acids, insulin, glucose and insulin-like growth factor-1 (IGF-1) concentrations in plasma were measured. Even though the EAA mixture and 5.0 g of the WPH contained similar amounts of EAA and leucine, the WPH was associated with significantly higher plasma EAA and leucine levels. These results suggest that the WPH can induce a higher level of aminoacidemia compared with a free amino acid mixture when both are ingested orally. PMID:29671767

Effects of Whey Protein Hydrolysate Ingestion on Postprandial Aminoacidemia Compared with a Free Amino Acid Mixture in Young Men.

PubMed

Nakayama, Kyosuke; Sanbongi, Chiaki; Ikegami, Shuji

2018-04-19

To stimulate muscle protein synthesis, it is important to increase the plasma levels of essential amino acids (EAA), especially leucine, by ingesting proteins. Protein hydrolysate ingestion can induce postprandial hyperaminoacidemia; however, it is unclear whether protein hydrolysate is associated with higher levels of aminoacidemia compared with a free amino acid mixture when both are ingested orally. We assessed the effects of whey protein hydrolysate (WPH) ingestion on postprandial aminoacidemia, especially plasma leucine levels, compared to ingestion of a free amino acid mixture. This study was an open-label, randomized, 4 × 4 Latin square design. After 12⁻15 h of fasting, 11 healthy young men ingested the WPH (3.3, 5.0, or 7.5 g of protein) or the EAA mixture (2.5 g). Blood samples were collected before ingestion and at time points from 10 to 120 min after ingestion, and amino acids, insulin, glucose and insulin-like growth factor-1 (IGF-1) concentrations in plasma were measured. Even though the EAA mixture and 5.0 g of the WPH contained similar amounts of EAA and leucine, the WPH was associated with significantly higher plasma EAA and leucine levels. These results suggest that the WPH can induce a higher level of aminoacidemia compared with a free amino acid mixture when both are ingested orally.

NPIDB: Nucleic acid-Protein Interaction DataBase.

PubMed

Kirsanov, Dmitry D; Zanegina, Olga N; Aksianov, Evgeniy A; Spirin, Sergei A; Karyagina, Anna S; Alexeevski, Andrei V

2013-01-01

The Nucleic acid-Protein Interaction DataBase (http://npidb.belozersky.msu.ru/) contains information derived from structures of DNA-protein and RNA-protein complexes extracted from the Protein Data Bank (3846 complexes in October 2012). It provides a web interface and a set of tools for extracting biologically meaningful characteristics of nucleoprotein complexes. The content of the database is updated weekly. The current version of the Nucleic acid-Protein Interaction DataBase is an upgrade of the version published in 2007. The improvements include a new web interface, new tools for calculation of intermolecular interactions, a classification of SCOP families that contains DNA-binding protein domains and data on conserved water molecules on the DNA-protein interface.

Modulation of GDP-fucose level for generating proteins with reduced rate of fucosylation (WO2010141855).

PubMed

Taupin, Philippe

2011-09-01

The application (WO2010141855) is in the field of glycobiology, and involves the control of the rate of fucosylation of proteins by exogenous factors. It aims at controlling the rate of protein fucosylation with inhibitors (drugs or nucleic acid antagonists) of enzymes involved in the synthesis of GDP-fucose. Mammalian cell lines were cultured in the presence of inhibitors, for example, siRNA. The rates of GDP-fucose in cells and during protein fucosylation were characterized. The level of protein fucosylation decreases rapidly in response to a decrease in GDP-fucose level. The relationship between the rate of fucosylation of proteins and the level of GDP-fucose in a cell is non-linear. Reduction in the rate of protein fucosylation can be achieved with a minimal reduction of the level of GDP-fucose in cells. The paradigm may be used to synthesize proteins and antibodies, with a reduced rate of fucosylation. The application claims that the use of drugs or nucleic acid antagonists that inhibit the enzymes involved in GDP-fucose biosynthesis optimizes the level of GDP-fucose present in cells, and reduces the rate of fucosylation of glycoproteins.

Glutamic Acid as a Precursor to N-Terminal Pyroglutamic Acid in Mouse Plasmacytoma Protein

PubMed Central

Twardzik, Daniel R.; Peterkofsky, Alan

1972-01-01

Cell suspensions derived from a mouse plasmacytoma (RPC-20) that secretes an immunoglobulin light chain containing N-terminal pyroglutamic acid can synthesize protein in vitro. Chromatographic examination of an enzymatic digest of protein labeled with glutamic acid shows only labeled glutamic acid and pyroglutamic acid; hydrolysis of protein from cells labeled with glutamine, however, yields substantial amounts of glutamic acid in addition to glutamine and pyroglutamic acid. The absence of glutamine synthetase and presence of glutaminase in plasmacytoma homogenates is consistent with these findings. These data indicate that N-terminal pyroglutamic acid can be derived from glutamic acid without prior conversion of glutamic acid to glutamine. Since free or bound forms of glutamine cyclize nonezymatically to pyroglutamate with ease, while glutamic acid does not, the data suggest that N-terminal pyroglutamic acid formation from glutamic acid is enzymatic rather than spontaneous. Images PMID:4400295

Automated protein hydrolysis delivering sample to a solid acid catalyst for amino acid analysis.

PubMed

Masuda, Akiko; Dohmae, Naoshi

2010-11-01

In this study, we developed an automatic protein hydrolysis system using strong cation-exchange resins as solid acid catalysts. Examining several kinds of inorganic solid acids and cation-exchange resins, we found that a few cation-exchange resins worked as acid catalysts for protein hydrolysis when heated in the presence of water. The most efficient resin yielded amounts of amino acids that were over 70% of those recovered after conventional hydrolysis with hydrochloric acid and resulted in amino acid compositions matching the theoretical values. The solid-acid hydrolysis was automated by packing the resin into columns, combining the columns with a high-performance liquid chromatography system, and heating them. The amino acids that constitute a protein can thereby be determined, minimizing contamination from the environment.

Trends of amino acid usage in the proteins from the unicellular parasite Giardia lamblia.

PubMed

Garat, B; Musto, H

2000-12-29

Correspondence analysis of amino acid frequencies was applied to 75 complete coding sequences from the unicellular parasite Giardia lamblia, and it was found that three major factors influence the variability of amino acidic composition of proteins. The first trend strongly correlated with (a) the cysteine content and (b) the mean weight of the amino acids used in each protein. The second trend correlated with the global levels of hydropathy and aromaticity of each protein. Both axes might be related with the defense of the parasite to oxygen free radicals. Finally, the third trend correlated with the expressivity of each gene, indicating that in G. lamblia highly expressed sequences display a tendency to preferentially use a subset of the total amino acids.

Serum levels of Glial fibrillary acidic protein in Chinese children with autism spectrum disorders.

PubMed

Wang, Jingwei; Zou, Qiuyan; Han, Renfeng; Li, Yupeng; Wang, Yulin

2017-04-01

Glial fibrillary acidic protein (GFAP) has been studied in many neurological diseases. The purpose of this study is to investigate the potential role of GFAP in Chinese children with autism spectrum disorders (ASD) by measuring serum circulating levels of GFAP and comparing them with age and gender-matched typical development children. A total of one hundred and fifty 2-6 years old Chinese children (75 confirmed autism cases and 75 their age-gender matched typical development children) participated in this study. Serum levels of GFAP were assayed with enzyme-linked immunosorbent assay methods, and severity of ASD was evaluated with the Childhood Autism Rating Scale (CARS) Score. The results indicated that the mean serum GFAP level was significantly (P<0.001) higher in autistic children as compared to controls (1.71±0.53ng/ml vs. 0.99±0.25ng/ml). There was a significant positive association between serum GFAP levels and CARS scores (r [Pearson]=0.390, P=0.001). Based on the Receiver operating characteristic (ROC) curve, the optimal cut-off value of serum GFAP levels as an indicator for auxiliary diagnosis of autism was projected to be 1.28ng/ml which yielded a sensitivity of 77.3% and a specificity of 88.4%, the area under the curve was 0.895(95%CI, 0.844-0.947). Further, an increased risk of ASD was associated with GFAP levels >1.28ng/ml (adjusted OR 9.88, 95% CI: 3.32-17.82) in the multivariate logistic analysis model. The data indicates that serum GFAP levels may be associated with severity of ASD among Chinese children, suggesting the hypothesis that increased serum levels of GFAP could be implicated in the pathophysiology of autism in Chinese children. Copyright © 2017 ISDN. Published by Elsevier Ltd. All rights reserved.

Fatty Acid Binding Protein 5 Modulates Docosahexaenoic Acid-Induced Recovery in Rats Undergoing Spinal Cord Injury

PubMed Central

Figueroa, Johnny D.; Serrano-Illan, Miguel; Licero, Jenniffer; Cordero, Kathia; Miranda, Jorge D.

2016-01-01

Abstract Omega-3 polyunsaturated fatty acids (n-3 PUFAs) promote functional recovery in rats undergoing spinal cord injury (SCI). However, the precise molecular mechanism coupling n-3 PUFAs to neurorestorative responses is not well understood. The aim of the present study was to determine the spatiotemporal expression of fatty acid binding protein 5 (FABP5) after contusive SCI and to investigate whether this protein plays a role in n-3 PUFA–mediated functional recovery post-SCI. We found that SCI resulted in a robust spinal cord up-regulation in FABP5 mRNA levels (556 ± 187%) and protein expression (518 ± 195%), when compared to sham-operated rats, at 7 days post-injury (dpi). This upregulation coincided with significant alterations in the metabolism of fatty acids in the injured spinal cord, as revealed by metabolomics-based lipid analyses. In particular, we found increased levels of the n-3 series PUFAs, particularly docosahexaenoic acid (DHA; 22:6 n-3) and eicosapentaenoic acid (EPA; 20:5 n-3) at 7 dpi. Animals consuming a diet rich in DHA and EPA exhibited a significant upregulation in FABP5 mRNA levels at 7 dpi. Immunofluorescence showed low basal FABP5 immunoreactivity in spinal cord ventral gray matter NeuN+ neurons of sham-operated rats. SCI resulted in a robust induction of FABP5 in glial (GFAP+, APC+, and NG2+) and precursor cells (DCX+, nestin+). We found that continuous intrathecal administration of FABP5 silencing with small interfering RNA (2 μg) impaired spontaneous open-field locomotion post-SCI. Further, FABP5 siRNA administration hindered the beneficial effects of DHA to ameliorate functional recovery at 7 dpi. Altogether, our findings suggest that FABP5 may be an important player in the promotion of cellular uptake, transport, and/or metabolism of DHA post-SCI. Given the beneficial roles of n-3 PUFAs in ameliorating functional recovery, we propose that FABP5 is an important contributor to basic repair mechanisms in the

Effects of alpha-lipoic acid supplementation on C-reactive protein level: A systematic review and meta-analysis of randomized controlled clinical trials.

PubMed

Saboori, S; Falahi, E; Eslampour, E; Zeinali Khosroshahi, M; Yousefi Rad, E

2018-04-17

The aim of this meta-analysis was to assess effects of alpha-lipoic acid supplementation on C-reactive protein (CRP) levels in clinical trial studies. A systematic search was carried out on clinical trial studies published in PubMed, ISI Web of Science, Cochrane Library and Scopus databases completed by manual search on reference list of eligible studies accomplished by November 4, 2017. Of a total number of 508 studies found in the first step of literature search, only 11 were included with 264 participants in supplementation groups and 287 in control groups. Estimated pooled random effects size analysis showed a significant reducing effect of alpha-lipoic acid supplementation on CRP level (-0.72 mg/l, 95% CI; -1.4, -0.04; P = 0.03) with a significant heterogeneity between the selected studies. Sub-group analysis showed that alpha-lipoic acid supplementation could significantly reduce serum CRP level when the baseline CRP level was greater than 3 mg/l (-1.02 mg/l, 95% CI: -1.3, -0.73) and when trial duration was >8 weeks (-0.99 mg/l, 95% CI: -1.29, -0.70). Results of subgroup analysis also showed that alpha lipoic acid supplementation could decrease CRP level only in non-diabetic patients (-1.02 mg/l, 95% CI: -1.31, -0.74). Results of the current meta-analysis study showed that alpha-lipoic acid supplementation could significantly decrease CRP level in patients with elevated levels of this inflammatory marker. Copyright © 2018 The Italian Society of Diabetology, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition, and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.

Intake of branched-chain amino acids influences the levels of MAFbx mRNA and MuRF-1 total protein in resting and exercising human muscle.

PubMed

Borgenvik, Marcus; Apró, William; Blomstrand, Eva

2012-03-01

Resistance exercise and amino acids are two major factors that influence muscle protein turnover. Here, we examined the effects of resistance exercise and branched-chain amino acids (BCAA), individually and in combination, on the expression of anabolic and catabolic genes in human skeletal muscle. Seven subjects performed two sessions of unilateral leg press exercise with randomized supplementation with BCAA or flavored water. Biopsies were collected from the vastus lateralis muscle of both the resting and exercising legs before and repeatedly after exercise to determine levels of mRNA, protein phosphorylation, and amino acid concentrations. Intake of BCAA reduced (P < 0.05) MAFbx mRNA by 30 and 50% in the resting and exercising legs, respectively. The level of MuRF-1 mRNA was elevated (P < 0.05) in the exercising leg two- and threefold under the placebo and BCAA conditions, respectively, whereas MuRF-1 total protein increased by 20% (P < 0.05) only in the placebo condition. Phosphorylation of p70(S6k) increased to a larger extent (∼2-fold; P < 0.05) in the early recovery period with BCAA supplementation, whereas the expression of genes regulating mTOR activity was not influenced by BCAA. Muscle levels of phenylalanine and tyrosine were reduced (13-17%) throughout recovery (P < 0.05) in the placebo condition and to a greater extent (32-43%; P < 0.05) following BCAA supplementation in both resting and exercising muscle. In conclusion, BCAA ingestion reduced MAFbx mRNA and prevented the exercise-induced increase in MuRF-1 total protein in both resting and exercising leg. Further-more, resistance exercise differently influenced MAFbx and MuRF-1 mRNA expression, suggesting both common and divergent regulation of these two ubiquitin ligases.

Effect of temperature and concentration on benzoyl peroxide bleaching efficacy and benzoic acid levels in whey protein concentrate.

PubMed

Smith, T J; Gerard, P D; Drake, M A

2015-11-01

Much of the fluid whey produced in the United States is a by-product of Cheddar cheese manufacture and must be bleached. Benzoyl peroxide (BP) is currently 1 of only 2 legal chemical bleaching agents for fluid whey in the United States, but benzoic acid is an unavoidable by-product of BP bleaching. Benzoyl peroxide is typically a powder, but new liquid BP dispersions are available. A greater understanding of the bleaching characteristics of BP is necessary. The objective of the study was to compare norbixin destruction, residual benzoic acid, and flavor differences between liquid whey and 80% whey protein concentrates (WPC80) bleached at different temperatures with 2 different benzoyl peroxides (soluble and insoluble). Two experiments were conducted in this study. For experiment 1, 3 factors (temperature, bleach type, bleach concentration) were evaluated for norbixin destruction using a response surface model-central composite design in liquid whey. For experiment 2, norbixin concentration, residual benzoic acid, and flavor differences were explored in WPC80 from whey bleached by the 2 commercially available BP (soluble and insoluble) at 5 mg/kg. In liquid whey, soluble BP bleached more norbixin than insoluble BP, especially at lower concentrations (5 and 10 mg/kg) at both cold (4°C) and hot (50°C) temperatures. The WPC80 from liquid whey bleached with BP at 50°C had lower norbixin concentration, benzoic acid levels, cardboard flavor, and aldehyde levels than WPC80 from liquid whey bleached with BP at 4°C. Regardless of temperature, soluble BP destroyed more norbixin at lower concentrations than insoluble BP. The WPC80 from soluble-BP-bleached wheys had lower cardboard flavor and lower aldehyde levels than WPC80 from insoluble-BP-bleached whey. This study suggests that new, soluble (liquid) BP can be used at lower concentrations than insoluble BP to achieve equivalent bleaching and that less residual benzoic acid remains in WPC80 powder from liquid whey

NMR studies of protein-nucleic acid interactions.

PubMed

Varani, Gabriele; Chen, Yu; Leeper, Thomas C

2004-01-01

Protein-DNA and protein-RNA complexes play key functional roles in every living organism. Therefore, the elucidation of their structure and dynamics is an important goal of structural and molecular biology. Nuclear magnetic resonance (NMR) studies of protein and nucleic acid complexes have common features with studies of protein-protein complexes: the interaction surfaces between the molecules must be carefully delineated, the relative orientation of the two species needs to be accurately and precisely determined, and close intermolecular contacts defined by nuclear Overhauser effects (NOEs) must be obtained. However, differences in NMR properties (e.g., chemical shifts) and biosynthetic pathways for sample productions generate important differences. Chemical shift differences between the protein and nucleic acid resonances can aid the NMR structure determination process; however, the relatively limited dispersion of the RNA ribose resonances makes the process of assigning intermolecular NOEs more difficult. The analysis of the resulting structures requires computational tools unique to nucleic acid interactions. This chapter summarizes the most important elements of the structure determination by NMR of protein-nucleic acid complexes and their analysis. The main emphasis is on recent developments (e.g., residual dipolar couplings and new Web-based analysis tools) that have facilitated NMR studies of these complexes and expanded the type of biological problems to which NMR techniques of structural elucidation can now be applied.

Studies on the protein and sulfur amino acid requirements of young bobwhite quail

USGS Publications Warehouse

Serafin, J.A.

1977-01-01

Four experiments were conducted with purified diets to examine the influence of protein level and to estimate the sulfur amino acid (S.A.A.) requirement of young Bobwhite quail (Colinus virginianus). These studies demonstrated (I) that 26% protein was sufficient for rapid growth when the diet was supplemented with methionine; (2) that diets containing higher levels of protein (29.3% and 31.3%) failed to support satisfactory growth unless they contained supplemental methionine; and (3) that young Bobwhite quail require no more than 1.0% sulfur-containing amino acids for optimal growth and efficiency of feed utilization. A fifth experiment was conducted to examine the protein and S.A.A. requirements of young Bobwhite quail using practical rations and to compare results with those obtained with purified diets. Diets containing 24%, 26% and 28% protein were supplied with and without supplemental methionine in a five week study. Results showed significant growth responses to protein and supplemental methionine. Responses showed that Bobwhite quail require no more than 26% protein for maximum growth and efficiency of feed utilization when the S.A.A. level of the diet was approximately 1.0%. The results were in close agreement with those obtained with purified diets. These findings define more precisely than had been known the quantitative requirements of young Bobwhite quail for protein and for the S.A.A. necessary for optimal growth.

Lower glutamic acid decarboxylase 65kD mRNA and protein levels in the prefrontal cortex in schizoaffective disorder but not schizophrenia

PubMed Central

Glausier, JR; Kimoto, S; Fish, KN; Lewis, DA

2014-01-01

Background Altered GABA signaling in the prefrontal cortex (PFC) has been associated with cognitive dysfunction in schizophrenia and schizoaffective disorder. PFC levels of the GABA-synthesizing enzyme glutamic acid decarboxylase 67kD (GAD67) has been consistently reported to be lower in these disorders, but the status of the second GABA-synthesizing enzyme, GAD65, remains unclear. Methods GAD65 mRNA levels were quantified in PFC area 9 by quantitative polymerase chain reaction from 62 subjects with schizophrenia or schizoaffective disorder and 62 matched healthy comparison subjects. GAD65 relative protein levels were quantified in a subset of subject pairs by confocal immunofluorescence microscopy. Results Mean GAD65 mRNA levels were 13.6% lower in schizoaffective disorder subjects, but did not differ in schizophrenia subjects, relative to their matched healthy comparison subjects. In the subjects with schizoaffective disorder, mean GAD65 protein levels were 19.4% lower and were correlated with GAD65 mRNA levels. Lower GAD65 mRNA and protein measures within schizoaffective disorder subjects was not attributable to factors commonly comorbid with the diagnosis. Conclusions In concert with previous studies, these findings suggest that schizoaffective disorder is associated with lower levels of both GAD65 and GAD67 mRNA and protein in the PFC, whereas subjects with schizophrenia have lower mean levels of only GAD67 mRNA and protein. Because cognitive function is generally better preserved in subjects with schizoaffective disorder relative to subjects with schizophrenia, these findings may support an interpretation that GAD65 down-regulation provides a homeostatic response complementary to GAD67 down-regulation expression that serves to reduce inhibition in the face of lower PFC network activity. PMID:24993056

Protein-membrane interaction and fatty acid transfer from intestinal fatty acid-binding protein to membranes. Support for a multistep process.

PubMed

Falomir-Lockhart, Lisandro J; Laborde, Lisandro; Kahn, Peter C; Storch, Judith; Córsico, Betina

2006-05-19

Fatty acid transfer from intestinal fatty acid-binding protein (IFABP) to phospholipid membranes occurs during protein-membrane collisions. Electrostatic interactions involving the alpha-helical "portal" region of the protein have been shown to be of great importance. In the present study, the role of specific lysine residues in the alpha-helical region of IFABP was directly examined. A series of point mutants in rat IFABP was engineered in which the lysine positive charges in this domain were eliminated or reversed. Using a fluorescence resonance energy transfer assay, we analyzed the rates and mechanism of fatty acid transfer from wild type and mutant proteins to acceptor membranes. Most of the alpha-helical domain mutants showed slower absolute fatty acid transfer rates to zwitterionic membranes, with substitution of one of the lysines of the alpha2 helix, Lys27, resulting in a particularly dramatic decrease in the fatty acid transfer rate. Sensitivity to negatively charged phospholipid membranes was also reduced, with charge reversal mutants in the alpha2 helix the most affected. The results support the hypothesis that the portal region undergoes a conformational change during protein-membrane interaction, which leads to release of the bound fatty acid to the membrane and that the alpha2 segment is of particular importance in the establishment of charge-charge interactions between IFABP and membranes. Cross-linking experiments with a phospholipid-photoactivable reagent underscored the importance of charge-charge interactions, showing that the physical interaction between wild-type intestinal fatty acid-binding protein and phospholipid membranes is enhanced by electrostatic interactions. Protein-membrane interactions were also found to be enhanced by the presence of ligand, suggesting different collisional complex structures for holo- and apo-IFABP.

Nucleic acid programmable protein array a just-in-time multiplexed protein expression and purification platform.

PubMed

Qiu, Ji; LaBaer, Joshua

2011-01-01

Systematic study of proteins requires the availability of thousands of proteins in functional format. However, traditional recombinant protein expression and purification methods have many drawbacks for such study at the proteome level. We have developed an innovative in situ protein expression and capture system, namely NAPPA (nucleic acid programmable protein array), where C-terminal tagged proteins are expressed using an in vitro expression system and efficiently captured/purified by antitag antibodies coprinted at each spot. The NAPPA technology presented in this chapter enable researchers to produce and display fresh proteins just in time in a multiplexed high-throughput fashion and utilize them for various downstream biochemical researches of interest. This platform could revolutionize the field of functional proteomics with it ability to produce thousands of spatially separated proteins in high density with narrow dynamic rand of protein concentrations, reproducibly and functionally. Copyright © 2011 Elsevier Inc. All rights reserved.

Seasonal changes in amino acids, protein and total nitrogen in needles of fertilized Scots pine trees.

PubMed

Näsholm, T; Ericsson, A

1990-09-01

Seasonal changes in amino acids, protein and total nitrogen in needles of 30-year-old, fertilized Scots pine (Pinus sylvestris L.) trees growing in Northern Sweden were investigated over two years in field experiments. The studied plots had been fertilized annually for 17 years with (i) a high level of N, (ii) a medium level of N, or (iii) a medium level of N, P and K. Trees growing on unfertilized plots served as controls. In control trees, glutamine, glutamic acid, gamma-aminobutyric acid, aspartic acid and proline represented 50-70% of the total free amino acids determined. Arginine was present only in low concentrations in control trees throughout the year, but it was usually the most abundant amino acid in fertilized trees. Glutamine concentrations were high during the spring and summer in both years of study, whereas proline concentrations were high in the spring but otherwise low throughout the year. In the first year of study, glutamic acid concentrations were high during the spring and summer, whereas gamma-aminobutyric acid was present in high concentrations during the winter months. This pattern was less pronounced in the second year of investigation. The concentrations of most amino acids, except glutamic acid, increased in response to fertilization. Nitrogen fertilization increased the foliar concentration of arginine from < 1 micromol g(dw) (-1) in control trees to a maximum of 110 micromol g(dw) (-1). Trees fertilized with nitrogen, phosphorus and potassium had significantly lower arginine concentrations than trees fertilized with the same amount of nitrogen only. Protein concentrations were similar in all fertilized trees but higher than those in control trees. For all treatments, protein concentrations were high in winter and at a minimum in early spring. In summer, the protein concentration remained almost constant except for a temporary decrease which coincided with the expansion of new shoots. Apart from arginine, the amino acid composition of

A fish protein hydrolysate alters fatty acid composition in liver and adipose tissue and increases plasma carnitine levels in a mouse model of chronic inflammation.

PubMed

Bjørndal, Bodil; Berge, Christ; Ramsvik, Marie Sannes; Svardal, Asbjørn; Bohov, Pavol; Skorve, Jon; Berge, Rolf K

2013-10-07

There is growing evidence that fish protein hydrolysate (FPH) diets affect mitochondrial fatty acid metabolism in animals. The aim of the study was to determine if FPH could influence fatty acid metabolism and inflammation in transgene mice expressing human tumor necrosis factor alpha (hTNFα). hTNFα mice (C57BL/6 hTNFα) were given a high-fat (23%, w/w) diet containing 20% casein (control group) or 15% FPH and 5% casein (FPH group) for two weeks. After an overnight fast, blood, adipose tissue, and liver samples were collected. Gene expression and enzyme activity was analysed in liver, fatty acid composition was analyzed in liver and ovarian white adipose tissue, and inflammatory parameters, carnitine, and acylcarnitines were analyzed in plasma. The n-3/n-6 fatty acid ratio was higher in mice fed the FPH diet than in mice fed the control diet in both adipose tissue and liver, and the FPH diet affected the gene expression of ∆6 and ∆9 desaturases. Mice fed this diet also demonstrated lower hepatic activity of fatty acid synthase. Concomitantly, a lower plasma INF-γ level was observed. Plasma carnitine and the carnitine precursor γ-butyrobetaine was higher in the FPH-group compared to control, as was plasma short-chained and medium-chained acylcarnitine esters. The higher level of plasma acetylcarnitine may reflect a stimulated mitochondrial and peroxisomal β-oxidation of fatty acids, as the hepatic activities of peroxisomal acyl-CoA oxidase 1 and mitochondrial carnitine palmitoyltransferase-II were higher in the FPH-fed mice. The FPH diet was shown to influence hepatic fatty acid metabolism and fatty acid composition. This indicates that effects on fatty acid metabolism are important for the bioactivity of protein hydrolysates of marine origin.

Studies on fatty acid-binding proteins. The detection and quantification of the protein from rat liver by using a fluorescent fatty acid analogue.

PubMed Central

Wilkinson, T C; Wilton, D C

1986-01-01

Fatty acid-binding protein from rat liver is shown to bind the fluorescent fatty acid probe dansyl undecanoic acid. Binding is accompanied by a shift in the fluorescence emission maximum from 550 nm to 500 nm and a 60-fold fluorescence enhancement at 500 nm. These spectral properties have allowed the use of this probe to detect and quantify microgram amounts of liver fatty acid-binding protein during purification procedures. In conjunction with h.p.l.c. the method allows the rapid estimation of liver fatty acid-binding protein in biological samples. The validity of the method is demonstrated by measuring the concentration of fatty acid-binding protein in livers from control and hypolipidaemic-drug-treated rats. The dramatic diurnal rhythm previously reported for this protein [Dempsey (1984) Curr. Top. Cell. Regul. 24, 63-86] was not observed with this method. Images Fig. 1. PMID:3800946

Effect of experimental diabetes on the levels of aromatic and branched-chain amino acids in rat blood and brain.

PubMed

Crandall, E A; Fernstrom, J D

1983-03-01

Male rats treated 3 wk earlier with streptozotocin showed abnormally high blood levels of leucine, isoleucine, and valine throughout the 24-h period. Serum phenylalanine levels were slightly increased, while those of tryptophan and tyrosine were occasionally reduced. In brain, the level of each branched-chain amino acid was significantly increased above normal at all times. The brain concentration of each aromatic amino acid was always below normal. These changes were restored almost to normal by exogenous insulin therapy. Since the ingestion of protein is normally a major factor influencing blood amino acid levels, the effect of ingesting single, protein-containing meals on the blood and brain levels of these amino acids was also studied. After an overnight fast, the ingestion of a protein-containing meal by diabetic rats increased substantially both blood and brain levels of each branched-chain amino acid. No such increases occurred in normal rats. Ingestion of this meal produced only small changes in the brain and blood levels of the aromatic amino acids in both diabetic and normal rats. The changes in the brain level of each large neutral amino acid in some cases paralleled those in its blood level. More often, they paralleled the changes in the blood ratio of each amino acid to the sum of the other aromatic and branched-chain amino acids. This ratio is often a good predictor of the competitive transport of these amino acids into brain (Fernstrom and Faller, 1978). The observed changes in the brain levels of these amino acids in diabetes may influence the rates at which they are consumed in metabolic pathways within this organ.

Suppression of muscle protein turnover and amino acid degradation by dietary protein deficiency

NASA Technical Reports Server (NTRS)

Tawa, N. E. Jr; Goldberg, A. L.

1992-01-01

To define the adaptations that conserve amino acids and muscle protein when dietary protein intake is inadequate, rats (60-70 g final wt) were fed a normal or protein-deficient (PD) diet (18 or 1% lactalbumin), and their muscles were studied in vitro. After 7 days on the PD diet, both protein degradation and synthesis fell 30-40% in skeletal muscles and atria. This fall in proteolysis did not result from reduced amino acid supply to the muscle and preceded any clear decrease in plasma amino acids. Oxidation of branched-chain amino acids, glutamine and alanine synthesis, and uptake of alpha-aminoisobutyrate also fell by 30-50% in muscles and adipose tissue of PD rats. After 1 day on the PD diet, muscle protein synthesis and amino acid uptake decreased by 25-40%, and after 3 days proteolysis and leucine oxidation fell 30-45%. Upon refeeding with the normal diet, protein synthesis also rose more rapidly (+30% by 1 day) than proteolysis, which increased significantly after 3 days (+60%). These different time courses suggest distinct endocrine signals for these responses. The high rate of protein synthesis and low rate of proteolysis during the first 3 days of refeeding a normal diet to PD rats contributes to the rapid weight gain ("catch-up growth") of such animals.

Olive oils modulate fatty acid content and signaling protein expression in apolipoprotein E knockout mice brain.

PubMed

Alemany, Regina; Navarro, María A; Vögler, Oliver; Perona, Javier S; Osada, Jesús; Ruiz-Gutiérrez, Valentina

2010-01-01

Atherosclerosis contributes to disruption of neuronal signaling pathways by producing lipid-dependent modifications of brain plasma membranes, neuroinflammation and oxidative stress. We investigated whether long-term (11 weeks) consumption of refined- (ROO) and pomace- (POO) olive oil modulated the fatty acid composition and the levels of membrane signaling proteins in the brain of apolipoprotein E (apoE) knockout (KO) mice, an animal model of atherosclerosis. Both of these oils are rich in bioactive molecules with anti-inflammatory and antioxidant effects. ROO and POO long-term consumption increased the proportion of monounsaturated fatty acids (MUFAs), particularly of oleic acid, while reducing the level of the saturated fatty acids (SFAs) palmitic and stearic acid. As a result, the MUFA:SFA ratio was higher in apoE KO mice brain fed with ROO and POO. Furthermore, both oils reduced the level of arachidonic and eicosapentaenoic acid, suggesting a decrease in the generation of pro- and anti-inflammatory eicosanoids. Finally, ROO and POO induced an increase in the density of membrane proteins implicated in both the Galphas/PKA and Galphaq/PLCbeta1/PKCalpha signaling pathways. The combined effects of long-term ROO and POO consumption on fatty acid composition and the level of signaling proteins involved in PKA and PKC activation, suggest positive effects on neuroinflammation and brain function in apoE KO mice brain, and convert these oils into promising functional foods in diseases involving apoE deficiency.

Protein-bound D-amino acids, and to a lesser extent lysinoalanine, decrease true ileal protein digestibility in minipigs as determined with (15)N-labeling.

PubMed

de Vrese, M; Frik, R; Roos, N; Hagemeister, H

2000-08-01

Heat and alkali treatment of food may increase the concentrations of protein-bound D-amino acids and cross-links such as lysinoalanine (LAL). To examine how protein treatment affects digestibility, purified test meals [total protein 150 g/kg dry matter (DM), 0.44 MJ/(kg BW(0.75). d)] were prepared, containing (g/kg DM) casein, 75; beta-lactoglobulin, 50; or wheat protein, 40. Each was (15)N-labeled. Test proteins were used either in their native form or after treatment for 6 or 24 h at 65 degrees C, pH 10.5-11.5. Each meal was fed to nine adult miniature pigs (twofold complete cross-classification). Chyme was collected continuously over 33 h postprandially via T-fistulas in the terminal ileum, and digestibilities of test proteins and individual L- and D-amino acids were calculated on the basis of recovery of (15)N and the respective amino acids in the chyme. Treatment of casein, beta-lactoglobulin or wheat protein for 24 h increased levels of D-amino acid residues. L-Asparagine and aspartate (L-Asx) were particularly susceptible; 14. 7 +/- 0.4, 11.7 +/- 0.2 and 11.0 +/- 0.9%, respectively, underwent racemization. LAL levels increased in parallel; 11.7 +/- 0.3, 13.6 +/- 0 and 14.8 +/- 0.0%, respectively, of total lysine was converted to LAL. At the same time, prececal protein digestibility was decreased by 13.4 +/- 2.3, 15.3 +/- 1.4 and 17.8 +/- 1.2% units, respectively (P < 0.05; mean +/- SEM, n = 9). Digestibility of individual L-amino acids decreased by 10-15%, but L-amino acids prone to peptic cleavage, such as L-phenylalanine and L-tyrosine, were not affected. Digestibilities of D-amino acids and LAL were approximately 35%. It seems that mainly D-amino acids, and to a lesser extent LAL, were responsible for lower digestibility by interfering with peptic cleavage.

Long-Term Intake of Uncaria rhynchophylla Reduces S100B and RAGE Protein Levels in Kainic Acid-Induced Epileptic Seizures Rats

PubMed Central

Tang, Nou-Ying; Ho, Tin-Yun; Chen, Chao-Hsiang

2017-01-01

Epileptic seizures are crucial clinical manifestations of recurrent neuronal discharges in the brain. An imbalance between the excitatory and inhibitory neuronal discharges causes brain damage and cell loss. Herbal medicines offer alternative treatment options for epilepsy because of their low cost and few side effects. We established a rat epilepsy model by injecting kainic acid (KA, 12 mg/kg, i.p.) and subsequently investigated the effect of Uncaria rhynchophylla (UR) and its underlying mechanisms. Electroencephalogram and epileptic behaviors revealed that the KA injection induced epileptic seizures. Following KA injection, S100B levels increased in the hippocampus. This phenomenon was attenuated by the oral administration of UR and valproic acid (VA, 250 mg/kg). Both drugs significantly reversed receptor potentiation for advanced glycation end product proteins. Rats with KA-induced epilepsy exhibited no increase in the expression of metabotropic glutamate receptor 3, monocyte chemoattractant protein 1, and chemokine receptor type 2, which play a role in inflammation. Our results provide novel and detailed mechanisms, explaining the role of UR in KA-induced epileptic seizures in hippocampal CA1 neurons. PMID:28386293

Long-Term Intake of Uncaria rhynchophylla Reduces S100B and RAGE Protein Levels in Kainic Acid-Induced Epileptic Seizures Rats.

PubMed

Tang, Nou-Ying; Lin, Yi-Wen; Ho, Tin-Yun; Cheng, Chin-Yi; Chen, Chao-Hsiang; Hsieh, Ching-Liang

2017-01-01

Epileptic seizures are crucial clinical manifestations of recurrent neuronal discharges in the brain. An imbalance between the excitatory and inhibitory neuronal discharges causes brain damage and cell loss. Herbal medicines offer alternative treatment options for epilepsy because of their low cost and few side effects. We established a rat epilepsy model by injecting kainic acid (KA, 12 mg/kg, i.p.) and subsequently investigated the effect of Uncaria rhynchophylla (UR) and its underlying mechanisms. Electroencephalogram and epileptic behaviors revealed that the KA injection induced epileptic seizures. Following KA injection, S100B levels increased in the hippocampus. This phenomenon was attenuated by the oral administration of UR and valproic acid (VA, 250 mg/kg). Both drugs significantly reversed receptor potentiation for advanced glycation end product proteins. Rats with KA-induced epilepsy exhibited no increase in the expression of metabotropic glutamate receptor 3, monocyte chemoattractant protein 1, and chemokine receptor type 2, which play a role in inflammation. Our results provide novel and detailed mechanisms, explaining the role of UR in KA-induced epileptic seizures in hippocampal CA1 neurons.

Comparison of Bile Acids and Acetaminophen Protein Adducts in Children and Adolescents with Acetaminophen Toxicity.

PubMed

James, Laura; Yan, Ke; Pence, Lisa; Simpson, Pippa; Bhattacharyya, Sudeepa; Gill, Pritmohinder; Letzig, Lynda; Kearns, Gregory; Beger, Richard

2015-01-01

Metabolomics approaches have enabled the study of new mechanisms of liver injury in experimental models of drug toxicity. Disruption of bile acid homeostasis is a known mechanism of drug induced liver injury. The relationship of individual bile acids to indicators of oxidative drug metabolism (acetaminophen protein adducts) and liver injury was examined in children with acetaminophen overdose, hospitalized children with low dose exposure to acetaminophen, and children with no recent exposure to acetaminophen. Nine bile acids were quantified through targeted metabolomic analysis in the serum samples of the three groups. Bile acids were compared to serum levels of acetaminophen protein adducts and alanine aminotransferase. Glycodeoxycholic acid, taurodeoxycholic acid, and glycochenodeoxycholic acid were significantly increased in children with acetaminophen overdose compared to healthy controls. Among patients with acetaminophen overdose, bile acids were higher in subjects with acetaminophen protein adduct values > 1.0 nmol/mL and modest correlations were noted for three bile acids and acetaminophen protein adducts as follows: taurodeoxycholic acid (R=0.604; p<0.001), glycodeoxycholic acid (R=0.581; p<0.001), and glycochenodeoxycholic acid (R=0.571; p<0.001). Variability in bile acids was greater among hospitalized children receiving low doses of acetaminophen than in healthy children with no recent acetaminophen exposure. Compared to bile acids, acetaminophen protein adducts more accurately discriminated among children with acetaminophen overdose, children with low dose exposure to acetaminophen, and healthy control subjects. In children with acetaminophen overdose, elevations of conjugated bile acids were associated with specific indicators of acetaminophen metabolism and non-specific indicators of liver injury.

Meal composition and plasma amino acid ratios: Effect of various proteins or carbohydrates, and of various protein concentrations

NASA Technical Reports Server (NTRS)

Yokogoshi, Hidehiko; Wurtman, Richard J.

1986-01-01

The effects of meals containing various proteins and carbohydrates, and of those containing various proportions of protein (0 percent to 20 percent of a meal, by weight) or of carbohydrate (0 percent to 75 percent), on plasma levels of certain large neutral amino acids (LNAA) in rats previously fasted for 19 hours were examined. Also the plasma tryptophan ratios (the ratio of the plasma trytophan concentration to the summed concentrations of the other large neutral amino acids) and other plasma amino acid ratios were calculated. (The plasma tryptophan ratio has been shown to determine brain tryptophan levels and, thereby, to affect the synthesis and release of the neurotransmitter serotonin). A meal containing 70 percent to 75 percent of an insulin-secreting carbohydrate (dextrose or dextrin) increased plasma insulin levels and the tryptophan ratio; those containing 0 percent or 25 percent carbohydrate failed to do so. Addition of as little as 5 percent casein to a 70 percent carbohydrate meal fully blocked the increase in the plasma tryptophan ratio without affecting the secretion of insulin - probably by contributing much larger quantities of the other LNAA than of tryptophan to the blood. Dietary proteins differed in their ability to suppress the carbohydrate-induced rise in the plasma tryptophan ratio. Addition of 10 percent casein, peanut meal, or gelatin fully blocked this increase, but lactalbumin failed to do so, and egg white did so only partially. (Consumption of the 10 percent gelatin meal also produced a major reduction in the plasma tyrosine ratio, and may thereby have affected brain tyrosine levels and catecholamine synthesis.) These observations suggest that serotonin-releasing neurons in brains of fasted rats are capable of distinguishing (by their metabolic effects) between meals poor in protein but rich in carbohydrates that elicit insulin secretion, and all other meals. The changes in brain serotonin caused by carbohydrate-rich, protein

Sunflower (Helianthus annuus) fatty acid synthase complex: β-hydroxyacyl-[acyl carrier protein] dehydratase genes.

PubMed

González-Thuillier, Irene; Venegas-Calerón, Mónica; Sánchez, Rosario; Garcés, Rafael; von Wettstein-Knowles, Penny; Martínez-Force, Enrique

2016-02-01

Two sunflower hydroxyacyl-[acyl carrier protein] dehydratases evolved into two different isoenzymes showing distinctive expression levels and kinetics' efficiencies. β-Hydroxyacyl-[acyl carrier protein (ACP)]-dehydratase (HAD) is a component of the type II fatty acid synthase complex involved in 'de novo' fatty acid biosynthesis in plants. This complex, formed by four intraplastidial proteins, is responsible for the sequential condensation of two-carbon units, leading to 16- and 18-C acyl-ACP. HAD dehydrates 3-hydroxyacyl-ACP generating trans-2-enoyl-ACP. With the aim of a further understanding of fatty acid biosynthesis in sunflower (Helianthus annuus) seeds, two β-hydroxyacyl-[ACP] dehydratase genes have been cloned from developing seeds, HaHAD1 (GenBank HM044767) and HaHAD2 (GenBank GU595454). Genomic DNA gel blot analyses suggest that both are single copy genes. Differences in their expression patterns across plant tissues were detected. Higher levels of HaHAD2 in the initial stages of seed development inferred its key role in seed storage fatty acid synthesis. That HaHAD1 expression levels remained constant across most tissues suggest a housekeeping function. Heterologous expression of these genes in E. coli confirmed both proteins were functional and able to interact with the bacterial complex 'in vivo'. The large increase of saturated fatty acids in cells expressing HaHAD1 and HaHAD2 supports the idea that these HAD genes are closely related to the E. coli FabZ gene. The proposed three-dimensional models of HaHAD1 and HaHAD2 revealed differences at the entrance to the catalytic tunnel attributable to Phe166/Val1159, respectively. HaHAD1 F166V was generated to study the function of this residue. The 'in vitro' enzymatic characterization of the three HAD proteins demonstrated all were active, with the mutant having intermediate K m and V max values to the wild-type proteins.

Designing medical foods for inherited metabolic disorders: why intact protein is superior to amino acids.

PubMed

Ney, Denise Marie; Etzel, Mark Raymond

2017-04-01

Phenylketonuria and tyrosinemia are inherited metabolic disorders characterized by high blood levels of phenylalanine (Phe) or tyrosine (Tyr), due to mutations in genes affecting Phe and Tyr metabolism, respectively. The primary management is a lifelong diet restricted in protein from natural foods in combination with medical foods comprised mixtures of synthetic amino acids. Compliance is often poor after childhood leading to neuropsychological sequela. Glycomacropeptide, an intact 64 amino acid glycophosphopeptide isolated from cheese whey, provides a new paradigm for the management of phenylketonuria and tyrosinemia because glycomacropeptide contains no Phe and Tyr in its pure form, and is also a prebiotic. Medical foods made from glycomacropeptide have been used successfully for the management of phenylketonuria and tyrosinemia. Preclinical and clinical studies demonstrate that intact protein from glycomacropeptide provides a more acceptable and physiologic source of defined protein compared to amino acids in medical foods. For example, harmful gut bacteria were reduced, beneficial short chain fatty acids increased, renal workload decreased, protein utilization increased, and bone fragility decreased using intact protein versus amino acids. Advances in biotechnology will propel the transition from synthetic amino acids to intact proteins for the management of inherited metabolic disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

Echinococcus granulosus fatty acid binding proteins subcellular localization.

PubMed

Alvite, Gabriela; Esteves, Adriana

2016-05-01

Two fatty acid binding proteins, EgFABP1 and EgFABP2, were isolated from the parasitic platyhelminth Echinococcus granulosus. These proteins bind fatty acids and have particular relevance in flatworms since de novo fatty acids synthesis is absent. Therefore platyhelminthes depend on the capture and intracellular distribution of host's lipids and fatty acid binding proteins could participate in lipid distribution. To elucidate EgFABP's roles, we investigated their intracellular distribution in the larval stage by a proteomic approach. Our results demonstrated the presence of EgFABP1 isoforms in cytosolic, nuclear, mitochondrial and microsomal fractions, suggesting that these molecules could be involved in several cellular processes. Copyright © 2016 Elsevier Inc. All rights reserved.

Non-protein amino acids in Australian acacia seed: implications for food security and recommended processing methods to reduce djenkolic acid.

PubMed

Boughton, Berin A; Reddy, Priyanka; Boland, Martin P; Roessner, Ute; Yates, Peter

2015-07-15

Seed of Australian acacia species, Acacia colei, Acacia elecantha, Acacia torulosa, Acacia turmida and Acacia saligna, were analysed for the presence of toxic non-protein amino acids and the levels of essential amino acids. Amines were derivatised with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate before analysis using liquid chromatography electrospray ionisation triple quadrupole mass spectrometry (LC-ESI-QQQ-MS). Multiple reaction monitoring (MRM) with optimised transitions and collision energies for each analyte were employed. The known nephrotoxic compound djenkolic acid was found to be present at elevated levels in all species tested. The lowest levels were in A. colei (0.49% w/w) and the highest in A. saligna (1.85% w/w). Observed levels of djenkolic acid are comparable to measured and reported levels found in the djenkol bean. Subsequent testing of seed processing methods showed djenkolic acid levels can be significantly reduced by over 90% by dry roasting at 180 °C rendering the seed safe for human consumption. Copyright © 2015 Elsevier Ltd. All rights reserved.

A dominant conformational role for amino acid diversity in minimalist protein–protein interfaces

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gilbreth, Ryan N.; Esaki, Kaori; Koide, Akiko

Recent studies have shown that highly simplified interaction surfaces consisting of combinations of just two amino acids, Tyr and Ser, exhibit high affinity and specificity. The high functional levels of such minimalist interfaces might thus indicate small contributions of greater amino acid diversity seen in natural interfaces. Toward addressing this issue, we have produced a pair of binding proteins built on the fibronectin type III scaffold, termed “monobodies.” One monobody contains the Tyr/Ser binary-code interface (termed YS) and the other contains an expanded amino acid diversity interface (YSX), but both bind to an identical target, maltose-binding protein. The YSX monobodymore » bound with higher affinity, a slower off rate and a more favorable enthalpic contribution than the YS monobody. High-resolution X-ray crystal structures revealed that both proteins bound to an essentially identical epitope, providing a unique opportunity to directly investigate the role of amino acid diversity in a protein interaction interface. Surprisingly, Tyr still dominates the YSX paratope and the additional amino acid types are primarily used to conformationally optimize contacts made by tyrosines. Scanning mutagenesis showed that while all contacting Tyr side chains are essential in the YS monobody, the YSX interface was more tolerant to mutations. These results suggest that the conformational, not chemical, diversity of additional types of amino acids provided higher functionality and evolutionary robustness, supporting the dominant role of Tyr and the importance of conformational diversity in forming protein interaction interfaces.« less

Mapping Hydrophobicity on the Protein Molecular Surface at Atom-Level Resolution

PubMed Central

Nicolau Jr., Dan V.; Paszek, Ewa; Fulga, Florin; Nicolau, Dan V.

2014-01-01

A precise representation of the spatial distribution of hydrophobicity, hydrophilicity and charges on the molecular surface of proteins is critical for the understanding of the interaction with small molecules and larger systems. The representation of hydrophobicity is rarely done at atom-level, as this property is generally assigned to residues. A new methodology for the derivation of atomic hydrophobicity from any amino acid-based hydrophobicity scale was used to derive 8 sets of atomic hydrophobicities, one of which was used to generate the molecular surfaces for 35 proteins with convex structures, 5 of which, i.e., lysozyme, ribonuclease, hemoglobin, albumin and IgG, have been analyzed in more detail. Sets of the molecular surfaces of the model proteins have been constructed using spherical probes with increasingly large radii, from 1.4 to 20 Å, followed by the quantification of (i) the surface hydrophobicity; (ii) their respective molecular surface areas, i.e., total, hydrophilic and hydrophobic area; and (iii) their relative densities, i.e., divided by the total molecular area; or specific densities, i.e., divided by property-specific area. Compared with the amino acid-based formalism, the atom-level description reveals molecular surfaces which (i) present an approximately two times more hydrophilic areas; with (ii) less extended, but between 2 to 5 times more intense hydrophilic patches; and (iii) 3 to 20 times more extended hydrophobic areas. The hydrophobic areas are also approximately 2 times more hydrophobicity-intense. This, more pronounced “leopard skin”-like, design of the protein molecular surface has been confirmed by comparing the results for a restricted set of homologous proteins, i.e., hemoglobins diverging by only one residue (Trp37). These results suggest that the representation of hydrophobicity on the protein molecular surfaces at atom-level resolution, coupled with the probing of the molecular surface at different geometric resolutions

High Dietary Protein Intake and Protein-Related Acid Load on Bone Health.

PubMed

Cao, Jay J

2017-12-01

Consumption of high-protein diets is increasingly popular due to the benefits of protein on preserving lean mass and controlling appetite and satiety. The paper is to review recent clinical research assessing dietary protein on calcium metabolism and bone health. Epidemiological studies show that long-term, high-protein intake is positively associated with bone mineral density and reduced risk of bone fracture incidence. Short-term interventional studies demonstrate that a high-protein diet does not negatively affect calcium homeostasis. Existing evidence supports that the negative effects of the acid load of protein on urinary calcium excretion are offset by the beneficial skeletal effects of high-protein intake. Future research should focus on the role and the degree of contribution of other dietary and physiological factors, such as intake of fruits and vegetables, in reducing the acid load and further enhancing the anabolic effects of protein on the musculoskeletal system.

Common FABP4 genetic variants and plasma levels of fatty acid binding protein 4 in older adults.

PubMed

Mukamal, Kenneth J; Wilk, Jemma B; Biggs, Mary L; Jensen, Majken K; Ix, Joachim H; Kizer, Jorge R; Tracy, Russell P; Zieman, Susan J; Mozaffarian, Dariush; Psaty, Bruce M; Siscovick, David S; Djoussé, Luc

2013-11-01

We examined common variants in the fatty acid binding protein 4 gene (FABP4) and plasma levels of FABP4 in adults aged 65 and older from the Cardiovascular Health Study. We genotyped rs16909187, rs1054135, rs16909192, rs10808846, rs7018409, rs2290201, and rs6992708 and measured circulating FABP4 levels among 3190 European Americans and 660 African Americans. Among European Americans, the minor alleles of six single nucleotide polymorphisms (SNP) were associated with lower FABP4 levels (all p ≤ 0.01). Among African Americans, the SNP with the lowest minor allele frequency was associated with lower FABP4 levels (p = 0.015). The C-A haplotype of rs16909192 and rs2290201 was associated with lower FABP4 levels in both European Americans (frequency = 16 %; p = 0.001) and African Americans (frequency = 8 %; p = 0.04). The haplotype combined a SNP in the first intron with one in the 3'untranslated region. However, the alleles associated with lower FABP4 levels were associated with higher fasting glucose in meta-analyses from the MAGIC consortium. These results demonstrate associations of common SNP and haplotypes in the FABP4 gene with lower plasma FABP4 but higher fasting glucose levels.

Serum creatinine and uric acid levels in pregnant urban African and Caucasian women.

PubMed

Nduka, N; Ekeke, G I

1986-12-01

Serum creatinine and uric acid concentrations were determined for 117 pregnant urban African women and 109 pregnant Caucasian women covering the three trimesters of pregnancy. The creatinine levels for Caucasians rose as pregnancy progressed, exceeding the normal adult level, while that for Africans rose only slightly and only during the last trimester of pregnancy. A similar observation was made for uric acid levels except that the rise in Africans commenced earlier than it did for creatinine, commencing from about the second trimester. Also, the levels in both groups of people remained within the normal adult range. The increased levels of these non-protein nitrogen compounds during pregnancy was attributable to increased metabolism associated with the condition. The difference observed between the Caucasians and Africans was attributed to the better protein nutritional status of the former. It is suggested that the creatinine "curve" especially for the Caucasians could be utilized in the monitoring of foetal well-being.

Exogenous FABP4 increases breast cancer cell proliferation and activates the expression of fatty acid transport proteins.

PubMed

Guaita-Esteruelas, Sandra; Bosquet, Alba; Saavedra, Paula; Gumà, Josep; Girona, Josefa; Lam, Eric W-F; Amillano, Kepa; Borràs, Joan; Masana, Lluís

2017-01-01

Adipose tissue plays an important role in tumor progression, because it provides nutrients and adipokines to proliferating cells. Fatty acid binding protein 4 (FABP4) is a key adipokine for fatty acid transport. In metabolic pathologies, plasma levels of FABP4 are increased. However, the role of this circulating protein is unknown. Recent studies have demonstrated that FABP4 might have a role in tumor progression, but the molecular mechanisms involved are still unclear. In this study, we analysed the role of eFABP4 (exogenous FABP4) in breast cancer progression. MCF-7 and MDA-MB-231 breast cancer cells did not express substantial levels of FABP4 protein, but intracellular FABP4 levels increased after eFABP4 incubation. Moreover, eFABP4 enhanced the proliferation of these breast cancer cells but did not have any effect on MCF-7 and MDA-MB-231 cell migration. Additionally, eFABP4 induced the AKT and MAPK signaling cascades in breast cancer cells, and the inhibition of these pathways reduced the eFBAP4-mediated cell proliferation. Interestingly, eFABP4 treatment in MCF-7 cells increased levels of the transcription factor FoxM1 and the fatty acid transport proteins CD36 and FABP5. In summary, we showed that eFABP4 plays a key role in tumor proliferation and activates the expression of fatty acid transport proteins in MCF-7 breast cancer cells. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Protein-based materials, toward a new level of structural control.

PubMed

van Hest, J C; Tirrell, D A

2001-10-07

Through billions of years of evolution nature has created and refined structural proteins for a wide variety of specific purposes. Amino acid sequences and their associated folding patterns combine to create elastic, rigid or tough materials. In many respects, nature's intricately designed products provide challenging examples for materials scientists, but translation of natural structural concepts into bio-inspired materials requires a level of control of macromolecular architecture far higher than that afforded by conventional polymerization processes. An increasingly important approach to this problem has been to use biological systems for production of materials. Through protein engineering, artificial genes can be developed that encode protein-based materials with desired features. Structural elements found in nature, such as beta-sheets and alpha-helices, can be combined with great flexibility, and can be outfitted with functional elements such as cell binding sites or enzymatic domains. The possibility of incorporating non-natural amino acids increases the versatility of protein engineering still further. It is expected that such methods will have large impact in the field of materials science, and especially in biomedical materials science, in the future.

Association Between Serum Levels of Adipocyte Fatty Acid-binding Protein and Free Thyroxine

PubMed Central

Tseng, Fen-Yu; Chen, Pei-Lung; Chen, Yen-Ting; Chi, Yu-Chao; Shih, Shyang-Ron; Wang, Chih-Yuan; Chen, Chi-Ling; Yang, Wei-Shiung

2015-01-01

Abstract Adipocyte fatty acid-binding protein (AFABP) has been shown to be a biomarker of body weight change and atherosclerosis. Changes in thyroid function are associated with changes in body weight and risks of cardiovascular diseases. The association between AFABP and thyroid function status has been seldom evaluated. The aim of this study was to compare the serum AFABP concentrations in hyperthyroid patients and those in euthyroid individuals, and to evaluate the associations between serum AFABP and free thyroxine (fT4) levels. For this study, 30 hyperthyroid patients and 30 euthyroid individuals at a referral medical center were recruited. The patients with hyperthyroidism were treated with antithyroid regimens as clinically indicated. No medication was given to the euthyroid individuals. The body weight, body mass index, thyroid function, serum levels of AFABP, and biochemical data of both groups at baseline and at the 6th month were compared. Associations between AFABP and fT4 levels were also analyzed. At the baseline, the hyperthyroid patients had significantly higher serum AFABP levels than the euthyroid individuals (median [Q1, Q3]: 22.8 [19.4, 30.6] ng/mL vs 18.6 [15.3, 23.2] ng/mL; P = 0.038). With the antithyroid regimens, the AFABP serum levels of the hyperthyroid patients decreased to 16.6 (15.0, 23.9) ng/mL at the 6th month. No difference in the AFABP level was found between the hyperthyroid and the euthyroid groups at the 6th month. At baseline, sex (female vs male, ß = 7.65, P = 0.022) and fT4 level (ß = 2.51, P = 0.018) were significantly associated with AFABP levels in the univariate regression analysis. At the 6th month, sex and fT4 level (ß = 8.09, P < 0.001 and ß = 3.61, P = 0.005, respectively) were also significantly associated with AFABP levels. The associations between sex and fT4 level with AFABP levels remained significant in the stepwise multivariate regression analysis, both at baseline and at

Imbalance of plasma amino acids, metabolites and lipids in patients with lysinuric protein intolerance (LPI).

PubMed

Kurko, Johanna; Tringham, Maaria; Tanner, Laura; Näntö-Salonen, Kirsti; Vähä-Mäkilä, Mari; Nygren, Heli; Pöhö, Päivi; Lietzen, Niina; Mattila, Ismo; Olkku, Anu; Hyötyläinen, Tuulia; Orešič, Matej; Simell, Olli; Niinikoski, Harri; Mykkänen, Juha

2016-09-01

Lysinuric protein intolerance (LPI [MIM 222700]) is an aminoaciduria with defective transport of cationic amino acids in epithelial cells in the small intestine and proximal kidney tubules due to mutations in the SLC7A7 gene. LPI is characterized by protein malnutrition, failure to thrive and hyperammonemia. Many patients also suffer from combined hyperlipidemia and chronic kidney disease (CKD) with an unknown etiology. Here, we studied the plasma metabolomes of the Finnish LPI patients (n=26) and healthy control individuals (n=19) using a targeted platform for analysis of amino acids as well as two analytical platforms with comprehensive coverage of molecular lipids and polar metabolites. Our results demonstrated that LPI patients have a dichotomy of amino acid profiles, with both decreased essential and increased non-essential amino acids. Altered levels of metabolites participating in pathways such as sugar, energy, amino acid and lipid metabolism were observed. Furthermore, of these metabolites, myo-inositol, threonic acid, 2,5-furandicarboxylic acid, galactaric acid, 4-hydroxyphenylacetic acid, indole-3-acetic acid and beta-aminoisobutyric acid associated significantly (P<0.001) with the CKD status. Lipid analysis showed reduced levels of phosphatidylcholines and elevated levels of triacylglycerols, of which long-chain triacylglycerols associated (P<0.01) with CKD. This study revealed an amino acid imbalance affecting the basic cellular metabolism, disturbances in plasma lipid composition suggesting hepatic steatosis and fibrosis and novel metabolites correlating with CKD in LPI. In addition, the CKD-associated metabolite profile along with increased nitrite plasma levels suggests that LPI may be characterized by increased oxidative stress and apoptosis, altered microbial metabolism in the intestine and uremic toxicity. Copyright © 2016 Elsevier Inc. All rights reserved.

FAX1, a Novel Membrane Protein Mediating Plastid Fatty Acid Export

PubMed Central

Li, Nannan; Gügel, Irene Luise; Giavalisco, Patrick; Zeisler, Viktoria; Schreiber, Lukas; Soll, Jürgen; Philippar, Katrin

2015-01-01

Fatty acid synthesis in plants occurs in plastids, and thus, export for subsequent acyl editing and lipid assembly in the cytosol and endoplasmatic reticulum is required. Yet, the transport mechanism for plastid fatty acids still remains enigmatic. We isolated FAX1 (fatty acid export 1), a novel protein, which inserts into the chloroplast inner envelope by α-helical membrane-spanning domains. Detailed phenotypic and ultrastructural analyses of FAX1 mutants in Arabidopsis thaliana showed that FAX1 function is crucial for biomass production, male fertility and synthesis of fatty acid-derived compounds such as lipids, ketone waxes, or pollen cell wall material. Determination of lipid, fatty acid, and wax contents by mass spectrometry revealed that endoplasmatic reticulum (ER)-derived lipids decreased when FAX1 was missing, but levels of several plastid-produced species increased. FAX1 over-expressing lines showed the opposite behavior, including a pronounced increase of triacyglycerol oils in flowers and leaves. Furthermore, the cuticular layer of stems from fax1 knockout lines was specifically reduced in C29 ketone wax compounds. Differential gene expression in FAX1 mutants as determined by DNA microarray analysis confirmed phenotypes and metabolic imbalances. Since in yeast FAX1 could complement for fatty acid transport, we concluded that FAX1 mediates fatty acid export from plastids. In vertebrates, FAX1 relatives are structurally related, mitochondrial membrane proteins of so-far unknown function. Therefore, this protein family might represent a powerful tool not only to increase lipid/biofuel production in plants but also to explore novel transport systems involved in vertebrate fatty acid and lipid metabolism. PMID:25646734

Oleic acid levels regulated by glycerolipid metabolism modulate defense gene expression in Arabidopsis

PubMed Central

Kachroo, Aardra; Venugopal, Srivathsa C.; Lapchyk, Ludmila; Falcone, Deane; Hildebrand, David; Kachroo, Pradeep

2004-01-01

Stearoyl-acyl-carrier-protein-desaturase-mediated conversion of stearic acid (18:0) to oleic acid (18:1) is a key step, which regulates levels of unsaturated fatty acids in cells. We previously showed that stearoyl-acyl-carrier-protein-desaturase mutants ssi2/fab2 carrying a loss-of-function mutation in the plastidial glycerol-3-phosphate (G3P) acyltransferase (act1) have elevated 18:1 levels and are restored in their altered defense signaling. Because G3P is required for the acylation of 18:1 by G3P acyltransferase, it was predicted that reduction of G3P levels should increase 18:1 levels and thereby revert ssi2-triggered phenotypes. Here we show that a mutation in G3P dehydrogenase restores both salicylic acid- and jasmonic acid-mediated phenotypes of ssi2 plants. The G3P dehydrogenase gene was identified by map-based cloning of the ssi2 suppressor mutant rdc8 (gly1-3) and confirmed by epistatic analysis of ssi2 with gly1-1. Restoration of ssi2-triggered phenotypes by the gly1-3 mutation was age-dependent and correlated with the levels of 18:1. Regeneration of G3P pools by glycerol application in ssi2 and ssi2 gly1-3 plants caused a marked reduction in the 18:1 levels, which rendered these plants hypersensitive to glycerol. This hypersensitivity in ssi2 was rescued by the act1 mutation. Furthermore, overexpression of the ACT1 gene resulted in enhanced sensitivity to glycerol. Glycerol application also lowered the 18:1 content in SSI2 plants and converted these into ssi2-mimics. Our results show that 18:1 levels in plastids are regulated by means of acylation with G3P, and a balance between G3P and 18:1 is critical for the regulation of salicylic acid- and jasmonic acid-mediated signaling pathways. PMID:15044700

Proximate composition, fatty acid analysis and protein digestibility-corrected amino acid score of three Mediterranean cephalopods.

PubMed

Zlatanos, Spiros; Laskaridis, Kostas; Feist, Christian; Sagredos, Angelos

2006-10-01

Proximate composition, fatty acid analysis and protein digestibility-corrected amino acid score (PDCAAS) in three commercially important cephalopods of the Mediterranean sea (cuttlefish, octopus and squid) were determined. The results of the proximate analysis showed that these species had very high protein:fat ratios similar to lean beef. Docosahexaenoic, palmitic and eicosipentaenoic acid were the most abundant fatty acids among analyzed species. The amount of n-3 fatty acids was higher than that of saturated, monounsaturated and n-6 fatty acids. Despite the fact that cephalopods contain small amounts of fat they were found quite rich in n-3 fatty acids. Finally, PDCAAS indicated that these organisms had a very good protein quality.

Decreased eicosapentaenoic acid levels in acne vulgaris reveals the presence of a proinflammatory state.

PubMed

Aslan, İbrahim; Özcan, Filiz; Karaarslan, Taner; Kıraç, Ebru; Aslan, Mutay

2017-01-01

This study aimed to determine circulating levels of polyunsaturated fatty acids (PUFAs), secretory phospholipase A2 (sPLA2), lipoprotein lipase (LPL) and measure circulating protein levels of angiopoietin-like protein 3 (ANGPTL3), ANGPTL4, cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) in patients with acne vulgaris. Serum from 21 control subjects and 31 acne vulgaris patients were evaluated for levels of arachidonic acid (AA, C20:4n- 6), dihomo-gamma-linolenic acid (DGLA, C20:3n-6), eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3). PUFA levels were determined by an optimized multiple reaction monitoring (MRM) method using ultra fast-liquid chromatography (UFLC) coupled with tandem mass spectrometry (MS/MS). Lipid profile, routine biochemical and hormone parameters were assayed by standard kit methods Serum EPA levels were significantly decreased while AA/EPA and DGLA/EPA ratio were significantly increased in acne vulgaris patients compared to controls. Serum levels of AA, DGLA and DHA showed no significant difference while activity of sPLA2 and LPL were significantly increased in acne vulgaris compared to controls. Results of this study reveal the presence of a proinflammatory state in acne vulgaris as shown by significantly decreased serum EPA levels and increased activity of sPLA2, AA/EPA and DGLA/EPA ratio. Increased LPL activity in the serum of acne vulgaris patients can be protective through its anti-dyslipidemic actions. This is the first study reporting altered EPA levels and increased sPLA2 activity in acne vulgaris and supports the use of omega-3 fatty acids as adjuvant treatment for acne patients. Copyright © 2016 Elsevier Inc. All rights reserved.

Association of serum uric acid with high-sensitivity C-reactive protein in postmenopausal women.

PubMed

Raeisi, A; Ostovar, A; Vahdat, K; Rezaei, P; Darabi, H; Moshtaghi, D; Nabipour, I

2017-02-01

To explore the independent correlation between serum uric acid and low-grade inflammation (measured by high-sensitivity C-reactive protein, hs-CRP) in postmenopausal women. A total of 378 healthy Iranian postmenopausal women were randomly selected in a population-based study. Circulating hs-CRP levels were measured by highly specific enzyme-linked immunosorbent assay method and an enzymatic calorimetric method was used to measure serum levels of uric acid. Pearson correlation coefficient, multiple linear regression and logistic regression models were used to analyze the association between uric acid and hs-CRP levels. A statistically significant correlation was seen between serum levels of uric acid and log-transformed circulating hs-CRP (r = 0.25, p < 0.001). After adjustment for age and cardiovascular risk factors (according to NCEP ATP III criteria), circulating hs-CRP levels were significantly associated with serum uric acid levels (β = 0.20, p < 0.001). After adjustment for age and cardiovascular risk factors, hs-CRP levels ≥3 mg/l were significantly associated with higher uric acid levels (odds ratio =1.52, 95% confidence interval 1.18-1.96). Higher serum uric acid levels were positively and independently associated with circulating hs-CRP in healthy postmenopausal women.

Non-standard amino acid incorporation into proteins using Escherichia coli cell-free protein synthesis

NASA Astrophysics Data System (ADS)

Hong, Seok Hoon; Kwon, Yong-Chan; Jewett, Michael

2014-06-01

Incorporating non-standard amino acids (NSAAs) into proteins enables new chemical properties, new structures, and new functions. In recent years, improvements in cell-free protein synthesis (CFPS) systems have opened the way to accurate and efficient incorporation of NSAAs into proteins. The driving force behind this development has been three-fold. First, a technical renaissance has enabled high-yielding (>1 g/L) and long-lasting (>10 h in batch operation) CFPS in systems derived from Escherichia coli. Second, the efficiency of orthogonal translation systems has improved. Third, the open nature of the CFPS platform has brought about an unprecedented level of control and freedom of design. Here, we review recent developments in CFPS platforms designed to precisely incorporate NSAAs. In the coming years, we anticipate that CFPS systems will impact efforts to elucidate structure/function relationships of proteins and to make biomaterials and sequence-defined biopolymers for medical and industrial applications.

Interaction of milk whey protein with common phenolic acids

NASA Astrophysics Data System (ADS)

Zhang, Hao; Yu, Dandan; Sun, Jing; Guo, Huiyuan; Ding, Qingbo; Liu, Ruihai; Ren, Fazheng

2014-01-01

Phenolics-rich foods such as fruit juices and coffee are often consumed with milk. In this study, the interactions of α-lactalbumin and β-lactoglobulin with the phenolic acids (chlorogenic acid, caffeic acid, ferulic acid, and coumalic acid) were examined. Fluorescence, CD, and FTIR spectroscopies were used to analyze the binding modes, binding constants, and the effects of complexation on the conformation of whey protein. The results showed that binding constants of each whey protein-phenolic acid interaction ranged from 4 × 105 to 7 × 106 M-n and the number of binding sites n ranged from 1.28 ± 0.13 to 1.54 ± 0.34. Because of these interactions, the conformation of whey protein was altered, with a significant reduction in the amount of α-helix and an increase in the amounts of β-sheet and turn structures.

Proteomic investigation of protein profile changes and amino acid residue-level modification in cooked lamb longissimus thoracis et lumborum: The effect of roasting.

PubMed

Yu, Tzer-Yang; Morton, James D; Clerens, Stefan; Dyer, Jolon M

2016-09-01

Protein modifications of meat cooked by typical dry-heat methods (e.g., roasting) are currently not well understood. The present study utilised a shotgun proteomic approach to examine the molecular-level effect of roasting on thin lamb longissimus thoracis et lumborum patties, in terms of changes to both the protein profile and amino acid residue side-chain modifications. Cooking caused aggregation of actin, myosin heavy chains and sarcoplasmic proteins. Longer roasting time resulted in significantly reduced protein extractability as well as protein truncation involving particularly a number of myofibrillar and sarcoplasmic proteins, e.g., 6-phosphofructokinase, beta-enolase, l-lactate dehydrogenase A chain, alpha-actinin-3, actin and possibly myosin heavy chains. Modifications that have potential influence on nutritional properties, including carboxyethyllysine and a potentially glucose-derived N-terminal Amadori compound, were observed in actin and myoglobin after roasting. This study provided new insights into molecular changes resulting from the dry-heat treatment of meat, such as commonly used in food preparation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Site specific incorporation of keto amino acids into proteins

DOEpatents

Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA

2011-03-22

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

Site specific incorporation of keto amino acids into proteins

DOEpatents

Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA

2008-10-07

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

Site specific incorporation of keto amino acids into proteins

DOEpatents

Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA

2011-12-06

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

Site specific incorporation of keto amino acids into proteins

DOEpatents

Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA

2012-02-14

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

Amino acid repletion does not decrease muscle protein catabolism during hemodialysis.

PubMed

Raj, Dominic S C; Adeniyi, Oladipo; Dominic, Elizabeth A; Boivin, Michel A; McClelland, Sandra; Tzamaloukas, Antonios H; Morgan, Nancy; Gonzales, Lawrence; Wolfe, Robert; Ferrando, Arny

2007-06-01

Intradialytic protein catabolism is attributed to loss of amino acids in the dialysate. We investigated the effect of amino acid infusion during hemodialysis (HD) on muscle protein turnover and amino acid transport kinetics by using stable isotopes of phenylalanine, leucine, and lysine in eight patients with end-stage renal disease (ESRD). Subjects were studied at baseline (pre-HD), 2 h of HD without amino acid infusion (HD-O), and 2 h of HD with amino acid infusion (HD+AA). Amino acid depletion during HD-O augmented the outward transport of amino acids from muscle into the vein. Increased delivery of amino acids to the leg during HD+AA facilitated the transport of amino acids from the artery into the intracellular compartment. Increase in muscle protein breakdown was more than the increase in synthesis during HD-O (46.7 vs. 22.3%, P < 0.001). Net balance (nmol.min(-1).100 ml (-1)) was more negative during HD-O compared with pre-HD (-33.7 +/- 1.5 vs. -6.0 +/- 2.3, P < 0.001). Despite an abundant supply of amino acids, the net balance (-16.9 +/- 1.8) did not switch from net release to net uptake. HD+AA induced a proportional increase in muscle protein synthesis and catabolism. Branched chain amino acid catabolism increased significantly from baseline during HD-O and did not decrease during HD+AA. Protein synthesis efficiency, the fraction of amino acid in the intracellular pool that is utilized for muscle protein synthesis decreased from 42.1% pre-HD to 33.7 and 32.6% during HD-O and HD+AA, respectively (P < 0.01). Thus amino acid repletion during HD increased muscle protein synthesis but did not decrease muscle protein breakdown.

LC-MS display of the total modified amino acids in cataract lens proteins and in lens proteins glycated by ascorbic acid in vitro.

PubMed

Cheng, Rongzhu; Feng, Qi; Ortwerth, Beryl J

2006-05-01

We previously reported chromatographic evidence supporting the similarity of yellow chromophores isolated from aged human lens proteins, early brunescent cataract lens proteins and calf lens proteins ascorbylated in vitro [Cheng, R. et al. Biochimica et Biophysica Acta 1537, 14-26, 2001]. In this paper, new evidence supporting the chemical identity of the modified amino acids in these protein populations were collected by using a newly developed two-dimensional LC-MS mapping technique supported by tandem mass analysis of the major species. The pooled water-insoluble proteins from aged normal human lenses, early stage brunescent cataract lenses and calf lens proteins reacted with or without 20 mM ascorbic acid in air for 4 weeks were digested with a battery of proteolytic enzymes under argon to release the modified amino acids. Aliquots equivalent to 2.0 g of digested protein were subjected to size-exclusion chromatography on a Bio-Gel P-2 column and four major A330nm-absorbing peaks were collected. Peaks 1, 2 and 3, which contained most of the modified amino acids were concentrated and subjected to RP-HPLC/ESI-MS, and the mass elution maps were determined. The samples were again analyzed and those peaks with a 10(4) - 10(6) response factor were subjected to MS/MS analysis to identify the daughter ions of each modification. Mass spectrometric maps of peaks 1, 2 and 3 from cataract lenses showed 58, 40 and 55 mass values, respectively, ranging from 150 to 600 Da. Similar analyses of the peaks from digests of the ascorbylated calf lens proteins gave 81, 70 and 67 mass values, respectively, of which 100 were identical to the peaks in the cataract lens proteins. A total of 40 of the major species from each digest were analyzed by LC-MS/MS and 36 were shown to be identical. Calf lens proteins incubated without ascorbic acid showed several similar mass values, but the response factors were 100 to 1000-fold less for every modification. Based upon these data, we conclude

Understanding of how Propionibacterium acidipropionici respond to propionic acid stress at the level of proteomics

PubMed Central

Guan, Ningzi; Shin, Hyun-dong; Chen, Rachel R.; Li, Jianghua; Liu, Long; Du, Guocheng; Chen, Jian

2014-01-01

Propionic acid (PA) is an important platform chemical in the food, agriculture, and pharmaceutical industries and is mainly biosynthesized by propionibacteria. Acid tolerance in PA-producing strains is crucial. In previous work, we investigated the acid tolerance mechanism of Propionibacterium acidipropionici at microenvironmental levels by analyzing physiological changes in the parental strain and three PA-tolerant mutants obtained by genome shuffling. However, the molecular mechanism of PA tolerance in P. acidipropionici remained unclear. Here, we performed a comparative proteomics study of P. acidipropionici CGMCC 1.2230 and the acid-tolerant mutant P. acidipropionici WSH1105; MALDI-TOF/MS identified 24 proteins that significantly differed between the parental and shuffled strains. The differentially expressed proteins were mainly categorized as key components of crucial biological processes and the acid stress response. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was used to confirm differential expression of nine key proteins. Overexpression of the secretory protein glyceraldehyde-3-phosphate dehydrogenase and ATP synthase subunit α in Escherichia coli BL21 improved PA and acetic acid tolerance; overexpression of NADH dehydrogenase and methylmalonyl-CoA epimerase improved PA tolerance. These results provide new insights into the acid tolerance of P. acidipropionici and will facilitate the development of PA production through fermentation by propionibacteria. PMID:25377721

PLASMA PROTEIN AND HEMOGLOBIN PRODUCTION : DELETION OF INDIVIDUAL AMINO ACIDS FROM GROWTH MIXTURE OF TEN ESSENTIAL AMINO ACIDS. SIGNIFICANT CHANGES IN URINARY NITROGEN.

PubMed

Robscheit-Robbins, F S; Miller, L L; Whipple, G H

1947-02-28

Given healthy dogs fed abundant iron and protein-free or low protein diets with sustained anemia and hypoproteinemia, we can study the capacity of these animals to produce simultaneously new hemoglobin and plasma protein. Reserve stores of blood protein-building materials are measurably depleted and levels of 6 to 8 gm. per cent for hemoglobin and 4 to 5 gm. per cent for plasma protein can be maintained for weeks or months depending upon the intake of food proteins or amino acid mixtures. These dogs are very susceptible to infection and various poisons. Dogs tire of these diets and loss of appetite terminates many experiments. Under these conditions (double depletion) standard growth mixtures of essential amino acids are tested to show the response in blood protein output and urinary nitrogen balance. As a part of each tabulated experiment one of the essential amino acids is deleted from the complete growth mixture to compare such response with that of the whole mixture. Methionine, threonine, phenylalanine, and tryptophane when singly eliminated from the complete amino acid mixture do effect a sharp rise in urinary nitrogen. This loss of urinary nitrogen is corrected when the individual amino acid is replaced in the mixture. Histidine, lysine, and valine have a moderate influence upon urinary nitrogen balance toward nitrogen conservation. Leucine, isoleucine, and arginine have minimal or no effect upon urinary nitrogen balance when these individual amino acids are deleted from the complete growth mixture of amino acids during 3 to 4 week periods. Tryptophane and to a less extent phenylalanine and threonine when returned to the amino acid mixture are associated with a conspicuous preponderance of plasma protein output over the hemoglobin output (Table 4). Arginine, lysine, and histidine when returned to the amino acid mixture are associated with a large preponderance of hemoglobin output. Various amino acid mixtures under these conditions may give a positive

The pathogen-related yeast protein Pry1, a member of the CAP protein superfamily, is a fatty acid-binding protein

PubMed Central

Darwiche, Rabih; Mène-Saffrané, Laurent; Gfeller, David; Asojo, Oluwatoyin A.; Schneiter, Roger

2017-01-01

Members of the CAP superfamily (cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins), also known as SCP superfamily (sperm-coating proteins), have been implicated in many physiological processes, including immune defenses, venom toxicity, and sperm maturation. Their mode of action, however, remains poorly understood. Three proteins of the CAP superfamily, Pry1, -2, and -3 (pathogen related in yeast), are encoded in the Saccharomyces cerevisiae genome. We have shown previously that Pry1 binds cholesterol in vitro and that Pry function is required for sterol secretion in yeast cells, indicating that members of this superfamily may generally bind sterols or related small hydrophobic compounds. On the other hand, tablysin-15, a CAP protein from the horsefly Tabanus yao, has been shown to bind leukotrienes and free fatty acids in vitro. Therefore, here we assessed whether the yeast Pry1 protein binds fatty acids. Computational modeling and site-directed mutagenesis indicated that the mode of fatty acid binding is conserved between tablysin-15 and Pry1. Pry1 bound fatty acids with micromolar affinity in vitro, and its function was essential for fatty acid export in cells lacking the acyl-CoA synthetases Faa1 and Faa4. Fatty acid binding of Pry1 is independent of its capacity to bind sterols, and the two sterol- and fatty acid-binding sites are nonoverlapping. These results indicate that some CAP family members, such as Pry1, can bind different lipids, particularly sterols and fatty acids, at distinct binding sites, suggesting that the CAP domain may serve as a stable, secreted protein domain that can accommodate multiple ligand-binding sites. PMID:28365570

The influences of ambient temperature and crude protein levels on performance and serum biochemical parameters in broilers.

PubMed

Liu, Q W; Feng, J H; Chao, Z; Chen, Y; Wei, L M; Wang, F; Sun, R P; Zhang, M H

2016-04-01

This study was undertaken to investigate the effects of ambient temperature, crude protein levels and their interaction on performance and serum biochemical parameters of broiler chickens. A total of 216 Arbor Acre broiler chickens (108 males and 108 females) were used in a 2 × 3 factorial arrangement and randomly reared at two temperatures (normal temperature: 23 °C; daily cyclic high temperature: 28-32 °C) and fed on three diets with different crude protein levels (153.3, 183.3 or 213.3 g/kg, with constant essential amino acids) from 28 to 42 days of age. Daily cyclic high ambient temperature decreased final body weight, average daily weight gain, average daily feed intake and serum total protein contents (p < 0.001, p < 0.001, p < 0.001, p = 0.008 respectively), but increased feed/gain, mortality, respiratory rate, rectal temperature, serum uric acid contents and serum creatine kinase activity (p = 0.008, p = 0.003, p < 0.0001, p < 0.0001, p < 0.0001, p = 0.003 respectively), irrespective of crude protein levels. At the ambient temperature, reducing crude protein levels resulted in an increase in feed/gain (p < 0.001), but a decrease in serum total protein and uric acid contents. Only serum creatine kinase activity in broiler chickens was interacted by daily cyclic high ambient temperature and dietary crude protein levels (p = 0.003). These results indicated that daily cyclic high ambient temperature had a great effect on performance and serum biochemical parameters in broiler chickens, whereas dietary crude protein levels affected them partially. Journal of Animal Physiology and Animal Nutrition © 2015 Blackwell Verlag GmbH.

Uric acid contributes greatly to hepatic antioxidant capacity besides protein.

PubMed

Mikami, T; Sorimachi, M

2017-12-20

Uric acid is the end-product of purine nucleotide metabolism and an increase in uric acid concentration in the body results in hyperuricemia, ultimately leading to gout. However, uric acid is a potent antioxidant and interacts with reactive oxygen species (ROS) to be non-enzymatically converted to allantoin. Uric acid accounts for approximately 60 % of antioxidant capacity in the plasma; however, its contribution to tissue antioxidant capacity is unknown. In this study, the contribution of uric acid to tissue antioxidant capacity and its conversion to allantoin by scavenging ROS in tissue were examined. The results showed that a decrease in hepatic uric acid content via allopurinol administration significantly reduced hepatic total-radical trapping antioxidant parameter (TRAP) content in protein-free cytosol. Additionally, treating protein-free cytosol with uricase led to a further reduction of hepatic TRAP content. Allantoin was also detected in the solution containing protein-free cytosol that reacted with ROS. These findings suggest that in the absence of protein, uric acid contributes greatly to antioxidant capacity in the liver, where uric acid is converted to allantoin by scavenging ROS.

Conformational Entropy of Intrinsically Disordered Proteins from Amino Acid Triads

PubMed Central

Baruah, Anupaul; Rani, Pooja; Biswas, Parbati

2015-01-01

This work quantitatively characterizes intrinsic disorder in proteins in terms of sequence composition and backbone conformational entropy. Analysis of the normalized relative composition of the amino acid triads highlights a distinct boundary between globular and disordered proteins. The conformational entropy is calculated from the dihedral angles of the middle amino acid in the amino acid triad for the conformational ensemble of the globular, partially and completely disordered proteins relative to the non-redundant database. Both Monte Carlo (MC) and Molecular Dynamics (MD) simulations are used to characterize the conformational ensemble of the representative proteins of each group. The results show that the globular proteins span approximately half of the allowed conformational states in the Ramachandran space, while the amino acid triads in disordered proteins sample the entire range of the allowed dihedral angle space following Flory’s isolated-pair hypothesis. Therefore, only the sequence information in terms of the relative amino acid triad composition may be sufficient to predict protein disorder and the backbone conformational entropy, even in the absence of well-defined structure. The predicted entropies are found to agree with those calculated using mutual information expansion and the histogram method. PMID:26138206

Influence of dietary protein level on the broiler chicken's response to methionine and betaine supplements.

PubMed

Garcia Neto, M; Pesti, G M; Bakalli, R I

2000-10-01

Two experiments were conducted to compare broiler chicken responses to methionine and betaine supplements when fed diets with low protein and relatively high metabolizable energy levels (17%, 3.3 kcal/g) or moderate protein and lower metabolizable energy levels (24%, 3.0 kcal/g), resulting in different levels of carcass fat. In Experiment 1, the basal diets were formulated with corn, soybean meal, poultry by-product meal, and poultry oil. In Experiment 2, glucose monohydrate was also added, so that identical amino acid profiles could be maintained in the 17 and 24% protein diets. On average, feeding the 17 vs. 24% protein diet decreased 21-d body weight gain by 20%, increased feed conversion ratio (FCR) by 13%, and increased abdominal fat pad weight by 104%. Methionine and betaine supplements improved the performance of chicks fed the 24% protein diet in both experiments, as indicated by body weight gain and FCR. Only supplementary methionine increased performance of chicks fed 17% protein diets, and then only in Experiment 2. Neither methionine nor betaine decreased abdominal fat pad size in either experiment. Methionine supplementation decreased relative liver size and increased breast muscle protein. Both methionine and betaine increased sample feather weight, but when expressed as a percentage of body weight, no significant differences were detected. It is concluded that increasing carcass fat by manipulating percentage dietary protein level or amino acid balance does not influence betaine's activity as a lipotropic agent.

Rewiring protein synthesis: From natural to synthetic amino acids.

PubMed

Fan, Yongqiang; Evans, Christopher R; Ling, Jiqiang

2017-11-01

The protein synthesis machinery uses 22 natural amino acids as building blocks that faithfully decode the genetic information. Such fidelity is controlled at multiple steps and can be compromised in nature and in the laboratory to rewire protein synthesis with natural and synthetic amino acids. This review summarizes the major quality control mechanisms during protein synthesis, including aminoacyl-tRNA synthetases, elongation factors, and the ribosome. We will discuss evolution and engineering of such components that allow incorporation of natural and synthetic amino acids at positions that deviate from the standard genetic code. The protein synthesis machinery is highly selective, yet not fixed, for the correct amino acids that match the mRNA codons. Ambiguous translation of a codon with multiple amino acids or complete reassignment of a codon with a synthetic amino acid diversifies the proteome. Expanding the genetic code with synthetic amino acids through rewiring protein synthesis has broad applications in synthetic biology and chemical biology. Biochemical, structural, and genetic studies of the translational quality control mechanisms are not only crucial to understand the physiological role of translational fidelity and evolution of the genetic code, but also enable us to better design biological parts to expand the proteomes of synthetic organisms. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2017 Elsevier B.V. All rights reserved.

Regulation of protein synthesis by amino acids in muscle of neonates

PubMed Central

Suryawan, Agus; Davis, Teresa A.

2011-01-01

The marked increase in skeletal muscle mass during the neonatal period is largely due to a high rate of postprandial protein synthesis that is modulated by an enhanced sensitivity to insulin and amino acids. The amino acid signaling pathway leading to the stimulation of protein synthesis has not been fully elucidated. Among the amino acids, leucine is considered to be a principal anabolic agent that regulates protein synthesis. mTORC1, which controls protein synthesis, has been implicated as a target for leucine. Until recently, there have been few studies exploring the role of amino acids in enhancing muscle protein synthesis in vivo. In this review, we discuss amino acid-induced protein synthesis in muscle in the neonate, focusing on current knowledge of the role of amino acids in the activation of mTORC1 leading to mRNA translation. The role of the amino acid transporters, SNAT2, LAT1, and PAT, in the modulation of mTORC1 activation and the role of amino acids in the activation of putative regulators of mTORC1, i.e., raptor, Rheb, MAP4K3, Vps34, and Rag GTPases, are discussed. PMID:21196241

Comparative proteomic analysis of differentially expressed proteins in β-aminobutyric acid enhanced Arabidopsis thaliana tolerance to simulated acid rain.

PubMed

Liu, Tingwu; Jiang, Xinwu; Shi, Wuliang; Chen, Juan; Pei, Zhenming; Zheng, Hailei

2011-05-01

Acid rain is a worldwide environmental issue that has seriously destroyed forest ecosystems. As a highly effective and broad-spectrum plant resistance-inducing agent, β-aminobutyric acid could elevate the tolerance of Arabidopsis when subjected to simulated acid rain. Using comparative proteomic strategies, we analyzed 203 significantly varied proteins of which 175 proteins were identified responding to β-aminobutyric acid in the absence and presence of simulated acid rain. They could be divided into ten groups according to their biological functions. Among them, the majority was cell rescue, development and defense-related proteins, followed by transcription, protein synthesis, folding, modification and destination-associated proteins. Our conclusion is β-aminobutyric acid can lead to a large-scale primary metabolism change and simultaneously activate antioxidant system and salicylic acid, jasmonic acid, abscisic acid signaling pathways. In addition, β-aminobutyric acid can reinforce physical barriers to defend simulated acid rain stress. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hepatic fatty acid biosynthesis is more responsive to protein than carbohydrate in rainbow trout during acute stimulations.

PubMed

Dai, Weiwei; Panserat, Stéphane; Kaushik, Sadasivam; Terrier, Frédéric; Plagnes-Juan, Elisabeth; Seiliez, Iban; Skiba-Cassy, Sandrine

2016-01-01

The link between dietary carbohydrate/protein and de novo lipogenesis (DNL) remains debatable in carnivorous fish. We aimed to evaluate and compare the response of hepatic lipogenic gene expression to dietary carbohydrate intake/glucose and dietary protein intake/amino acids (AAs) during acute stimulations using both in vivo and in vitro approaches. For the in vivo trial, three different diets and a controlled-feeding method were employed to supply fixed amount of dietary protein or carbohydrate in a single meal; for the in vitro trial, primary hepatocytes were stimulated with a low or high level of glucose (3 mM or 20 mM) and a low or high level of AAs (one-fold or four-fold concentrated AAs). In vitro data showed that a high level of AAs upregulated the expression of enzymes involved in DNL [fatty acid synthase (FAS) and ATP citrate lyase (ACLY)], lipid bioconversion [elongation of very long chain fatty acids like-5 (Elovl5), Elovl2, Δ6 fatty acyl desaturase (D6D) and stearoyl-CoA desaturase-1 (SCD1)], NADPH production [glucose-6-phosphate dehydrogenase (G6PDH) and malic enzyme (ME)], and transcriptional factor sterol regulatory element binding protein 1-like, while a high level of glucose only elevated the expression of ME. Data in trout liver also showed that high dietary protein intake induced higher lipogenic gene expression (FAS, ACLY, and Elovl2) regardless of dietary carbohydrate intake, while high carbohydrate intake markedly suppressed the expression of acetyl-CoA carboxylase (ACC) and Elovl5. Overall, we conclude that, unlike rodents or humans, hepatic fatty acid biosynthetic gene expression in rainbow trout is more responsive to dietary protein intake/AAs than dietary carbohydrate intake/glucose during acute stimulations. This discrepancy probably represents one important physiological and metabolic difference between carnivores and omnivores. Copyright © 2016 the American Physiological Society.

G-protein beta3 subunit gene variant is unlikely to have a significant influence on serum uric acid level in Japanese workers.

PubMed

Suwazono, Yasushi; Kobayashi, Etsuko; Uetani, Mirei; Miura, Katsuyuki; Morikawa, Yuko; Ishizaki, Masao; Kido, Teruhiko; Nakagawa, Hideaki; Nogawa, Koji

2006-06-01

The C825T variant of the G-protein beta3 subunit (GNB3) gene has attracted renewed attention as a candidate gene for obesity, hypertension and hyperuricemia. The main role of G-protein is to translate signals from the cell surface into a cellular response. The 825T allele is associated with a splice variant of GNB3 protein and enhanced G-protein activation. We examined the relationship between this variant and the risk of hyperuricemia in Japanese workers. The study subjects were 1,452 men and 1,169 women selected from 3,834 men and 2,591 women in 1997. On the basis of common clinical criteria, hyperuricemia I was defined as serum uric acid >or= 7.0 mg/dl in men and 6.0 mg/dl in women or taking antihyperuricemic medication. The hyperuricemia I group consisted of 186 men and 20 women and its control of 1,266 men and 1,149 women. Hyperuricemia II was defined as serum uric acid > 5.7 mg/dl (median) in men and 3.9 mg/dl (median) in women or taking antihyperuricemic medication. The hyperuricemic II group consisted of 684 men and 570 women and its control of 768 men and 599 women. To replicate previous significant results in young Caucasian men, we selected these criteria because the authors of the study in young Caucasian men adopted the median in their subjects as a cut-off. The statistical power was estimated as 99% based on the significant results in Caucasians. Genotype and allele distributions in men and women with hyperuricemia I and II were not significantly different from those in the corresponding control groups. Logistic regression analysis on hyperuricemia I and II, and multiple regression on serum uric acid level demonstrated no significant effect of the C825T genotype. Despite the sufficient statistical power, this study could not demonstrate the significant influence of C825T on hyperuricemia or serum uric acid. The targeting of this polymorphism is unlikely to be beneficial in the prevention of hyperuricemia in the general Japanese population.

Modulating protein adsorption onto hydroxyapatite particles using different amino acid treatments

PubMed Central

Lee, Wing-Hin; Loo, Ching-Yee; Van, Kim Linh; Zavgorodniy, Alexander V.; Rohanizadeh, Ramin

2012-01-01

Hydroxyapatite (HA) is a material of choice for bone grafts owing to its chemical and structural similarities to the mineral phase of hard tissues. The combination of osteogenic proteins with HA materials that carry and deliver the proteins to the bone-defective areas will accelerate bone regeneration. The study investigated the treatment of HA particles with different amino acids such as serine (Ser), asparagine (Asn), aspartic acid (Asp) and arginine (Arg) to enhance the adsorption ability of HA carrier for delivering therapeutic proteins to the body. The crystallinity of HA reduced when amino acids were added during HA preparation. Depending on the types of amino acid, the specific surface area of the amino acid-functionalized HA particles varied from 105 to 149 m2 g–1. Bovine serum albumin (BSA) and lysozyme were used as model proteins for adsorption study. The protein adsorption onto the surface of amino acid-functionalized HA depended on the polarities of HA particles, whereby, compared with lysozyme, BSA demonstrated higher affinity towards positively charged Arg-HA. Alternatively, the binding affinity of lysozyme onto the negatively charged Asp-HA was higher when compared with BSA. The BSA and lysozyme adsorptions onto the amino acid-functionalized HA fitted better into the Freundlich than Langmuir model. The amino acid-functionalized HA particles that had higher protein adsorption demonstrated a lower protein-release rate. PMID:21957116

Computationally Efficient Multiscale Reactive Molecular Dynamics to Describe Amino Acid Deprotonation in Proteins

PubMed Central

2016-01-01

An important challenge in the simulation of biomolecular systems is a quantitative description of the protonation and deprotonation process of amino acid residues. Despite the seeming simplicity of adding or removing a positively charged hydrogen nucleus, simulating the actual protonation/deprotonation process is inherently difficult. It requires both the explicit treatment of the excess proton, including its charge defect delocalization and Grotthuss shuttling through inhomogeneous moieties (water and amino residues), and extensive sampling of coupled condensed phase motions. In a recent paper (J. Chem. Theory Comput.2014, 10, 2729−273725061442), a multiscale approach was developed to map high-level quantum mechanics/molecular mechanics (QM/MM) data into a multiscale reactive molecular dynamics (MS-RMD) model in order to describe amino acid deprotonation in bulk water. In this article, we extend the fitting approach (called FitRMD) to create MS-RMD models for ionizable amino acids within proteins. The resulting models are shown to faithfully reproduce the free energy profiles of the reference QM/MM Hamiltonian for PT inside an example protein, the ClC-ec1 H+/Cl– antiporter. Moreover, we show that the resulting MS-RMD models are computationally efficient enough to then characterize more complex 2-dimensional free energy surfaces due to slow degrees of freedom such as water hydration of internal protein cavities that can be inherently coupled to the excess proton charge translocation. The FitRMD method is thus shown to be an effective way to map ab initio level accuracy into a much more computationally efficient reactive MD method in order to explicitly simulate and quantitatively describe amino acid protonation/deprotonation in proteins. PMID:26734942

Computationally Efficient Multiscale Reactive Molecular Dynamics to Describe Amino Acid Deprotonation in Proteins.

PubMed

Lee, Sangyun; Liang, Ruibin; Voth, Gregory A; Swanson, Jessica M J

2016-02-09

An important challenge in the simulation of biomolecular systems is a quantitative description of the protonation and deprotonation process of amino acid residues. Despite the seeming simplicity of adding or removing a positively charged hydrogen nucleus, simulating the actual protonation/deprotonation process is inherently difficult. It requires both the explicit treatment of the excess proton, including its charge defect delocalization and Grotthuss shuttling through inhomogeneous moieties (water and amino residues), and extensive sampling of coupled condensed phase motions. In a recent paper (J. Chem. Theory Comput. 2014, 10, 2729-2737), a multiscale approach was developed to map high-level quantum mechanics/molecular mechanics (QM/MM) data into a multiscale reactive molecular dynamics (MS-RMD) model in order to describe amino acid deprotonation in bulk water. In this article, we extend the fitting approach (called FitRMD) to create MS-RMD models for ionizable amino acids within proteins. The resulting models are shown to faithfully reproduce the free energy profiles of the reference QM/MM Hamiltonian for PT inside an example protein, the ClC-ec1 H(+)/Cl(-) antiporter. Moreover, we show that the resulting MS-RMD models are computationally efficient enough to then characterize more complex 2-dimensional free energy surfaces due to slow degrees of freedom such as water hydration of internal protein cavities that can be inherently coupled to the excess proton charge translocation. The FitRMD method is thus shown to be an effective way to map ab initio level accuracy into a much more computationally efficient reactive MD method in order to explicitly simulate and quantitatively describe amino acid protonation/deprotonation in proteins.

Elevation of synaptic protein is associated with the antidepressant-like effects of ferulic acid in a chronic model of depression.

PubMed

Liu, Ya-Min; Hu, Chun-Yue; Shen, Ji-Duo; Wu, Su-Hui; Li, Yu-Cheng; Yi, Li-Tao

2017-02-01

Ferulic acid is a hydroxycinnamic acid that widely presents in plant cell wall components. It has been demonstrated that ferulic acid can reverse depressive-like behaviors in both forced swimming test and tail suspension test. However, it is unclear whether chronic ferulic acid treatment can ameliorate the depressive-like behaviors in chronic unpredictable mild stress (CUMS). Because of the putative relationship between neurotrophic system and antidepressant-like activity, we also investigated the effects of chronic ferulic acid on the brain-derived neurotrophic factor (BDNF), postsynaptic protein PSD95, presynaptic protein synapsin I in both prefrontal cortex and hippocampus. The results showed that ferulic acid significantly alleviated CUMS-induced depressive-like behaviors in sucrose preference test and forced swimming test. In addition, ferulic acid significantly up-regulated the levels of BDNF, PSD95 and synapsin I in the prefrontal cortex and hippocampus. The present data indicated that ferulic acid exerted the antidepressant-like effects on behaviors by increasing neurotrophin-related synaptic protein levels in CUMS mice. Copyright © 2016. Published by Elsevier Inc.

Effect of baseline plasma fatty acids on eicosapentaenoic acid levels in individuals supplemented with alpha-linolenic acid.

PubMed

DeFilippis, Andrew P; Harper, Charles R; Cotsonis, George A; Jacobson, Terry A

2009-01-01

We previously reported a >50% increase in mean plasma eicosapentaenoic acid levels in a general medicine clinic population after supplementation with alpha-linolenic acid. In the current analysis, we evaluate the variability of changes in eicosapentaenoic acid levels among individuals supplemented with alpha-linolenic acid and evaluated the impact of baseline plasma fatty acids levels on changes in eicosapentaenoic acid levels in these individuals. Changes in eicosapentaenoic acid levels among individuals supplemented with alpha-linolenic acid ranged from a 55% decrease to a 967% increase. Baseline plasma fatty acids had no statistically significant effect on changes in eicosapentaenoic levels acid after alpha-linolenic acid supplementation. Changes in eicosapentaenoic acid levels varied considerably in a general internal medicine clinic population supplemented with alpha-linolenic acid. Factors that may impact changes in plasma eicosapentaenoic acid levels after alpha-linolenic acid supplementation warrant further study.

The Citrus transcription factor, CitERF13, regulates citric acid accumulation via a protein-protein interaction with the vacuolar proton pump, CitVHA-c4

PubMed Central

Li, Shao-jia; Yin, Xue-ren; Xie, Xiu-lan; Allan, Andrew C.; Ge, Hang; Shen, Shu-ling; Chen, Kun-song

2016-01-01

Organic acids are essential to fruit flavor. The vacuolar H+ transporting adenosine triphosphatase (V-ATPase) plays an important role in organic acid transport and accumulation. However, less is known of V-ATPase interacting proteins and their relationship with organic acid accumulation. The relationship between V-ATPase and citric acid was investigated, using the citrus tangerine varieties ‘Ordinary Ponkan (OPK)’ and an early maturing mutant ‘Zaoshu Ponkan (ZPK)’. Five V-ATPase genes (CitVHA) were predicted as important to citric acid accumulation. Among the genes, CitVHA-c4 was observed, using a yeast two-hybrid screen, to interact at the protein level with an ethylene response factor, CitERF13. This was verified using bimolecular fluorescence complementation assays. A similar interaction was also observed between Arabidopsis AtERF017 (a CitERF13 homolog) and AtVHA-c4 (a CitVHA-c4 homolog). A synergistic effect on citric acid levels was observed between V-ATPase proteins and interacting ERFs when analyzed using transient over-expression in tobacco and Arabidopsis mutants. Furthermore, the transcript abundance of CitERF13 was concomitant with CitVHA-c4. CitERF13 or AtERF017 over-expression leads to significant citric acid accumulation. This accumulation was abolished in an AtVHA-c4 mutant background. ERF-VHA interactions appear to be involved in citric acid accumulation, which was observed in both citrus and Arabidopsis. PMID:26837571

The Citrus transcription factor, CitERF13, regulates citric acid accumulation via a protein-protein interaction with the vacuolar proton pump, CitVHA-c4.

PubMed

Li, Shao-jia; Yin, Xue-ren; Xie, Xiu-lan; Allan, Andrew C; Ge, Hang; Shen, Shu-ling; Chen, Kun-song

2016-02-03

Organic acids are essential to fruit flavor. The vacuolar H(+) transporting adenosine triphosphatase (V-ATPase) plays an important role in organic acid transport and accumulation. However, less is known of V-ATPase interacting proteins and their relationship with organic acid accumulation. The relationship between V-ATPase and citric acid was investigated, using the citrus tangerine varieties 'Ordinary Ponkan (OPK)' and an early maturing mutant 'Zaoshu Ponkan (ZPK)'. Five V-ATPase genes (CitVHA) were predicted as important to citric acid accumulation. Among the genes, CitVHA-c4 was observed, using a yeast two-hybrid screen, to interact at the protein level with an ethylene response factor, CitERF13. This was verified using bimolecular fluorescence complementation assays. A similar interaction was also observed between Arabidopsis AtERF017 (a CitERF13 homolog) and AtVHA-c4 (a CitVHA-c4 homolog). A synergistic effect on citric acid levels was observed between V-ATPase proteins and interacting ERFs when analyzed using transient over-expression in tobacco and Arabidopsis mutants. Furthermore, the transcript abundance of CitERF13 was concomitant with CitVHA-c4. CitERF13 or AtERF017 over-expression leads to significant citric acid accumulation. This accumulation was abolished in an AtVHA-c4 mutant background. ERF-VHA interactions appear to be involved in citric acid accumulation, which was observed in both citrus and Arabidopsis.

TMV-Cg Coat Protein stabilizes DELLA proteins and in turn negatively modulates salicylic acid-mediated defense pathway during Arabidopsis thaliana viral infection.

PubMed

Rodriguez, Maria Cecilia; Conti, Gabriela; Zavallo, Diego; Manacorda, Carlos Augusto; Asurmendi, Sebastian

2014-08-03

Plant viral infections disturb defense regulatory networks during tissue invasion. Emerging evidence demonstrates that a significant proportion of these alterations are mediated by hormone imbalances. Although the DELLA proteins have been reported to be central players in hormone cross-talk, their role in the modulation of hormone signaling during virus infections remains unknown. This work revealed that TMV-Cg coat protein (CgCP) suppresses the salicylic acid (SA) signaling pathway without altering defense hormone SA or jasmonic acid (JA) levels in Arabidopsis thaliana. Furthermore, it was observed that the expression of CgCP reduces plant growth and delays the timing of floral transition. Quantitative RT-qPCR analysis of DELLA target genes showed that CgCP alters relative expression of several target genes, indicating that the DELLA proteins mediate transcriptional changes produced by CgCP expression. Analyses by fluorescence confocal microscopy showed that CgCP stabilizes DELLA proteins accumulation in the presence of gibberellic acid (GA) and that the DELLA proteins are also stabilized during TMV-Cg virus infections. Moreover, DELLA proteins negatively modulated defense transcript profiles during TMV-Cg infection. As a result, TMV-Cg accumulation was significantly reduced in the quadruple-DELLA mutant Arabidopsis plants compared to wild type plants. Taken together, these results demonstrate that CgCP negatively regulates the salicylic acid-mediated defense pathway by stabilizing the DELLA proteins during Arabidopsis thaliana viral infection, suggesting that CgCP alters the stability of DELLAs as a mechanism of negative modulation of antiviral defense responses.

Amino acid composition and biological effects of supplementing broad bean and corn proteins with Nigella sativa (black cumin) cake protein.

PubMed

al-Gaby, A M

1998-10-01

The biological effects of supplementing broad bean (Vicia faba) or corn (Zea maize) meal protein with black cumin (Nigella sativa) cake protein as well as their amino acid composition were investigated. The percentage of total protein content of Nigella cake was 22.7%. Lysine is existent in abundant amounts in faba meal protein, while leucine is the most abundant in corn meal protein (chemical score = 156) and valine is higher in Nagella cake protein. compared with rats fed sole corn or faba meal protein, substitution of 25% of corn or faba meal protein with Nigella cake protein in the diet remarkably raised the growth rate of rats and resulted in significant higher levels of rat total serum lipids and triglycerides. Also, the supplemented diet caused significant increases in serum total protein and its two fractions albumin and globulin and insignificantly increase the activity of serum phosphatases and transaminases within normal ranges. The supplementation did not have any adverse nutritional effects in the levels of lipid fractions in the serum.

Detection of nucleic acid-protein interactions in plant leaves using fluorescence lifetime imaging microscopy.

PubMed

Camborde, Laurent; Jauneau, Alain; Brière, Christian; Deslandes, Laurent; Dumas, Bernard; Gaulin, Elodie

2017-09-01

DNA-binding proteins (DNA-BPs) and RNA-binding proteins (RNA-BPs) have critical roles in living cells in all kingdoms of life. Various experimental approaches exist for the study of nucleic acid-protein interactions in vitro and in vivo, but the detection of such interactions at the subcellular level remains challenging. Here we describe how to detect nucleic acid-protein interactions in plant leaves by using a fluorescence resonance energy transfer (FRET) approach coupled to fluorescence lifetime imaging microscopy (FLIM). Proteins of interest (POI) are tagged with a GFP and transiently expressed in plant cells to serve as donor fluorophore. After sample fixation and cell wall permeabilization, leaves are treated with Sytox Orange, a nucleic acid dye that can function as a FRET acceptor. Upon close association of the GFP-tagged POI with Sytox-Orange-stained nucleic acids, a substantial decrease of the GFP lifetime due to FRET between the donor and the acceptor can be monitored. Treatment with RNase before FRET-FLIM measurements allows determination of whether the POI associates with DNA and/or RNA. A step-by-step protocol is provided for sample preparation, data acquisition and analysis. We describe how to calibrate the equipment and include a tutorial explaining the use of the FLIM software. To illustrate our approach, we provide experimental procedures to detect the interaction between plant DNA and two proteins (the AeCRN13 effector from the oomycete Aphanomyces euteiches and the AtWRKY22 defensive transcription factor from Arabidopsis). This protocol allows the detection of protein-nucleic acid interactions in plant cells and can be completed in <2 d.

Protein and metabolic engineering for the production of organic acids.

PubMed

Liu, Jingjing; Li, Jianghua; Shin, Hyun-Dong; Liu, Long; Du, Guocheng; Chen, Jian

2017-09-01

Organic acids are natural metabolites of living organisms. They have been widely applied in the food, pharmaceutical, and bio-based materials industries. In recent years, biotechnological routes to organic acids production from renewable raw materials have been regarded as very promising approaches. In this review, we provide an overview of current developments in the production of organic acids using protein and metabolic engineering strategies. The organic acids include propionic acid, pyruvate, itaconic acid, succinic acid, fumaric acid, malic acid and citric acid. We also expect that rapid developments in the fields of systems biology and synthetic biology will accelerate protein and metabolic engineering for microbial organic acid production in the future. Copyright © 2017. Published by Elsevier Ltd.

Acid-degradable polyurethane particles for protein-based vaccines

PubMed Central

Bachelder, Eric M.; Beaudette, Tristan T.; Broaders, Kyle E.; Paramonov, Sergey E.; Dashe, Jesse; Fréchet, Jean M. J.

2009-01-01

Acid-degradable particles containing a model protein antigen, ovalbumin, were prepared from a polyurethane with acetal moieties embedded throughout the polymer, and characterized by dynamic light scattering and transmission electron microscopy. The small molecule degradation by-product of the particles was synthesized and tested in vitro for toxicity indicating an LC50 of 12,500 μg/ml. A new liquid chromatography-mass spectrometry technique was developed to monitor the in vitro degradation of these particles. The degradation by-product inside RAW macrophages was at its highest level after 24 hours of culture and was efficiently exocytosed until it was no longer detectable after four days. When tested in vitro, these particles induced a substantial increase in the presentation of the immunodominant ovalbumin-derived peptide SIINFEKL in both macrophages and dendritic cells. In addition, vaccination with these particles generated a cytotoxic T-lymphocyte response that was superior to both free ovalbumin and particles made from an analogous but slower-degrading acid-labile polyurethane polymer. Overall, we present a fully degradable polymer system with non-toxic by-products, which may find use in various biomedical applications including protein-based vaccines. PMID:18710254

Induced oxidative stress management in wounds through phenolic acids engineered fibrous protein: An in vitro assessment using polymorphonuclear (PMN) cells.

PubMed

Thiruselvi, T; Thirupathi Kumara Raja, S; Shanuja, S K; Iswarya, S; Gnanamani, A

2017-03-01

The present study explores the preparation, characterization and the role of phenolic acid tethered fibrous protein in the management of induced oxidative stress studied under in vitro conditions. In brief, the biomaterial is prepared by engineering the fibrous protein with dihydroxy and trihydroxy phenolic acid moieties and subjected to characterization to ensure the tethering. The resultant biomaterial studied for its efficacy as a free radical scavenger using polymorphonuclear (PMN) cells with induced oxidative stress and also as an agent for cell migration using fibroblasts cells. Results revealed that induced oxidative stress in PMN cells after exposure to UVB radiation managed well with the prepared biomaterial by reducing the levels of superoxide anion, oxygen and hydroxyl radicals. Further, the protein and the phenolic acid interaction supports the cell migration as evidenced from the scratch assay. In conclusion, though phenolic acids are well known for their antimicrobial and antioxidant potential, indenting these acids directly to the wounds is not sensible, but tethering to protein explored the scavenging activity as expected. The present study infers that phenolic acid engineered protein has a significant role in managing the imbalance in the redox state prevailing in wounds and supports the healing at appreciable level. Copyright © 2016 Elsevier B.V. All rights reserved.

Pretreatment levels of the fatty acid handling proteins H-FABP and CD36 predict response to olanzapine in recent-onset schizophrenia patients.

PubMed

Tomasik, Jakub; Schwarz, Emanuel; Lago, Santiago G; Rothermundt, Matthias; Leweke, F Markus; van Beveren, Nico J M; Guest, Paul C; Rahmoune, Hassan; Steiner, Johann; Bahn, Sabine

2016-02-01

Traditional schizophrenia pharmacotherapy remains a subjective trial and error process involving administration, titration and switching of drugs multiple times until an adequate response is achieved. Despite this time-consuming and costly process, not all patients show an adequate response to treatment. As a consequence, relapse is a common occurrence and early intervention is hampered. Here, we have attempted to identify candidate blood biomarkers associated with drug response in 121 initially antipsychotic-free recent-onset schizophrenia patients treated with widely-used antipsychotics, namely olanzapine (n=40), quetiapine (n=23), risperidone (n=30) and a mixture of these drugs (n=28). Patients were recruited and investigated as two separate cohorts to allow biomarker validation. Data analysis showed the most significant relationship between pre-treatment levels of heart-type fatty acid binding protein (H-FABP) and response to olanzapine (p=0.008, F=8.6, β=70.4 in the discovery cohort and p=0.003, F=15.2, β=24.4 in the validation cohort, adjusted for relevant confounding variables). In a functional follow-up analysis of this finding, we tested an independent cohort of 10 patients treated with olanzapine and found that baseline levels of plasma H-FABP and expression of the binding partner for H-FABP, fatty acid translocase (CD36), on monocytes predicted the reduction of psychotic symptoms (p=0.040, F=6.0, β=116.3 and p=0.012, F=11.9, β=-0.0054, respectively). We also identified a set of serum molecules changed after treatment with antipsychotic medication, in particular olanzapine. These molecules are predominantly involved in cellular development and metabolism. Taken together, our findings suggest an association between biomarkers involved in fatty acid metabolism and response to olanzapine, while other proteins may serve as surrogate markers associated with drug efficacy and side effects. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights

Utilizing knowledge base of amino acids structural neighborhoods to predict protein-protein interaction sites.

PubMed

Jelínek, Jan; Škoda, Petr; Hoksza, David

2017-12-06

Protein-protein interactions (PPI) play a key role in an investigation of various biochemical processes, and their identification is thus of great importance. Although computational prediction of which amino acids take part in a PPI has been an active field of research for some time, the quality of in-silico methods is still far from perfect. We have developed a novel prediction method called INSPiRE which benefits from a knowledge base built from data available in Protein Data Bank. All proteins involved in PPIs were converted into labeled graphs with nodes corresponding to amino acids and edges to pairs of neighboring amino acids. A structural neighborhood of each node was then encoded into a bit string and stored in the knowledge base. When predicting PPIs, INSPiRE labels amino acids of unknown proteins as interface or non-interface based on how often their structural neighborhood appears as interface or non-interface in the knowledge base. We evaluated INSPiRE's behavior with respect to different types and sizes of the structural neighborhood. Furthermore, we examined the suitability of several different features for labeling the nodes. Our evaluations showed that INSPiRE clearly outperforms existing methods with respect to Matthews correlation coefficient. In this paper we introduce a new knowledge-based method for identification of protein-protein interaction sites called INSPiRE. Its knowledge base utilizes structural patterns of known interaction sites in the Protein Data Bank which are then used for PPI prediction. Extensive experiments on several well-established datasets show that INSPiRE significantly surpasses existing PPI approaches.

Salivary Sialic Acid Levels in Smokeless Tobacco Users

PubMed Central

Farhad Mollashahi, Leila; Honarmand, Marieh; Nakhaee, Alireza; Mollashahi, Ghasem

2016-01-01

Background Smokeless tobacco chewing is one of the known risk factors for oral cancer. It is consumed widely by residents of southeastern Iran. Objectives In this study, salivary free and total sialic acid, and total protein were compared in paan consumers and non-consumers. Patients and Methods In this cross-sectional study, unstimulated saliva of 94 subjects (44 paan consumers and 50 non-consumers) who were referred to the oral medicine department of the dentistry school of Zahedan were collected. Salivary free and total sialic acid, and total protein concentration were measured by standard biochemical methods, and the obtained data were analyzed using SPSS 20 through the non-parametric Mann-Whitney test. Results The concentration of salivary free sialic acid (23.21 ± 18.98 mg/L) was significantly increased in paan consumers. The concentration of salivary Total sialic acid (TSA) (39.57 ± 26.58 mg/L) and total protein (0.77 ± 0.81 mg/mL) showed increases in paan consumers, however, the results were not statistically significant. Conclusions Salivary free and total sialic acid, and total protein were higher in the paan consumers compared to non-consumers. Due to the carcinogenic effect of smokeless tobacco, measurement of these parameters in saliva may be useful in early detection of oral cancer. PMID:27622172

Relation of soya bean meal level to the concentration of plasma free amino acids and body growth in white rats.

PubMed

Mandal, Tapas K; Parvin, Nargish; Mondal, Santanu; Saxena, Vijaylaxmi; Saxena, Ashok K; Sarkar, Sabyasachi; Saha, Mitali

2012-04-01

Amino acid (AA) levels in plasma and body growth were determined in rats (n20) fed diets with different soya bean meal levels. Free AA in plasma was determined by reversed-phase high-pressure liquid chromatography. We have used four levels of protein diets like 8%, 15%, 23% and 35% in this trial. Rats which were fed the low-protein (8%) diet with low percentage of soya bean meal were found to be growth-retarded. The body weight gain of high protein group (35%) was lower than that of the 23% groups. In the rats fed with the low-soya bean meal diet, some nonessential AA (NEAA) in plasma like asparagine, aspartic acid, cysteine, glutamic acid and serine increased, whereas the essential AA (EAA), with the exception of arginine, methionine and valine decreased. Here, plasma EAA-to-NEAA ratios were not correlated to growth and experimental diet. We hypothesize that AA metabolism is associated to changes in growth in rats on different protein intake. This study has showed the sensitivity of body mass gain, feed intake, feed conversion rate of rats to four levels of protein in the diet under controlled experimental conditions. © 2011 Blackwell Verlag GmbH.

Postprandial Levels of Branch Chained and Aromatic Amino Acids Associate with Fasting Glycaemia.

PubMed

Ottosson, Filip; Ericson, Ulrika; Almgren, Peter; Nilsson, Jeanette; Magnusson, Martin; Fernandez, Céline; Melander, Olle

2016-01-01

High fasting plasma concentrations of isoleucine, phenylalanine, and tyrosine have been associated with increased risk of hyperglycaemia and incidence of type 2 diabetes. Whether these associations are diet or metabolism driven is unknown. We examined how the dietary protein source affects the postprandial circulating profile of these three diabetes associated amino acids (DMAAs) and tested whether the postprandial DMAA profiles are associated with fasting glycaemia. We used a crossover design with twenty-one healthy individuals and four different isocaloric test meals, containing proteins from different dietary sources (dairy, fish, meat, and plants). Analysis of the postprandial DMAAs concentrations was performed using targeted mass spectrometry. A DMAA score was defined as the sum of all the three amino acid concentrations. The postprandial area under the curve (AUC) of all the three amino acids and the DMAA score was significantly greater after intake of the meal with dairy protein compared to intake of the three other meals. The postprandial AUC for the DMAA score and all the three amino acids strongly associated with fasting glucose level and insulin resistance. This indicates the importance of the postprandial kinetics and metabolism of DMAAs in understanding the overall association between DMAAs and glycaemia.

Long-Term Effect of Docosahexaenoic Acid Feeding on Lipid Composition and Brain Fatty Acid-Binding Protein Expression in Rats

PubMed Central

Elsherbiny, Marwa E.; Goruk, Susan; Monckton, Elizabeth A.; Richard, Caroline; Brun, Miranda; Emara, Marwan; Field, Catherine J.; Godbout, Roseline

2015-01-01

Arachidonic (AA) and docosahexaenoic acid (DHA) brain accretion is essential for brain development. The impact of DHA-rich maternal diets on offspring brain fatty acid composition has previously been studied up to the weanling stage; however, there has been no follow-up at later stages. Here, we examine the impact of DHA-rich maternal and weaning diets on brain fatty acid composition at weaning and three weeks post-weaning. We report that DHA supplementation during lactation maintains high DHA levels in the brains of pups even when they are fed a DHA-deficient diet for three weeks after weaning. We show that boosting dietary DHA levels for three weeks after weaning compensates for a maternal DHA-deficient diet during lactation. Finally, our data indicate that brain fatty acid binding protein (FABP7), a marker of neural stem cells, is down-regulated in the brains of six-week pups with a high DHA:AA ratio. We propose that elevated levels of DHA in developing brain accelerate brain maturation relative to DHA-deficient brains. PMID:26506385

CMP‑N‑acetylneuraminic acid synthetase interacts with fragile X related protein 1.

PubMed

Ma, Yun; Tian, Shuai; Wang, Zongbao; Wang, Changbo; Chen, Xiaowei; Li, Wei; Yang, Yang; He, Shuya

2016-08-01

Fragile X mental retardation protein (FMRP), fragile X related 1 protein (FXR1P) and FXR2P are the members of the FMR protein family. These proteins contain two KH domains and a RGG box, which are characteristic of RNA binding proteins. The absence of FMRP, causes fragile X syndrome (FXS), the leading cause of hereditary mental retardation. FXR1P is expressed throughout the body and important for normal muscle development, and its absence causes cardiac abnormality. To investigate the functions of FXR1P, a screen was performed to identify FXR1P‑interacting proteins and determine the biological effect of the interaction. The current study identified CMP‑N‑acetylneuraminic acid synthetase (CMAS) as an interacting protein using the yeast two‑hybrid system, and the interaction between FXR1P and CMAS was validated in yeast using a β‑galactosidase assay and growth studies with selective media. Furthermore, co‑immunoprecipitation was used to analyze the FXR1P/CMAS association and immunofluorescence microscopy was performed to detect expression and intracellular localization of the proteins. The results of the current study indicated that FXR1P and CMAS interact, and colocalize in the cytoplasm and the nucleus of HEK293T and HeLa cells. Accordingly, a fragile X related 1 (FXR1) gene overexpression vector was constructed to investigate the effect of FXR1 overexpression on the level of monosialotetrahexosylganglioside 1 (GM1). The results of the current study suggested that FXR1P is a tissue‑specific regulator of GM1 levels in SH‑SY5Y cells, but not in HEK293T cells. Taken together, the results initially indicate that FXR1P interacts with CMAS, and that FXR1P may enhance the activation of sialic acid via interaction with CMAS, and increase GM1 levels to affect the development of the nervous system, thus providing evidence for further research into the pathogenesis of FXS.

Enhanced Bio-hydrogen Production from Protein Wastewater by Altering Protein Structure and Amino Acids Acidification Type

PubMed Central

Xiao, Naidong; Chen, Yinguang; Chen, Aihui; Feng, Leiyu

2014-01-01

Enhanced bio-hydrogen production from protein wastewater by altering protein structure and amino acids acidification type via pH control was investigated. The hydrogen production reached 205.2 mL/g-protein when protein wastewater was pretreated at pH 12 and then fermented at pH 10. The mechanism studies showed that pH 12 pretreatment significantly enhanced protein bio-hydrolysis during the subsequent fermentation stage as it caused the unfolding of protein, damaged the protein hydrogen bonding networks, and destroyed the disulfide bridges, which increased the susceptibility of protein to protease. Moreover, pH 10 fermentation produced more acetic but less propionic acid during the anaerobic fermentation of amino acids, which was consistent with the theory of fermentation type affecting hydrogen production. Further analyses of the critical enzymes, genes, and microorganisms indicated that the activity and abundance of hydrogen producing bacteria in the pH 10 fermentation reactor were greater than those in the control. PMID:24495932

Enhanced bio-hydrogen production from protein wastewater by altering protein structure and amino acids acidification type.

PubMed

Xiao, Naidong; Chen, Yinguang; Chen, Aihui; Feng, Leiyu

2014-02-05

Enhanced bio-hydrogen production from protein wastewater by altering protein structure and amino acids acidification type via pH control was investigated. The hydrogen production reached 205.2 mL/g-protein when protein wastewater was pretreated at pH 12 and then fermented at pH 10. The mechanism studies showed that pH 12 pretreatment significantly enhanced protein bio-hydrolysis during the subsequent fermentation stage as it caused the unfolding of protein, damaged the protein hydrogen bonding networks, and destroyed the disulfide bridges, which increased the susceptibility of protein to protease. Moreover, pH 10 fermentation produced more acetic but less propionic acid during the anaerobic fermentation of amino acids, which was consistent with the theory of fermentation type affecting hydrogen production. Further analyses of the critical enzymes, genes, and microorganisms indicated that the activity and abundance of hydrogen producing bacteria in the pH 10 fermentation reactor were greater than those in the control.

Enhanced Bio-hydrogen Production from Protein Wastewater by Altering Protein Structure and Amino Acids Acidification Type

NASA Astrophysics Data System (ADS)

Xiao, Naidong; Chen, Yinguang; Chen, Aihui; Feng, Leiyu

2014-02-01

Enhanced bio-hydrogen production from protein wastewater by altering protein structure and amino acids acidification type via pH control was investigated. The hydrogen production reached 205.2 mL/g-protein when protein wastewater was pretreated at pH 12 and then fermented at pH 10. The mechanism studies showed that pH 12 pretreatment significantly enhanced protein bio-hydrolysis during the subsequent fermentation stage as it caused the unfolding of protein, damaged the protein hydrogen bonding networks, and destroyed the disulfide bridges, which increased the susceptibility of protein to protease. Moreover, pH 10 fermentation produced more acetic but less propionic acid during the anaerobic fermentation of amino acids, which was consistent with the theory of fermentation type affecting hydrogen production. Further analyses of the critical enzymes, genes, and microorganisms indicated that the activity and abundance of hydrogen producing bacteria in the pH 10 fermentation reactor were greater than those in the control.

Protein and Amino Acid Requirements during Pregnancy.

PubMed

Elango, Rajavel; Ball, Ronald O

2016-07-01

Protein forms an essential component of a healthy diet in humans to support both growth and maintenance. During pregnancy, an exceptional stage of life defined by rapid growth and development, adequate dietary protein is crucial to ensure a healthy outcome. Protein deposition in maternal and fetal tissues increases throughout pregnancy, with most occurring during the third trimester. Dietary protein intake recommendations are based on factorial estimates because the traditional method of determining protein requirements, nitrogen balance, is invasive and undesirable during pregnancy. The current Estimated Average Requirement and RDA recommendations of 0.88 and 1.1 g · kg(-1) · d(-1), respectively, are for all stages of pregnancy. The single recommendation does not take into account the changing needs during different stages of pregnancy. Recently, with the use of the minimally invasive indicator amino acid oxidation method, we defined the requirements to be, on average, 1.2 and 1.52 g · kg(-1) · d(-1) during early (∼16 wk) and late (∼36 wk) stages of pregnancy, respectively. Although the requirements are substantially higher than current recommendations, our values are ∼14-18% of total energy and fit within the Acceptable Macronutrient Distribution Range. Using swine as an animal model we showed that the requirements for several indispensable amino acids increase dramatically during late gestation compared with early gestation. Additional studies should be conducted during pregnancy to confirm the newly determined protein requirements and to determine the indispensable amino acid requirements during pregnancy in humans. © 2016 American Society for Nutrition.

The Prognostic Value of Serum Levels of Heart-Type Fatty Acid Binding Protein and High Sensitivity C-Reactive Protein in Patients With Increased Levels of Amino-Terminal Pro-B Type Natriuretic Peptide.

PubMed

Jeong, Ji Hun; Seo, Yiel Hea; Ahn, Jeong Yeal; Kim, Kyung Hee; Seo, Ja Young; Kim, Moon Jin; Lee, Hwan Tae; Park, Pil Whan

2016-09-01

Amino-terminal pro-B type natriuretic peptide (NT-proBNP) is a well-established prognostic factor in heart failure (HF). However, numerous causes may lead to elevations in NT-proBNP, and thus, an increased NT-proBNP level alone is not sufficient to predict outcome. The aim of this study was to evaluate the utility of two acute response markers, high sensitivity C-reactive protein (hsCRP) and heart-type fatty acid binding protein (H-FABP), in patients with an increased NT-proBNP level. The 278 patients were classified into three groups by etiology: 1) acute coronary syndrome (ACS) (n=62), 2) non-ACS cardiac disease (n=156), and 3) infectious disease (n=60). Survival was determined on day 1, 7, 14, 21, 28, 60, 90, 120, and 150 after enrollment. H-FABP (P<0.001), NT-proBNP (P=0.006), hsCRP (P<0.001) levels, and survival (P<0.001) were significantly different in the three disease groups. Patients were divided into three classes by using receiver operating characteristic curves for NT-proBNP, H-FABP, and hsCRP. Patients with elevated NT-proBNP (≥3,856 pg/mL) and H-FABP (≥8.8 ng/mL) levels were associated with higher hazard ratio for mortality (5.15 in NT-proBNP and 3.25 in H-FABP). Area under the receiver operating characteristic curve analysis showed H-FABP was a better predictor of 60-day mortality than NT-proBNP. The combined measurement of H-FABP with NT-proBNP provides a highly reliable means of short-term mortality prediction for patients hospitalized for ACS, non-ACS cardiac disease, or infectious disease.

Parsing the life-shortening effects of dietary protein: effects of individual amino acids

PubMed Central

Bouchebti, Sofia; Bazazi, Sepideh; Le Hesran, Sophie; Puga, Camille; Latil, Gérard; Simpson, Stephen J.

2017-01-01

High-protein diets shorten lifespan in many organisms. Is it because protein digestion is energetically costly or because the final products (the amino acids) are harmful? To answer this question while circumventing the life-history trade-off between reproduction and longevity, we fed sterile ant workers on diets based on whole proteins or free amino acids. We found that (i) free amino acids shortened lifespan even more than proteins; (ii) the higher the amino acid-to-carbohydrate ratio, the shorter ants lived and the lower their lipid reserves; (iii) for the same amino acid-to-carbohydrate ratio, ants eating free amino acids had more lipid reserves than those eating whole proteins; and (iv) on whole protein diets, ants seem to regulate food intake by prioritizing sugar, while on free amino acid diets, they seem to prioritize amino acids. To test the effect of the amino acid profile, we tested diets containing proportions of each amino acid that matched the ant's exome; surprisingly, longevity was unaffected by this change. We further tested diets with all amino acids under-represented except one, finding that methionine, serine, threonine and phenylalanine are especially harmful. All together, our results show certain amino acids are key elements behind the high-protein diet reduction in lifespan. PMID:28053059

Parsing the life-shortening effects of dietary protein: effects of individual amino acids.

PubMed

Arganda, Sara; Bouchebti, Sofia; Bazazi, Sepideh; Le Hesran, Sophie; Puga, Camille; Latil, Gérard; Simpson, Stephen J; Dussutour, Audrey

2017-01-11

High-protein diets shorten lifespan in many organisms. Is it because protein digestion is energetically costly or because the final products (the amino acids) are harmful? To answer this question while circumventing the life-history trade-off between reproduction and longevity, we fed sterile ant workers on diets based on whole proteins or free amino acids. We found that (i) free amino acids shortened lifespan even more than proteins; (ii) the higher the amino acid-to-carbohydrate ratio, the shorter ants lived and the lower their lipid reserves; (iii) for the same amino acid-to-carbohydrate ratio, ants eating free amino acids had more lipid reserves than those eating whole proteins; and (iv) on whole protein diets, ants seem to regulate food intake by prioritizing sugar, while on free amino acid diets, they seem to prioritize amino acids. To test the effect of the amino acid profile, we tested diets containing proportions of each amino acid that matched the ant's exome; surprisingly, longevity was unaffected by this change. We further tested diets with all amino acids under-represented except one, finding that methionine, serine, threonine and phenylalanine are especially harmful. All together, our results show certain amino acids are key elements behind the high-protein diet reduction in lifespan. © 2017 The Author(s).

Measuring protein-protein and protein-nucleic Acid interactions by biolayer interferometry.

PubMed

Sultana, Azmiri; Lee, Jeffrey E

2015-02-02

Biolayer interferometry (BLI) is a simple, optical dip-and-read system useful for measuring interactions between proteins, peptides, nucleic acids, small molecules, and/or lipids in real time. In BLI, a biomolecular bait is immobilized on a matrix at the tip of a fiber-optic sensor. The binding between the immobilized ligand and another molecule in an analyte solution produces a change in optical thickness at the tip and results in a wavelength shift proportional to binding. BLI provides direct binding affinities and rates of association and dissociation. This unit describes an efficient approach using streptavidin-based BLI to analyze DNA-protein and protein-protein interactions. A quantitative set of equilibrium binding affinities (K(d)) and rates of association and dissociation (k(a)/k(d)) can be measured in minutes using nanomole quantities of sample. Copyright © 2015 John Wiley & Sons, Inc.

Alkali-catalyzed low temperature wet crosslinking of plant proteins using carboxylic acids.

PubMed

Reddy, Narendra; Li, Ying; Yang, Yiqi

2009-01-01

We report the development of a new method of alkali-catalyzed low temperature wet crosslinking of plant proteins to improve their breaking tenacity without using high temperatures or phosphorus-containing catalysts used in conventional poly(carboxylic acid) crosslinking of cellulose and proteins. Carboxylic acids are preferred over aldehyde-containing crosslinkers for crosslinking proteins and cellulose because of their low toxicity and cost and ability to improve the desired properties of the materials. However, current knowledge in carboxylic acid crosslinking of proteins and cellulose requires the use of carboxylic acids with at least three carboxylic groups, toxic phosphorous-containing catalysts and curing at high temperatures (150-185 degrees C). The use of high temperatures and low pH in conventional carboxylic acid crosslinking has been reported to cause substantial strength loss and/or undesired changes in the properties of the crosslinked materials. In this research, gliadin, soy protein, and zein fibers have been crosslinked with malic acid, citric acid, and butanetetracarboxylic acid to improve the tenacity of the fibers without using high temperatures and phosphorus-containing catalysts. The new method of wet crosslinking using carboxylic acids containing two or more carboxylic groups will be useful to crosslink proteins for various industrial applications.

Uric acid level and erectile dysfunction in patients with coronary artery disease.

PubMed

Solak, Yalcin; Akilli, Hakan; Kayrak, Mehmet; Aribas, Alpay; Gaipov, Abduzhappar; Turk, Suleyman; Perez-Pozo, Santos E; Covic, Adrian; McFann, Kim; Johnson, Richard J; Kanbay, Mehmet

2014-01-01

Erectile dysfunction (ED) is a frequent complaint of elderly subjects and is closely associated with endothelial dysfunction and cardiovascular disease (CVD). Uric acid is also associated with endothelial dysfunction, oxidative stress, and CVD, raising the hypothesis that an increased serum uric acid might predict ED in patients who are at risk for coronary artery disease (CAD). This study aims to evaluate the association of serum uric acid levels with presence and severity of ED in patients presenting with chest pain of presumed cardiac origin. This is a cross-sectional study of 312 adult male patients with suspected CAD who underwent exercise stress test (EST) for workup of chest pain and completed a sexual health inventory for men survey form to determine the presence and severity of ED. Routine serum biochemistry (and uric acid levels) were measured. Logistic regression analysis was used to assess risk factors for ED. The short version of the International Index of Erectile Function questionnaire diagnosed ED (cutoff score ≤ 21). Serum uric acid levels were determined. Patients with chest pain of suspected cardiac origin underwent an EST. One hundred forty-nine of 312 (47.7%) male subjects had ED by survey criteria. Patients with ED were older and had more frequent CAD, hypertension, diabetes and impaired renal function, and also had significantly higher levels of uric acid, fibrinogen, glucose, C-reactive protein, triglycerides compared with patients without ED. Uric acid levels were associated with ED by univariate analysis (odds ratio = 1.36, P = 0.002); however, this association was not observed in multivariate analysis adjusted for estimated glomerular filtration rate. Subjects presenting with chest pain of presumed cardiac origin are more likely to have ED if they have elevated uric acid levels. © 2013 International Society for Sexual Medicine.

Effects of conjugated linoleic acid supplementation and feeding level on dairy performance, milk fatty acid composition, and body fat changes in mid-lactation goats.

PubMed

Ghazal, S; Berthelot, V; Friggens, N C; Schmidely, P

2014-11-01

The objective of this trial was to study the interaction between the supplementation of lipid-encapsulated conjugated linoleic acid (CLA; 4.5 g of cis-9,trans-11 C18:2 and 4.5 g of trans-10,cis-12 C18:2) and feeding level to test if milk performance or milk fatty acid (FA) profile are affected by the interaction between CLA and feeding level. Twenty-four dairy goats were used in an 8-wk trial with a 3-wk adaptation to the experimental ration that contained corn silage, beet pulp, barley, and a commercial concentrate. During the third week, goats were assigned into blocks of 2 goats according to their dry matter intake (DMI), raw milk yield, and fat yield. Each block was randomly allocated to control (45 g of Ca salt of palm oil/d) or CLA treatment. Within each block, one goat was fed to cover 100% (FL100) of the calculated energy requirements and the other was fed 85% of the DMI of the first goat (FL85). Individual milk production and composition were recorded weekly, and milk FA composition was analyzed in wk 3, 5, and 7. Conjugated linoleic acid supplementation reduced milk fat content and fat yield by 17 and 19%, respectively, independent of the feeding level. It reduced both the secretion of milk FA synthesized de novo, and those taken up from the blood. No interaction between CLA and feeding level was observed on milk secretion of any group of FA. The CLA supplementation had no effect on DMI, milk yield, protein, and lactose yields but it improved calculated net energy for lactation balance. Goats fed the FL100 × CLA diet tended to have the highest DMI and protein yield. The interaction between CLA and feeding level was not significant for any other variables. Compared with the goats fed FL100, those fed FL85 had lower DMI, lower net energy for lactation balance, and lower digestible protein in the intestine balance. The body weight; milk yield; milk fat, protein, and lactose yields; and fat, protein, lactose, and urea contents in milk were not affected by

Site-specific incorporation of redox active amino acids into proteins

DOEpatents

Alfonta, Lital [San Diego, CA; Schultz, Peter G [La Jolla, CA; Zhang, Zhiwen [Austin, TX

2011-08-30

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

Site-specific incorporation of redox active amino acids into proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alfonta, Lital; Schultz, Peter G.; Zhang, Zhiwen

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

Site-specific incorporation of redox active amino acids into proteins

DOEpatents

Alfonta, Lital [San Diego, CA; Schultz, Peter G [La Jolla, CA; Zhang, Zhiwen [San Diego, CA

2012-02-14

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

Site-specific incorporation of redox active amino acids into proteins

DOEpatents

Alfonta; Lital , Schultz; Peter G. , Zhang; Zhiwen

2010-10-12

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

Site-specific incorporation of redox active amino acids into proteins

DOEpatents

Alfonta, Lital [San Diego, CA; Schultz, Peter G [La Jolla, CA; Zhang, Zhiwen [San Diego, CA

2009-02-24

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

Multiple Amino Acid Supplementations to Reduce Dietary Protein in Plant-Based Rainbow Trout, Oncorhynchus mykiss, Feeds

USDA-ARS?s Scientific Manuscript database

Reducing dietary protein in trout feeds will reduce production costs if growth performance can be maintained. A study was conducted to determine if balancing plant-based diets on an available amino acid basis would result in a reduction in total protein level. The diets were formulated to contain ...

Crystal structure of axolotl (Ambystoma mexicanum) liver bile acid-binding protein bound to cholic and oleic acid.

PubMed

Capaldi, Stefano; Guariento, Mara; Perduca, Massimiliano; Di Pietro, Santiago M; Santomé, José A; Monaco, Hugo L

2006-07-01

The family of the liver bile acid-binding proteins (L-BABPs), formerly called liver basic fatty acid-binding proteins (Lb-FABPs) shares fold and sequence similarity with the paralogous liver fatty acid-binding proteins (L-FABPs) but has a different stoichiometry and specificity of ligand binding. This article describes the first X-ray structure of a member of the L-BABP family, axolotl (Ambystoma mexicanum) L-BABP, bound to two different ligands: cholic and oleic acid. The protein binds one molecule of oleic acid in a position that is significantly different from that of either of the two molecules that bind to rat liver FABP. The stoichiometry of binding of cholate is of two ligands per protein molecule, as observed in chicken L-BABP. The cholate molecule that binds buried most deeply into the internal cavity overlaps well with the analogous bound to chicken L-BABP, whereas the second molecule, which interacts with the first only through hydrophobic contacts, is more external and exposed to the solvent. (c) 2006 Wiley-Liss, Inc.

Measurement of amino acid levels in the vitreous humor of rats after chronic intraocular pressure elevation or optic nerve transection.

PubMed

Levkovitch-Verbin, Hana; Martin, Keith R G; Quigley, Harry A; Baumrind, Lisa A; Pease, Mary Ellen; Valenta, Danielle

2002-10-01

To investigate whether the levels of free amino acids and protein in the vitreous of rat eyes are altered with chronic intraocular pressure (IOP) elevation or after optic nerve transection. The concentrations of 20 amino acids in the vitreous humor were measured by high-performance liquid chromatography in both eyes of 41 rats with unilateral IOP elevation induced by translimbal photocoagulation. Eyes were studied 1 day and 1, 2, 4, and 9 weeks after initial IOP elevation. The same amino acids were measured in 41 rats 1 day and 2, 4, and 9 weeks after unilateral transection of the orbital optic nerve. The intravitreal protein level was assayed in additional 22 rats with IOP elevation and 12 rats after nerve transection. Two masked observers evaluated the amount of optic nerve damage with a semiquantitative, light-microscopic technique. In rats with experimental glaucoma, amino acid concentrations were unchanged 1 day after treatment. At 1 week, 4 of 20 amino acids (aspartate, proline, alanine, and lysine) were higher than in control eyes ( < or = 0.01), but this difference was nonsignificant after Bonferroni correction for multiple simultaneous amino acid comparisons (none achieved < 0.0025). No amino acid was significantly different from control in the nerve transection groups (all > 0.05). Vitreous protein level was significantly higher in glaucomatous eyes than their paired controls at 1 day ( < 0.0001) and 1 week ( < 0.002). One day and 1 week after optic nerve transection, vitreal proteins were significantly elevated compared with control eyes from untreated animals ( < 0.0020 and < 0.0022, respectively), though not compared with their fellow eyes ( = 0.25 and 0.10). Chronic experimental glaucoma and transection of the optic nerve increase the amount of protein in the rat vitreous above control levels. In the vitreous of rats with experimental glaucoma, a number of free amino acids were transiently elevated to a modest degree, but no significant difference in

Fatty acid binding proteins have the potential to channel dietary fatty acids into enterocyte nuclei.

PubMed

Esteves, Adriana; Knoll-Gellida, Anja; Canclini, Lucia; Silvarrey, Maria Cecilia; André, Michèle; Babin, Patrick J

2016-02-01

Intracellular lipid binding proteins, including fatty acid binding proteins (FABPs) 1 and 2, are highly expressed in tissues involved in the active lipid metabolism. A zebrafish model was used to demonstrate differential expression levels of fabp1b.1, fabp1b.2, and fabp2 transcripts in liver, anterior intestine, and brain. Transcription levels of fabp1b.1 and fabp2 in the anterior intestine were upregulated after feeding and modulated according to diet formulation. Immunofluorescence and electron microscopy immunodetection with gold particles localized these FABPs in the microvilli, cytosol, and nuclei of most enterocytes in the anterior intestinal mucosa. Nuclear localization was mostly in the interchromatin space outside the condensed chromatin clusters. Native PAGE binding assay of BODIPY-FL-labeled FAs demonstrated binding of BODIPY-FLC(12) but not BODIPY-FLC(5) to recombinant Fabp1b.1 and Fabp2. The binding of BODIPY-FLC(12) to Fabp1b.1 was fully displaced by oleic acid. In vivo experiments demonstrated, for the first time, that intestinal absorption of dietary BODIPY-FLC(12) was followed by colocalization of the labeled FA with Fabp1b and Fabp2 in the nuclei. These data suggest that dietary FAs complexed with FABPs are able to reach the enterocyte nucleus with the potential to modulate nuclear activity. Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.

Electricity-Free, Sequential Nucleic Acid and Protein Isolation

PubMed Central

Pawlowski, David R.; Karalus, Richard J.

2012-01-01

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable 1. The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment 2. The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis. Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters 3. CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation4. By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification while

Electricity-free, sequential nucleic acid and protein isolation.

PubMed

Pawlowski, David R; Karalus, Richard J

2012-05-15

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable (1). The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment (2). The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis. Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters (3). CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation(4). By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification

Cortical deficits of glutamic acid decarboxylase 67 expression in schizophrenia: clinical, protein, and cell type-specific features.

PubMed

Curley, Allison A; Arion, Dominique; Volk, David W; Asafu-Adjei, Josephine K; Sampson, Allan R; Fish, Kenneth N; Lewis, David A

2011-09-01

Cognitive deficits in schizophrenia are associated with altered activity of the dorsolateral prefrontal cortex, which has been attributed to lower expression of the 67 kDa isoform of glutamic acid decarboxylase (GAD67), the major γ-aminobutyric acid (GABA)-synthesizing enzyme. However, little is known about the relationship of prefrontal GAD67 mRNA levels and illness severity, translation of the transcript into protein, and protein levels in axon terminals, the key site of GABA production and function. Quantitative polymerase chain reaction was used to measure GAD67 mRNA levels in postmortem specimens of dorsolateral prefrontal cortex from subjects with schizophrenia and matched comparison subjects with no known history of psychiatric or neurological disorders (N=42 pairs). In a subset of this cohort in which potential confounds of protein measures were controlled (N=19 pairs), Western blotting was used to quantify tissue levels of GAD67 protein in tissue. In five of these pairs, multilabel confocal immunofluorescence was used to quantify GAD67 protein levels in the axon terminals of parvalbumin-containing GABA neurons, which are known to have low levels of GAD67 mRNA in schizophrenia. GAD67 mRNA levels were significantly lower in schizophrenia subjects (by 15%), but transcript levels were not associated with predictors or measures of illness severity or chronicity. In schizophrenia subjects, GAD67 protein levels were significantly lower in total gray matter (by 10%) and in parvalbumin axon terminals (by 49%). The findings that lower GAD67 mRNA expression is common in schizophrenia, that it is not a consequence of having the illness, and that it leads to less translation of the protein, especially in the axon terminals of parvalbumin-containing neurons, support the hypothesis that lower GABA synthesis in parvalbumin neurons contributes to dorsolateral prefrontal cortex dysfunction and impaired cognition in schizophrenia.

The primary structure of fatty-acid-binding protein from nurse shark liver. Structural and evolutionary relationship to the mammalian fatty-acid-binding protein family.

PubMed

Medzihradszky, K F; Gibson, B W; Kaur, S; Yu, Z H; Medzihradszky, D; Burlingame, A L; Bass, N M

1992-02-01

The primary structure of a fatty-acid-binding protein (FABP) isolated from the liver of the nurse shark (Ginglymostoma cirratum) was determined by high-performance tandem mass spectrometry (employing multichannel array detection) and Edman degradation. Shark liver FABP consists of 132 amino acids with an acetylated N-terminal valine. The chemical molecular mass of the intact protein determined by electrospray ionization mass spectrometry (Mr = 15124 +/- 2.5) was in good agreement with that calculated from the amino acid sequence (Mr = 15121.3). The amino acid sequence of shark liver FABP displays significantly greater similarity to the FABP expressed in mammalian heart, peripheral nerve myelin and adipose tissue (61-53% sequence similarity) than to the FABP expressed in mammalian liver (22% similarity). Phylogenetic trees derived from the comparison of the shark liver FABP amino acid sequence with the members of the mammalian fatty-acid/retinoid-binding protein gene family indicate the initial divergence of an ancestral gene into two major subfamilies: one comprising the genes for mammalian liver FABP and gastrotropin, the other comprising the genes for mammalian cellular retinol-binding proteins I and II, cellular retinoic-acid-binding protein myelin P2 protein, adipocyte FABP, heart FABP and shark liver FABP, the latter having diverged from the ancestral gene that ultimately gave rise to the present day mammalian heart-FABP, adipocyte FABP and myelin P2 protein sequences. The sequence for intestinal FABP from the rat could be assigned to either subfamily, depending on the approach used for phylogenetic tree construction, but clearly diverged at a relatively early evolutionary time point. Indeed, sequences proximately ancestral or closely related to mammalian intestinal FABP, liver FABP, gastrotropin and the retinoid-binding group of proteins appear to have arisen prior to the divergence of shark liver FABP and should therefore also be present in elasmobranchs

Cardiotonic Steroids Stabilize Regulator of G Protein Signaling 2 Protein Levels

PubMed Central

Sjögren, Benita; Parra, Sergio; Heath, Lauren J.; Atkins, Kevin B.; Xie, Zie-Jian

2012-01-01

Regulator of G protein signaling 2 (RGS2), a Gq-specific GTPase-activating protein, is strongly implicated in cardiovascular function. RGS2(−/−) mice are hypertensive and prone to heart failure, and several rare human mutations that accelerate RGS2 degradation have been identified among patients with hypertension. Therefore, pharmacological up-regulation of RGS2 protein levels might be beneficial. We used a β-galactosidase complementation method to screen several thousand compounds with known pharmacological functions for those that increased RGS2 protein levels. Several cardiotonic steroids (CTSs), including ouabain and digoxin, increased RGS2 but not RGS4 protein levels. CTSs increased RGS2 protein levels through a post-transcriptional mechanism, by slowing protein degradation. RGS2 mRNA levels in primary vascular smooth muscle cells were unaffected by CTS treatment, whereas protein levels were increased 2- to 3-fold. Na+/K+-ATPase was required for the increase in RGS2 protein levels, because the effect was lost in Na+/K+-ATPase-knockdown cells. Furthermore, we demonstrated that CTS-induced increases in RGS2 levels were functional and reduced receptor-stimulated, Gq-dependent, extracellular signal-regulated kinase phosphorylation. Finally, we showed that in vivo treatment with digoxin led to increased RGS2 protein levels in heart and kidney. CTS-induced increases in RGS2 protein levels and function might modify several deleterious mechanisms in hypertension and heart failure. This novel CTS mechanism might contribute to the beneficial actions of low-dose digoxin treatment in heart failure. Our results support the concept of small-molecule modulation of RGS2 protein levels as a new strategy for cardiovascular therapy. PMID:22695717

Structure of the ordered hydration of amino acids in proteins: analysis of crystal structures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Biedermannová, Lada, E-mail: lada.biedermannova@ibt.cas.cz; Schneider, Bohdan

2015-10-27

The hydration of protein crystal structures was studied at the level of individual amino acids. The dependence of the number of water molecules and their preferred spatial localization on various parameters, such as solvent accessibility, secondary structure and side-chain conformation, was determined. Crystallography provides unique information about the arrangement of water molecules near protein surfaces. Using a nonredundant set of 2818 protein crystal structures with a resolution of better than 1.8 Å, the extent and structure of the hydration shell of all 20 standard amino-acid residues were analyzed as function of the residue conformation, secondary structure and solvent accessibility. Themore » results show how hydration depends on the amino-acid conformation and the environment in which it occurs. After conformational clustering of individual residues, the density distribution of water molecules was compiled and the preferred hydration sites were determined as maxima in the pseudo-electron-density representation of water distributions. Many hydration sites interact with both main-chain and side-chain amino-acid atoms, and several occurrences of hydration sites with less canonical contacts, such as carbon–donor hydrogen bonds, OH–π interactions and off-plane interactions with aromatic heteroatoms, are also reported. Information about the location and relative importance of the empirically determined preferred hydration sites in proteins has applications in improving the current methods of hydration-site prediction in molecular replacement, ab initio protein structure prediction and the set-up of molecular-dynamics simulations.« less

Fatty acid oxidation promotes reprogramming by enhancing oxidative phosphorylation and inhibiting protein kinase C.

PubMed

Lin, Zhaoyu; Liu, Fei; Shi, Peiliang; Song, Anying; Huang, Zan; Zou, Dayuan; Chen, Qin; Li, Jianxin; Gao, Xiang

2018-02-26

Changes in metabolic pathway preferences are key events in the reprogramming process of somatic cells to induced pluripotent stem cells (iPSCs). The optimization of metabolic conditions can enhance reprogramming; however, the detailed underlying mechanisms are largely unclear. By comparing the gene expression profiles of somatic cells, intermediate-phase cells, and iPSCs, we found that carnitine palmitoyltransferase (Cpt)1b, a rate-limiting enzyme in fatty acid oxidation, was significantly upregulated in the early stage of the reprogramming process. Mouse embryonic fibroblasts isolated from transgenic mice carrying doxycycline (Dox)-inducible Yamanaka factor constructs were used for reprogramming. Various fatty acid oxidation-related metabolites were added during the reprogramming process. Colony counting and fluorescence-activated cell sorting (FACS) were used to calculate reprogramming efficiency. Fatty acid oxidation-related metabolites were measured by liquid chromatography-mass spectrometry. Seahorse was used to measure the level of oxidative phosphorylation. We found that overexpression of cpt1b enhanced reprogramming efficiency. Furthermore, palmitoylcarnitine or acetyl-CoA, the primary and final products of Cpt1-mediated fatty acid oxidation, also promoted reprogramming. In the early reprogramming process, fatty acid oxidation upregulated oxidative phosphorylation and downregulated protein kinase C activity. Inhibition of protein kinase C also promoted reprogramming. We demonstrated that fatty acid oxidation promotes reprogramming by enhancing oxidative phosphorylation and inhibiting protein kinase C activity in the early stage of the reprogramming process. This study reveals that fatty acid oxidation is crucial for the reprogramming efficiency.

Etomoxir-induced increase in UCP3 supports a role of uncoupling protein 3 as a mitochondrial fatty acid anion exporter.

PubMed

Schrauwen, Patrick; Hinderling, Vera; Hesselink, Matthijs K C; Schaart, Gert; Kornips, Esther; Saris, Wim H M; Westerterp-Plantenga, Margriet; Langhans, Wolfgang

2002-10-01

The physiological function of human uncoupling protein-3 is still unknown. Uncoupling protein-3 is increased during fasting and high-fat feeding. In these situations the availability of fatty acids to the mitochondria exceeds the capacity to metabolize fatty acids, suggesting a role for uncoupling protein-3 in handling of non-metabolizable fatty acids. To test the hypothesis that uncoupling protein-3 acts as a mitochondrial exporter of non-metabolizable fatty acids from the mitochondrial matrix, we gave human subjects Etomoxir (which blocks mitochondrial entry of fatty acids) or placebo in a cross-over design during a 36-h stay in a respiration chamber. Etomoxir inhibited 24-h fat oxidation and fat oxidation during exercise by approximately 14-19%. Surprisingly, uncoupling protein-3 content in human vastus lateralis muscle was markedly up-regulated within 36 h of Etomoxir administration. Up-regulation of uncoupling protein-3 was accompanied by lowered fasting blood glucose and increased translocation of glucose transporter-4. These data support the hypothesis that the physiological function of uncoupling protein-3 is to facilitate the outward transport of non-metabolizable fatty acids from the mitochondrial matrix and thus prevents mitochondria from the potential deleterious effects of high fatty acid levels. In addition our data show that up-regulation of uncoupling protein-3 can be beneficial in the treatment of type 2 diabetes.

Leucine-Enriched Essential Amino Acids Augment Mixed Protein Synthesis, But Not Collagen Protein Synthesis, in Rat Skeletal Muscle after Downhill Running

PubMed Central

Kato, Hiroyuki; Suzuki, Hiromi; Inoue, Yoshiko; Suzuki, Katsuya; Kobayashi, Hisamine

2016-01-01

Mixed and collagen protein synthesis is elevated for as many as 3 days following exercise. Immediately after exercise, enhanced amino acid availability increases synthesis of mixed muscle protein, but not muscle collagen protein. However, the potential for synergic effects of amino acid ingestion with exercise on both mixed and collagen protein synthesis remains unclear. We investigated muscle collagen protein synthesis in rats following post-exercise ingestion of leucine-enriched essential amino acids. We determined fractional protein synthesis rates (FSR) at different time points following exercise. Mixed protein and collagen protein FSRs in skeletal muscle were determined by measuring protein-bound enrichments of hydroxyproline and proline, and by measuring the intracellular enrichment of proline, using injections of flooding d3-proline doses. A leucine-enriched mixture of essential amino acids (or distilled water as a control) was administrated 30 min or 1 day post-exercise. The collagen protein synthesis in the vastus lateralis was elevated for 2 days after exercise. Although amino acid administration did not increase muscle collagen protein synthesis, it did lead to augmented mixed muscle protein synthesis 1 day following exercise. Thus, contrary to the regulation of mixed muscle protein synthesis, muscle collagen protein synthesis is not affected by amino acid availability after damage-inducing exercise. PMID:27367725

NIAS-Server: Neighbors Influence of Amino acids and Secondary Structures in Proteins.

PubMed

Borguesan, Bruno; Inostroza-Ponta, Mario; Dorn, Márcio

2017-03-01

The exponential growth in the number of experimentally determined three-dimensional protein structures provide a new and relevant knowledge about the conformation of amino acids in proteins. Only a few of probability densities of amino acids are publicly available for use in structure validation and prediction methods. NIAS (Neighbors Influence of Amino acids and Secondary structures) is a web-based tool used to extract information about conformational preferences of amino acid residues and secondary structures in experimental-determined protein templates. This information is useful, for example, to characterize folds and local motifs in proteins, molecular folding, and can help the solution of complex problems such as protein structure prediction, protein design, among others. The NIAS-Server and supplementary data are available at http://sbcb.inf.ufrgs.br/nias .

Topology of RNA–protein nucleobase–amino acid π–π interactions and comparison to analogous DNA–protein π–π contacts

PubMed Central

Wilson, Katie A.; Holland, Devany J.; Wetmore, Stacey D.

2016-01-01

The present work analyzed 120 high-resolution X-ray crystal structures and identified 335 RNA–protein π-interactions (154 nonredundant) between a nucleobase and aromatic (W, H, F, or Y) or acyclic (R, E, or D) π-containing amino acid. Each contact was critically analyzed (including using a visual inspection protocol) to determine the most prevalent composition, structure, and strength of π-interactions at RNA–protein interfaces. These contacts most commonly involve F and U, with U:F interactions comprising one-fifth of the total number of contacts found. Furthermore, the RNA and protein π-systems adopt many different relative orientations, although there is a preference for more parallel (stacked) arrangements. Due to the variation in structure, the strength of the intermolecular forces between the RNA and protein components (as determined from accurate quantum chemical calculations) exhibits a significant range, with most of the contacts providing significant stability to the associated RNA–protein complex (up to −65 kJ mol−1). Comparison to the analogous DNA–protein π-interactions emphasizes differences in RNA– and DNA–protein π-interactions at the molecular level, including the greater abundance of RNA contacts and the involvement of different nucleobase/amino acid residues. Overall, our results provide a clearer picture of the molecular basis of nucleic acid–protein binding and underscore the important role of these contacts in biology, including the significant contribution of π–π interactions to the stability of nucleic acid–protein complexes. Nevertheless, more work is still needed in this area in order to further appreciate the properties and roles of RNA nucleobase–amino acid π-interactions in nature. PMID:26979279

Interaction of melanin with proteins--the importance of an acidic intramelanosomal pH.

PubMed

Mani, I; Sharma, V; Tamboli, I; Raman, G

2001-06-01

Melanin is a highly irregular heteropolymer consisting of monomeric units derived from the enzymatic oxidation of the amino acid tyrosine. The process of melanin formation takes place in specialized acidic organelles (melanosomes) in melanocytes. The process of melanin polymerization requires an alkaline pH in vitro, and therefore, the purpose of an acidic environment in vivo remains a mystery. It is known that melanin is always bound to protein in vivo. It is also seen that polymerization in vitro at an acidic pH necessarily requires the presence of proteins. The effect of various model proteins on melanin synthesis and their interaction with melanin was studied. It was seen that many proteins could increase melanin synthesis at an acidic pH, and that different proteins resulted in the formation of different states of melanin, i.e., a precipitate or a soluble, protein-bound form. We also present evidence to show that soluble protein-bound melanin is present in vivo (in B16 cells as well as in B16 melanoma tissue). An acidic pH appeared to be necessary to ensure the formation of a uniform, very high molecular weight melano-protein complex. The interaction between melanin and proteins appears to be largely charge-dependent as evidenced by zeta potential measurements, and this interaction is also increased in an acidic pH. Thus, it appears that an acidic intramelanosomal pH is essential to ensure maximum interaction between protein and melanin, and also to ensure that all the melanin formed is protein-bound.

Modeling of DNA and Protein Organization Levels with Cn3D Software

ERIC Educational Resources Information Center

Stasinakis, Panagiotis K.; Nicolaou, Despoina

2017-01-01

The molecular structure of living organisms and the complex interactions amongst its components are the basis for the diversity observed at the macroscopic level. Proteins and nucleic acids are some of the major molecular components, and play a key role in several biological functions, such as those of development and evolution. This article…

Predicting nucleic acid binding interfaces from structural models of proteins.

PubMed

Dror, Iris; Shazman, Shula; Mukherjee, Srayanta; Zhang, Yang; Glaser, Fabian; Mandel-Gutfreund, Yael

2012-02-01

The function of DNA- and RNA-binding proteins can be inferred from the characterization and accurate prediction of their binding interfaces. However, the main pitfall of various structure-based methods for predicting nucleic acid binding function is that they are all limited to a relatively small number of proteins for which high-resolution three-dimensional structures are available. In this study, we developed a pipeline for extracting functional electrostatic patches from surfaces of protein structural models, obtained using the I-TASSER protein structure predictor. The largest positive patches are extracted from the protein surface using the patchfinder algorithm. We show that functional electrostatic patches extracted from an ensemble of structural models highly overlap the patches extracted from high-resolution structures. Furthermore, by testing our pipeline on a set of 55 known nucleic acid binding proteins for which I-TASSER produces high-quality models, we show that the method accurately identifies the nucleic acids binding interface on structural models of proteins. Employing a combined patch approach we show that patches extracted from an ensemble of models better predicts the real nucleic acid binding interfaces compared with patches extracted from independent models. Overall, these results suggest that combining information from a collection of low-resolution structural models could be a valuable approach for functional annotation. We suggest that our method will be further applicable for predicting other functional surfaces of proteins with unknown structure. Copyright © 2011 Wiley Periodicals, Inc.

Predicting nucleic acid binding interfaces from structural models of proteins

PubMed Central

Dror, Iris; Shazman, Shula; Mukherjee, Srayanta; Zhang, Yang; Glaser, Fabian; Mandel-Gutfreund, Yael

2011-01-01

The function of DNA- and RNA-binding proteins can be inferred from the characterization and accurate prediction of their binding interfaces. However the main pitfall of various structure-based methods for predicting nucleic acid binding function is that they are all limited to a relatively small number of proteins for which high-resolution three dimensional structures are available. In this study, we developed a pipeline for extracting functional electrostatic patches from surfaces of protein structural models, obtained using the I-TASSER protein structure predictor. The largest positive patches are extracted from the protein surface using the patchfinder algorithm. We show that functional electrostatic patches extracted from an ensemble of structural models highly overlap the patches extracted from high-resolution structures. Furthermore, by testing our pipeline on a set of 55 known nucleic acid binding proteins for which I-TASSER produces high-quality models, we show that the method accurately identifies the nucleic acids binding interface on structural models of proteins. Employing a combined patch approach we show that patches extracted from an ensemble of models better predicts the real nucleic acid binding interfaces compared to patches extracted from independent models. Overall, these results suggest that combining information from a collection of low-resolution structural models could be a valuable approach for functional annotation. We suggest that our method will be further applicable for predicting other functional surfaces of proteins with unknown structure. PMID:22086767

N-3 fatty acids reduced trans fatty acids retention and increased docosahexaenoic acid levels in the brain.

PubMed

Lavandera, Jimena Verónica; Saín, Juliana; Fariña, Ana Clara; Bernal, Claudio Adrián; González, Marcela Aída

2017-09-01

The levels of docosahexaenoic acid (DHA, 22:6n-3) and arachidonic acid (AA, 20:4n-6) are critical for the normal structure and function of the brain. Trans fatty acids (TFA) and the source of the dietary fatty acids (FA) interfere with long-chain polyunsaturated fatty acids (LC-PUFA) biosynthesis. The aim of this study was to investigate the effect of TFA supplementation in diets containing different proportions of n-9, n-6, and n-3 FA on the brain FA profile, including the retention of TFA, LC-PUFA levels, and n-6/n-3 PUFA ratios. These parameters were also investigated in the liver, considering that LC-PUFA are mainly bioconverted from their dietary precursors in this tissue and transported by serum to the brain. Also, stearoyl-CoA desaturase-1 (SCD1) and sterol regulatory element-binding protein-1c (SREBP-1c) gene expressions were evaluated. Male CF1 mice were fed (16 weeks) diets containing different oils (olive, corn, and rapeseed) with distinct proportions of n-9, n-6, and n-3 FA (55.2/17.2/0.7, 32.0/51.3/0.9, and 61.1/18.4/8.6), respectively, substituted or not with 0.75% of TFA. FA composition of the brain, liver, and serum was assessed by gas chromatography. TFA were incorporated into, and therefore retained in the brain, liver, and serum. However, the magnitude of retention was dependent on the tissue and type of isomer. In the brain, total TFA retention was lower than 1% in all diets. Dietary n-3 PUFA decreased TFA retention and increased DHA accretion in the brain. The results underscore the importance of the type of dietary FA on the retention of TFA in the brain and also on the changes of the FA profile.

Database of amino acid-nucleotide contacts in contacts in DNA-homeodomain protein

NASA Astrophysics Data System (ADS)

Grokhlina, T. I.; Zrelov, P. V.; Ivanov, V. V.; Polozov, R. V.; Chirgadze, Yu. N.; Sivozhelezov, V. S.

2013-09-01

The analysis of amino acid-nucleotide contacts in interfaces of the protein-DNA complexes, intended to find consistencies in the protein-DNA recognition, is a complex problem that requires an analysis of the physicochemical characteristics of these contacts and the positions of the participating amino acids and nucleotides in the chains of the protein and the DNA, respectively, as well as conservatism of these contacts. Thus, those heterogeneous data should be systematized. For this purpose we have developed a database of amino acid-nucleotide contacts ANTPC (Amino acid Nucleotide Type Position Conservation) following the archetypal example of the proteins in the homeodomain family. We show that it can be used to compare and classify the interfaces of the protein-DNA complexes.

Improved plasma amino acids pattern following 12 months of supplemented low-protein diet in peritoneal dialysis patients.

PubMed

Jiang, Na; Qian, Jiaqi; Lin, Aiwu; Fang, Wei; Cao, Liou; Wang, Qin; Ni, Zhaohui; Lindholm, Bengt; Axelsson, Jonas; Yao, Qiang

2010-07-01

Decreased plasma essential amino acid (EAA) levels, increased nonessential amino acid (NEAA) levels, and low EAA to NEAA ratio (E/NEAA) are common in peritoneal dialysis (PD) patients and may impact uremic complications. In the present study, we investigate the impact of keto acids-supplemented low-protein (sLP) diet on plasma amino acids (AAs) patterns in stable PD patients. This is a supplemental analysis of a previously published prospective and randomized trial. Thirty-nine PD patients selected from the original population were divided to receive either low (LP: 0.6-0.8 g/kg ideal body weight [IBW]/d, n = 13), keto acids-supplemented low- (sLP: 0.6-0.8 g/kg IBW/d + 0.12 g/kg IBW/d of keto acids, n = 12), or high- (HP: 1.0-1.2 g/kg IBW/d, n = 14) protein diets and followed for 1 year. Plasma AA patterns were assessed at baseline and 12 months using high-performance liquid chromatography. Whereas there were no significant differences between the three groups at baseline, following 12 months, the E/NEAA had increased significantly in group sLP (0.58 +/- 0.16 to 0.83 +/- 0.20, p < 0.05), but was not different in either LP (0.62 +/- 0.20 to 0.72 +/- 0.13, p = ns) or HP (0.66 +/- 0.14 to 0.74 +/- 0.12, p = ns) group. This change in E/NEAA in group sLP was due to a significant decrease in NEAA concomitantly with maintained EAA levels, whereas in the other two groups, neither EAA nor NEAA changed significantly. A low-protein diet supplemented with keto acids significantly improved the pattern of plasma AA in prevalent PD patients.

Sink limitation induces the expression of multiple soybean vegetative lipoxygenase mRNAs while the endogenous jasmonic acid level remains low.

PubMed Central

Bunker, T W; Koetje, D S; Stephenson, L C; Creelman, R A; Mullet, J E; Grimes, H D

1995-01-01

The response of individual members of the lipoxygenase multigene family in soybeans to sink deprivation was analyzed. RNase protection assays indicated that a novel vegetative lipoxygenase gene, vlxC, and three other vegetative lipoxygenase mRNAs accumulated in mature leaves in response to a variety of sink limitations. These data suggest that several members of the lipoxygenase multigene family are involved in assimilate partitioning. The possible involvement of jasmonic acid as a signaling molecule regulating assimilate partitioning into the vegetative storage proteins and lipoxygenases was directly assessed by determining the endogenous level of jasmonic acid in leaves from plants with their pods removed. There was no rise in the level of endogenous jasmonic acid coincident with the strong increase in both vlxC and vegetative storage protein VspB transcripts in response to sink limitation. Thus, expression of the vegetative lipoxygenases and vegetative storage proteins is not regulated by jasmonic acid in sink-limited leaves. PMID:7549487

Sink limitation induces the expression of multiple soybean vegetative lipoxygenase mRNAs while the endogenous jasmonic acid level remains low.

PubMed

Bunker, T W; Koetje, D S; Stephenson, L C; Creelman, R A; Mullet, J E; Grimes, H D

1995-08-01

The response of individual members of the lipoxygenase multigene family in soybeans to sink deprivation was analyzed. RNase protection assays indicated that a novel vegetative lipoxygenase gene, vlxC, and three other vegetative lipoxygenase mRNAs accumulated in mature leaves in response to a variety of sink limitations. These data suggest that several members of the lipoxygenase multigene family are involved in assimilate partitioning. The possible involvement of jasmonic acid as a signaling molecule regulating assimilate partitioning into the vegetative storage proteins and lipoxygenases was directly assessed by determining the endogenous level of jasmonic acid in leaves from plants with their pods removed. There was no rise in the level of endogenous jasmonic acid coincident with the strong increase in both vlxC and vegetative storage protein VspB transcripts in response to sink limitation. Thus, expression of the vegetative lipoxygenases and vegetative storage proteins is not regulated by jasmonic acid in sink-limited leaves.

Differential Occurrence of Interactions and Interaction Domains in Proteins Containing Homopolymeric Amino Acid Repeats

PubMed Central

Pelassa, Ilaria; Fiumara, Ferdinando

2015-01-01

Homopolymeric amino acids repeats (AARs), which are widespread in proteomes, have often been viewed simply as spacers between protein domains, or even as “junk” sequences with no obvious function but with a potential to cause harm upon expansion as in genetic diseases associated with polyglutamine or polyalanine expansions, including Huntington disease and cleidocranial dysplasia. A growing body of evidence indicates however that at least some AARs can form organized, functional protein structures, and can regulate protein function. In particular, certain AARs can mediate protein-protein interactions, either through homotypic AAR-AAR contacts or through heterotypic contacts with other protein domains. It is still unclear however, whether AARs may have a generalized, proteome-wide role in shaping protein-protein interaction networks. Therefore, we have undertaken here a bioinformatics screening of the human proteome and interactome in search of quantitative evidence of such a role. We first identified the sets of proteins that contain repeats of any one of the 20 amino acids, as well as control sets of proteins chosen at random in the proteome. We then analyzed the connectivity between the proteins of the AAR-containing protein sets and we compared it with that observed in the corresponding control networks. We find evidence for different degrees of connectivity in the different AAR-containing protein networks. Indeed, networks of proteins containing polyglutamine, polyglutamate, polyproline, and other AARs show significantly increased levels of connectivity, whereas networks containing polyleucine and other hydrophobic repeats show lower degrees of connectivity. Furthermore, we observed that numerous protein-protein, -nucleic acid, and -lipid interaction domains are significantly enriched in specific AAR protein groups. These findings support the notion of a generalized, combinatorial role of AARs, together with conventional protein interaction domains, in

Utilization of dipeptide/protein based diets in larval and juvenile Koi carp--post-prandial free amino acid levels.

PubMed

Kwasek, K; Zhang, Y; Dabrowski, K

2010-02-01

We have shown previously that diets in which the nitrogen portion is based on synthetic dipeptides (PP) resulted in weight gain by rainbow trout alevins when free amino acid (FAA) based diets did not. However, the protein-based diet used as a control in the previous study resulted in a significantly better performance of fish than peptide- or FAA based diet fed fish. Therefore, the objectives of our study were (i) to test how stomachless fish respond to peptide-based diets, (ii) to evaluate PP and protein-PP mixture diets and (iii) to examine if post-prandial response to FAA concentrations in the fish body can be used as an indicator of the availability of dietary amino acid sources. The first experiment was conducted with a 4-day old Koi carp (Cyprinus carpio) larvae and included groups fed a casein-gelatin (CG) based diet, a commercial diet and live Artemia nauplii. In the second experiment, fish fed live Artemia for 3 weeks (69 +/- 12 mg) were placed in glass aquaria in triplicate per dietary treatment. Four diets provided equivalent amounts of nitrogen in the form of a CG, a PP, a 50% PP and 50% CG (PP50) mix and as FAA. Fish were fed at 1 and 3 h intervals, and the survival and growth were monitored during the second and third weeks of experiments 1 and 2 respectively. Following the completion of feeding, juvenile carp were sampled prior to feeding and 3 h after a meal. Whole body FAA analyses were carried out. We observed marginal suitability of the CG diet for larval Koi carp, as earlier indicated in common carp. Juvenile Koi carp fed the CG diet achieved 236 +/- 19 mg, whereas PP50, PP and FAA diet fed fish grew to 140 +/- 37, 70 +/- 8 and 73 +/- 5 mg respectively. Free amino acids in the fish body, and in particular indispensable amino acids (IDAA), were excellent indicators of dietary availability. The present experiment shows that a dietary 1:1 ratio of protein to synthetic PPs results in better growth, survival and whole body IDAA concentrations in

Improving protein complex classification accuracy using amino acid composition profile.

PubMed

Huang, Chien-Hung; Chou, Szu-Yu; Ng, Ka-Lok

2013-09-01

Protein complex prediction approaches are based on the assumptions that complexes have dense protein-protein interactions and high functional similarity between their subunits. We investigated those assumptions by studying the subunits' interaction topology, sequence similarity and molecular function for human and yeast protein complexes. Inclusion of amino acids' physicochemical properties can provide better understanding of protein complex properties. Principal component analysis is carried out to determine the major features. Adopting amino acid composition profile information with the SVM classifier serves as an effective post-processing step for complexes classification. Improvement is based on primary sequence information only, which is easy to obtain. Copyright © 2013 Elsevier Ltd. All rights reserved.

Acid extraction and purification of recombinant spider silk proteins.

PubMed

Mello, Charlene M; Soares, Jason W; Arcidiacono, Steven; Butler, Michelle M

2004-01-01

A procedure has been developed for the isolation of recombinant spider silk proteins based upon their unique stability and solubilization characteristics. Three recombinant silk proteins, (SpI)7, NcDS, and [(SpI)4/(SpII)1]4, were purified by extraction with organic acids followed by affinity or ion exchange chromatography resulting in 90-95% pure silk solutions. The protein yield of NcDS (15 mg/L culture) and (SpI)7 (35 mg/L) increased 4- and 5-fold, respectively, from previously reported values presumably due to a more complete solubilization of the expressed recombinant protein. [(SpI)4/(SpII)1]4, a hybrid protein based on the repeat sequences of spidroin I and spidroin II, had a yield of 12.4 mg/L. This method is an effective, reproducible technique that has broad applicability for a variety of silk proteins as well as other acid stable biopolymers.

Soy-dairy protein blend and whey protein ingestion after resistance exercise increases amino acid transport and transporter expression in human skeletal muscle.

PubMed

Reidy, P T; Walker, D K; Dickinson, J M; Gundermann, D M; Drummond, M J; Timmerman, K L; Cope, M B; Mukherjea, R; Jennings, K; Volpi, E; Rasmussen, B B

2014-06-01

Increasing amino acid availability (via infusion or ingestion) at rest or postexercise enhances amino acid transport into human skeletal muscle. It is unknown whether alterations in amino acid availability, from ingesting different dietary proteins, can enhance amino acid transport rates and amino acid transporter (AAT) mRNA expression. We hypothesized that the prolonged hyperaminoacidemia from ingesting a blend of proteins with different digestion rates postexercise would enhance amino acid transport into muscle and AAT expression compared with the ingestion of a rapidly digested protein. In a double-blind, randomized clinical trial, we studied 16 young adults at rest and after acute resistance exercise coupled with postexercise (1 h) ingestion of either a (soy-dairy) protein blend or whey protein. Phenylalanine net balance and transport rate into skeletal muscle were measured using stable isotopic methods in combination with femoral arteriovenous blood sampling and muscle biopsies obtained at rest and 3 and 5 h postexercise. Phenylalanine transport into muscle and mRNA expression of select AATs [system L amino acid transporter 1/solute-linked carrier (SLC) 7A5, CD98/SLC3A2, system A amino acid transporter 2/SLC38A2, proton-assisted amino acid transporter 1/SLC36A1, cationic amino acid transporter 1/SLC7A1] increased to a similar extent in both groups (P < 0.05). However, the ingestion of the protein blend resulted in a prolonged and positive net phenylalanine balance during postexercise recovery compared with whey protein (P < 0.05). Postexercise myofibrillar protein synthesis increased similarly between groups. We conclude that, while both protein sources enhanced postexercise AAT expression, transport into muscle, and myofibrillar protein synthesis, postexercise ingestion of a protein blend results in a slightly prolonged net amino acid balance across the leg compared with whey protein. Copyright © 2014 the American Physiological Society.

Soy-dairy protein blend and whey protein ingestion after resistance exercise increases amino acid transport and transporter expression in human skeletal muscle

PubMed Central

Reidy, P. T.; Walker, D. K.; Dickinson, J. M.; Gundermann, D. M.; Drummond, M. J.; Timmerman, K. L.; Cope, M. B.; Mukherjea, R.; Jennings, K.; Volpi, E.

2014-01-01

Increasing amino acid availability (via infusion or ingestion) at rest or postexercise enhances amino acid transport into human skeletal muscle. It is unknown whether alterations in amino acid availability, from ingesting different dietary proteins, can enhance amino acid transport rates and amino acid transporter (AAT) mRNA expression. We hypothesized that the prolonged hyperaminoacidemia from ingesting a blend of proteins with different digestion rates postexercise would enhance amino acid transport into muscle and AAT expression compared with the ingestion of a rapidly digested protein. In a double-blind, randomized clinical trial, we studied 16 young adults at rest and after acute resistance exercise coupled with postexercise (1 h) ingestion of either a (soy-dairy) protein blend or whey protein. Phenylalanine net balance and transport rate into skeletal muscle were measured using stable isotopic methods in combination with femoral arteriovenous blood sampling and muscle biopsies obtained at rest and 3 and 5 h postexercise. Phenylalanine transport into muscle and mRNA expression of select AATs [system L amino acid transporter 1/solute-linked carrier (SLC) 7A5, CD98/SLC3A2, system A amino acid transporter 2/SLC38A2, proton-assisted amino acid transporter 1/SLC36A1, cationic amino acid transporter 1/SLC7A1] increased to a similar extent in both groups (P < 0.05). However, the ingestion of the protein blend resulted in a prolonged and positive net phenylalanine balance during postexercise recovery compared with whey protein (P < 0.05). Postexercise myofibrillar protein synthesis increased similarly between groups. We conclude that, while both protein sources enhanced postexercise AAT expression, transport into muscle, and myofibrillar protein synthesis, postexercise ingestion of a protein blend results in a slightly prolonged net amino acid balance across the leg compared with whey protein. PMID:24699854

Fatty acid binding proteins have the potential to channel dietary fatty acids into enterocyte nuclei[S

PubMed Central

Esteves, Adriana; Knoll-Gellida, Anja; Canclini, Lucia; Silvarrey, Maria Cecilia; André, Michèle; Babin, Patrick J.

2016-01-01

Intracellular lipid binding proteins, including fatty acid binding proteins (FABPs) 1 and 2, are highly expressed in tissues involved in the active lipid metabolism. A zebrafish model was used to demonstrate differential expression levels of fabp1b.1, fabp1b.2, and fabp2 transcripts in liver, anterior intestine, and brain. Transcription levels of fabp1b.1 and fabp2 in the anterior intestine were upregulated after feeding and modulated according to diet formulation. Immunofluorescence and electron microscopy immunodetection with gold particles localized these FABPs in the microvilli, cytosol, and nuclei of most enterocytes in the anterior intestinal mucosa. Nuclear localization was mostly in the interchromatin space outside the condensed chromatin clusters. Native PAGE binding assay of BODIPY-FL-labeled FAs demonstrated binding of BODIPY-FLC12 but not BODIPY-FLC5 to recombinant Fabp1b.1 and Fabp2. The binding of BODIPY-FLC12 to Fabp1b.1 was fully displaced by oleic acid. In vivo experiments demonstrated, for the first time, that intestinal absorption of dietary BODIPY-FLC12 was followed by colocalization of the labeled FA with Fabp1b and Fabp2 in the nuclei. These data suggest that dietary FAs complexed with FABPs are able to reach the enterocyte nucleus with the potential to modulate nuclear activity. PMID:26658423

Amino acid contribution to protein solubility: Asp, Glu, and Ser contribute more favorably than the other hydrophilic amino acids in RNase Sa.

PubMed

Trevino, Saul R; Scholtz, J Martin; Pace, C Nick

2007-02-16

Poor protein solubility is a common problem in high-resolution structural studies, formulation of protein pharmaceuticals, and biochemical characterization of proteins. One popular strategy to improve protein solubility is to use site-directed mutagenesis to make hydrophobic to hydrophilic mutations on the protein surface. However, a systematic investigation of the relative contributions of all 20 amino acids to protein solubility has not been done. Here, 20 variants at the completely solvent-exposed position 76 of ribonuclease (RNase) Sa are made to compare the contributions of each amino acid. Stability measurements were also made for these variants, which occur at the i+1 position of a type II beta-turn. Solubility measurements in ammonium sulfate solutions were made at high positive net charge, low net charge, and high negative net charge. Surprisingly, there was a wide range of contributions to protein solubility even among the hydrophilic amino acids. The results suggest that aspartic acid, glutamic acid, and serine contribute significantly more favorably than the other hydrophilic amino acids especially at high net charge. Therefore, to increase protein solubility, asparagine, glutamine, or threonine should be replaced with aspartic acid, glutamic acid or serine.

Amino acid contribution to protein solubility: Asp, Glu, and Ser contribute more favorably than the other hydrophilic amino acids in RNase Sa

PubMed Central

Trevino, Saul R.; Scholtz, J. Martin; Pace, C. Nick

2009-01-01

SUMMARY Poor protein solubility is a common problem in high resolution structural studies, formulation of protein pharmaceuticals, and biochemical characterization of proteins. One popular strategy to improve protein solubility is to use site-directed mutagenesis to make hydrophobic to hydrophilic mutations on the protein surface. However, a systematic investigation of the relative contributions of all twenty amino acids to protein solubility has not been done. Here, twenty variants at the completely solvent-exposed position 76 of Ribonuclease (RNase) Sa are made to compare the contributions of each amino acid. Stability measurements were also made for these variants, which occur at the i+1 position of a type II β-turn. Solubility measurements in ammonium sulfate solutions were made at high positive net charge, low net charge, and high negative net charge. Surprisingly, there was a wide range of contributions to protein solubility even among the hydrophilic amino acids. The results suggest that aspartic acid, glutamic acid, and serine contribute significantly more favorably than the other hydrophilic amino acids especially at high net charge. Therefore, to increase protein solubility, asparagine, glutamine, or threonine should be replaced with aspartic acid, glutamic acid or serine. PMID:17174328

Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gangi Setty, Thanuja; Cho, Christine; Govindappa, Sowmya

2014-07-01

Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which oftenmore » takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.« less

Protein and Amino Acid Requirements during Pregnancy123

PubMed Central

Elango, Rajavel; Ball, Ronald O

2016-01-01

Protein forms an essential component of a healthy diet in humans to support both growth and maintenance. During pregnancy, an exceptional stage of life defined by rapid growth and development, adequate dietary protein is crucial to ensure a healthy outcome. Protein deposition in maternal and fetal tissues increases throughout pregnancy, with most occurring during the third trimester. Dietary protein intake recommendations are based on factorial estimates because the traditional method of determining protein requirements, nitrogen balance, is invasive and undesirable during pregnancy. The current Estimated Average Requirement and RDA recommendations of 0.88 and 1.1 g · kg−1 · d−1, respectively, are for all stages of pregnancy. The single recommendation does not take into account the changing needs during different stages of pregnancy. Recently, with the use of the minimally invasive indicator amino acid oxidation method, we defined the requirements to be, on average, 1.2 and 1.52 g · kg−1 · d−1 during early (∼16 wk) and late (∼36 wk) stages of pregnancy, respectively. Although the requirements are substantially higher than current recommendations, our values are ∼14–18% of total energy and fit within the Acceptable Macronutrient Distribution Range. Using swine as an animal model we showed that the requirements for several indispensable amino acids increase dramatically during late gestation compared with early gestation. Additional studies should be conducted during pregnancy to confirm the newly determined protein requirements and to determine the indispensable amino acid requirements during pregnancy in humans. PMID:27422521

Neuromuscular electrical stimulation prior to presleep protein feeding stimulates the use of protein-derived amino acids for overnight muscle protein synthesis.

PubMed

Dirks, Marlou L; Groen, Bart B L; Franssen, Rinske; van Kranenburg, Janneau; van Loon, Luc J C

2017-01-01

Short periods of muscle disuse result in substantial skeletal muscle atrophy. Recently, we showed that both neuromuscular electrical stimulation (NMES) as well as presleep dietary protein ingestion represent effective strategies to stimulate muscle protein synthesis rates. In this study, we test our hypothesis that NMES can augment the use of presleep protein-derived amino acids for overnight muscle protein synthesis in older men. Twenty healthy, older [69 ± 1 (SE) yr] men were subjected to 24 h of bed rest, starting at 8:00 AM. In the evening, volunteers were subjected to 70-min 1-legged NMES, while the other leg served as nonstimulated control (CON). Immediately following NMES, 40 g of intrinsically l-[1- 13 C]-phenylalanine labeled protein was ingested prior to sleep. Blood samples were taken throughout the night, and muscle biopsies were obtained from both legs in the evening and the following morning (8 h after protein ingestion) to assess dietary protein-derived l-[1- 13 C]-phenylalanine enrichments in myofibrillar protein. Plasma phenylalanine concentrations and plasma l-[1- 13 C]-phenylalanine enrichments increased significantly following protein ingestion and remained elevated for up to 6 h after protein ingestion (P < 0.05). During overnight sleep, myofibrillar protein-bound l-[1- 13 C]-phenylalanine enrichments (MPE) increased to a greater extent in the stimulated compared with the control leg (0.0344 ± 0.0019 vs. 0.0297 ± 0.0016 MPE, respectively; P < 0.01), representing 18 ± 6% greater incorporation of presleep protein-derived amino acids in the NMES compared with CON leg. In conclusion, application of NMES prior to presleep protein feeding stimulates the use of dietary protein-derived amino acids for overnight muscle protein synthesis in older men. Neuromuscular electrical stimulation (NMES) as well as presleep dietary protein ingestion represent effective strategies to stimulate muscle protein synthesis rates. Here we demonstrate that in older

Electrophoretic Mobility Shift Assay (EMSA) for Detecting Protein-Nucleic Acid Interactions

PubMed Central

Hellman, Lance M.; Fried, Michael G.

2009-01-01

The gel electrophoresis mobility shift assay (EMSA) is used to detect protein complexes with nucleic acids. It is the core technology underlying a wide range of qualitative and quantitative analyses for the characterization of interacting systems. In the classical assay, solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel. After electrophoresis, the distribution of species containing nucleic acid is determined, usually by autoradiography of 32P-labeled nucleic acid. In general, protein-nucleic acid complexes migrate more slowly than the corresponding free nucleic acid. In this article, we identify the most important factors that determine the stabilities and electrophoretic mobilities of complexes under assay conditions. A representative protocol is provided and commonly used variants are discussed. Expected outcomes are briefly described. References to extensions of the method and a troubleshooting guide are provided. PMID:17703195

Effect of dietary protein and GABA on food intake, growth and tissue amino acids in cats.

PubMed

Tews, J K; Rogers, Q R; Morris, J G; Harper, A E

1984-02-01

GABA at 5%, but not 3%, of a low protein diet depressed food intake and growth of kittens. Adaptation to high protein prevented these effects. When cats adapted to low or high protein were fed a meal containing GABA, plasma GABA concentration after 2 hr was 8-fold higher in the low than in the high protein group; clearance was almost complete within 6 hr. Concentrations of proline, branched-chain, other large neutral and basic (especially ornithine) amino acids increased more when cats were fed a high rather than a low protein meal; glycine decreased. At 6 hr, concentrations had consistently returned to initial levels only in the low protein group. Feeding the high protein diet ad lib increased tissue concentrations of threonine, proline and the branched-chain amino acids. Hepatic or renal GABA-aminotransferase activity was not altered in kittens fed the high protein diet. Kidney activity was 10-fold that of liver, which may contribute to the better tolerance of GABA by cats than by rats.

Enhanced butyric acid tolerance and production by Class I heat shock protein-overproducing Clostridium tyrobutyricum ATCC 25755.

PubMed

Suo, Yukai; Luo, Sheng; Zhang, Yanan; Liao, Zhengping; Wang, Jufang

2017-08-01

The response of Clostridium tyrobutyricum to butyric acid stress involves various stress-related genes, and therefore overexpression of stress-related genes can improve butyric acid tolerance and yield. Class I heat shock proteins (HSPs) play an important role in the process of protecting bacteria from sudden changes of extracellular stress by assisting protein folding correctly. The results of quantitative real-time PCR indicated that the Class I HSGs grpE, dnaK, dnaJ, groEL, groES, and htpG were significantly upregulated under butyric acid stress, especially the dnaK and groE operons. Overexpression of groESL and htpG could significantly improve the tolerance of C. tyrobutyricum to butyric acid, while overexpression of dnaK and dnaJ showed negative effects on butyric acid tolerance. Acid production was also significantly promoted by increased GroESL expression levels; the final butyric acid and acetic acid concentrations were 28.2 and 38% higher for C. tyrobutyricum ATCC 25755/groESL than for the wild-type strain. In addition, when fed-batch fermentation was carried out using cell immobilization in a fibrous-bed bioreactor, the butyric acid yield produced by C. tyrobutyricum ATCC 25755/groESL reached 52.2 g/L, much higher than that for the control. The improved butyric acid yield is probably attributable to the high GroES and GroEL levels, which can stabilize the biosynthetic machinery of C. tyrobutyricum under extracellular butyric acid stress.

Two-level QSAR network (2L-QSAR) for peptide inhibitor design based on amino acid properties and sequence positions.

PubMed

Du, Q S; Ma, Y; Xie, N Z; Huang, R B

2014-01-01

In the design of peptide inhibitors the huge possible variety of the peptide sequences is of high concern. In collaboration with the fast accumulation of the peptide experimental data and database, a statistical method is suggested for peptide inhibitor design. In the two-level peptide prediction network (2L-QSAR) one level is the physicochemical properties of amino acids and the other level is the peptide sequence position. The activity contributions of amino acids are the functions of physicochemical properties and the sequence positions. In the prediction equation two weight coefficient sets {ak} and {bl} are assigned to the physicochemical properties and to the sequence positions, respectively. After the two coefficient sets are optimized based on the experimental data of known peptide inhibitors using the iterative double least square (IDLS) procedure, the coefficients are used to evaluate the bioactivities of new designed peptide inhibitors. The two-level prediction network can be applied to the peptide inhibitor design that may aim for different target proteins, or different positions of a protein. A notable advantage of the two-level statistical algorithm is that there is no need for host protein structural information. It may also provide useful insight into the amino acid properties and the roles of sequence positions.

Acute supplementation of amino acids increases net protein accretion in IUGR fetal sheep

PubMed Central

Rozance, Paul J.; Thorn, Stephanie R.; Friedman, Jacob E.; Hay, William W.

2012-01-01

Placental insufficiency decreases fetal amino acid uptake from the placenta, plasma insulin concentrations, and protein accretion, thus compromising normal fetal growth trajectory. We tested whether acute supplementation of amino acids or insulin into the fetus with intrauterine growth restriction (IUGR) would increase net fetal protein accretion rates. Late-gestation IUGR and control (CON) fetal sheep received acute, 3-h infusions of amino acids (with euinsulinemia), insulin (with euglycemia and euaminoacidemia), or saline. Fetal leucine metabolism was measured under steady-state conditions followed by a fetal muscle biopsy to quantify insulin signaling. In CON, increasing amino acid delivery rates to the fetus by 100% increased leucine oxidation rates by 100%. In IUGR, amino acid infusion completely suppressed fetal protein breakdown rates but increased leucine oxidation rate by only 25%, resulting in increased protein accretion rates by 150%. Acute insulin infusion, however, had very little effect on amino acid delivery rates, fetal leucine disposal rates, or fetal protein accretion rates in CON or IUGR fetuses despite robust signaling of the fetal skeletal muscle insulin-signaling cascade. These results indicate that, when amino acids are given directly into the fetal circulation independently of changes in insulin concentrations, IUGR fetal sheep have suppressed protein breakdown rates, thus increasing net fetal protein accretion. PMID:22649066

Relationship between Acute Phase Proteins and Serum Fatty Acid Composition in Morbidly Obese Patients

PubMed Central

Fernandes, Ricardo; Beserra, Bruna Teles Soares; Cunha, Raphael Salles Granato; Hillesheim, Elaine; Camargo, Carolina de Quadros; Pequito, Danielle Cristina Tonello; de Castro, Isabela Coelho; Fernandes, Luiz Cláudio; Nunes, Everson Araújo; Trindade, Erasmo Benício Santos de Moraes

2013-01-01

Background. Obesity is considered a low-grade inflammatory state and has been associated with increased acute phase proteins as well as changes in serum fatty acids. Few studies have assessed associations between acute phase proteins and serum fatty acids in morbidly obese patients. Objective. To investigate the relationship between acute phase proteins (C-Reactive Protein, Orosomucoid, and Albumin) and serum fatty acids in morbidly obese patients. Methods. Twenty-two morbidly obese patients were enrolled in this study. Biochemical and clinical data were obtained before bariatric surgery, and fatty acids measured in preoperative serum. Results. Orosomucoid was negatively correlated with lauric acid (P = 0.027) and eicosapentaenoic acid (EPA) (P = 0.037) and positively with arachidonic acid (AA) (P = 0.035), AA/EPA ratio (P = 0.005), and n-6/n-3 polyunsaturated fatty acids ratio (P = 0.035). C-Reactive Protein (CRP) was negatively correlated with lauric acid (P = 0.048), and both CRP and CRP/Albumin ratio were negatively correlated with margaric acid (P = 0.010, P = 0.008, resp.). Albumin was positively correlated with EPA (P = 0.027) and margaric acid (P = 0.008). Other correlations were not statistically significant. Conclusion. Our findings suggest that serum fatty acids are linked to acute phase proteins in morbidly obese patients. PMID:24167354

Increased neuroinflammatory and arachidonic acid cascade markers, and reduced synaptic proteins, in the postmortem frontal cortex from schizophrenia patients

PubMed Central

Rao, Jagadeesh Sridhara; Kim, Hyung-Wook; Harry, Gaylia Jean; Rapoport, Stanley Isaac; Reese, Edmund Arthur

2013-01-01

Schizophrenia (SZ) is a progressive, neuropsychiatric disorder associated with cognitive impairment. A number of brain alterations have been linked to cognitive impairment, including neuroinflammation, excitotoxicity, increased arachidonic acid (AA) signaling and reduced synaptic protein. On this basis, we tested the hypothesis that SZ pathology is associated with these pathological brain changes. To do this, we examined postmortem frontal cortex from 10 SZ patients and 10 controls and measured protein and mRNA levels of cytokines, and astroglial, microglial, neuroinflammatory excitotoxic, AA cascade, apoptotic and synaptic markers. Mean protein and mRNA levels of interleukin-1β, tumor necrosis factor-α, glial acidic fibrillary protein (GFAP), a microglial marker CD11b, and nuclear factor kappa B subunits were significantly increased in SZ compared with control brain. Protein and mRNA levels of cytosolic and secretory phospholipase A2 and cyclooxygenase were significantly elevated in postmortem brains from SZ patients. N-methyl-D-aspartate receptor subunits 1 and 2B, inducible nitric oxide synthase and c-FOS were not significantly different. In addition, reduced protein and mRNA levels of brain-derived neurotrophic factor, synaptophysin and drebrin were found in SZ compared with control frontal cortex. Increased neuroinflammation and AA cascade enzyme markers with synaptic protein loss could promote disease progression and cognitive defects in SZ patients. Drugs that downregulate these changes might be considered for new therapies in SZ. PMID:23566496

Effect of tannic acid on the synthesis of protein and nucleic acid by rat liver

PubMed Central

Badawy, A. A.-B.; White, Audrey E.; Lathe, G. H.

1969-01-01

1. As early as 1hr. after the intraperitoneal administration of tannic acid to rats, it could be demonstrated in the liver. At 3hr. the nuclear fraction contained the largest amount of tannic acid. 2. Nuclear RNA synthesis was inhibited in vivo 2hr. after the administration of tannic acid. Induction by cortisol of tryptophan pyrrolase was 90% inhibited at 24hr. 3. Incorporation of [1-14C]leucine into protein by liver slices from treated rats was decreased by 50% after 24hr. Its incorporation into postmitochondrial supernatant from treated animals was not inhibited. Incorporation into slices and postmitochondrial supernatants were inhibited in vitro by tannic acid. 4. The sequence of events: concentration of tannic acid in nuclei, inhibition of nuclear RNA synthesis, inhibition of protein synthesis and production of necrosis, is discussed. PMID:5808319

Apparent growth phase-dependent phosphorylation of malonyl coenzyme A:acyl carrier protein transacylase (MCAT), a major fatty acid synthase II component in Mycobacterium bovis BCG.

PubMed

Sinha, Indrajit; Boon, Calvin; Dick, Thomas

2003-10-10

Probing protein extracts from exponentially growing and stationary phase cultures of Mycobacterium bovis BCG with anti-phospho amino acid antibodies revealed a 31-kDa anti-phospho threonine antibody-reactive protein specific to growing culture. The corresponding protein was purified via two-dimensional gel electrophoresis and identified via mass spectrometry to be malonyl coenzyme A:acyl carrier protein transacylase (MCAT), a component of the fatty acid biosynthetic pathway. MCAT tagged with histidine reacted with anti-phospho threonine antibody and was positive in an in-gel chemical assay for phospho proteins. Analysis of the growth phase dependence of MCAT-His phosphorylation and protein levels showed that phosphorylated MCAT-His can be detected only in growing culture. In contrast, MCAT-His protein level was growth phase-independent. These results suggest that MCAT may be a substrate of a protein kinase and phosphatase, and that aspects of fatty acid synthesis in tubercle bacilli are regulated by protein phosphorylation.

Molecular mechanisms of adaptation emerging from the physics and evolution of nucleic acids and proteins.

PubMed

Goncearenco, Alexander; Ma, Bin-Guang; Berezovsky, Igor N

2014-03-01

DNA, RNA and proteins are major biological macromolecules that coevolve and adapt to environments as components of one highly interconnected system. We explore here sequence/structure determinants of mechanisms of adaptation of these molecules, links between them, and results of their mutual evolution. We complemented statistical analysis of genomic and proteomic sequences with folding simulations of RNA molecules, unraveling causal relations between compositional and sequence biases reflecting molecular adaptation on DNA, RNA and protein levels. We found many compositional peculiarities related to environmental adaptation and the life style. Specifically, thermal adaptation of protein-coding sequences in Archaea is characterized by a stronger codon bias than in Bacteria. Guanine and cytosine load in the third codon position is important for supporting the aerobic life style, and it is highly pronounced in Bacteria. The third codon position also provides a tradeoff between arginine and lysine, which are favorable for thermal adaptation and aerobicity, respectively. Dinucleotide composition provides stability of nucleic acids via strong base-stacking in ApG dinucleotides. In relation to coevolution of nucleic acids and proteins, thermostability-related demands on the amino acid composition affect the nucleotide content in the second codon position in Archaea.

Molecular mechanisms of adaptation emerging from the physics and evolution of nucleic acids and proteins

PubMed Central

Goncearenco, Alexander; Ma, Bin-Guang; Berezovsky, Igor N.

2014-01-01

DNA, RNA and proteins are major biological macromolecules that coevolve and adapt to environments as components of one highly interconnected system. We explore here sequence/structure determinants of mechanisms of adaptation of these molecules, links between them, and results of their mutual evolution. We complemented statistical analysis of genomic and proteomic sequences with folding simulations of RNA molecules, unraveling causal relations between compositional and sequence biases reflecting molecular adaptation on DNA, RNA and protein levels. We found many compositional peculiarities related to environmental adaptation and the life style. Specifically, thermal adaptation of protein-coding sequences in Archaea is characterized by a stronger codon bias than in Bacteria. Guanine and cytosine load in the third codon position is important for supporting the aerobic life style, and it is highly pronounced in Bacteria. The third codon position also provides a tradeoff between arginine and lysine, which are favorable for thermal adaptation and aerobicity, respectively. Dinucleotide composition provides stability of nucleic acids via strong base-stacking in ApG dinucleotides. In relation to coevolution of nucleic acids and proteins, thermostability-related demands on the amino acid composition affect the nucleotide content in the second codon position in Archaea. PMID:24371267

Serum levels of fatty acid binding protein 4 and fat metabolic markers in relation to catecholamines following exercise.

PubMed

Iso, Tatsuya; Sunaga, Hiroaki; Matsui, Hiroki; Kasama, Shu; Oshima, Naomi; Haruyama, Hikari; Furukawa, Nozomi; Nakajima, Kiyomi; Machida, Tetsuo; Murakami, Masami; Yokoyama, Tomoyuki; Kurabayashi, Masahiko

2017-11-01

Lipolysis is stimulated by activation of adrenergic inputs to adipose tissues. Our recent study showed that serum concentrations of fatty acid binding protein 4 (FABP4) are robustly elevated in patients with acute myocardial infarction and ventricular tachyarrhythmia, that display a marked activation of the sympathetic nervous system (SNS). However, it remains unknown whether circulating FABP4 concentrations are associated with exercise-induced SNS activation. Thirty one healthy volunteers underwent cardiopulmonary exercise testing on a cycle ergometer up to the workload levels below and above anaerobic threshold, low- and high-intensity exercise, respectively. Serial blood samplings were performed before and after exercise. High-intensity exercise significantly increased serum concentrations of FABP4 and catecholamines, and their concentrations declined fast thereafter in a similar fashion. These changes were accompanied by little, if any, changes in other metabolic markers. Regardless of adiposity, percent change from baseline to peak FABP4 levels (%FABP4) was comparable in all subjects. Stepwise regression analysis revealed that %FABP4 was highly correlated with that in norepinephrine. Our study reveals the significant correlation between circulating FABP4 and norepinephrine levels during exercise testing. Together with the fact that FABP4 is secreted from adipocytes via β-adrenergic-mediated lipolytic mechanisms, this study suggests FABP4 as a potential biomarker for adrenergic overdrive. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Fiber type- and fatty acid composition-dependent effects of high-fat diets on rat muscle triacylglyceride and fatty acid transporter protein-1 content.

PubMed

Marotta, Mario; Ferrer-Martnez, Andreu; Parnau, Josep; Turini, Marco; Macé, Katherine; Gómez Foix, Anna M

2004-08-01

Intramuscular triacylglyceride (TAG) is considered an independent marker of insulin resistance in humans. Here, we examined the effect of high-fat diets, based on distinct fatty acid compositions (saturated, monounsaturated or n-6 polyunsaturated), on TAG levels and fatty acid transporter protein (FATP-1) expression in 2 rat muscles that differ in their fiber type, soleus, and gastrocnemius; the relationship to whole body glucose intolerance was also studied. Compared with carbohydrate-fed rats, the groups subjected to any one of the high-fat diets consistently exhibited enhanced body weight gain and adiposity, elevated plasma free fatty acids and TAG in the fed condition, hyperinsulinemia, and glucose intolerance. TAG content was consistently higher in soleus than in gastrocnemius, but was only significantly elevated by the n-6 polyunsaturated-based diet. FATP-1 levels in soleus were double those in gastrocnemius muscle in carbohydrate-fed animals. High-fat diets caused an elevation in FATP-1 protein content in soleus, but a reduction in gastrocnemius. In conclusion, the hyperinsulinemic hyperlipidemic condition upregulates FATP-1 expression in soleus and downregulates that of gastrocnemius. Hypercaloric saturated, monounsaturated, or n-6 polyunsaturated lipid diets cause equivalent whole body insulin resistance in rats, but only an n-6 polyunsaturated acid-based diet triggers intramuscular TAG accumulation. Copyright 2004 Elsevier Inc.

Nucleic acid chaperone activity of retroviral Gag proteins.

PubMed

Rein, Alan

2010-01-01

Retrovirus particles in which the Gag protein has not yet been cleaved by the viral protease are termed immature particles. The viral RNA within these particles shows clear evidence of the action of a nucleic acid chaperone (NAC): the genomic RNA is dimeric, and a cellular tRNA molecule is annealed, by its 3' 18 nucleotides, to a complementary stretch in the viral RNA, in preparation for priming reverse transcription in the next round of infection. It seems very likely that the NAC that has catalyzed dimerization and tRNA annealing is the NC domain of the Gag protein itself. However, neither the dimeric linkage nor the tRNA:viral RNA complex has the same structure as those in mature virus particles: thus the conformational effects of Gag within the particles are not equivalent to those of the free NC protein present in mature particles. It is not known whether these dissimilarities reflect intrinsic differences in the NAC activities of Gag and NC, or limitations on Gag imposed by the structure of the immature particle. Analysis of the interactions of recombinant Gag proteins with nucleic acids is complicated by the fact that they result in assembly of virus-like particles. Nevertheless, the available data indicates that the affinity of Gag for nucleic acids can be considerably higher than that of free NC. This enhanced affinity may be due to contributions of the matrix domain, a positively charged region at the N-terminus of Gag; interactions of neighboring Gag molecules with each other may also increase the affinity due to cooperativity of the binding. Recombinant HIV-1 Gag protein clearly exhibits NAC activity. In two well-studied experimental systems, Gag was more efficient than NC, as its NAC effects could be detected at a significantly lower molar ratio of protein to nucleotide than with NC. In one system, binding of nucleic acid by the matrix domain of Gag retarded the Gag-induced annealing of two RNAs; this effect could be ameliorated by the competitive

Cortical Deficits of Glutamic Acid Decarboxylase 67 Expression in Schizophrenia: Clinical, Protein, and Cell Type-Specific Features

PubMed Central

Curley, Allison A.; Arion, Dominique; Volk, David W.; Asafu-Adjei, Josephine K.; Sampson, Allan R.; Fish, Kenneth N.; Lewis, David A.

2012-01-01

Objective Cognitive deficits in schizophrenia are associated with altered activity of the dorsolateral prefrontal cortex, which has been attributed to lower expression of the 67 kDa isoform of glutamic acid decarboxylase (GAD67), the major γ-aminobutyric acid (GABA)-synthesizing enzyme. However, little is know n about the relationship of prefrontal GAD67 m RNA levels and illness severity, translation of the transcript into protein, and protein levels in axon terminals, the key site of GABA production and function. Method Quantitative polymerase chain reaction was used to measure GAD67 m RNA levels in postmortem specimens of dorsolateral prefrontal cortex from subjects with schizophrenia and matched comparison subjects with no know n history of psychiatric or neurological disorders (N=42 pairs). In a subset of this cohort in which potential confounds of protein measures were controlled (N=19 pairs), Western blotting was used to quantify tissue levels of GAD67 protein in tissue. In five of these pairs, multilabel confocalimm unofluorescence was used to quantify GAD67 protein levels in the axon terminals of parvalbumin-containing GABA neurons, which are know n to have low levels of GAD67 m RNA in schizophrenia. Results GAD67 m RNA levels were significantly lower in schizophrenia subjects (by 15%), but transcript levels were not associated with predictors or measures of illness severity or chronicity. In schizophrenia subjects, GAD67 protein levels were significantly lower in total gray matter (by 10%) and in parvalbumin axon terminals (by 49%). Conclusions The findings that lower GAD67 m RNA expression is com m on in schizophrenia, that it is not a consequence of having the illness, and that it leads to less translation of the protein, especially in the axon terminals of parvalbumin-containing neurons, support the hypothesis that lower GABA synthesis in parvalbumin neurons contributes to dorsolateral prefrontal cortex dysfunction and impaired cognition in

Folic acid-conjugated soybean protein-based nanoparticles mediate efficient antitumor ability in vitro.

PubMed

Yao, Weijing; Zha, Qian; Cheng, Xu; Wang, Xin; Wang, Jun; Tang, Rupei

2016-11-23

In this study, soy protein isolate was hydrolyzed by compound enzymes to give aqueous soy protein with low molecular weights. Folic acid modified and free soy protein nanoparticles were successfully prepared by a desolvation method as target-specific drug delivery, respectively. Ultraviolet spectrophotometry demonstrated that folic acid was successfully grafted onto soy protein. The shape and size of folic acid modified soy protein nanoparticles were detected by transmission electron microscopy, scanning electron microscope, and dynamic light scattering. In addition, a series of characteristics including kinetic stability, pH stability, and time stability were also performed. Doxorubicin was successfully loaded into folic acid modified soy protein nanoparticles, and the encapsulation and loading efficiencies were 96.7% and 23%, respectively. Doxorubicin-loaded folic acid modified soy protein nanoparticles exhibited faster drug release rate than soy protein nanoparticles in PBS solution (pH = 5). The tumor penetration and antitumor experiments were done using three-dimensional multicellular tumor spheroids as the in vitro model. The results proved that folic acid modified soy protein nanoparticles display higher penetration and accumulation than soy protein nanoparticles, therefore possessing efficient growth inhibitory ability against multicellular tumor spheroids. © The Author(s) 2016.

Attomole-level protein fingerprinting based on intrinsic peptide fluorescence.

PubMed

Okerberg, E; Shear, J B

2001-04-01

Protein identification has relied heavily on proteolytic analysis, but current techniques are often slow and generally consume large quantities of valuable protein sample. We report the development of a rapid, ultralow volume protein analysis strategy based on tryptic digestion within the tip of a 1.5-microm capillary channel followed by separation of the proteolytic fragments using capillary electrophoresis (CE). Two-photon excitation is used to probe the intrinsic fluorescence of peptide fragments through "deep-UV" excitation of aromatic amino acid residues at the outlet of the CE channel. Detection limits using this technique are 0.7, 2.4, and 23 amol for the aromatic amino acids tryptophan, tyrosine, and phenylalanine, respectively. In these studies, we demonstrate the capacity to differentiate bovine and yeast cytochrome c variants using less than 15 amol of protein through tryptic fingerprinting. Moreover, the detection of a single amino acid substitution between bovine and canine cytochrome c illustrates the sensitivity of this approach to minor differences in protein sequence. The 2-pL sample volume required for this on-column tryptic digestion is, to our knowledge, the smallest yet reported for a proteolytic assay.

Reduction of circulating FABP4 level by treatment with omega-3 fatty acid ethyl esters.

PubMed

Furuhashi, Masato; Hiramitsu, Shinya; Mita, Tomohiro; Omori, Akina; Fuseya, Takahiro; Ishimura, Shutaro; Watanabe, Yuki; Hoshina, Kyoko; Matsumoto, Megumi; Tanaka, Marenao; Moniwa, Norihito; Yoshida, Hideaki; Ishii, Junnichi; Miura, Tetsuji

2016-01-12

Fatty acid-binding protein 4 (FABP4/A-FABP/aP2) mainly expressed in adipocytes is secreted and acts as an adipokine. Increased circulating FABP4 level is associated with obesity, insulin resistance and atherosclerosis. However, little is known about the modulation of serum FABP4 level by drugs including anti-dyslipidemic agents. Patients with dyslipidemia were treated with omega-3 fatty acid ethyl esters (4 g/day; n = 14) containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) for 4 weeks. Serum FABP4 level was measured before and after treatment. Expression and secretion of FABP4 were also examined in mouse 3T3-L1 adipocytes treated with EPA or DHA. Treatment with omega-3 fatty acid ethyl esters significantly decreased triglycerides and serum FABP4 level (13.5 ± 1.5 vs. 11.5 ± 1.1 ng/ml, P = 0.017). Change in FABP4 level by omega-3 fatty acids was negatively correlated with change in levels of EPA + DHA (r = -0.643, P = 0.013), EPA (r = -0.540, P = 0.046) and DHA (r = -0.650, P = 0.011) but not change in the level of triglycerides or other fatty acid composition. Treatment of 3T3-L1 adipocytes with EPA or DHA had no effect on short-term (2 h) secretion of FABP4. However, gene expression and long-term (24 h) secretion of FABP4 were significantly reduced by treatment with EPA or DHA. Omega-3 fatty acids decrease circulating FABP4 level, possibly by reducing expression and consecutive secretion of FABP4 in adipocytes. Reducing FABP4 level might be involved in suppression of cardiovascular events by omega-3 fatty acids.

Effects of Long-Term Protein Restriction on Meat Quality, Muscle Amino Acids, and Amino Acid Transporters in Pigs.

PubMed

Yin, Jie; Li, Yuying; Zhu, Xiaotong; Han, Hui; Ren, Wenkai; Chen, Shuai; Bin, Peng; Liu, Gang; Huang, Xingguo; Fang, Rejun; Wang, Bin; Wang, Kai; Sun, Liping; Li, Tiejun; Yin, Yulong

2017-10-25

This study aimed to investigate the long-term effects of protein restriction from piglets to finishing pigs for 16 weeks on meat quality, muscle amino acids, and amino acid transporters. Thirty-nine piglets were randomly divided into three groups: a control (20-18-16% crude protein, CP) and two protein restricted groups (17-15-13% CP and 14-12-10% CP). The results showed that severe protein restriction (14-12-10% CP) inhibited feed intake and body weight, while moderate protein restriction (17-15-13% CP) had little effect on growth performance in pigs. Meat quality (i.e., pH, color traits, marbling, water-holding capacity, and shearing force) were tested, and the results exhibited that 14-12-10% CP treatment markedly improved muscle marbling score and increased yellowness (b*). pH value (45 min) was significantly higher in 17-15-13% CP group than that in other groups. In addition, protein restriction reduced muscle histone, arginine, valine, and isoleucine abundances and enhanced glycine and lysine concentrations compared with the control group, while the RT-PCR results showed that protein restriction downregulated amino acids transporters. Mechanistic target of rapamycin (mTOR) signaling pathway was inactivated in the moderate protein restricted group (17-15-13% CP), while severe protein restriction with dietary 14-12-10% CP markedly enhanced mTOR phosphorylation. In conclusion, long-term protein restriction affected meat quality and muscle amino acid metabolism in pigs, which might be associated with mTOR signaling pathway.

NR4A orphan nuclear receptors influence retinoic acid and docosahexaenoic acid signaling via up-regulation of fatty acid binding protein 5

DOE Office of Scientific and Technical Information (OSTI.GOV)

Volakakis, Nikolaos; Joodmardi, Eliza; Perlmann, Thomas, E-mail: thomas.perlmann@licr.ki.se

2009-12-25

The orphan nuclear receptor (NR) Nurr1 is expressed in the developing and adult nervous system and is also induced as an immediate early gene in a variety of cell types. In silico analysis of human promoters identified fatty acid binding protein 5 (FABP5), a protein shown to enhance retinoic acid-mediated PPAR{beta}/{delta} signaling, as a potential Nurr1 target gene. Nurr1 has previously been implicated in retinoid signaling via its heterodimerization partner RXR. Since NRs are commonly involved in cross-regulatory control we decided to further investigate the regulatory relationship between Nurr1 and FABP5. FABP5 expression was up-regulated by Nurr1 and other NR4Amore » NRs in HEK293 cells, and Nurr1 was shown to activate and bind to the FABP5 promoter, supporting that FABP5 is a direct downstream target of NR4A NRs. We also show that the RXR ligand docosahexaenoic acid (DHA) can induce nuclear translocation of FABP5. Moreover, via up-regulation of FABP5 Nurr1 can enhance retinoic acid-induced signaling of PPAR{beta}/{delta} and DHA-induced activation of RXR. We also found that other members of the NR4A orphan NRs can up-regulate FABP5. Thus, our findings suggest that NR4A orphan NRs can influence signaling events of other NRs via control of FABP5 expression levels.« less

[Metabolism of cholesterol and fatty acids in nephrotic syndrome and its regulation by sterol regulatory element binding proteins (SREBP's). Effect of soy protein consumption].

PubMed

Tovar, Armando; Manzano, Natalia; Torres, Nimbe

2005-01-01

Hyperlipidemia occurs during nephrotic syndrome (NS). It is known that cholesterol and fatty acid biosynthesis is controlled by the transcription factors sterol regulatory element binding proteins (SREBPs). Soy protein consumption reduces the concentration of these lipids, although its mechanism of action is not well known. The aim of the present study was to establish whether soy protein consumption reduces cholesterol and triglycerides levels by regulating of SREBPs. Male Wistar rats with experimental NS were studied for 64 days. The results showed that rats fed with soy protein had significantly lower plasma cholesterol and triglyceride concentrations as well as proteinuria than rats fed with casein diet. These decrements were associated with a decrease in the expression of SREBP-1 and fatty acid biosynthetic enzymes. In addition, Western blot analysis revealed that in nuclear extracts from hepatocytes of rats fed with soy protein, there was a lower concentration of SREBP-1 than in rats fed with casein. The results of this study indicate that consumption of a soy protein diet has beneficial effects on nephrotic syndrome.

[Effects of low-protein diet plus alpha-keto acid on micro-inflammation and the relationship between micro-inflammation and nutritional status in patients performing continuous ambulatory peritoneal dialysis: a randomized controlled trial].

PubMed

Chen, Wei; Guo, Zhi-Yong; Wu, Hao; Sun, Li-Jing; Cai, Li-Li; Xu, Hai-Yan

2008-05-01

To investigate the effects of the combination of alpha-keto acid and low-protein diet on the levels of serum cytokines in patients performing continuous ambulatory peritoneal dialysis (CAPD) and to explore the relationship between inflammation and malnutrition in CAPD patients. Eighty-nine CAPD patients were randomized into three groups, and 78 cases completed a one-year follow-up and with complete data. There were 31 cases in low-protein diet plus alpha-keto acid group, 26 cases in low-protein diet group and 21 cases in routine-protein diet group. The levels of serum albumin (Alb), prealbumin (PA), retinol-binding protein (RBP), transferrin (TRF), cholesterol (TC), triglycerides (TG), leptin, and triceps skinfold thickness (TSF), mid-arm muscle circumference (MAMC), body mass index (BMI) were measured. The changes of serum interleukin-1alpha (IL-1alpha), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and C-reactive protein (CRP) were also detected. Compared with low-protein diet group, serum levels of PA, RBP and TRF were significantly increased both in low-protein diet plus alpha-keto acid and routine-protein diet groups ( P<0.01), however, there was no significant difference in the levels of PA, RBP and TRF between low-protein diet plus alpha-keto acid group and routine-protein diet group. There was an increased tendency in the content of Alb, TC, TG, BMI, TSF and MAMC, but there were no significant differences. The plasma levels of IL-1alpha, IL-6 and TNF-alpha in low-protein diet plus alpha-keto acid group were decreased as compared with the routine-protein diet group, but there were no significant differences. The plasma level of CRP in low-protein diet plus alpha-keto acid group was lower than that in the routine-protein diet group ( P<0.01). The combination of alpha-keto acid and low-protein diet can ameliorate malnutrition and micro-inflammation in CAPD patients.

A 100-Year Review: Protein and amino acid nutrition in dairy cows.

PubMed

Schwab, Charles G; Broderick, Glen A

2017-12-01

Considerable progress has been made in understanding the protein and amino acid (AA) nutrition of dairy cows. The chemistry of feed crude protein (CP) appears to be well understood, as is the mechanism of ruminal protein degradation by rumen bacteria and protozoa. It has been shown that ammonia released from AA degradation in the rumen is used for bacterial protein formation and that urea can be a useful N supplement when lower protein diets are fed. It is now well documented that adequate rumen ammonia levels must be maintained for maximal synthesis of microbial protein and that a deficiency of rumen-degradable protein can decrease microbial protein synthesis, fiber digestibility, and feed intake. Rumen-synthesized microbial protein accounts for most of the CP flowing to the small intestine and is considered a high-quality protein for dairy cows because of apparent high digestibility and good AA composition. Much attention has been given to evaluating different methods to quantify ruminal protein degradation and escape and for measuring ruminal outflows of microbial protein and rumen-undegraded feed protein. The methods and accompanying results are used to determine the nutritional value of protein supplements and to develop nutritional models and evaluate their predictive ability. Lysine, methionine, and histidine have been identified most often as the most-limiting amino acids, with rumen-protected forms of lysine and methionine available for ration supplementation. Guidelines for protein feeding have evolved from simple feeding standards for dietary CP to more complex nutrition models that are designed to predict supplies and requirements for rumen ammonia and peptides and intestinally absorbable AA. The industry awaits more robust and mechanistic models for predicting supplies and requirements of rumen-available N and absorbed AA. Such models will be useful in allowing for feeding lower protein diets and increased efficiency of microbial protein synthesis

Identification of a Novel Hypocholesterolemic Protein, Major Royal Jelly Protein 1, Derived from Royal Jelly

PubMed Central

Asai, Saori; Kusada, Mio; Watanabe, Suzuyo; Kawashima, Takuji; Nakamura, Tadashi; Shimada, Masaya; Goto, Tsuyoshi; Nagaoka, Satoshi

2014-01-01

Royal jelly (RJ) intake lowers serum cholesterol levels in animals and humans, but the active component in RJ that lowers serum cholesterol level and its molecular mechanism are unclear. In this study, we set out to identify the bile acid-binding protein contained in RJ, because dietary bile acid-binding proteins including soybean protein and its peptide are effective in ameliorating hypercholesterolemia. Using a cholic acid-conjugated column, we separated some bile acid-binding proteins from RJ and identified the major RJ protein 1 (MRJP1), MRJP2, and MRJP3 as novel bile acid-binding proteins from RJ, based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Purified MRJP1, which is the most abundant protein of the bile acid-binding proteins in RJ, exhibited taurocholate-binding activity in vitro. The micellar solubility of cholesterol was significantly decreased in the presence of MRJP1 compared with casein in vitro. Liver bile acids levels were significantly increased, and cholesterol 7α-hydroxylase (CYP7A1) mRNA and protein tended to increase by MRJP1 feeding compared with the control. CYP7A1 mRNA and protein levels were significantly increased by MRJP1 tryptic hydrolysate treatment compared with that of casein tryptic hydrolysate in hepatocytes. MRJP1 hypocholesterolemic effect has been investigated in rats. The cholesterol-lowering action induced by MRJP1 occurs because MRJP1 interacts with bile acids induces a significant increase in fecal bile acids excretion and a tendency to increase in fecal cholesterol excretion and also enhances the hepatic cholesterol catabolism. We have identified, for the first time, a novel hypocholesterolemic protein, MRJP1, in RJ. Interestingly, MRJP1 exhibits greater hypocholesterolemic activity than the medicine β-sitosterol in rats. PMID:25144734

Serum adipocyte-fatty acid binding protein (FABP4) levels in women from Mexico exposed to polycyclic aromatic hydrocarbons (PAHs).

PubMed

Ochoa-Martínez, Ángeles C; Ruíz-Vera, Tania; Pruneda-Álvarez, Lucia G; González-Palomo, Ana K; Almendarez-Reyna, Claudia I; Pérez-Vázquez, Francisco J; Pérez-Maldonado, Iván N

2017-01-01

Recent studies indicate that exposure to polycyclic aromatic hydrocarbons (PAHs) is a very important risk factor for the development of cardiovascular diseases (CVDs). Correspondingly, adipocyte-fatty acid binding protein (FABP4, also known as aP2 and AFABP) has been proposed as a new, meaningful and useful biomarker to predict metabolic and cardiovascular diseases. Therefore, the aim of this study was to evaluate serum FABP4 levels in Mexican women exposed to PAHs. Urinary 1-hydroxypyrene ((1-OHP), exposure biomarker for PAHs) levels were quantified using a high-performance liquid chromatography (HPLC) technique, and serum FABP4 concentrations were analyzed using a commercially available ELISA kit. The mean urinary 1-OHP level found in women participating in this study was 1.30 ± 1.10 μmol/mol creatinine (2.45 ± 2.10 μg/g creatinine). Regarding serum FABP4 concentrations, the levels ranged from 3.80 to 62.5 ng/mL in the assessed population. Moreover, a significant association (p < 0.001) was found between urinary 1-OHP levels and serum FABP4 concentrations in women after adjusting for potential confounding variables. The presented data in this study can be considered only as a starting point for further studies. Then, in order to elucidate whether FABP4 represents a risk factor for CVD disease in humans exposed to air contaminants (such as PAHs), large epidemiological studies are necessary.

Genetically programmed expression of proteins containing the unnatural amino acid phenylselenocysteine

DOEpatents

Wang, Jiangyun; Schultz, Peter G.

2010-09-07

The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the unnatural amino acid phenylselenocysteine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal aminoacyl-tRNA synthetases, polynucleotides encoding the novel synthetase molecules, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid phenylselenocysteine and translation systems. The invention further provides methods for producing modified proteins (e.g., lipidated proteins) through targeted modification of the phenylselenocysteine residue in a protein.

Genetically programmed expression of proteins containing the unnatural amino acid phenylselenocysteine

DOEpatents

Wang, Jiangyun; Schultz, Peter G.

2012-07-10

The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the unnatural amino acid phenylselenocysteine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal aminoacyl-tRNA synthetases, polynucleotides encoding the novel synthetase molecules, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid phenylselenocysteine and translation systems. The invention further provides methods for producing modified proteins (e.g., lipidated proteins) through targeted modification of the phenylselenocysteine residue in a protein.

Total amino acid stabilization during cell-free protein synthesis reactions.

PubMed

Calhoun, Kara A; Swartz, James R

2006-05-17

Limitations in amino acid supply have been recognized as a substantial problem in cell-free protein synthesis reactions. Although enzymatic inhibitors and fed-batch techniques have been beneficial, the most robust way to stabilize amino acids is to remove the responsible enzymatic activities by genetically modifying the source strain used for cell extract preparation. Previous work showed this was possible for arginine, serine, and tryptophan, but cysteine degradation remained a major limitation in obtaining high protein synthesis yields. Through radiolabel techniques, we confirmed that cysteine degradation was caused by the activity of glutamate-cysteine ligase (gene gshA) in the cell extract. Next, we created Escherichia coli strain KC6 that combines a gshA deletion with previously described deletions for arginine, serine, and tryptophan stabilization. Strain KC6 grows well, and active cell extract can be produced from it for cell-free protein synthesis reactions. The extract from strain KC6 maintains stable amino acid concentrations of all 20 amino acids in a 3-h batch reaction. Yields for three different proteins improved 75-250% relative to cell-free expression using the control extract.

AMINO ACID MIXTURES EFFECTIVE PARENTERALLY FOR LONG CONTINUED PLASMA PROTEIN PRODUCTION. CASEIN DIGESTS COMPARED

PubMed Central

Madden, S. C.; Woods, R. R.; Shull, F. W.; Whipple, G. H.

1944-01-01

When blood plasma proteins are depleted by bleeding with return of red cells suspended in saline (plasmapheresis) it is possible to bring dogs to a steady state of hypoproteinemia and a constant level of plasma protein production if the diet nitrogen intake is controlled and limited. Such dogs are outwardly normal but have a lowered resistance to infection and to certain intoxications. The ten growth essential amino acids of Rose plus glycine will maintain nitrogen balance and produce as much new plasma protein as will good diet proteins. This good utilization is demonstrated over periods of several months when the amino acids are given either orally or parenterally. There is no evidence of toxicity in general nor to unnatural forms of these synthetic amino acids in particular. Given parenterally appropriate mixtures of these amino acids are well tolerated even upon rapid injection. The minimal daily requirements for a 10 kilo dog may be given intravenously in 10 minutes without reaction. Subcutaneously a 10 per cent solution may be given rapidly without reaction. Among various mixtures tested Vt approximates a minimum for a 10 kilo dog. It contains in grams (dl-threonine 0.7, dl-valine 1.5, l-(-) leucine 1.5, dl-isoleucine 1.4, dl-lysine hydrochloride 1.5, l(-) tryptophane 0.4, dl-phenylalanine 1.0, dl-methionine 0.6, l(+)-histidine hydrochloride 0.5, l(+)-arginine hydrochloride 0.5, and glycine 1.0. The presence of glycine improves tolerance to rapid intravenous injection, but excess glycine does not improve utilization of the mixture. Over a long period this mixture appears suboptimal in quantity. Doubled it is more than ample. Of two casein digests tested the one prepared by enzymatic hydrolysis provided good nitrogen retention and fairly good plasma protein production but was much less tolerable upon intravenous injection than certain mixtures of pure amino acids. The other one prepared by acid hydrolysis and tryptophane fortification afforded bare nitrogen

The effects of high dietary protein and nitrogen levels on the preformed methyl group requirement and methionine-induced growth depression in chicks.

PubMed

Pesti, G M; Benevenga, N J; Harper, A E; Sunde, M L

1981-02-01

The chick's choline and methionine requirements are both increased by high dietary protein level. Studies were conducted to test the hypothesis that the chicks' need for preformed methyl groups is increased by high protein diets (not methionine or choline per se). Chicks fed 25% isolated soybean protein (ISP) diets responded to methionine supplementation (162 vs 110 g gained in 14 days) but not to choline (119 g vs. 110 g), while those fed 50% ISP responded to either methionine (174 g vs. 126 g) or choline (181 g vs. 126 g) supplementation. Further, neither cystine nor homocystine could replace methionine in improving the growth of chicks fed the high protein diet. In other experiments, L-methionine and betaine HCl were found to alleviate the growth depression caused by excessive levels of L-glutamic acid. Excessive levels of L-methionine had a protective effect against growth depression caused by L-glutamate and diammonium citrate, and conversely, supplementary L-serine and sodium formate were not protective against glutamic acid- or arginine-induced growth depression. The results are consistent with the hypothesis that the preformed methyl group requirement is increased by high levels of dietary protein and excessive nitrogen from a single amino acid.

Lithium Decreases Glial Fibrillary Acidic Protein in a Mouse Model of Alexander Disease

PubMed Central

LaPash Daniels, Christine M.; Paffenroth, Elizabeth; Austin, Elizabeth V.; Glebov, Konstantin; Lewis, Diana; Walter, Jochen; Messing, Albee

2015-01-01

Alexander disease is a fatal neurodegenerative disease caused by mutations in the astrocyte intermediate filament glial fibrillary acidic protein (GFAP). The disease is characterized by elevated levels of GFAP and the formation of protein aggregates, known as Rosenthal fibers, within astrocytes. Lithium has previously been shown to decrease protein aggregates by increasing the autophagy pathway for protein degradation. In addition, lithium has also been reported to decrease activation of the transcription factor STAT3, which is a regulator of GFAP transcription and astrogliogenesis. Here we tested whether lithium treatment would decrease levels of GFAP in a mouse model of Alexander disease. Mice with the Gfap-R236H point mutation were fed lithium food pellets for 4 to 8 weeks. Four weeks of treatment with LiCl at 0.5% in food pellets decreased GFAP protein and transcripts in several brain regions, although with mild side effects and some mortality. Extending the duration of treatment to 8 weeks resulted in higher mortality, and again with a decrease in GFAP in the surviving animals. Indicators of autophagy, such as LC3, were not increased, suggesting that lithium may decrease levels of GFAP through other pathways. Lithium reduced the levels of phosphorylated STAT3, suggesting this as one pathway mediating the effects on GFAP. In conclusion, lithium has the potential to decrease GFAP levels in Alexander disease, but with a narrow therapeutic window separating efficacy and toxicity. PMID:26378915

AFAL: a web service for profiling amino acids surrounding ligands in proteins

NASA Astrophysics Data System (ADS)

Arenas-Salinas, Mauricio; Ortega-Salazar, Samuel; Gonzales-Nilo, Fernando; Pohl, Ehmke; Holmes, David S.; Quatrini, Raquel

2014-11-01

With advancements in crystallographic technology and the increasing wealth of information populating structural databases, there is an increasing need for prediction tools based on spatial information that will support the characterization of proteins and protein-ligand interactions. Herein, a new web service is presented termed amino acid frequency around ligand (AFAL) for determining amino acids type and frequencies surrounding ligands within proteins deposited in the Protein Data Bank and for assessing the atoms and atom-ligand distances involved in each interaction (availability: http://structuralbio.utalca.cl/AFAL/index.html). AFAL allows the user to define a wide variety of filtering criteria (protein family, source organism, resolution, sequence redundancy and distance) in order to uncover trends and evolutionary differences in amino acid preferences that define interactions with particular ligands. Results obtained from AFAL provide valuable statistical information about amino acids that may be responsible for establishing particular ligand-protein interactions. The analysis will enable investigators to compare ligand-binding sites of different proteins and to uncover general as well as specific interaction patterns from existing data. Such patterns can be used subsequently to predict ligand binding in proteins that currently have no structural information and to refine the interpretation of existing protein models. The application of AFAL is illustrated by the analysis of proteins interacting with adenosine-5'-triphosphate.

AFAL: a web service for profiling amino acids surrounding ligands in proteins.

PubMed

Arenas-Salinas, Mauricio; Ortega-Salazar, Samuel; Gonzales-Nilo, Fernando; Pohl, Ehmke; Holmes, David S; Quatrini, Raquel

2014-11-01

With advancements in crystallographic technology and the increasing wealth of information populating structural databases, there is an increasing need for prediction tools based on spatial information that will support the characterization of proteins and protein-ligand interactions. Herein, a new web service is presented termed amino acid frequency around ligand (AFAL) for determining amino acids type and frequencies surrounding ligands within proteins deposited in the Protein Data Bank and for assessing the atoms and atom-ligand distances involved in each interaction (availability: http://structuralbio.utalca.cl/AFAL/index.html ). AFAL allows the user to define a wide variety of filtering criteria (protein family, source organism, resolution, sequence redundancy and distance) in order to uncover trends and evolutionary differences in amino acid preferences that define interactions with particular ligands. Results obtained from AFAL provide valuable statistical information about amino acids that may be responsible for establishing particular ligand-protein interactions. The analysis will enable investigators to compare ligand-binding sites of different proteins and to uncover general as well as specific interaction patterns from existing data. Such patterns can be used subsequently to predict ligand binding in proteins that currently have no structural information and to refine the interpretation of existing protein models. The application of AFAL is illustrated by the analysis of proteins interacting with adenosine-5'-triphosphate.

Selective Cooperation between Fatty Acid Binding Proteins and Peroxisome Proliferator-Activated Receptors in Regulating Transcription

PubMed Central

Tan, Nguan-Soon; Shaw, Natacha S.; Vinckenbosch, Nicolas; Liu, Peng; Yasmin, Rubina; Desvergne, Béatrice; Wahli, Walter; Noy, Noa

2002-01-01

Lipophilic compounds such as retinoic acid and long-chain fatty acids regulate gene transcription by activating nuclear receptors such as retinoic acid receptors (RARs) and peroxisome proliferator-activated receptors (PPARs). These compounds also bind in cells to members of the family of intracellular lipid binding proteins, which includes cellular retinoic acid-binding proteins (CRABPs) and fatty acid binding proteins (FABPs). We previously reported that CRABP-II enhances the transcriptional activity of RAR by directly targeting retinoic acid to the receptor. Here, potential functional cooperation between FABPs and PPARs in regulating the transcriptional activities of their common ligands was investigated. We show that adipocyte FABP and keratinocyte FABP (A-FABP and K-FABP, respectively) selectively enhance the activities of PPARγ and PPARβ, respectively, and that these FABPs massively relocate to the nucleus in response to selective ligands for the PPAR isotype which they activate. We show further that A-FABP and K-FABP interact directly with PPARγ and PPARβ and that they do so in a receptor- and ligand-selective manner. Finally, the data demonstrate that the presence of high levels of K-FABP in keratinocytes is essential for PPARβ-mediated induction of differentiation of these cells. Taken together, the data establish that A-FABP and K-FABP govern the transcriptional activities of their ligands by targeting them to cognate PPARs in the nucleus, thereby enabling PPARs to exert their biological functions. PMID:12077340

Reasons for the occurrence of the twenty coded protein amino acids

NASA Technical Reports Server (NTRS)

Weber, A. L.; Miller, S. L.

1981-01-01

Factors involved in the selection of the 20 protein L-alpha-amino acids during chemical evolution and the early stages of Darwinian evolution are discussed. The selection is considered on the basis of the availability in the primitive ocean, function in proteins, the stability of the amino acid and its peptides, stability to racemization, and stability on the transfer RNA. It is concluded that aspartic acid, glutamic acid, arginine, lysine, serine and possibly threonine are the best choices for acidic, basic and hydroxy amino acids. The hydrophobic amino acids are reasonable choices, except for the puzzling absences of alpha-amino-n-butyric acid, norvaline and norleucine. The choices of the sulfur and aromatic amino acids seem reasonable, but are not compelling. Asparagine and glutamine are apparently not primitive. If life were to arise on another planet, it would be expected that the catalysts would be poly-alpha-amino acids and that about 75% of the amino acids would be the same as on the earth.

Microwave-assisted acid and base hydrolysis of intact proteins containing disulfide bonds for protein sequence analysis by mass spectrometry.

PubMed

Reiz, Bela; Li, Liang

2010-09-01

Controlled hydrolysis of proteins to generate peptide ladders combined with mass spectrometric analysis of the resultant peptides can be used for protein sequencing. In this paper, two methods of improving the microwave-assisted protein hydrolysis process are described to enable rapid sequencing of proteins containing disulfide bonds and increase sequence coverage, respectively. It was demonstrated that proteins containing disulfide bonds could be sequenced by MS analysis by first performing hydrolysis for less than 2 min, followed by 1 h of reduction to release the peptides originally linked by disulfide bonds. It was shown that a strong base could be used as a catalyst for microwave-assisted protein hydrolysis, producing complementary sequence information to that generated by microwave-assisted acid hydrolysis. However, using either acid or base hydrolysis, amide bond breakages in small regions of the polypeptide chains of the model proteins (e.g., cytochrome c and lysozyme) were not detected. Dynamic light scattering measurement of the proteins solubilized in an acid or base indicated that protein-protein interaction or aggregation was not the cause of the failure to hydrolyze certain amide bonds. It was speculated that there were some unknown local structures that might play a role in preventing an acid or base from reacting with the peptide bonds therein. 2010 American Society for Mass Spectrometry. Published by Elsevier Inc. All rights reserved.

Designing Light-Activated Charge-Separating Proteins with a Naphthoquinone Amino Acid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lichtenstein, Bruce R.; Bialas, Chris; Cerda, José F.

2015-09-14

The first principles design of manmade redox-protein maquettes is used to clarify the physical/chemical engineering supporting the mechanisms of natural enzymes with a view to recapitulate and surpass natural performance. Herein, we use intein-based protein semisynthesis to pair a synthetic naphthoquinone amino acid (Naq) with histidine-ligated photoactive metal–tetrapyrrole cofactors, creating a 100 μs photochemical charge separation unit akin to photosynthetic reaction centers. By using propargyl groups to protect the redox-active para-quinone during synthesis and assembly while permitting selective activation, we gain the ability to employ the quinone amino acid redox cofactor with the full set of natural amino acids inmore » protein design. Direct anchoring of quinone to the protein backbone permits secure and adaptable control of intraprotein electron-tunneling distances and rates.« less

Stabilization of Proteins and Noncovalent Protein Complexes during Electrospray Ionization by Amino Acid Additives.

PubMed

Zhang, Hua; Lu, Haiyan; Chingin, Konstantin; Chen, Huanwen

2015-07-21

Ionization of proteins and noncovalent protein complexes with minimal disturbance to their native structure presents a great challenge for biological mass spectrometry (MS). In living organisms, the native structure of intracellular proteins is commonly stabilized by solute amino acids (AAs) accumulated in cells at very high concentrations. Inspired by nature, we hypothesized that AAs could also pose a stabilizing effect on the native structure of proteins and noncovalent protein complexes during ionization. To test this hypothesis, here we explored MS response for various protein complexes upon the addition of free AAs at mM concentrations into the electrospray ionization (ESI) solution. Thermal activation of ESI droplets in the MS inlet capillary was employed as a model destabilizing factor during ionization. Our results indicate that certain AAs, in particular proline (Pro), pose considerable positive effect on the stability of noncovalent protein complexes in ESI-MS without affecting the signal intensity of protein ions and original protein-ligand equilibrium, even when added at the 20 mM concentration. The data suggest that the degree of protein stabilization is primarily determined by the osmolytic and ampholytic characteristics of AA solutes. The highest stability and visibility of noncovalent protein complexes in ESI-MS are achieved using AA additives with neutral isoelectric point, moderate proton affinity, and unfavorable interaction with the native protein state. Overall, our results indicate that the simple addition of free amino acids into the working solution can notably improve the stability and accuracy of protein analysis by native ESI-MS.

Engineering acyl carrier protein to enhance production of shortened fatty acids.

PubMed

Liu, Xueliang; Hicks, Wade M; Silver, Pamela A; Way, Jeffrey C

2016-01-01

The acyl carrier protein (ACP) is an essential and ubiquitous component of microbial synthesis of fatty acids, the natural precursor to biofuels. Natural fatty acids usually contain long chains of 16 or more carbon atoms. Shorter carbon chains, with increased fuel volatility, are desired for internal combustion engines. Engineering the length specificity of key proteins in fatty acid metabolism, such as ACP, may enable microbial synthesis of these shorter chain fatty acids. We constructed a homology model of the Synechococcus elongatus ACP, showing a hydrophobic pocket harboring the growing acyl chain. Amino acids within the pocket were mutated to increase steric hindrance to the acyl chain. Certain mutant ACPs, when over-expressed in Escherichia coli, increased the proportion of shorter chain lipids; I75 W and I75Y showed the strongest effects. Expression of I75 W and I75Y mutant ACPs also increased production of lauric acid in E. coli that expressed the C12-specific acyl-ACP thioesterase from Cuphea palustris. We engineered the specificity of the ACP, an essential protein of fatty acid metabolism, to alter the E. coli lipid pool and enhance production of medium-chain fatty acids as biofuel precursors. These results indicate that modification of ACP itself could be combined with enzymes affecting length specificity in fatty acid synthesis to enhance production of commodity chemicals based on fatty acids.

Auxins differentially regulate root system architecture and cell cycle protein levels in maize seedlings.

PubMed

Martínez-de la Cruz, Enrique; García-Ramírez, Elpidio; Vázquez-Ramos, Jorge M; Reyes de la Cruz, Homero; López-Bucio, José

2015-03-15

Maize (Zea mays) root system architecture has a complex organization, with adventitious and lateral roots determining its overall absorptive capacity. To generate basic information about the earlier stages of root development, we compared the post-embryonic growth of maize seedlings germinated in water-embedded cotton beds with that of plants obtained from embryonic axes cultivated in liquid medium. In addition, the effect of four different auxins, namely indole-3-acetic acid (IAA), 1-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA) and 2,4-dichlorophenoxyacetic acid (2,4-D) on root architecture and levels of the heat shock protein HSP101 and the cell cycle proteins CKS1, CYCA1 and CDKA1 were analyzed. Our data show that during the first days after germination, maize seedlings develop several root types with a simultaneous and/or continuous growth. The post-embryonic root development started with the formation of the primary root (PR) and seminal scutellar roots (SSR) and then continued with the formation of adventitious crown roots (CR), brace roots (BR) and lateral roots (LR). Auxins affected root architecture in a dose-response fashion; whereas NAA and IBA mostly stimulated crown root formation, 2,4-D showed a strong repressing effect on growth. The levels of HSP101, CKS1, CYCA1 and CDKA in root and leaf tissues were differentially affected by auxins and interestingly, HSP101 registered an auxin-inducible and root specific expression pattern. Taken together, our results show the timing of early branching patterns of maize and indicate that auxins regulate root development likely through modulation of the HSP101 and cell cycle proteins. Copyright © 2014 Elsevier GmbH. All rights reserved.

Overexpression of hydroxynitrile lyase in cassava roots elevates protein and free amino acids while reducing residual cyanogen levels.

PubMed

Narayanan, Narayanan N; Ihemere, Uzoma; Ellery, Claire; Sayre, Richard T

2011-01-01

Cassava is the major source of calories for more than 250 million Sub-Saharan Africans, however, it has the lowest protein-to-energy ratio of any major staple food crop in the world. A cassava-based diet provides less than 30% of the minimum daily requirement for protein. Moreover, both leaves and roots contain potentially toxic levels of cyanogenic glucosides. The major cyanogen in cassava is linamarin which is stored in the vacuole. Upon tissue disruption linamarin is deglycosylated by the apolplastic enzyme, linamarase, producing acetone cyanohydrin. Acetone cyanohydrin can spontaneously decompose at pHs >5.0 or temperatures >35°C, or is enzymatically broken down by hydroxynitrile lyase (HNL) to produce acetone and free cyanide which is then volatilized. Unlike leaves, cassava roots have little HNL activity. The lack of HNL activity in roots is associated with the accumulation of potentially toxic levels of acetone cyanohydrin in poorly processed roots. We hypothesized that the over-expression of HNL in cassava roots under the control of a root-specific, patatin promoter would not only accelerate cyanogenesis during food processing, resulting in a safer food product, but lead to increased root protein levels since HNL is sequestered in the cell wall. Transgenic lines expressing a patatin-driven HNL gene construct exhibited a 2-20 fold increase in relative HNL mRNA levels in roots when compared with wild type resulting in a threefold increase in total root protein in 7 month old plants. After food processing, HNL overexpressing lines had substantially reduced acetone cyanohydrin and cyanide levels in roots relative to wild-type roots. Furthermore, steady state linamarin levels in intact tissues were reduced by 80% in transgenic cassava roots. These results suggest that enhanced linamarin metabolism contributed to the elevated root protein levels.

Saturated fatty acid palmitate negatively regulates autophagy by promoting ATG5 protein degradation in meniscus cells.

PubMed

Mallik, Aritra; Yammani, Raghunatha R

2018-07-20

Obesity and associated metabolic factors are major risk factors for the development of osteoarthritis. Previously, we have shown that the free fatty acid palmitate induces endoplasmic reticulum (ER) stress and induces apoptosis in meniscus cells. However, the molecular mechanisms involved in these effects are not clearly understood. In our current study, we found that palmitate inhibits autophagy by modulating the protein levels of autophagy-related genes-5 (ATG5) that is associated with decreased lipidation of LC3 and increased activation of cleaved caspase 3. Pretreatment of meniscus cells with 4-phenyl butyric acid, a small molecule chemical chaperone that alleviates ER stress, or with MG-132, a proteasome inhibitor, restored normal levels of ATG5 and autophagosome formation, and decreased expression of cleaved caspase 3. Thus, our data suggest that palmitate downregulates autophagy in meniscus cells by degrading ATG5 protein via ER-associated protein degradation, and thus promotes apoptosis. This is the first study to demonstrate that palmitate-induced endoplasmic reticulum stress negatively regulates autophagy. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Immunization with a recombinant vaccinia virus that encodes nonstructural proteins of the hepatitis C virus suppresses viral protein levels in mouse liver.

PubMed

Sekiguchi, Satoshi; Kimura, Kiminori; Chiyo, Tomoko; Ohtsuki, Takahiro; Tobita, Yoshimi; Tokunaga, Yuko; Yasui, Fumihiko; Tsukiyama-Kohara, Kyoko; Wakita, Takaji; Tanaka, Toshiyuki; Miyasaka, Masayuki; Mizuno, Kyosuke; Hayashi, Yukiko; Hishima, Tsunekazu; Matsushima, Kouji; Kohara, Michinori

2012-01-01

Chronic hepatitis C, which is caused by infection with the hepatitis C virus (HCV), is a global health problem. Using a mouse model of hepatitis C, we examined the therapeutic effects of a recombinant vaccinia virus (rVV) that encodes an HCV protein. We generated immunocompetent mice that each expressed multiple HCV proteins via a Cre/loxP switching system and established several distinct attenuated rVV strains. The HCV core protein was expressed consistently in the liver after polyinosinic acid-polycytidylic acid injection, and these mice showed chronic hepatitis C-related pathological findings (hepatocyte abnormalities, accumulation of glycogen, steatosis), liver fibrosis, and hepatocellular carcinoma. Immunization with one rVV strain (rVV-N25), which encoded nonstructural HCV proteins, suppressed serum inflammatory cytokine levels and alleviated the symptoms of pathological chronic hepatitis C within 7 days after injection. Furthermore, HCV protein levels in liver tissue also decreased in a CD4 and CD8 T-cell-dependent manner. Consistent with these results, we showed that rVV-N25 immunization induced a robust CD8 T-cell immune response that was specific to the HCV nonstructural protein 2. We also demonstrated that the onset of chronic hepatitis in CN2-29((+/-))/MxCre((+/-)) mice was mainly attributable to inflammatory cytokines, (tumor necrosis factor) TNF-α and (interleukin) IL-6. Thus, our generated mice model should be useful for further investigation of the immunological processes associated with persistent expression of HCV proteins because these mice had not developed immune tolerance to the HCV antigen. In addition, we propose that rVV-N25 could be developed as an effective therapeutic vaccine.

Glutamic Acid - Amino Acid, Neurotransmitter, and Drug - Is Responsible for Protein Synthesis Rhythm in Hepatocyte Populations in vitro and in vivo.

PubMed

Brodsky, V Y; Malchenko, L A; Konchenko, D S; Zvezdina, N D; Dubovaya, T K

2016-08-01

Primary cultures of rat hepatocytes were studied in serum-free media. Ultradian protein synthesis rhythm was used as a marker of cell synchronization in the population. Addition of glutamic acid (0.2 mg/ml) to the medium of nonsynchronous sparse cultures resulted in detection of a common protein synthesis rhythm, hence in synchronization of the cells. The antagonist of glutamic acid metabotropic receptors MCPG (0.01 mg/ml) added together with glutamic acid abolished the synchronization effect; in sparse cultures, no rhythm was detected. Feeding rats with glutamic acid (30 mg with food) resulted in protein synthesis rhythm in sparse cultures obtained from the rats. After feeding without glutamic acid, linear kinetics of protein synthesis was revealed. Thus, glutamic acid, a component of blood as a non-neural transmitter, can synchronize the activity of hepatocytes and can form common rhythm of protein synthesis in vitro and in vivo. This effect is realized via receptors. Mechanisms of cell-cell communication are discussed on analyzing effects of non-neural functions of neurotransmitters. Glutamic acid is used clinically in humans. Hence, a previously unknown function of this drug is revealed.

Fluorescence Modulation of Green Fluorescent Protein Using Fluorinated Unnatural Amino Acids.

PubMed

Villa, Jordan K; Tran, Hong-Anh; Vipani, Megha; Gianturco, Stephanie; Bhasin, Konark; Russell, Brent L; Harbron, Elizabeth J; Young, Douglas D

2017-07-16

The ability to modulate protein function through minimal perturbations to amino acid structure represents an ideal mechanism to engineer optimized proteins. Due to the novel spectroscopic properties of green fluorescent protein, it has found widespread application as a reporter protein throughout the fields of biology and chemistry. Using site-specific amino acid mutagenesis, we have incorporated various fluorotyrosine residues directly into the fluorophore of the protein, altering the fluorescence and shifting the pKa of the phenolic proton associated with the fluorophore. Relative to wild type GFP, the fluorescence spectrum of the protein is altered with each additional fluorine atom, and the mutant GFPs have the potential to be employed as pH sensors due to the altered electronic properties of the fluorine atoms.

Dietary protein deficiency affects n-3 and n-6 polyunsaturated fatty acids hepatic storage and very low density lipoprotein transport in rats on different diets.

PubMed

Bouziane, M; Prost, J; Belleville, J

1994-04-01

Fatty livers and the similarity between the skin lesions in kwashiorkor and those described in experimental essential fatty acid (EFA) deficiency have led to the hypothesis that protein and EFA deficiencies may both occur in chronic malnutrition. The relationship between serum very low density lipoprotein (VLDL) and hepatic lipid composition was studied after 28 d of protein depletion to determine the interactions between dietary protein levels and EFA availability. Rats were fed purified diets containing 20 or 2% casein and 5% fat as either soybean oil rich in EFA, or salmon oil rich in eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, or hydrogenated coconut oil poor in EFA. Animals were divided into six groups, SOC (20% casein + 5% soybean oil), SOd (2% casein + 5% soybean oil), COC (20% casein + 5% hydrogenated coconut oil), COd (2% casein + 5% hydrogenated coconut oil), SAC (20% casein + 5% salmon oil) and SAd (2% casein + 5% salmon oil). After 28 d, liver steatosis and reduced VLDL-phospholipid contents (P < 0.001) were observed in protein-deficient rats. In protein deficiency, triacylglycerol and phospholipid fatty acid compositions in both liver and VLDL showed a decreased polyunsaturated-to-saturated fatty acid ratio. This ratio was higher with the salmon oil diets and lower with the hydrogenated coconut oil diets. Furthermore, independent of the oil in the diet, protein deficiency decreased linoleic and arachidonic acids in VLDL phospholipids. Conversely, despite decreased proportions of EPA at low protein levels, DHA levels remained higher in rats fed salmon oil diets.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetically programmed expression of proteins containing the unnatural amino acid phenylselenocysteine

DOEpatents

Wang, Jiangyun; Schultz, Peter G.

2013-03-12

The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetase that can incorporate the unnatural amino acid phenylselenocysteine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal aminoacyl-tRNA synthetases, polynucleotides encoding the novel sythetases molecules, methods for identifying and making the novel synthetases, methods for producing containing the unnatural amino acid phenylselenocysteine and translation systems. The invention further provides methods for producing modified proteins (e.g., lapidated proteins) through targeted modification of the phenylselenocysteine residue in a protein.

Cellular nucleic acid binding protein binds G-rich single-stranded nucleic acids and may function as a nucleic acid chaperone.

PubMed

Armas, Pablo; Nasif, Sofía; Calcaterra, Nora B

2008-02-15

Cellular nucleic acid binding protein (CNBP) is a small single-stranded nucleic acid binding protein made of seven Zn knuckles and an Arg-Gly rich box. CNBP is strikingly conserved among vertebrates and was reported to play broad-spectrum functions in eukaryotic cells biology. Neither its biological function nor its mechanisms of action were elucidated yet. The main goal of this work was to gain further insights into the CNBP biochemical and molecular features. We studied Bufo arenarum CNBP (bCNBP) binding to single-stranded nucleic acid probes representing the main reported CNBP putative targets. We report that, although bCNBP is able to bind RNA and single-stranded DNA (ssDNA) probes in vitro, it binds RNA as a preformed dimer whereas both monomer and dimer are able to bind to ssDNA. A systematic analysis of variant probes shows that the preferred bCNBP targets contain unpaired guanosine-rich stretches. These data expand the knowledge about CNBP binding stoichiometry and begins to dissect the main features of CNBP nucleic acid targets. Besides, we show that bCNBP presents a highly disordered predicted structure and promotes the annealing and melting of nucleic acids in vitro. These features are typical of proteins that function as nucleic acid chaperones. Based on these data, we propose that CNBP may function as a nucleic acid chaperone through binding, remodeling, and stabilizing nucleic acids secondary structures. This novel CNBP biochemical activity broadens the field of study about its biological function and may be the basis to understand the diverse ways in which CNBP controls gene expression. Copyright 2007 Wiley-Liss, Inc.

Influence of the Amino Acid Sequence on Protein-Mineral Interactions in Soil

NASA Astrophysics Data System (ADS)

Chacon, S. S.; Reardon, P. N.; Purvine, S.; Lipton, M. S.; Washton, N.; Kleber, M.

2017-12-01

The intimate associations between protein and mineral surfaces have profound impacts on nutrient cycling in soil. Proteins are an important source of organic C and N, and a subset of proteins, extracellular enzymes (EE), can catalyze the depolymerization of soil organic matter (SOM). Our goal was to determine how variation in the amino acid sequence could influence a protein's susceptibility to become chemically altered by mineral surfaces to infer the fate of adsorbed EE function in soil. We hypothesized that (1) addition of charged amino acids would enhance the adsorption onto oppositely charged mineral surfaces (2) addition of aromatic amino acids would increase adsorption onto zero charged surfaces (3) Increase adsorption of modified proteins would enhance their susceptibility to alterations by redox active minerals. To test these hypotheses, we generated three engineered proxies of a model protein Gb1 (IEP 4.0, 6.2 kDA) by inserting either negatively charged, positively charged or aromatic amino acids in the second loop. These modified proteins were allowed to interact with functionally different mineral surfaces (goethite, montmorillonite, kaolinite and birnessite) at pH 5 and 7. We used LC-MS/MS and solution-state Heteronuclear Single Quantum Coherence Spectroscopy NMR to observe modifications on engineered proteins as a consequence to mineral interactions. Preliminary results indicate that addition of any amino acids to a protein increase its susceptibility to fragmentation and oxidation by redox active mineral surfaces, and alter adsorption to the other mineral surfaces. This suggest that not all mineral surfaces in soil may act as sorbents for EEs and chemical modification of their structure should also be considered as an explanation for decrease in EE activity. Fragmentation of proteins by minerals can bypass the need to produce proteases, but microbial acquisition of other nutrients that require enzymes such as cellulases, ligninases or phosphatases

Solubilization of protein aggregates by the acid stress chaperones HdeA and HdeB.

PubMed

Malki, Abderrahim; Le, Hai-Tuong; Milles, Sigrid; Kern, Renée; Caldas, Teresa; Abdallah, Jad; Richarme, Gilbert

2008-05-16

The acid stress chaperones HdeA and HdeB of Escherichia coli prevent the aggregation of periplasmic proteins at acidic pH. We show in this report that they also form mixed aggregates with proteins that have failed to be solubilized at acidic pH and allow their subsequent solubilization at neutral pH. HdeA, HdeB, and HdeA and HdeB together display an increasing efficiency for the solubilization of protein aggregates at pH 3. They are less efficient for the solubilization of aggregates at pH 2, whereas HdeB is the most efficient. Increasing amounts of periplasmic proteins draw increasing amounts of chaperone into pellets, suggesting that chaperones co-aggregate with their substrate proteins. We observed a decrease in the size of protein aggregates in the presence of HdeA and HdeB, from very high molecular mass aggregates to 100-5000-kDa species. Moreover, a marked decrease in the exposed hydrophobicity of aggregated proteins in the presence of HdeA and HdeB was revealed by 1,1'-bis(4-anilino)naphtalene-5,5'-disulfonic acid binding experiments. In vivo, during the recovery at neutral pH of acid stressed bacterial cells, HdeA and HdeB allow the solubilization and renaturation of protein aggregates, including those formed by the maltose receptor MalE, the oligopeptide receptor OppA, and the histidine receptor HisJ. Thus, HdeA and HdeB not only help to maintain proteins in a soluble state during acid treatment, as previously reported, but also assist, both in vitro and in vivo, in the solubilization at neutral pH of mixed protein-chaperone aggregates formed at acidic pH, by decreasing the size of protein aggregates and the exposed hydrophobicity of aggregated proteins.

A sensitive method for measuring protein turnover based on the measurement of 2-3H-labelled amino acids in protein.

PubMed Central

Humphrey, T J; Davies, D D

1976-01-01

A method for measuring the rate of protein degradation is described. The method measures the change in 2-3H content of protein with time by racemization of the protein hydrolysate with acetic anhydride. The 3H on C-2 of amino acids is stable in proteins but becomes labile, owing to the action of transaminases, once the amino acids are released by proteolysis. The specific measurement of 2-3H in amino acids largely overcomes problems due to compartmentation and isotope recycling and evidence to support this claim is presented. Values for the half-life of Lemna minor (duckweed) protein determined by the new method are compared with values obtained by other methods. PMID:949338

pH shift protein recovery with organic acids on texture and color of cooked gels.

PubMed

Paker, Ilgin; Beamer, Sarah; Jaczynski, Jacek; Matak, Kristen E

2015-01-01

Isoelectric solubilization and precipitation (ISP) processing uses pH shifts to separate protein from fish frames, which may increase commercial interest for silver carp. Texture and color properties of gels made from silver carp protein recovered at different pH strategies and organic acid types were compared. ISP was applied to headed gutted silver carp using 10 mol L(-1) sodium hydroxide (NaOH) and either glacial acetic acid (AA) or a (1:1) formic and lactic acid combination (F&L). Protein gels were made with recovered protein and standard functional additives. Texture profile analysis and the Kramer shear test showed that protein gels made from protein solubilized at basic pH values were firmer, harder, more cohesive, gummier and chewier (P < 0.05) than proteins solubilized under acidic conditions. Acidic solubilization led to whiter (P < 0.05) gels, and using F&L during ISP yielded whiter gels under all treatments (P < 0.05). Gels made from ISP-recovered silver carp protein using organic acids show potential for use as a functional ingredient in restructured foods. © 2014 Society of Chemical Industry.

Epidermal fatty acid-binding protein protects nerve growth factor-differentiated PC12 cells from lipotoxic injury

PubMed Central

Liu, Jo-Wen; Montero, Manuel; Bu, Liming; De Leon, Marino

2015-01-01

Epidermal fatty acid-binding protein (E-FABP/FABP5/DA11) binds and transport long-chain fatty acids in the cytoplasm and may play a protecting role during neuronal injury. We examined whether E-FABP protects nerve growth factor-differentiated PC12 cells (NGFDPC12 cells) from lipotoxic injury observed after palmitic acid (C16:0; PAM) overload. NGFDPC12 cells cultures treated with PAM/bovine serum albumin at 0.3 mM/0.15 mM show PAM-induced lipotoxicity (PAM-LTx) and apoptosis. The apoptosis was preceded by a cellular accumulation of reactive oxygen species (ROS) and higher levels of E-FABP. Antioxidants MCI-186 and N-acetyl cysteine prevented E-FABP's induction in expression by PAM-LTx, while tert-butyl hydroperoxide increased ROS and E-FABP expression. Non-metabolized methyl ester of PAM, methyl palmitic acid (mPAM), failed to increase cellular ROS, E-FABP gene expression, or trigger apoptosis. Treatment of NGFDPC12 cultures with siE-FABP showed reduced E-FABP levels correlating with higher accumulation of ROS and cell death after exposure to PAM. In contrast, increasing E-FABP cellular levels by pre-loading the cells with recombinant E-FABP diminished the PAM-induced ROS and cell death. Finally, agonists for PPARβ (GW0742) or PPARγ (GW1929) increased E-FABP expression and enhanced the resistance of NGFDPC12 cells to PAM-LTx. We conclude that E-FABP protects NGFDPC12 cells from lipotoxic injury through mechanisms that involve reduction of ROS. Epidermal fatty acid-binding protein (E-FABP) may protect nerve cells from the damaging exposure to high levels of free fatty acids (FA). We show that E-FABP can neutralize the effects of reactive oxygen species (ROS) generated by the high levels of FA in the cell and protect PC12 cells from lipotoxic injuries common in Type 2 diabetes neuropathy. Potentially, E-FABP gene up-regulation may be mediated through the NFkB pathway and future studies are needed to further evaluate this proposition. PMID:25147052

Effects of Dietary Crude Protein Levels and Cysteamine Supplementation on Protein Synthetic and Degradative Signaling in Skeletal Muscle of Finishing Pigs.

PubMed

Zhou, Ping; Zhang, Lin; Li, Jiaolong; Luo, Yiqiu; Zhang, Bolin; Xing, Shen; Zhu, Yuping; Sun, Hui; Gao, Feng; Zhou, Guanghong

2015-01-01

Dietary protein levels and cysteamine (CS) supplementation can affect growth performance and protein metabolism of pigs. However, the influence of dietary protein intake on the growth response of CS-treated pigs is unclear, and the mechanisms involved in protein metabolism remain unknown. Hence, we investigated the interactions between dietary protein levels and CS supplementation and the effects of dietary crude protein levels and CS supplementation on protein synthetic and degradative signaling in skeletal muscle of finishing pigs. One hundred twenty barrows (65.84 ± 0.61 kg) were allocated to a 2 × 2 factorial arrangement with five replicates of six pigs each. The primary variations were dietary crude protein (CP) levels (14% or 10%) and CS supplemental levels (0 or 700 mg/kg). The low-protein (LP) diets (10% CP) were supplemented with enough essential amino acids (EAA) to meet the NRC AA requirements of pigs and maintain the balanced supply of eight EAA including lysine, methionine, threonine, tryptophan, valine, phenylalanine, isoleucine, and leucine. After 41 days, 10 pigs per treatment were slaughtered. We found that LP diets supplemented with EAA resulted in decreased concentrations of plasma somatostatin (SS) (P<0.01) and plasma urea nitrogen (PUN) (P<0.001), while dietary protein levels did not affect other traits. However, CS supplementation increased the average daily gain (P<0.001) and lean percentage (P<0.05), and decreased the feed conversion ratio (P<0.05) and back fat (P<0.05). CS supplementation also increased the concentrations of plasma insulin-like growth factor 1 (IGF-1) (P<0.001), and reduced the concentrations of leptin, SS, and PUN (P<0.001). Increased mRNA abundance of Akt1 and IGF-1 signaling (P<0.001) and decreased mRNA abundance of Forkhead Box O (FOXO) 4 (P<0.01) and muscle atrophy F-box (P<0.001) were observed in pigs receiving CS. Additionally, CS supplementation increased the protein levels for the phosphorylated mammalian target

Protein Quantification by Derivatization-Free High-Performance Liquid Chromatography of Aromatic Amino Acids

PubMed Central

Hesse, Almut

2016-01-01

Amino acid analysis is considered to be the gold standard for quantitative peptide and protein analysis. Here, we would like to propose a simple HPLC/UV method based on a reversed-phase separation of the aromatic amino acids tyrosine (Tyr), phenylalanine (Phe), and optionally tryptophan (Trp) without any derivatization. The hydrolysis of the proteins and peptides was performed by an accelerated microwave technique, which needs only 30 minutes. Two internal standard compounds, homotyrosine (HTyr) and 4-fluorophenylalanine (FPhe) were used for calibration. The limit of detection (LOD) was estimated to be 0.05 µM (~10 µg/L) for tyrosine and phenylalanine at 215 nm. The LOD for a protein determination was calculated to be below 16 mg/L (~300 ng BSA absolute). Aromatic amino acid analysis (AAAA) offers excellent accuracy and a precision of about 5% relative standard deviation, including the hydrolysis step. The method was validated with certified reference materials (CRM) of amino acids and of a pure protein (bovine serum albumin, BSA). AAAA can be used for the quantification of aromatic amino acids, isolated peptides or proteins, complex peptide or protein samples, such as serum or milk powder, and peptides or proteins immobilized on solid supports. PMID:27559481

Evaluation of the relationship between passive smoking and salivary electrolytes, protein, secretory IgA, sialic acid and amylase in young children.

PubMed

Avşar, Aysun; Darka, Ozge; Bodrumlu, Ebru Hazar; Bek, Yüksel

2009-05-01

To evaluate the relationship between passive smoking as determined by salivary cotinine levels and salivary electrolytes, protein, secretory IgA, sialic acid and amylase in children. Saliva was collected from 90 passive smoker (PS) subjects (the study group) and 90 healthy age-matched children (the control group). The study group was divided into three subgroups according the number of cigarettes smoked. Socio-economic status, dental and dietary habits were recorded by questionnaire. Stimulated salivary calcium (Ca), phosphate (P), sodium (Na), potassium (P), total protein, amylase activity, sialic acid level, secretory IgA concentration and cotinine level were analysed. All data were analysed using SPSS, version 13.0. Socio-economic status, dental and dietary habits were similar between the two groups. The salivary electrolytes concentrations did not reveal significant difference between the two groups (p>0.05). The mean cotinine levels of PS children were 1.58+/-4.3 ng/mL. The salivary concentrations of protein were similar between the two groups (p>0.05). The salivary secretory IgA concentration was significantly lower in the PS group than controls. The sialic acid level and amylase activity in PS group were found significantly higher compared with the controls (p<0.05). No difference was observed for all these parameters with sex (p>0.05). When saliva samples were analysed for output, the sialic acid level and amylase activity increased significantly in PS subjects (p<0.05). Further, the output of secretory IgA concentration was found significantly lower compared with the controls (p<0.05). In conclusion, we show that passive smoking was associated with a decrease in secretory IgA concentration, whereas with increase in amylase activity and sialic acid level of stimulated whole saliva in young children.

Proteome-Level Analysis of Metabolism- and Stress-Related Proteins during Seed Dormancy and Germination in Gnetum parvifolium.

PubMed

Chang, Ermei; Deng, Nan; Zhang, Jin; Liu, Jianfeng; Chen, Lanzhen; Zhao, Xiulian; Abbas, M; Jiang, Zeping; Shi, Shengqing

2018-03-21

Gnetum parvifolium is a rich source of materials for traditional medicines, food, and oil, but little is known about the mechanism underlying its seed dormancy and germination. In this study, we analyzed the proteome-level changes in its seeds during germination using isobaric tags for relative and absolute quantitation. In total, 1,040 differentially expressed proteins were identified, and cluster analysis revealed the distinct time points during which signal transduction and oxidation-reduction activity changed. Gene Ontology analysis showed that "carbohydrate metabolic process" and "response to oxidative stress" were the main enriched terms. Proteins associated with starch degradation and antioxidant enzymes were important for dormancy-release, while proteins associated with energy metabolism and protein synthesis were up-regulated during germination. Moreover, protein-interaction networks were mainly associated with heat-shock proteins. Furthermore, in accord with changes in the energy metabolism- and antioxidant-related proteins, indole-3-acetic acid, Peroxidase, and soluble sugar content increased, and the starch content decreased in almost all six stages of dormancy and germination analyzed (S1-S6). The activity of superoxide dismutase, abscisic acid, and malondialdehyde content increased in the dormancy stages (S1-S3) and then decreased in the germination stages (S4-S6). Our results provide new insights into G. parvifolium seed dormancy and germination at the proteome and physiological levels, with implications for improving seed propagation.

Modular Organization of Residue-Level Contacts Shapes the Selection Pressure on Individual Amino Acid Sites of Ribosomal Proteins.

PubMed

Mallik, Saurav; Kundu, Sudip

2017-04-01

Understanding the molecular evolution of macromolecular complexes in the light of their structure, assembly, and stability is of central importance. Here, we address how the modular organization of native molecular contacts shapes the selection pressure on individual residue sites of ribosomal complexes. The bacterial ribosomal complex is represented as a residue contact network where nodes represent amino acid/nucleotide residues and edges represent their van der Waals interactions. We find statistically overrepresented native amino acid-nucleotide contacts (OaantC, one amino acid contacts one or multiple nucleotides, internucleotide contacts are disregarded). Contact number is defined as the number of nucleotides contacted. Involvement of individual amino acids in OaantCs with smaller contact numbers is more random, whereas only a few amino acids significantly contribute to OaantCs with higher contact numbers. An investigation of structure, stability, and assembly of bacterial ribosome depicts the involvement of these OaantCs in diverse biophysical interactions stabilizing the complex, including high-affinity protein-RNA contacts, interprotein cooperativity, intersubunit bridge, packing of multiple ribosomal RNA domains, etc. Amino acid-nucleotide constituents of OaantCs with higher contact numbers are generally associated with significantly slower substitution rates compared with that of OaantCs with smaller contact numbers. This evolutionary rate heterogeneity emerges from the strong purifying selection pressure that conserves the respective amino acid physicochemical properties relevant to the stabilizing interaction with OaantC nucleotides. An analysis of relative molecular orientations of OaantC residues and their interaction energetics provides the biophysical ground of purifying selection conserving OaantC amino acid physicochemical properties. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and

Fatty acid-amino acid conjugates are essential for systemic activation of salicylic acid-induced protein kinase and accumulation of jasmonic acid in Nicotiana attenuata.

PubMed

Hettenhausen, Christian; Heinrich, Maria; Baldwin, Ian T; Wu, Jianqiang

2014-11-28

Herbivory induces the activation of mitogen-activated protein kinases (MAPKs), the accumulation of jasmonates and defensive metabolites in damaged leaves and in distal undamaged leaves. Previous studies mainly focused on individual responses and a limited number of systemic leaves, and more research is needed for a better understanding of how different plant parts respond to herbivory. In the wild tobacco Nicotiana attenuata, FACs (fatty acid-amino acid conjugates) in Manduca sexta oral secretions (OS) are the major elicitors that induce herbivory-specific signaling but their role in systemic signaling is largely unknown. Here, we show that simulated herbivory (adding M. sexta OS to fresh wounds) dramatically increased SIPK (salicylic acid-induced protein kinase) activity and jasmonic acid (JA) levels in damaged leaves and in certain (but not all) undamaged systemic leaves, whereas wounding alone had no detectable systemic effects; importantly, FACs and wounding are both required for activating these systemic responses. In contrast to the activation of SIPK and elevation of JA in specific systemic leaves, increases in the activity of an important anti-herbivore defense, trypsin proteinase inhibitor (TPI), were observed in all systemic leaves after simulated herbivory, suggesting that systemic TPI induction does not require SIPK activation and JA increases. Leaf ablation experiments demonstrated that within 10 minutes after simulated herbivory, a signal (or signals) was produced and transported out of the treated leaves, and subsequently activated systemic responses. Our results reveal that N. attenuata specifically recognizes herbivore-derived FACs in damaged leaves and rapidly send out a long-distance signal to phylotactically connected leaves to activate MAPK and JA signaling, and we propose that FACs that penetrated into wounds rapidly induce the production of another long-distance signal(s) which travels to all systemic leaves and activates TPI defense.

Pericardial fluid level of heart-type cytoplasmic fatty acid-binding protein (H-FABP) is an indicator of severe myocardial ischemia.

PubMed

Tambara, Keiichi; Fujita, Masatoshi; Miyamoto, Shoichi; Doi, Kazuhiko; Nishimura, Kazunobu; Komeda, Masashi

2004-02-01

Heart-type cytoplasmic fatty acid-binding protein (H-FABP) has been reported as a sensitive and specific marker for the early diagnosis of acute myocardial infarction. Our hypothesis was that serum or pericardial fluid levels of H-FABP can reflect not only myocardial infarction but also myocardial ischemia. A total of 34 patients with unstable angina, who had anginal symptoms and/or ST-changes in ECG monitoring within 24 h before operation, were classified into group A (n=17), and those without these symptoms and changes into group B (n=17). Blood and pericardial fluid samples were obtained immediately after median sternotomy, and serum and pericardial fluid levels of creatine kinase-MB, cardiac troponin-T, and H-FABP were measured. Serum H-FABP levels were slightly elevated compared with their normal values in both groups. While they showed no difference between groups A and B (group A vs. B: 8.5+/-1.0 vs. 7.1+/-0.7 ng/ml, P=0.25), pericardial fluid levels of H-FABP were significantly higher in group A than in group B (16.3+/-2.0 vs. 9.6+/-1.0 ng/ml, P=0.0046). H-FABP showed a weak correlation between its serum levels and pericardial fluid levels (r=0.40). Pericardial fluid levels of H-FABP reflect myocardial ischemia occurring within 24 h of their measurements. H-FABP may be secreted into the interstitial space by increased permeability of the myocardial cell membrane associated with severe myocardial ischemia. Thus, pericardial fluid reflects pathophysiological conditions of cardiomyocytes more sensitively than circulating blood.

[Analysis of proteins, amino acids and inorganic elements in Holotrichia diomphalia from different areas].

PubMed

Cao, Wei; Liu, Dan; Zhang, Yi-Kai; Wang, Xiao-Yu; Chang, Yan-Rong; Yang, Qian; Wang, Si-Wang

2010-10-01

To analyze the content of proteins,amino acids and inorganic elements of Holotrichia diomphalia in different growing areas as the references for quality evaluation and reasonable application of them. The contents of proteins were determined using semi-micro Kjeldahl method. The contents of seventeen amino acids and inorganic elements were determined with amino acid analyzer and atomic absorption spectrometer and elemental analyzer, respectively. The contents of protein were 33.4%-44.4%, and that in Jiangxi were the highest in five different areas. There were seventeen kinds of amino acids in Holotrichia diomphalia. Among them, seven amino acids were essential to human life. The content of glutamic acid was the highest in seventeen amino acids. In inorganic elements, the content of Mg, Ca was higher in macroelements and Fe, Zn was higher in microelements. There are many kinds of necessary amino acids and inorganic elements for man kind in Holotrichia diomphalia. The contents of proteins, amino acids and inorganic elements have some difference in Holotrichia diomphalia of different growing areas.

Comparative characterization of random-sequence proteins consisting of 5, 12, and 20 kinds of amino acids

PubMed Central

Tanaka, Junko; Doi, Nobuhide; Takashima, Hideaki; Yanagawa, Hiroshi

2010-01-01

Screening of functional proteins from a random-sequence library has been used to evolve novel proteins in the field of evolutionary protein engineering. However, random-sequence proteins consisting of the 20 natural amino acids tend to aggregate, and the occurrence rate of functional proteins in a random-sequence library is low. From the viewpoint of the origin of life, it has been proposed that primordial proteins consisted of a limited set of amino acids that could have been abundantly formed early during chemical evolution. We have previously found that members of a random-sequence protein library constructed with five primitive amino acids show high solubility (Doi et al., Protein Eng Des Sel 2005;18:279–284). Although such a library is expected to be appropriate for finding functional proteins, the functionality may be limited, because they have no positively charged amino acid. Here, we constructed three libraries of 120-amino acid, random-sequence proteins using alphabets of 5, 12, and 20 amino acids by preselection using mRNA display (to eliminate sequences containing stop codons and frameshifts) and characterized and compared the structural properties of random-sequence proteins arbitrarily chosen from these libraries. We found that random-sequence proteins constructed with the 12-member alphabet (including five primitive amino acids and positively charged amino acids) have higher solubility than those constructed with the 20-member alphabet, though other biophysical properties are very similar in the two libraries. Thus, a library of moderate complexity constructed from 12 amino acids may be a more appropriate resource for functional screening than one constructed from 20 amino acids. PMID:20162614

Isolation and characterization of a novel acidic matrix protein hic22 from the nacreous layer of the freshwater mussel, Hyriopsis cumingii.

PubMed

Liu, X J; Jin, C; Wu, L M; Dong, S J; Zeng, S M; Li, J L

2016-07-29

Matrix proteins that either weakly acidic or unusually highly acidic have important roles in shell biomineralization. In this study, we have identified and characterized hic22, a weakly acidic matrix protein, from the nacreous layer of Hyriopsis cumingii. Total protein was extracted from the nacre using 5 M EDTA and hic22 was purified using a DEAE-sepharose column. The N-terminal amino acid sequence of hic22 was determined and the complete cDNA encoding hic22 was cloned and sequenced by rapid amplification of cDNA ends-polymerase chain reaction. Finally, the localization and distribution of hic22 was determined by in situ hybridization. Our results revealed that hic22 encodes a 22-kDa protein composed of 185 amino acids. Tissue expression analysis and in situ hybridization indicated that hic22 is expressed in the dorsal epithelial cells of the mantle pallial; moreover, significant expression levels of hic22 were observed after the early formation of the pearl sac (days 19-77), implying that hic22 may play an important role in biomineralization of the nacreous layer.

Solution Structure and Backbone Dynamics of Human Liver Fatty Acid Binding Protein: Fatty Acid Binding Revisited

PubMed Central

Cai, Jun; Lücke, Christian; Chen, Zhongjing; Qiao, Ye; Klimtchuk, Elena; Hamilton, James A.

2012-01-01

Liver fatty acid binding protein (L-FABP), a cytosolic protein most abundant in liver, is associated with intracellular transport of fatty acids, nuclear signaling, and regulation of intracellular lipolysis. Among the members of the intracellular lipid binding protein family, L-FABP is of particular interest as it can i), bind two fatty acid molecules simultaneously and ii), accommodate a variety of bulkier physiological ligands such as bilirubin and fatty acyl CoA. To better understand the promiscuous binding and transport properties of L-FABP, we investigated structure and dynamics of human L-FABP with and without bound ligands by means of heteronuclear NMR. The overall conformation of human L-FABP shows the typical β-clam motif. Binding of two oleic acid (OA) molecules does not alter the protein conformation substantially, but perturbs the chemical shift of certain backbone and side-chain protons that are involved in OA binding according to the structure of the human L-FABP/OA complex. Comparison of the human apo and holo L-FABP structures revealed no evidence for an “open-cap” conformation or a “swivel-back” mechanism of the K90 side chain upon ligand binding, as proposed for rat L-FABP. Instead, we postulate that the lipid binding process in L-FABP is associated with backbone dynamics. PMID:22713574

A mutation in the Arabidopsis HYL1 gene encoding a dsRNA binding protein affects responses to abscisic acid, auxin, and cytokinin

NASA Technical Reports Server (NTRS)

Lu, C.; Fedoroff, N.

2000-01-01

Both physiological and genetic evidence indicate interconnections among plant responses to different hormones. We describe a pleiotropic recessive Arabidopsis transposon insertion mutation, designated hyponastic leaves (hyl1), that alters the plant's responses to several hormones. The mutant is characterized by shorter stature, delayed flowering, leaf hyponasty, reduced fertility, decreased rate of root growth, and an altered root gravitropic response. It also exhibits less sensitivity to auxin and cytokinin and hypersensitivity to abscisic acid (ABA). The auxin transport inhibitor 2,3,5-triiodobenzoic acid normalizes the mutant phenotype somewhat, whereas another auxin transport inhibitor, N-(1-naph-thyl)phthalamic acid, exacerbates the phenotype. The gene, designated HYL1, encodes a 419-amino acid protein that contains two double-stranded RNA (dsRNA) binding motifs, a nuclear localization motif, and a C-terminal repeat structure suggestive of a protein-protein interaction domain. We present evidence that the HYL1 gene is ABA-regulated and encodes a nuclear dsRNA binding protein. We hypothesize that the HYL1 protein is a regulatory protein functioning at the transcriptional or post-transcriptional level.

Probing Protein Structure by Amino Acid-Specific Covalent Labeling and Mass Spectrometry

PubMed Central

Mendoza, Vanessa Leah; Vachet, Richard W.

2009-01-01

For many years, amino acid-specific covalent labeling has been a valuable tool to study protein structure and protein interactions, especially for systems that are difficult to study by other means. These covalent labeling methods typically map protein structure and interactions by measuring the differential reactivity of amino acid side chains. The reactivity of amino acids in proteins generally depends on the accessibility of the side chain to the reagent, the inherent reactivity of the label and the reactivity of the amino acid side chain. Peptide mass mapping with ESI- or MALDI-MS and peptide sequencing with tandem MS are typically employed to identify modification sites to provide site-specific structural information. In this review, we describe the reagents that are most commonly used in these residue-specific modification reactions, details about the proper use of these covalent labeling reagents, and information about the specific biochemical problems that have been addressed with covalent labeling strategies. PMID:19016300

Values for digestible indispensable amino acid scores (DIAAS) for some dairy and plant proteins may better describe protein quality than values calculated using the concept for protein digestibility-corrected amino acid scores (PDCAAS).

PubMed

Mathai, John K; Liu, Yanhong; Stein, Hans H

2017-02-01

An experiment was conducted to compare values for digestible indispensable amino acid scores (DIAAS) for four animal proteins and four plant proteins with values calculated as recommended for protein digestibility-corrected amino acid scores (PDCAAS), but determined in pigs instead of in rats. Values for standardised total tract digestibility (STTD) of crude protein (CP) and standardised ileal digestibility (SID) of amino acids (AA) were calculated for whey protein isolate (WPI), whey protein concentrate (WPC), milk protein concentrate (MPC), skimmed milk powder (SMP), pea protein concentrate (PPC), soya protein isolate (SPI), soya flour and whole-grain wheat. The PDCAAS-like values were calculated using the STTD of CP to estimate AA digestibility and values for DIAAS were calculated from values for SID of AA. Results indicated that values for SID of most indispensable AA in WPI, WPC and MPC were greater (P<0·05) than for SMP, PPC, SPI, soya flour and wheat. With the exception of arginine and tryptophan, the SID of all indispensable AA in SPI was greater (P<0·05) than in soya flour, and with the exception of threonine, the SID of all indispensable AA in wheat was less (P<0·05) than in all other ingredients. If the same scoring pattern for children between 6 and 36 months was used to calculate PDCAAS-like values and DIAAS, PDCAAS-like values were greater (P<0·05) than DIAAS values for SMP, PPC, SPI, soya flour and wheat indicating that PDCAAS-like values estimated in pigs may overestimate the quality of these proteins.

Association of Serum Uric Acid Levels with Leg Ischemia in Patients with Peripheral Arterial Disease after Treatment.

PubMed

Sotoda, Yoko; Hirooka, Shigeki; Orita, Hiroyuki; Wakabayashi, Ichiro

2017-07-01

We investigated the relationships of serum uric acid levels with the progression of atherosclerosis in patients with peripheral arterial disease (PAD) after treatment. Subjects were male patients diagnosed with PAD. Atherosclerosis at the common carotid artery was evaluated based on its intima-media thickness (IMT). Leg arterial flow was evaluated by measuring ankle-brachial index (ABI) and exercise-induced decrease in ABI. Among various risk factors including age, blood pressure, adiposity, estimated glomerular filtration rate, and blood lipid, blood glucose, uric acid, fibrinogen and C-reactive protein levels, only uric acid levels showed significant correlations with ABI [Pearson's correlation coefficient, -0.292 (p＜0.01)] and leg exercise-induced decrease in ABI [Pearson's correlation coefficient, 0.236 (p＜ 0.05)]. However, there was no significant correlation between uric acid levels and maximum or mean IMT. Odds ratios of subjects with the 3rd tertile versus subjects with the 1st tertile for uric acid levels were significantly higher than the reference level of 1.00 for low ABI [4.44 (95% confidence interval, 1.45-13.65, p＜0.01)] and for high % decrease in ABI after exercise [4.31 (95% confidence interval, 1.34-13.82, p＜0.05)]. The associations of uric acid levels with the indicators of leg ischemia were also found after adjustment for age, history of revascularization therapy, diabetes, smoking, alcohol consumption, body mass index, triglyceride levels, and renal function. Uric acid levels are associated with the degree of leg ischemia in patients with PAD. Further interventional studies are needed to determine whether the correction of uric acid levels is effective in preventing the progression of PAD.

A Complex Prime Numerical Representation of Amino Acids for Protein Function Comparison.

PubMed

Chen, Duo; Wang, Jiasong; Yan, Ming; Bao, Forrest Sheng

2016-08-01

Computationally assessing the functional similarity between proteins is an important task of bioinformatics research. It can help molecular biologists transfer knowledge on certain proteins to others and hence reduce the amount of tedious and costly benchwork. Representation of amino acids, the building blocks of proteins, plays an important role in achieving this goal. Compared with symbolic representation, representing amino acids numerically can expand our ability to analyze proteins, including comparing the functional similarity of them. Among the state-of-the-art methods, electro-ion interaction pseudopotential (EIIP) is widely adopted for the numerical representation of amino acids. However, it could suffer from degeneracy that two different amino acid sequences have the same numerical representation, due to the design of EIIP. In light of this challenge, we propose a complex prime numerical representation (CPNR) of amino acids, inspired by the similarity between a pattern among prime numbers and the number of codons of amino acids. To empirically assess the effectiveness of the proposed method, we compare CPNR against EIIP. Experimental results demonstrate that the proposed method CPNR always achieves better performance than EIIP. We also develop a framework to combine the advantages of CPNR and EIIP, which enables us to improve the performance and study the unique characteristics of different representations.

The bisphosphonate zoledronic acid effectively targets lung cancer cells by inhibition of protein prenylation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xie, Fan; Li, Pengcheng; Gong, Jianhua

Aberrant activation of oncoproteins such as members of the Ras family is common in human lung cancers. The proper function of Ras largely depends on a post-translational modification termed prenylation. Bisphosphonates have been shown to inhibit prenylation in cancer cells. In this study, we show that zoledronic acid, a third generation bisphosphonate, is effective in targeting lung cancer cells. This is achieved by the induction of apoptosis and inhibition of proliferation, through suppressing the activation of downstream Ras and EGFR signalling by zoledronic acid. The combination of zoledronic acid and paclitaxel or cisplatin (commonly used chemotherapeutic drugs for lung cancer)more » augmented the activity of either drug alone in in vitro lung cancer cellular system and in vivo lung xenograft mouse model. Importantly, zoledronic acid inhibits protein prenylation as shown by the increased levels of unprenylated Ras and Rap1A. In addition, the effects of zoledronic acid were reversed in the presence of geranylgeraniol and farnesol, further confirming that mechanism of zoledroinc acid's action in lung cancer cells is through prenylation inhibition. Since zoledronic acid is already available for clinic use, these results suggest that it may be an effective addition to the armamentarium of drugs for the treatment of lung cancer. - Highlights: • Zoledronic acid (ZA) is effectively against lung cancer cells in vitro and in vivo. • ZA acts on lung cancer cells through inhibition of protein prenylation. • ZA suppresses global downstream phosphorylation of Ras signalling. • ZA enhances the effects of chemotherapeutic drugs in lung cancer cells.« less

Effect of short-term low-protein diet supplemented with keto acids on hyperphosphatemia in maintenance hemodialysis patients.

PubMed

Li, Haiming; Long, Quan; Shao, Chunhai; Fan, Hong; Yuan, Li; Huang, Bihong; Gu, Yong; Lin, Shanyan; Hao, Chuanming; Chen, Jing

2011-01-01

To evaluate the effects of short-term restriction of dietary protein intake (DPI) supplemented with keto acids on hyperphosphatemia in maintenance hemodialysis (MHD) patients. Forty MHD patients with uncontrolled hyperphosphatemia were randomized to either low DPI with keto acid-supplemented (sLP) or normal DPI (NP) group for 8 weeks. After 8 weeks, the sLP group was shifted to NP for another 8 weeks. Low-protein diet (LPD) was individualized with total caloric intake 30-35 kcal/kg/day, protein intake of 0.8 g/kg/day and phosphate intake of 500 mg/day. Keto acids were supplied in a dosage of 12 pills per day. Calcium phosphorous metabolism index and nutritional index (serum albumin, total protein, somatometric measurements, 3-day diaries and Mini-Nutritional Assessment score) were recorded. C-reactive protein, CO(2) combining power and Kt/V were measured to evaluate the inflammation, metabolic acidosis and dialysis adequacy, respectively. Serum phosphorus level and calcium-phosphate product were significantly decreased at the end of the first 8 weeks in the sLP group compared to the basal value and the NP group (p < 0.001). No difference was observed in C-reactive protein, Kt/V and nutritional index, while CO(2) combining power was significantly higher at week 8 in the sLP group (p < 0.001). Short-term restriction of DPI supplemented with keto acids could decrease hyperphosphatemia and calcium-phosphate product, while keeping stable nutritional status among MHD patients. Copyright © 2010 S. Karger AG, Basel.

Taurine decreased uric acid levels in hyperuricemic rats and alleviated kidney injury.

PubMed

Feng, Ying; Sun, Fang; Gao, Yongchao; Yang, Jiancheng; Wu, Gaofeng; Lin, Shumei; Hu, Jianmin

2017-07-29

Hyperuricemia can lead to direct kidney damage. Taurine participates in several renal physiological processes and has been shown as a renoprotective agent. It has been reported that taurine could reduce uric acid levels in diabetic rats, but to date there was no research on the effects of taurine on hyperuricemic rats with kidney injury. In present study, hyperuricemic rat models were induced by intragastric administration of adenine and ethambutol hydrochloride for 10 days, and taurine (1% or 2%) were added in the drinking water 7 days in advance for consecutively 17 days. The results showed that taurine alleviated renal morphological and pathological changes as well as kidney dysfunction in hyperuricemic rats. Taurine could efficiently decrease the elevated xanthine oxidase activities in hyperuricemic rats, indicating its effect on the regulation of uric acid formation. The reabsorption and secretion of uric acid are dependent on a number of urate transporters. Expressions of three urate transporters were significantly down-regulated in hyperuricemic rats, while taurine prevented the decrease of mRNA and protein expression levels of these urate transporters. The results indicate that taurine might play a role in the regulation of renal uric acid excretion. Therefore, taurine could be a promising agent for the treatment of hyperuricemia. Copyright © 2017 Elsevier Inc. All rights reserved.

Rapid protein disappearance rates along the small intestine advantage poultry performance and influence the post-enteral availability of amino acids.

PubMed

Truong, Ha H; Chrystal, Peter V; Moss, Amy F; Selle, Peter H; Liu, Sonia Yun

2017-12-01

A foundation diet, an intermediate blend and a summit diet were formulated with different levels of soyabean meal, casein and crystalline amino acids to compare 'slow' and 'rapid' protein diets. The diets were offered to male Ross 308 chicks from 7 to 28 d post-hatch and assessed parameters included growth performance, nutrient utilisation, apparent digestibility coefficients and disappearance rates of starch and protein (N) in four small intestinal segments. Digestibility coefficients and disappearance rates of sixteen amino acids in three small intestinal segments and amino acid concentrations in plasma from portal and systemic circulations from the foundation and summit diets were determined. The dietary transition significantly accelerated protein (N) disappearance rates in the distal jejunum and ileum. The transition from foundation to summit diets significantly increased starch digestibility coefficients in the ileum and disappearance rates in all four small intestinal segments. These starch responses were associated with significant enhancements in nutrient utilisation. The dietary transition linearly increased digestibility coefficients and disappearance rates of amino acids in the majority of cases. The summit diet increased plasma concentrations of five amino acids but decreased those of four amino acids relative to the foundation diet to significant extents. Plasma concentrations of free amino acids were higher in the portal than systemic circulations. Rapid protein disappearance rates advantaged poultry performance and influenced post-enteral availability of amino acids. If the underlying mechanisms are to be identified, further research into the impact of protein digestive dynamics on broiler performance is required but appears justified.

Elevated tropospheric ozone increased grain protein and amino acid content of a hybrid rice without manipulation by planting density.

PubMed

Zhou, Xiaodong; Zhou, Juan; Wang, Yunxia; Peng, Bin; Zhu, Jianguo; Yang, Lianxin; Wang, Yulong

2015-01-01

Rising tropospheric ozone affects crop yield and quality. Rice protein concentration, which is closely associated with eating/cooking quality, is of critical importance to nutritional quality. The ozone effect on amino acids of rice grains was little known, especially grown under different cultivation conditions. A hybrid rice cultivar Shanyou 63 was grown in 2010 and 2011 to investigate the interactive effect of ozone exposure and planting density on rice protein quality in a free-air ozone enrichment system. The content of protein, total amino acids (TAA), total essential (TEAA) and non-essential amino acids (TNEAA) in rice grain was increased by 12-14% with elevated ozone. A similar significant response to ozone was observed for concentrations of the seven essential and eight non-essential amino acids. In contrast, elevated ozone caused a small but significant decrease in percentage of TEAA to TAA. The year effect was significant for all measured traits; however, interactions of ozone with year or planting density were not detected. The study suggested that season-long elevation of ozone concentration to projected 2050 levels will increase protein and amino acids of Shanyou 63, and crop management such as changing planting density might not alter the impact. © 2014 Society of Chemical Industry.

Gross and true ileal digestible amino acid contents of several animal body proteins and their hydrolysates.

PubMed

Cui, J; Chong, B; Rutherfurd, S M; Wilkinson, B; Singh, H; Moughan, P J

2013-07-01

Amino acid compositions of ovine muscle, ovine myofibrillar protein, ovine spleen, ovine liver, bovine blood plasma, bovine blood globulins and bovine serum albumin and the amino acid compositions and in vivo (laboratory rat) true ileal amino acid digestibilities of hydrolysates (sequential hydrolysis with Neutrase, Alcalase and Flavourzyme) of these protein sources were determined. True ileal amino acid digestibility differed (P<0.05) among the seven protein hydrolysates. The ovine myofibrillar protein and liver hydrolysates were the most digestible, with a mean true ileal digestibility across all amino acids of 99%. The least digestible protein hydrolysate was bovine serum albumin with a comparable mean true ileal digestibility of 93%. When the digestible amino acid contents were expressed as proportions relative to lysine, considerable differences, across the diverse protein sources, were found in the pattern of predicted absorbed amino acids. Copyright © 2013 Elsevier Ltd. All rights reserved.

Overcoming the Refractory Expression of Secreted Recombinant Proteins in Mammalian Cells through Modification of the Signal Peptide and Adjacent Amino Acids.

PubMed

Güler-Gane, Gülin; Kidd, Sara; Sridharan, Sudharsan; Vaughan, Tristan J; Wilkinson, Trevor C I; Tigue, Natalie J

2016-01-01

The expression and subsequent purification of mammalian recombinant proteins is of critical importance to many areas of biological science. To maintain the appropriate tertiary structure and post-translational modifications of such proteins, transient mammalian expression systems are often adopted. The successful utilisation of these systems is, however, not always forthcoming and some recombinant proteins prove refractory to expression in mammalian hosts. In this study we focussed on the role of different N-terminal signal peptides and residues immediately downstream, in influencing the level of secreted recombinant protein obtained from suspension HEK293 cells. Using secreted alkaline phosphatase (SEAP) as a model protein, we identified that the +1/+2 downstream residues flanking a heterologous signal peptide significantly affect secreted levels. By incorporating these findings we conducted a comparison of different signal peptide sequences and identified the most productive as secrecon, a computationally-designed sequence. Importantly, in the context of the secrecon signal peptide and SEAP, we also demonstrated a clear preference for specific amino acid residues at the +1 position (e.g. alanine), and a detrimental effect of others (cysteine, proline, tyrosine and glutamine). When proteins that naturally contain these "undesirable" residues at the +1 position were expressed with their native signal peptide, the heterologous secrecon signal peptide, or secrecon with an additional alanine at the +1 or +1 and +2 position, the level of expression differed significantly and in an unpredictable manner. For each protein, however, at least one of the panel of signal peptide/adjacent amino acid combinations enabled successful recombinant expression. In this study, we highlight the important interplay between a signal peptide and its adjacent amino acids in enabling protein expression, and we describe a strategy that could enable recombinant proteins that have so far

Alterations in protein and amino acid metabolism in rats fed a branched-chain amino acid- or leucine-enriched diet during postprandial and postabsorptive states.

PubMed

Holecek, Milan; Siman, Pavel; Vodenicarovova, Melita; Kandar, Roman

2016-01-01

Many people believe in favourable effects of branched-chain amino acids (BCAAs; valine, leucine, and isoleucine), especially leucine, on muscle protein balance and consume BCAAs for many years. We determined the effects of the chronic intake of a BCAA- or leucine-enriched diet on protein and amino acid metabolism in fed and postabsorptive states. Rats were fed a standard diet, a diet with a high content of valine, leucine, and isoleucine (HVLID), or a high content of leucine (HLD) for 2 months. Half of the animals in each group were sacrificed in the fed state on the last day, and the other half were sacrificed after overnight fast. Protein synthesis was assessed using the flooding dose method (L-[3,4,5-(3)H]phenylalanine), proteolysis on the basis of chymotrypsin-like activity (CHTLA) of proteasome and cathepsin B and L activities. Chronic intake of HVLID or HLD enhanced plasma levels of urea, alanine and glutamine. HVLID also increased levels of all three BCAA and branched-chain keto acids (BCKA), HLD increased leucine, ketoisocaproate and alanine aminotransferase and decreased valine, ketovaline, isoleucine, ketoisoleucine, and LDL cholesterol. Tissue weight and protein content were lower in extensor digitorum longus muscles in the HLD group and higher in kidneys in the HVLID and HLD groups. Muscle protein synthesis in postprandial state was higher in the HVLID group, and CHTLA was lower in muscles of the HVLID and HLD groups compared to controls. Overnight starvation enhanced alanine aminotransferase activity in muscles, and decreased protein synthesis in gastrocnemius (in HVLID group) and extensor digitorum longus (in HLD group) muscles more than in controls. Effect of HVLID and HLD on CHTLA in muscles in postabsorptive state was insignificant. The results failed to demonstrate positive effects of the chronic consumption of a BCAA-enriched diet on protein balance in skeletal muscle and indicate rather negative effects from a leucine-enriched diet. The primary

Isolation of complementary DNA clones encoding pathogenesis-related proteins P and Q, two acidic chitinases from tobacco.

PubMed Central

Payne, G; Ahl, P; Moyer, M; Harper, A; Beck, J; Meins, F; Ryals, J

1990-01-01

Complementary DNA clones encoding two isoforms of the acidic endochitinase (chitinase, EC 3.2.1.14) from tobacco were isolated. Comparison of amino acid sequences deduced from the cDNA clones and the sequence of peptides derived from purified proteins show that these clones encode the pathogenesis-related proteins PR-P and PR-Q. The cDNA inserts were not homologous to either the bacterial form of chitinase or the form from cucumber but shared significant homology to the basic form of chitinase from tobacco and bean. The acidic isoforms of tobacco chitinase did not contain the amino-terminal, cysteine-rich "hevein" domain found in the basic isoforms, indicating that this domain, which binds chitin, is not essential for chitinolytic activity. The accumulation of mRNA for the pathogenesis-related proteins PR-1, PR-R, PR-P, and PR-Q in Xanthi.nc tobacco leaves following infection with tobacco mosaic virus was measured by primer extension. The results indicate that the induction of these proteins during the local necrotic lesion response to the virus is coordinated at the mRNA level. Images PMID:2296608

Enamel proteins mitigate mechanical and structural degradations in mature human enamel during acid attack

NASA Astrophysics Data System (ADS)

Lubarsky, Gennady V.; Lemoine, Patrick; Meenan, Brian J.; Deb, Sanjukta; Mutreja, Isha; Carolan, Patrick; Petkov, Nikolay

2014-04-01

A hydrazine deproteination process was used to investigate the role of enamel proteins in the acid erosion of mature human dental enamel. Bright field high resolution transmission electron micrographs and x-ray diffraction analysis show no crystallographic changes after the hydrazine treatment with similar nanoscale hydroxyapatite crystallite size and orientation for sound and de-proteinated enamel. However, the presence of enamel proteins reduces the erosion depth, the loss of hardness and the loss of structural order in enamel, following exposure to citric acid. Nanoindentation creep is larger for sound enamel than for deproteinated enamel but it reduces in sound enamel after acid attack. These novel results are consistent with calcium ion-mediated visco-elasticty in enamel matrix proteins as described previously for nacre, bone and dental proteins. They are also in good agreement with a previous double layer force spectroscopy study by the authors which found that the proteins electrochemically buffer enamel against acid attack. Finally, this suggests that acid attack, and more specifically dental erosion, is influenced by ionic permeation through the enamel layer and that it is mitigated by the enamel protein matrix.

Decreased serum protein binding of diazepam and its major metabolite in the neonate during the first postnatal week relate to increased free fatty acid levels.

PubMed Central

Nau, H; Luck, W; Kuhnz, W

1984-01-01

The protein binding of diazepam (D) and its major active metabolite N-desmethyl diazepam (DD) was investigated in vitro in the serum of 14 mothers at birth, 21 foetuses at birth, in 100 neonates between 1 and 11 days of age and in 16 control subjects. The free (unbound) fractions of D and DD in the foetus were similar to those in the controls, but lower than those in the mothers. During the first day of life the free fractions of D and DD doubled in the neonates and subsequently declined slowly to reach near control levels at 1 week of age. The sharp increase and slow decrease of the free fractions of D and DD during the first postnatal week was closely paralleled by sharply increasing and decreasing free fatty acid (FFA) concentrations. Bilirubin and albumin levels were of lower importance in regard to the protein binding of D and DD. These results indicate that the greatly increased FFA levels shortly after birth result in increased free fractions of D and DD. Because of the known immaturity of the neonatal hepatic elimination capacity, these elevated free fractions may result in elevated free concentrations of the two compounds, which may help to explain the adverse effects observed clinically in some D-exposed neonates. PMID:6419763

Factor VII and protein C are phosphatidic acid-binding proteins.

PubMed

Tavoosi, Narjes; Smith, Stephanie A; Davis-Harrison, Rebecca L; Morrissey, James H

2013-08-20

Seven proteins in the human blood clotting cascade bind, via their GLA (γ-carboxyglutamate-rich) domains, to membranes containing exposed phosphatidylserine (PS), although with membrane binding affinities that vary by 3 orders of magnitude. Here we employed nanodiscs of defined phospholipid composition to quantify the phospholipid binding specificities of these seven clotting proteins. All bound preferentially to nanobilayers in which PS headgroups contained l-serine versus d-serine. Surprisingly, however, nanobilayers containing phosphatidic acid (PA) bound substantially more of two of these proteins, factor VIIa and activated protein C, than did equivalent bilayers containing PS. Consistent with this finding, liposomes containing PA supported higher proteolytic activity by factor VIIa and activated protein C toward their natural substrates (factors X and Va, respectively) than did PS-containing liposomes. Moreover, treating activated human platelets with phospholipase D enhanced the rates of factor X activation by factor VIIa in the presence of soluble tissue factor. We hypothesize that factor VII and protein C bind preferentially to the monoester phosphate of PA because of its accessibility and higher negative charge compared with the diester phosphates of most other phospholipids. We further found that phosphatidylinositol 4-phosphate, which contains a monoester phosphate attached to its myo-inositol headgroup, also supported enhanced enzymatic activity of factor VIIa and activated protein C. We conclude that factor VII and protein C bind preferentially to monoester phosphates, which may have implications for the function of these proteases in vivo.

Protein and protein hydrolysates in sports nutrition.

PubMed

van Loon, Luc J C; Kies, Arie K; Saris, Wim H M

2007-08-01

With the increasing knowledge about the role of nutrition in increasing exercise performance, it has become clear over the last 2 decades that amino acids, protein, and protein hydrolysates can play an important role. Most of the attention has been focused on their effects at a muscular level. As these nutrients are ingested, however, it also means that gastrointestinal digestibility and absorption can modulate their efficacy significantly. Therefore, discussing the role of amino acids, protein, and protein hydrolysates in sports nutrition entails holding a discussion on all levels of the metabolic route. On May 28-29, 2007, a small group of researchers active in the field of exercise science and protein metabolism presented an overview of the different aspects of the application of protein and protein hydrolysates in sports nutrition. In addition, they were asked to share their opinions on the future progress in their fields of research. In this overview, an introduction to the workshop and a short summary of its outcome is provided.

Multi-level machine learning prediction of protein-protein interactions in Saccharomyces cerevisiae.

PubMed

Zubek, Julian; Tatjewski, Marcin; Boniecki, Adam; Mnich, Maciej; Basu, Subhadip; Plewczynski, Dariusz

2015-01-01

Accurate identification of protein-protein interactions (PPI) is the key step in understanding proteins' biological functions, which are typically context-dependent. Many existing PPI predictors rely on aggregated features from protein sequences, however only a few methods exploit local information about specific residue contacts. In this work we present a two-stage machine learning approach for prediction of protein-protein interactions. We start with the carefully filtered data on protein complexes available for Saccharomyces cerevisiae in the Protein Data Bank (PDB) database. First, we build linear descriptions of interacting and non-interacting sequence segment pairs based on their inter-residue distances. Secondly, we train machine learning classifiers to predict binary segment interactions for any two short sequence fragments. The final prediction of the protein-protein interaction is done using the 2D matrix representation of all-against-all possible interacting sequence segments of both analysed proteins. The level-I predictor achieves 0.88 AUC for micro-scale, i.e., residue-level prediction. The level-II predictor improves the results further by a more complex learning paradigm. We perform 30-fold macro-scale, i.e., protein-level cross-validation experiment. The level-II predictor using PSIPRED-predicted secondary structure reaches 0.70 precision, 0.68 recall, and 0.70 AUC, whereas other popular methods provide results below 0.6 threshold (recall, precision, AUC). Our results demonstrate that multi-scale sequence features aggregation procedure is able to improve the machine learning results by more than 10% as compared to other sequence representations. Prepared datasets and source code for our experimental pipeline are freely available for download from: http://zubekj.github.io/mlppi/ (open source Python implementation, OS independent).

Inferring Selection on Amino Acid Preference in Protein Domains

PubMed Central

Durbin, Richard

2009-01-01

Models that explicitly account for the effect of selection on new mutations have been proposed to account for “codon bias” or the excess of “preferred” codons that results from selection for translational efficiency and/or accuracy. In principle, such models can be applied to any mutation that results in a preferred allele, but in most cases, the fitness effect of a specific mutation cannot be predicted. Here we show that it is possible to assign preferred and unpreferred states to amino acid changing mutations that occur in protein domains. We propose that mutations that lead to more common amino acids (at a given position in a domain) can be considered “preferred alleles” just as are synonymous mutations leading to codons for more abundant tRNAs. We use genome-scale polymorphism data to show that alleles for preferred amino acids in protein domains occur at higher frequencies in the population, as has been shown for preferred codons. We show that this effect is quantitative, such that there is a correlation between the shift in frequency of preferred alleles and the predicted fitness effect. As expected, we also observe a reduction in the numbers of polymorphisms and substitutions at more important positions in domains, consistent with stronger selection at those positions. We examine the derived allele frequency distribution and polymorphism to divergence ratios of preferred and unpreferred differences and find evidence for both negative and positive selections acting to maintain protein domains in the human population. Finally, we analyze a model for selection on amino acid preferences in protein domains and find that it is consistent with the quantitative effects that we observe. PMID:19095755

DNA-dependent homodimerization, sub-cellular partitioning, and protein destabilization control WUSCHEL levels and spatial patterning

PubMed Central

Rodriguez, Kevin; Perales, Mariano; Snipes, Stephen; Yadav, Ram Kishor; Diaz-Mendoza, Mercedes; Reddy, G. Venugopala

2016-01-01

The homeodomain transcription factor WUSCHEL (WUS) promotes stem cell maintenance in inflorescence meristems of Arabidopsis thaliana. WUS, which is synthesized in the rib meristem, migrates and accumulates at lower levels in adjacent cells. Maintenance of WUS protein levels and spatial patterning distribution is not well-understood. Here, we show that the last 63-aa stretch of WUS is necessary for maintaining different levels of WUS protein in the rib meristem and adjacent cells. The 63-aa region contains the following transcriptional regulatory domains: the acidic region, the WUS-box, which is conserved in WUS-related HOMEOBOX family members, and the ethylene-responsive element binding factor-associated amphiphilic repression (EAR-like) domain. Our analysis reveals that the opposing functions of WUS-box, which is required for nuclear retention, and EAR-like domain, which participates in nuclear export, are necessary to maintain higher nuclear levels of WUS in cells of the rib meristem and lower nuclear levels in adjacent cells. We also show that the N-terminal DNA binding domain, which is required for both DNA binding and homodimerization, along with the homodimerization sequence located in the central part of the protein, restricts WUS from spreading excessively and show that the homodimerization is critical for WUS function. Our analysis also reveals that a higher level of WUS outside the rib meristem leads to protein destabilization, suggesting a new tier of regulation in WUS protein regulation. Taken together our data show that processes that influence WUS protein levels and spatial distribution are highly coupled to its transcriptional activity. PMID:27671631