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Sample records for acid total protein

  1. Total protein

    MedlinePlus

    ... 2016:chap 215. Read More Agammaglobulinemia Albumin - blood (serum) test Amino acids Antibody Burns Chronic Congenital nephrotic syndrome Fibrinogen blood test Glomerulonephritis Hemoglobin Liver disease Malabsorption Multiple myeloma Polycythemia vera Protein in diet ...

  2. Total nitrogen vs. amino-acid profile as indicator of protein content of beef.

    PubMed

    Hall, Nicolette G; Schönfeldt, Hettie C

    2013-10-01

    In most cited food composition studies and tables, the proximate system measures protein as total nitrogen (N) (determined by Kjeldahl or Dumas method) multiplied by a specific factor. A factor of 6.25 is used for determining total protein from total N (Jones, Munsey, & Walker, 1942). Although more expensive, it is considered more accurate to base protein content of foods on amino acid data (Greenfield & Southgate, 2003). A study on the nutrient composition of beef analysed the full amino-acid profile of fifteen retail cuts from three age groups and six fat codes, as well as determined total nitrogen content to determine proximate protein composition. For all cuts, the correlation coefficient of total amino acids to protein (N×6.25) was 0.635. This indicates a poor correlation for predicting actual protein content (as determined by total amino acid count), based on the nitrogen factor of 6.25. On average, the sum of amino acids per cut amounted to 91% of total determined protein (N×6.25) for the same cut.

  3. Bioprocess monitoring: minimizing sample matrix effects for total protein quantification with bicinchoninic acid assay.

    PubMed

    Reichelt, Wieland N; Waldschitz, Daniel; Herwig, Christoph; Neutsch, Lukas

    2016-09-01

    Determining total protein content is a routine operation in many laboratories. Despite substantial work on assay optimization interferences, the widely used bicinchoninic acid (BCA) assay remains widely recognized for its robustness. Especially in the field of bioprocess engineering the inaccuracy caused by interfering substances remains hardly predictable and not well understood. Since the introduction of the assay, sample pre-treatment by trichloroacetic acid (TCA) precipitation has been indicated as necessary and sufficient to minimize interferences. However, the sample matrix in cultivation media is not only highly complex but also dynamically changing over process time in terms of qualitative and quantitative composition. A significant misestimation of the total protein concentration of bioprocess samples is often observed when following standard work-up schemes such as TCA precipitation, indicating that this step alone is not an adequate means to avoid measurement bias. Here, we propose a modification of the BCA assay, which is less influenced by sample complexity. The dynamically changing sample matrix composition of bioprocessing samples impairs the conventional approach of compensating for interfering substances via a static offset. Hence, we evaluated the use of a correction factor based on an internal spike measurement for the respective samples. Using protein spikes, the accuracy of the BCA protein quantification could be improved fivefold, taking the BCA protein quantification to a level of accuracy comparable to other, more expensive methods. This will allow reducing expensive iterations in bioprocess development to due inaccurate total protein analytics.

  4. Total protein quantitation using the bicinchoninic acid assay and gradient elution moving boundary electrophoresis.

    PubMed

    Kralj, Jason G; Munson, Matthew S; Ross, David

    2014-07-01

    We investigated the ability of gradient elution moving boundary electrophoresis (GEMBE) with capacitively coupled contactless conductivity detection (C(4) D) to assay total protein concentration using the bicinchoninic acid (BCA) reaction. We chose this format because GEMBE-C(4) D behaves as a concentration dependent detection system, unlike optical methods that also rely on pathlength (due to Beer's law). This system tolerates proteins well compared with other capillary electrophoretic methods, allowing the capillary to be reused without coatings or additional hydroxide wash steps. The typical reaction protocol was modified by reducing the pH slightly from 11.25 to 9.4, which enabled elimination of tartrate from the reagents. We estimated that copper (I) could be detected at approximately 3.0 μmol/L, which agrees with similar GEMBE and CZE systems utilizing C(4) D. Under conditions similar to the BCA "micro method" assay, we determined the LOD for three common proteins (insulin, BSA, and bovine gamma globulin) and found that they agree well with the existing spectroscopic detection methods. Further, we investigated how long reaction times impact the LOD and found that the conversion was proportional to log(time). This indicated that little sensitivity is gained by extending the reaction past 1 h. Hence, GEMBE provides an alternative platform for total protein assays while maintaining the excellent sensitivity of the optical-based methods.

  5. Isotopomer distributions in amino acids from a highly expressed protein as a proxy for those from total protein

    SciTech Connect

    Shaikh, Afshan; Shaikh, Afshan S.; Tang, Yinjie; Mukhopadhyay, Aindrila; Keasling, Jay D.

    2008-06-27

    {sup 13}C-based metabolic flux analysis provides valuable information about bacterial physiology. Though many biological processes rely on the synergistic functions of microbial communities, study of individual organisms in a mixed culture using existing flux analysis methods is difficult. Isotopomer-based flux analysis typically relies on hydrolyzed amino acids from a homogeneous biomass. Thus metabolic flux analysis of a given organism in a mixed culture requires its separation from the mixed culture. Swift and efficient cell separation is difficult and a major hurdle for isotopomer-based flux analysis of mixed cultures. Here we demonstrate the use of a single highly-expressed protein to analyze the isotopomer distribution of amino acids from one organism. Using the model organism E. coli expressing a plasmid-borne, his-tagged Green Fluorescent Protein (GFP), we show that induction of GFP does not affect E. coli growth kinetics or the isotopomer distribution in nine key metabolites. Further, the isotopomer labeling patterns of amino acids derived from purified GFP and total cell protein are indistinguishable, indicating that amino acids from a purified protein can be used to infer metabolic fluxes of targeted organisms in a mixed culture. This study provides the foundation to extend isotopomer-based flux analysis to study metabolism of individual strains in microbial communities.

  6. Comparative Analysis of Urinary Total Proteins by Bicinchoninic Acid and Pyrogallol Red Molybdate Methods

    PubMed Central

    Bhongir, Aparna Varma; Karra, Madhulatha; Beedu, Sashidhar Rao

    2015-01-01

    Background The concentration of total proteins in urine is a good index of renal function, but its determination is found to be unreliable. The pyrogallol red molybdate (PRM) method for urine total proteins is being widely used in most of the hospitals because of its high sensitivity, better precision and its practicability. Bicinchoninic acid method (BCA) is also used for protein estimation and there have been no studies comparing this method with the PRM method in human urine samples. BCA method overestimates the urinary protein concentration in the presence of interfering substances. After removing the interfering substances present in the human urine samples the results of BCA method were compared with the PRM method. Aim The purpose of the study is to identify whether the results of urine total proteins by BCA method are comparable to PRM method and can be used as an alternative to the PRM method. Setting and Design This is a cross-sectional study done on fresh urine specimens from the hospital laboratory, covering a wide range of protein concentrations. Material and Methods Fresh urine specimens covering a wide range of protein concentrations (urine dipstick: nil, trace, 1+, 2+ and ≥ 3+) of 36 patients were analysed by both the methods. Statistical Analysis Imprecision was determined by repeated analysis study and Inaccuracy was assessed by comparing the results of the patient’s urine samples by both the methods using correlation plots, Bland and Altman, and Passing and Bablok regression analyses. Results The coefficient of variation and mean (SD) for the BCA method were 4.6% and 799.1 (882.5) mg/L and for the PRM method were 5.1% and 802.1 (911.9) mg/L. The Pearson correlation coefficient, r was 0.93 (p < 0.0001). Method agreement studies showed no significant constant and proportional bias between both the methods. Conclusion In urine which is subjected to removal of interfering substances, the BCA results are comparable to PRM method. PMID:26435938

  7. Comparisons between true digestibility of total nitrogen and limiting amino acids in vegetable proteins fed to rats.

    PubMed

    Sarwar, G; Peace, R W

    1986-07-01

    Values (%) for true digestibility (TD) of protein and individual amino acids in some vegetable proteins were determined by the rat balance (fecal) method. Diets containing 8% crude protein (N X 6.25) from soaked and autoclaved samples of Trapper and Century field peas, lentil, pinto bean, seafarer bean, black bean or fababean and autoclaved samples of soybean, peanut, sunflower, rolled oat, rice + soybean and corn + pea were tested in two rat balance studies. In the case of blends, each protein source provided 50% of total protein. The beans, peas and lentil proteins were limiting in sulphur amino acids, tryptophan and threonine, whereas sunflower and rolled oat were most limiting in lysine. In beans, peas and lentil, the TD values of methionine (51-82), cystine (46-85), tryptophan (47-90) and threonine (62-84) were considerably lower than the TD values of total nitrogen (72-90). Similarly, in sunflower and rolled oat, the TD values of lysine (81-83) were lower than the TD values of total nitrogen (90-91). These data suggested that crude protein digestibility may not be a good predictor of bioavailability of limiting amino acids in vegetable proteins. Amino acid scores of the vegetable proteins were 62-96%. The corrections for true digestibility of protein and individual amino acids lowered the scores by 6-15 and 11-47 percentage units, respectively.

  8. 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

  9. Total leaf crude protein, amino acid composition and elemental content in the USDA-ARS bamboo germplasm collection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bamboo shoots and leaves are valuable food sources for both humans and livestock. The USDA-ARS National Plant Germplasm System (NPGS) collections hold 93 bamboo species in 20 genera. Total leaf protein, amino acid composition and elemental content for these important genetic resources had never bee...

  10. CSF total protein

    MedlinePlus

    CSF total protein is a test to determine the amount of protein in your spinal fluid, also called cerebrospinal fluid (CSF). ... The normal protein range varies from lab to lab, but is typically about 15 to 60 milligrams per deciliter (mg/dL) ...

  11. Combined nitrogen limitation and cadmium stress stimulate total carbohydrates, lipids, protein and amino acid accumulation in Chlorella vulgaris (Trebouxiophyceae).

    PubMed

    Chia, Mathias Ahii; Lombardi, Ana Teresa; da Graça Gama Melão, Maria; Parrish, Christopher C

    2015-03-01

    Metals have interactive effects on the uptake and metabolism of nutrients in microalgae. However, the effect of trace metal toxicity on amino acid composition of Chlorella vulgaris as a function of varying nitrogen concentrations is not known. In this research, C. vulgaris was used to investigate the influence of cadmium (10(-7) and 2.0×10(-8)molL(-1) Cd) under varying nitrogen (2.9×10(-6), 1.1×10(-5) and 1.1×10(-3)molL(-1)N) concentrations on its growth rate, biomass and biochemical composition. Total carbohydrates, total proteins, total lipids, as well as individual amino acid proportions were determined. The combination of Cd stress and N limitation significantly inhibited growth rate and cell density of C. vulgaris. However, increasing N limitation and Cd stress stimulated higher dry weight and chlorophyll a production per cell. Furthermore, biomolecules like total proteins, carbohydrates and lipids increased with increasing N limitation and Cd stress. Ketogenic and glucogenic amino acids were accumulated under the stress conditions investigated in the present study. Amino acids involved in metal chelation like proline, histidine and glutamine were significantly increased after exposure to combined Cd stress and N limitation. We conclude that N limitation and Cd stress affects the physiology of C. vulgaris by not only decreasing its growth but also stimulating biomolecule production.

  12. Effect of the Kinetin-Naphthaleneacetic Acid Interaction Upon Total RNA and Protein in Senescing Detached Leaves 1

    PubMed Central

    Vonabrams, G. J.; Pratt, Harlan K.

    1968-01-01

    The interaction between kinetin and naphthaleneacetic acid in the regulation of senescence of excised tissue of mature broccoli leaves has been used to examine the extent of synchrony between changes in chlorophyll, RNA, and protein. Kinetin increased the net uptake of 14C-labeled orotic acid and leucine. Naphthaleneacetic acid decreased the effect of kinetin on net uptake after long treatment, but in short-time treatments the auxin increased the effect of kinetin on net uptake. Results of long (24 hr) treatments indicated a general synchrony between the loss of RNA, protein, and chlorophyll. Naphthaleneacetic acid reduced the stabilizing effect of kinetin upon chlorophyll content and upon the content and synthesis of RNA. In short-time experiments, however, RNA content and synthesis were transiently increased by kinetin, and further increased by kinetin plus naphthaleneacetic acid, while chlorophyll content decreased in the presence of kinetin and decreased further in the presence of kinetin plus naphthaleneacetic acid. Actinomycin-D accelerated the loss of chlorophyll, RNA and protein and strongly depressed the rate of RNA synthesis. In the presence of actinomycin-D the stabilizing effect of kinetin upon RNA was substantially reduced. In contrast, the chlorophyll and protein contents remained higher than in the control. Actinomycin-D did not nullify the basal incorporation of orotic acid into RNA, nor did it negate the effect of kinetin upon incorporation. The failure of synchrony between changes in chlorophyll and RNA does not substantiate the proposal that kinetin regulates senescence by a direct effect upon DNA-dependent RNA synthesis. Images PMID:5700020

  13. [Total synthesis of nordihydroguaiaretic acid].

    PubMed

    Wu, A X; Zhao, Y R; Chen, N; Pan, X F

    1997-04-01

    beta-Keto ester(5) was obtained from vanilin through etherification, oxidation and condensation with acetoacetic ester, (5) on oxidative coupling reaction by NaOEt/I2 produced dimer (6) in high yield. Acid catalyzed cyclodehydration of (6) gave the furan derivative(7), and by a series of selective hydrogenation nordihydroguaiaretic acid, furoguaiacin dimethyl ether and dihydroguaiaretic acid dimethyl ether were synthesized.

  14. Influences of dietary protein level, amino acid supplementation and environmental temperature on performance, body composition, organ weights and total heat production of growing pigs.

    PubMed

    Kerr, B J; Yen, J T; Nienaber, J A; Easter, R A

    2003-08-01

    The study was conducted to determine the effects of feeding a 16% CP diet, a 12% CP diet, or a 12% CP diet supplemented with crystalline Lys, Trp, and Thr (12% CP + AA diet) in a thermal-neutral (23 degrees C) or heat-stressed (33 degrees C) environment on various body and physiological measurements in growing pigs. Heat-stressed pigs were given a 15% lower daily feed allowance than thermal-neutral pigs to remove the confounding effect of feed intake caused by high temperature. No diet x temperature interaction was observed for any variables (P > 0.09) except for pig activity and pancreas weight. At 33 degrees C, pig activity and pancreas weight did not differ among dietary treatments (P > 0.05). In contrast, at 23 degrees C, pigs fed the 12% CP diet had greater activity than those fed the 16% CP diet or the 12% CP + AA diet (P < 0.05). Pancreas weight was greater for pigs fed the 12% CP + AA diet than those fed the 12% CP diet (P < 0.05) when maintained at 23 degrees C. Compared with 23 degrees C, the 33 degrees C temperature decreased pig activity, heat production, daily gain, feed efficiency, and affected the concentration and accretion of empty body protein and ash, as well as weights of heart, pancreas, stomach, and large intestine (P < 0.05). Pigs fed the 12% CP + AA diet attained similar levels of performance and rates of empty body water, protein, lipid, and ash deposition as pigs fed the 16% CP diet (P > 0.10). Pigs fed the 12% CP + AA diet had lower serum urea plus ammonia nitrogen concentrations (P < 0.01) and total heat production (P < 0.05) compared with those fed the 16% CP diet or the 12% CP diet. These results confirm that, with crystalline AA supplementation, growing pigs fed a 12% CP diet will perform similar to pigs fed a 16% CP diet. The data further indicate that lowering dietary CP and supplementing crystalline AA will decrease total heat production in growing pigs whether they are housed in a thermal-neutral or heat-stressed environment.

  15. Asymmetric Total Syntheses of Megacerotonic Acid and Shimobashiric Acid A

    PubMed Central

    Krabbe, Scott W.; Johnson, Jeffrey S.

    2015-01-01

    The asymmetric total syntheses of the α-benzylidene-γ-butyrolactone natural products megacerotonic acid and shimobashiric acid A have been accomplished in nine and 11 steps, respectively, from simple, commercially available starting materials. The key step for each synthesis is the (arene)RuCl(monosulfonamide)-catalyzed dynamic kinetic resolution-asymmetric transfer hydrogenation (DKR-ATH) of racemic α,δ-diketo-β-aryl esters to establish the absolute stereochemistry. Intramolecular diastereoselective Dieckmann cyclization forms the lactone core, and ketone reduction/alcohol elimination installs the α-arylidene. PMID:25699999

  16. 21 CFR 862.1635 - Total protein test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Total protein test system. 862.1635 Section 862....1635 Total protein test system. (a) Identification. A total protein test system is a device intended to measure total protein(s) in serum or plasma. Measurements obtained by this device are used in...

  17. 21 CFR 862.1635 - Total protein test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Total protein test system. 862.1635 Section 862....1635 Total protein test system. (a) Identification. A total protein test system is a device intended to measure total protein(s) in serum or plasma. Measurements obtained by this device are used in...

  18. 21 CFR 862.1635 - Total protein test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Total protein test system. 862.1635 Section 862....1635 Total protein test system. (a) Identification. A total protein test system is a device intended to measure total protein(s) in serum or plasma. Measurements obtained by this device are used in...

  19. 21 CFR 862.1635 - Total protein test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Total protein test system. 862.1635 Section 862....1635 Total protein test system. (a) Identification. A total protein test system is a device intended to measure total protein(s) in serum or plasma. Measurements obtained by this device are used in...

  20. [Acids in coffee. XI. The proportion of individual acids in the total titratable acid].

    PubMed

    Engelhardt, U H; Maier, H G

    1985-07-01

    22 acids in ground roast coffees and instant coffees were determined by GLC of their silyl derivatives (after preseparation by gel electrophoresis) or isotachophoresis. The contribution to the total acidity (which was estimated by titration to pH 8 after cation exchange of the coffee solutions) was calculated for each individual acid. The mentioned acids contribute with 67% (roast coffee) and 72% (instant coffee) to the total acidity. In the first place citric acid (12.2% in roast coffee/10.7% in instant coffee), acetic acid (11.2%/8.8%) and the high molecular weight acids (8%/9%) contribute to the total acidity. Also to be mentioned are the shares of chlorogenic acids (9%/4.8%), formic acid (5.3%/4.6%), quinic acid (4.7%/5.9%), malic acid (3.9%/3%) and phosphoric acid (2.5%/5.2%). A notable difference in the contribution to total acidity between roast and instant coffee was found for phosphoric acid and pyrrolidonecarboxylic acid (0.7%/1.9%). It can be concluded that those two acids are formed or released from e.g. their esters in higher amounts than other acids during the production of instant coffee.

  1. Protein and amino acid nutrition

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  2. A comparison of methods for determining total body protein.

    PubMed

    Brooks, S P; Lampi, B J; Sarwar, G; Botting, H G

    1995-03-20

    The aim of the study was to find the optimal method (with respect to convenience and accuracy) for determining total protein in whole-body homogenates of rats. Three different protein extraction methods and five different protein concentration methods were assessed. The results were compared against a reference value measured by complete amino acid analysis after acid hydrolysis. The data demonstrated that extraction with 5% (w/v) sodium dodecylsulfate (SDS) in 0.5 N NaOH was far superior to that with water alone or to 6 N guanidine-HCl. A comparison of the Biuret, Bradford, and bicinchoninic acid methods on the SDS-NaOH-extracted samples showed that the Biuret method was optimal, giving a value that was 90% of the reference value with a small variation (2.4% of the mean). The Kjeldahl method gave the correct protein concentration only when a nitrogen factor of 5.51 +/- 0.03 (N = 5) was applied. The results suggest that extraction with SDS-NaOH followed by the Biuret procedure is a good method for measuring protein concentrations in whole body rat homogenates.

  3. Formulation of plant based Rainbow trout feeds on an Ideal Protein Basis can reduce total dietary protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fish meal has been a primary protein source in trout feeds and any changes that can reduce fish meal levels and total costs are beneficial. Replacing fish meal with plant protein is a first step, but amino acid content of plant based diets can be limiting. Amino acids are needed for many metabolic...

  4. A procedure for total protein determination with special application to allergenic extract standardization.

    PubMed

    Richman, P G; Cissel, D S

    1988-10-01

    A method for total protein determination of allergenic extracts has been developed and evaluated. Samples were hydrolyzed with 5 M NaOH followed by colorimetric determination with ninhydrin of the released amino acids using bovine serum albumin as the standard. The entire procedure was carried out in disposable plastic tubes. Substances (glycerol, phenol and mannitol) commonly present in allergenic extracts manufactured for human use did not affect the assay results. Analyses of four different pollen extracts by the method gave good agreement with amino acid analyses. Other methods of analysis (total N, protein N unit assay, Lowry) gave more variable results compared with amino acid analysis. Analysis of the total protein content of 53 different lots of allergenic extracts gave narrow ranges of values for each species. Standardized mite extracts analyzed for total protein by US FDA-licensed manufacturers using this assay showed a good correlation of biological activity with total protein.

  5. Short-chain fatty acid-supplemented total parenteral nutrition alters intestinal structure, glucose transporter 2 (GLUT2) mRNA and protein, and proglucagon mRNA abundance in normal rats.

    PubMed

    Tappenden, K A; Drozdowski, L A; Thomson, A B; McBurney, M I

    1998-07-01

    Intestinal adaptation is a complex physiologic process that is not completely understood. Intravenous short-chain fatty acids (SCFAs) enhance intestinal adaptation after 80% enterectomy in rats. The purpose of this study was to examine rapid responses to SCFA-supplemented total parenteral nutrition (TPN) in the normal small intestine. After jugular catheterization, 31 Sprague-Dawley rats (weighing 258 +/- 3 g) were randomly assigned to receive standard TPN or an isoenergetic, isonitrogenous TPN solution supplemented with SCFAs (TPN+SCFA). Intestinal samples were obtained after 24 or 72 h of nutrient infusion. TPN+SCFA for 24 h increased (P < 0.05) the ileal RNA concentration (microg RNA/mg ileum) whereas TPN+SCFA for 72 h increased (P < 0.05) the ileal DNA concentration (microg DNA/mg ileum) and decreased (P < 0.05) the ileal protein concentration (microg protein/mg ileum). Ileal proglucagon mRNA abundance was elevated (P < 0.05) after 24 h of TPN+SCFA infusion and returned to levels seen with control TPN by 72 h. Glucose transporter 2 (GLUT2) mRNA was significantly higher (P < 0.05) in the TPN+SCFA groups at both time points when compared with control TPN groups. Ileal GLUT2 protein abundance in the 72-h TPN+SCFA group was significantly higher (P < 0.05) than that of all other groups. Sodium-glucose cotransporter (SGLT-1) mRNA and protein abundance and uptake of D-fructose and D-glucose did not differ between groups. Jejunal uptake of L-glucose and lauric acid was significantly higher (P < 0.05) after 72 h of TPN+SCFA than after 24 h, whereas the 24- and 72-h TPN groups did not differ. In summary, SCFAs led to rapid changes in ileal proglucagon and glucose transporter expression in rats receiving TPN and provide insights into therapeutic management of individuals with short bowel syndrome or intestinal malabsorption syndromes.

  6. Alimentary proteins, amino acids and cholesterolemia.

    PubMed

    Blachier, François; Lancha, Antonio H; Boutry, Claire; Tomé, Daniel

    2010-01-01

    Numerous data from both epidemiological and experimental origins indicate that some alimentary proteins and amino acids in supplements can modify the blood LDL cholesterol, HDL cholesterol and total cholesterol. After an initial approval of the health claim for soy protein consumption for the prevention of coronary heart disease, more recently it has been concluded from an overall analysis of literature that isolated soy protein with isoflavones only slightly decrease LDL and total cholesterol. Other plant extracts and also some proteins from animal origin have been reported to exert a lowering effect on blood cholesterol when compared with a reference protein (often casein). The underlying mechanisms are still little understood. Individual amino acids and mixture of amino acids have also been tested (mostly in animal studies) for their effects on cholesterol parameters and on cholesterol metabolism. Methionine, lysine, cystine, leucine, aspartate and glutamate have been tested individually and in combination in different models of either normo or hypercholesterolemic animals and found to be able to modify blood cholesterol and/or LDL cholesterol and/or HDL cholesterol. It is however not known if these results are relevant to human nutrition.

  7. 21 CFR 862.1635 - Total protein test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... measure total protein(s) in serum or plasma. Measurements obtained by this device are used in the diagnosis and treatment of a variety of diseases involving the liver, kidney, or bone marrow as well...

  8. Quantifying protein by bicinchoninic Acid.

    PubMed

    Simpson, Richard J

    2008-08-01

    INTRODUCTIONThis protocol describes a method of quantifying protein that is a variation of the Lowry assay. It uses bicinchoninic acid (BCA) to enhance the detection of Cu(+) generated under alkaline conditions at sites of complexes between Cu(2+) and protein. The resulting chromophore absorbs at 562 nm. This technique is divided into three parts: Standard Procedure, Microprocedure, and 96-Well Microtiter Plate Procedure. For each procedure, test samples are assayed in parallel with protein standards that are used to generate a calibration curve, and the exact concentration of protein in the test samples is interpolated. The standard BCA assay uses large volumes of both reagents and samples and cannot easily be automated. If these issues are important, the Microprocedure is recommended. This in turn can be adapted for use with a microplate reader in the 96-Well Microtiter Plate Procedure. If the microplate reader is interfaced with a computer, more than 1000 samples can be read per hour.

  9. Simplified quantitative determination of total fecal bile acids.

    PubMed

    de Wael, J; Raaymakers, C E; Endeman, H J

    1977-09-01

    To determine total fecal bile acids, these are extracted with diethyl ether after boiling with a solution of potassium hydroxide in ethanediol. After evaporating the ether and dissolving the residue in methanol, the bile acids are directly determined with 3 alpha-hydroxysteroid dehydrogenase. Values for 9 normals are given.

  10. Benefits of pomegranate (Punica granatum Linn) fruit extracts to weight changes, total protein, and uric acid in white rats (Rattus norvegicus) as an animal model of acute renal failure

    PubMed Central

    Primarizky, Hardany; Yuniarti, Wiwik Misaco; Lukiswanto, Bambang Sektiari

    2016-01-01

    Aim: The occurrence of acute renal failure (ARF) cases continues to increase every year. Some of the cases are due to nephrotoxic effect caused by overdose of antibiotic consumption or abuse of the drug, gentamicin. An antibiotic therapy that can be used to overcome in such a case is the pomegranate extracts. However, until now, studies using pomegranate for cases of ARF have not been done. This study aims to determine changes in body weight, the levels of total protein (TP), and the levels of serum uric acid (UA) as a result of the pomegranate extract consumption. Materials and Methods: A total number of 32 rats (Rattus norvegicus) were divided into four groups randomly. One group was assigned as the control group (P0) and given intraperitoneal (i.p.) saline and 0.3% carboxy methyl cellulose sodium (CMC) Na; P1 was provided with 80 mg/kg bw/i.p. gentamicin and 0.3% CMC Na orally, P2 was supplied with 80 mg/kg bw/i.p. gentamicin and ellagic acid in 0.3% CMC Na, and P3 was given 80 mg/kg bw/i.p. gentamicin and 150 mg/kg bw pomegranate extract in 0.3% CMC Na. The provision of treatment was carried out in 8 days and followed by making the overthrow of body weight and blood sampling for the examination of study variables. Results: The results taken by doing the analysis of variance method for the four treatment groups show that the control group (P0) has significant differences from P1, P2, and P3 (p<0.05), but there are no significant differences among the other three treatment groups. Meanwhile, the average values of serum UA levels among P1, P2, and P3 indicate significant differences. Conclusion: In conclusion, the administration of pomegranate extracts in the treatment of nephrotoxicity toward rats is effective to maintain normal body weight, normal TP levels, and the UA blood serum of the rats. As this study is a preventive therapy, it needs further researches about the effective dose as a curative therapy, its level of effectiveness and its long-term side

  11. Protein and Calorie Requirements with Total Parenteral Nutrition

    PubMed Central

    Shizgal, Harry M.; Forse, R. Armour

    1980-01-01

    Body composition measurements, performed by multiple isotope dilution, were used to determine the protein and caloric requirements of patients receiving total parenteral nutrition (TPN). In addition the relative efficacy of lipid as opposed to carbohydrate calories were evaluated. Patients requiring TPN were randomly allocated to receive one of the following TPN solutions: a) 2.5% amino acid with 25% dextrose b) 5% amino acid with 25% dextrose c) 2.5% amino acid with 12.5% dextrose and a 5% lipid emulsion. The efficacy of each solution was evaluated by determining body composition at the onset,and at two week intervals during the course of TPN. In 204 patients who received TPN for 4447 days, 533 body composition studies were performed to evaluate 308 periods of TPN. In the normally nourished patient, as defined by the pre-TPN body composition, the body composition remained unchanged and normal with the three solutions. In the presence of preexisting malnutrition, two weeks of TPN resulted in a significant increase in body weight, arising primarily from an increase in the body cell mass. To evaluate the relative importance of the various factors responsible for the increase in the body cell mass, a multiple linear regression analysis was performed. The mean daily change in the body cell mass was correlated with the carbohydrate, protein and lipid calories infused and with the nutritional state. The resulting regression equation, which was statistically significant, indicated that the rate at which a depleted body cell mass was restored was related to the lipid and carbohydrate calories infused and to the nutritional state of the patient. Carbohydrate calories were more efficient than lipid calories. However increasing the amino acid concentration from 2.5 to 5% had no effect on the rate at which the body cell mass increased. The repletion rate was also directly related to the severity of malnutrition. Thus the correction of a malnourished individual with TPN is

  12. Total Synthesis of (±)- and (−)-Actinophyllic Acid

    PubMed Central

    Martin, Connor L.; Overman, Larry E.; Rohde, Jason M.

    2010-01-01

    Development of efficient sequences for the total syntheses of (±)-actinophyllic acid (rac-1) and (−)-actinophyllic acid (1) are described. The central step in these syntheses is the aza-Cope/Mannich reaction, which constructs the previously unknown hexacyclic ring system of actinophyllic acid in one step from much simpler tetracyclic precursors. The tetracyclic hexahydro-1,5-methano-1H-azocino[4,3-b]indole ketone rac-37 is assembled from o-nitrophenylacetic acid in four steps, with oxidative cyclization of a dienolate derivative of tricyclic precursor rac-35 being the central step. In the first-generation synthesis, this intermediate is transformed in two steps to homoallyl amine rac-43, whose formaldiminium derivative undergoes efficient aza-Cope/Mannich reaction to give pentacyclic ketone rac-44. In four additional steps, this intermediate is advanced to (±)-actinophyllic acid. The synthesis is streamlined by elaborating ketone rac-37 to β-hydroxyester intermediate rac-53, which is directly transformed to (±)-actinophyllic acid upon exposure to HCl and paraformaldehyde. This concise second-generation total synthesis of (±)-actinophyllic acid is realized in 22% overall yield from commercially available di-tert-butylmalonate and o-nitrophenylacetic acid by a sequence that proceeds by way of only six isolated intermediates. The first enantioselective total synthesis of (−)-actinophyllic acid (1) is accomplished by this direct sequence from tricyclic keto malonate (S)-35. Catalytic enantioselective reduction of α,β-unsaturated ketone 66 is the key step in the preparation of intermediate (S)-35 from the commercially available Boc-amino acid 65. Discussed also is the possibility that the aza-Cope/Mannich reaction might be involved in the biosynthesis of (−)-actinophyllic acid. PMID:20218696

  13. Protein and sulfur amino acid requirements of broiler breeder hens.

    PubMed

    Harms, R H; Wilson, H R

    1980-02-01

    Two experiments were conducted with Cobb color-sexed broiler breeder hens to determine their protein and sulfur amino acid requirement. A daily intake between 400 and 478 mg of methionine and between 722 and 839 mg of total sulfur amino acids was necessary for maximum egg production, the latter in a diet of 13.07% protein. Slightly lower levels supported maximum body weights. Hens laying at the highest rate consumed 23.4 g of protein per day.

  14. Quantitative analysis of total flavonoids and total phenolic acids in thirty Hypericum taxa.

    PubMed

    Pilepić, Kroata Hazler; Males, Zeljan; Crkvencić, Maja

    2013-03-01

    The content of total flavonoids and total phenolic acids in the aerial parts of thirty Hypericum taxa collected over two consecutive seasons was investigated by spectrophotometric methods. All taxa examined contained flavonoids and phenolic acids in different quantities, although the differences between species and year's harvest were not found to be significant. The quantity of total flavonoids ranged from 0.1 to 1.6%. The highest content of flavonoids was found in the samples of H. japonicum (1.6%) and H. perforatum (1.5%), while the samples of H. androsaemum and H. balearicum comprised the lowest flavonoid amount (0.13%). The content of total phenolic acids in the investigated samples was found to be between 1.1-10.4%. The samples of H. perforatum were found to contain the highest quantity of phenolic acids (10.4% and 10.2%), whereas the sample ofH. linarifolium showed the lowest amount of phenolic acids (1.1%).

  15. A modified gelatin zymography technique incorporating total protein normalization.

    PubMed

    Raykin, Julia; Snider, Eric; Bheri, Sruti; Mulvihill, John; Ethier, C Ross

    2017-03-15

    Gelatinase zymography is a commonly used laboratory procedure; however, variability in sample loading and concentration reduce the accuracy of quantitative results obtained from this technique. To facilitate normalization of gelatinase activity by loaded protein amount, we developed a protocol using the trihalocompound 2,2,2-trichloroethanol to allow for gelatin zymography and total protein labeling within the same gel. We showed that detected protein levels increased linearly with loading, and describe a loading concentration range over which normalized gelatinase activity was constant. We conclude that in-gel total protein detection is feasible in gelatin zymography and greatly improves comparison of gelatinase activity between samples.

  16. Phenol-Sulfuric Acid Method for Total Carbohydrates

    NASA Astrophysics Data System (ADS)

    Nielsen, S. Suzanne

    The phenol-sulfuric acid method is a simple and rapid colorimetric method to determine total carbohydrates in a sample. The method detects virtually all classes of carbohydrates, including mono-, di-, oligo-, and polysaccharides. Although the method detects almost all carbohydrates, the absorptivity of the different carbohydrates varies. Thus, unless a sample is known to contain only one carbohydrate, the results must be expressed arbitrarily in terms of one carbohydrate.

  17. Distinguishing Proteins From Arbitrary Amino Acid Sequences

    PubMed Central

    Yau, Stephen S.-T.; Mao, Wei-Guang; Benson, Max; He, Rong Lucy

    2015-01-01

    What kinds of amino acid sequences could possibly be protein sequences? From all existing databases that we can find, known proteins are only a small fraction of all possible combinations of amino acids. Beginning with Sanger's first detailed determination of a protein sequence in 1952, previous studies have focused on describing the structure of existing protein sequences in order to construct the protein universe. No one, however, has developed a criteria for determining whether an arbitrary amino acid sequence can be a protein. Here we show that when the collection of arbitrary amino acid sequences is viewed in an appropriate geometric context, the protein sequences cluster together. This leads to a new computational test, described here, that has proved to be remarkably accurate at determining whether an arbitrary amino acid sequence can be a protein. Even more, if the results of this test indicate that the sequence can be a protein, and it is indeed a protein sequence, then its identity as a protein sequence is uniquely defined. We anticipate our computational test will be useful for those who are attempting to complete the job of discovering all proteins, or constructing the protein universe. PMID:25609314

  18. The total protein content, protein fractions and proteases activities of drone prepupae of Apis mellifera due to varrosis.

    PubMed

    Zółtowska, Krystyna; Lipiński, Zbigniew; Dmitryjuk, Małgorzata

    2005-01-01

    The proteins level and activities of acid and alkaline proteases in whole body extracts of drone prepupae of Apis mellifera naturally infested with Varroa destructor were studied. The infested and a non-infested group did not differ significantly in their total protein content. However, some differences in protein profiles were found. A lack of three protein fractions of moderate and lower molecular weight in infested prepupae was noted. Moreover, some differences in the quantity of protein in most of the fractions were observed. The activity of acid proteases from infested prepupae was lower (p < 0.05) compared with the activity of these proteases from the non-infested one group. The infested drone had higher activity of alkaline proteases than non-infested but this difference was not statisticaly significant.

  19. Protein and Amino Acid Profiles of Different Whey Protein Supplements.

    PubMed

    Almeida, Cristine C; Alvares, Thiago S; Costa, Marion P; Conte-Junior, Carlos A

    2016-01-01

    Whey protein (WP) supplements have received increasing attention by consumers due to the high nutritional value of the proteins and amino acids they provide. However, some WP supplements may not contain the disclosed amounts of the ingredients listed on the label, compromising the nutritional quality and the effectiveness of these supplements. The aim of this study was to evaluate and compare the contents of total protein (TP), α-lactalbumin (α-LA), β-lactoglobulin (β-LG), free essential amino acids (free EAA), and free branched-chain amino acids (free BCAA), amongst different WP supplements produced by U.S. and Brazilian companies. Twenty commercial brands of WP supplements were selected, ten manufactured in U.S. (WP-USA) and ten in Brazil (WP-BRA). The TP was analyzed using the Kjeldahl method, while α-LA, β-LG, free EAA, and free BCAA were analyzed using HPLC system. There were higher (p < 0.05) concentrations of TP, α-LA, β-LG, and free BCAA in WP-USA supplements, as compared to the WP-BRA supplements; however, there was no difference (p > 0.05) in the content of free EAA between WP-USA and WP-BRA. Amongst the 20 brands evaluated, four WP-USA and seven WP-BRA had lower (p < 0.05) values of TP than those specified on the label. In conclusion, the WP-USA supplements exhibited better nutritional quality, evaluated by TP, α-LA, β-LG, and free BCAA when compared to WP-BRA.

  20. A METHOD FOR DETERMINING TOTAL PROTEIN OF ISOLATED CELLULAR ELEMENTS AND CORRESPONDING TRITIUM RADIOACTIVITY

    PubMed Central

    Koenig, Edward

    1968-01-01

    A method is described for the microanalysis of protein, obtained from isolated tissue elements, in the range of 500 µµg-500 mµg. The method entails solubilization of cellular protein with phosphoric acid and heat after extraction of acid-soluble compounds, lipids, and RNA. A procedure for the extraction and recovery of cellular RNA by the use of 40% trichloroacetic acid is presented. The solubilized protein, in the form of a microdroplet, is photomicrographed with monochromatic light at 230 mµ. Total density in the microdroplet is determined from calibrated photographic plates by microdensitometry, and is converted to protein mass by using an experimentally determined average specific absorbance value. A solubilized protein labeled with tritium can be recovered after photomicrography, combusted, and reduced to generate tritiated gas for high-efficiency tritium radiometry. Total protein was analyzed in (a) nerve cells of three different sizes from Deiters' nucleus of the rabbit; and the whole rod cell and rod cell nucleus of the rabbit retina. PMID:5664225

  1. Gas chromatographic analysis of infant formulas for total fatty acids, including trans fatty acids.

    PubMed

    Satchithanandam, Subramaniam; Fritsche, Jan; Rader, Jeanne I

    2002-01-01

    Twelve powdered and 13 liquid infant formulas were analyzed by using an extension of AOAC Official Method 996.01 for fat analysis in cereal products. Samples were hydrolyzed with 8 N HCl and extracted with ethyl and petroleum ethers. Fatty acid methyl esters were prepared by refluxing the mixed ether extracts with methanolic sodium hydroxide in the presence of 14% boron trifluoride in methanol. The extracts were analyzed by gas chromatography. In powdered formulas, saturated fatty acid (SFA) content (mean +/- SD; n = 12) was 41.05 +/- 3.94%, monounsaturated fatty acid (MUFA) content was 36.97 +/- 3.38%, polyunsaturated fatty acid (PUFA) content was 20.07 +/- 3.08%, and total trans fatty acid content was 1.30 +/- 1.27%. In liquid formulas, SFA content (mean +/- SD; n = 13) was 42.29 +/- 2.98%, MUFA content was 36.05 +/- 2.47%, PUFA content was 20.65 +/- 2.40%, and total trans fatty acid content was 0.88 +/- 0.54%. Total fat content in powdered formulas ranged from 4.4 to 5.5 g/100 kcal and linoleic acid content ranged from 868 to 1166 mg/100 kcal. In liquid formulas, total fat content ranged from 4.1 to 5.1 g/100 kcal and linoleic acid content ranged from 820 to 1100 mg/100 kcal. There were no significant differences between powdered and liquid infant formulas in concentrations of total fat, SFA, MUFA, PUFA, or trans fatty acids.

  2. SLC27 fatty acid transport proteins.

    PubMed

    Anderson, Courtney M; Stahl, Andreas

    2013-01-01

    The uptake and metabolism of long chain fatty acids (LCFA) are critical to many physiological and cellular processes. Aberrant accumulation or depletion of LCFA underlie the pathology of numerous metabolic diseases. Protein-mediated transport of LCFA has been proposed as the major mode of LCFA uptake and activation. Several proteins have been identified to be involved in LCFA uptake. This review focuses on the SLC27 family of fatty acid transport proteins, also known as FATPs, with an emphasis on the gain- and loss-of-function animal models that elucidate the functions of FATPs in vivo and how these transport proteins play a role in physiological and pathological situations.

  3. Total syntheses of cis-cyclopropane fatty acids: dihydromalvalic acid, dihydrosterculic acid, lactobacillic acid, and 9,10-methylenehexadecanoic acid.

    PubMed

    Shah, Sayali; White, Jonathan M; Williams, Spencer J

    2014-12-14

    cis-Cyclopropane fatty acids (cis-CFAs) are widespread constituents of the seed oils of subtropical plants, membrane components of bacteria and protozoa, and the fats and phospholipids of animals. We describe a systematic approach to the synthesis of enantiomeric pairs of four cis-CFAs: cis-9,10-methylenehexadecanoic acid, lactobacillic acid, dihydromalvalic acid, and dihydrosterculic acid. The approach commences with Rh2(OAc)4-catalyzed cyclopropenation of 1-octyne and 1-decyne, and hinges on the preparative scale chromatographic resolution of racemic 2-alkylcycloprop-2-ene-1-carboxylic acids using a homochiral Evan's auxiliary. Saturation of the individual diastereomeric N-cycloprop-2-ene-1-carbonylacyloxazolidines, followed by elaboration to alkylcyclopropylmethylsulfones, allowed Julia-Kocienski olefination with various ω-aldehyde-esters. Finally, saponification and diimide reduction afforded the individual cis-CFA enantiomers.

  4. Total Synthesis of Five Lipoteichoic acids of Clostridium difficile.

    PubMed

    Hogendorf, Wouter F J; Gisch, Nicolas; Schwudke, Dominik; Heine, Holger; Bols, Mikael; Pedersen, Christian Marcus

    2014-10-13

    The emergence of hypervirulent resistant strains have made Clostridium difficile a notorious nosocomial pathogen and has resulted in a renewed interest in preventive strategies, such as vaccines based on (synthetic) cell wall antigens. Recently, the structure of the lipoteichoic acid (LTA) of this species has been elucidated. Additionally, this LTA was found to induce the formation of protective antibodies against C. difficile in rabbits and mice. The LTA from C. difficile is isolated as a microheterogenous mixture, differing in size and composition, impeding any structure-activity relationship studies. To ensure reliable biological results, pure and well-defined synthetic samples are required. In this work the total synthesis of LTAs from C. difficile with defined chain length is described and the initial biological results are presented.

  5. Total parenteral nutrition-associated cholestasis in rats: comparison of different amino acid mixtures.

    PubMed

    Belli, D C; Fournier, L A; Lepage, G; Yousef, I; Weber, A M; Tuchweber, B; Roy, C C

    1987-01-01

    It has been suggested that the quantity of amino acids perfused is a pathogenetic factor in total parenteral nutrition (TPN)-associated hepatotoxicity. However, the effect of the qualitative pattern of amino acid solutions has not been studied. Rats on parenteral nutrition for 5 days received 10.2 g of dextrose and 3.4 g of amino acids daily. Bile flow (microliter/min/g liver protein) after administration of Vamin was 16.2 +/- 0.8, which was similar to that in controls given chow and dextrose iv, but it was significantly higher (p less than 0.001) than those on Travasol (12.3 +/- 0.8). The decrease in bile flow was not related to the large concentrations of alanine and glycine present in Travasol. However, the addition to Travasol of serine present only in Vamin increased bile flow significantly. Bile acid secretion rate, biliary lipid constituents, calcium, sodium, and glucose showed little change. In contrast, alpha-amino nitrogen was increased (p less than 0.05) in Vamin-perfused animals. Steatosis was noted in only a few animals in the Travasol group, and was not associated with an increase in the triglycerides content of the liver. Glycogen and protein content of the livers did not differ. The data show that the composition of amino acid solutions may be a determinant of TPN-induced cholestasis and suggest that the presence of methyl donor amino acids may have a protective effect.

  6. Standards for total serum protein assays--a collaborative study.

    PubMed

    Doumas, B T

    1975-07-01

    We have studied the standardization of total serum protein assay with the biuret reaction. Standard solutions were prepared from lyophilized preparations of human serum albumin and bovine serum albumin, with corrections made for volatile material and ash contents. These solutions and a solution of crystalline albumin standard were analyzed with a new stable biuret reagent, to establish absorptivity values (values for the absorbance of a 1 g/liter final reaction mixture). The mean values obtained were 0.302, 0.292, and 0.290 for human serum albumin, bovine serum albumin, and the crystalline albumin, respectively. We believe that the established absorptivity value will improve the accuracy of serum protein determinations. We studied the linearity of the relation between color produced and protein concentration, with use of the solutions described above and a serum pool. The color adheres to Beer's law up to the highest concentration tested: 3 g/liter for HSA and BSA, and 2.8 g/liter for serum in the final reaction mixture. The new biuret reagent has been stable for one year at room temperature. We recommend the use of bovine serum albumin as a primary standard for serum protein assays. It is inexpensive, easily available, and exhibits the best linearity in the biuret reaction.

  7. Evaluation of standardized ileal digestible valine:lysine, total lysine:crude protein, and replacing fish meal, meat and bone meal, and poultry byproduct meal with crystalline amino acids on growth performance of nursery pigs from seven to twelve kilograms.

    PubMed

    Nemechek, J E; Tokach, M D; Dritz, S S; Goodband, R D; DeRouchey, J M

    2014-04-01

    Five experiments were conducted to evaluate replacing fish meal, meat and bone meal, and poultry byproduct meal with crystalline AA for 7- to 12-kg pigs. In all experiments, pigs (PIC TR4 × 1050) were fed a common diet for 3 d postweaning, treatment diets for 14 d (d 0 to 14), and, again, a common diet for 14 d (d 14 to 28). Treatment diets were corn-soybean meal based and formulated to contain 1.30% standardized ileal digestible (SID) Lys. Experiment 1 evaluated replacing dietary fish meal with crystalline AA. For the 6 treatments, crystalline Lys, Met, Thr, Trp, Ile, Val, Gln, and Gly all increased to maintain minimum AA ratios as fish meal decreased (4.50, 3.60, 2.70, 1.80, and 0.90 to 0.00%). There was no difference in ADG, ADFI, or G:F among treatments, validating a low-CP, AA-fortified diet for subsequent experiments. Experiment 2 evaluated deleting crystalline AA from a low-CP, AA-fortified diet with 6 treatments: 1) a positive control similar to the diet validated in Exp. 1, 2) positive control with l-Ile deleted, 3) positive control with l-Trp deleted, 4) positive control with l-Val deleted, 5) positive control with l-Gln and l-Gly deleted, and 6) positive control with l-Ile, l-Trp, l-Val, l-Gln, and l-Gly deleted (NC). Pigs fed the positive control or Ile deleted diet had improved (P < 0.05) ADG and ADFI during d 0 to 14 compared with pigs fed diets with l-Trp or l-Val deleted or NC. Experiment 3 evaluated 6 treatments with total Lys:CP of 6.79, 6.92, 7.06, 7.20, 7.35, and 7.51%. Fish meal was adjusted as a source of dispensable N to achieve the target Lys:CP. There were no differences in growth performance among pigs fed different Lys:CP diets. Experiment 4 evaluated increasing SID Val:Lys with Val at 57.4, 59.9, 62.3, 64.7, 67.2, and 69.6% of Lys. Average daily gain and ADFI increased (quadratic, P < 0.01) and G:F improved (linear, P = 0.02) during d 0 to 14 as Val:Lys increased from 57.4 to 64.7%. Experiment 5 was a 2 × 3 factorial arrangement of

  8. Use of tranexamic acid in total knee arthroplasty

    PubMed Central

    MARRA, FRANCESCO; ROSSO, FEDERICA; BRUZZONE, MATTEO; BONASIA, DAVIDE EDOARDO; DETTONI, FEDERICO; ROSSI, ROBERTO

    2016-01-01

    Purpose different strategies have been developed to reduce blood loss in total knee arthroplasty (TKA). The efficacy of both systemic and local tranexamic acid (TXA) administration is demonstrated in the literature. The aim of the present study was to compare the efficacy of systemic, local and combined (systemic + local) administration of TXA in reducing blood loss after TKA. Methods we enrolled all patients submitted to a primary TKA in our department between November 2014 and August 2015. They were divided into three groups corresponding to the method of TXA administration used: intravenous (IV), intra-articular (IA), and a combination of the two. Demographic data, as well as preoperative hemoglobin and platelet levels, were collected. The primary outcome was the maximum hemoglobin loss, while the secondary outcomes were the amount of blood in the drain (cc/hour) and the rate of transfusions; postoperative pain was also assessed. Student’s t-test or a χ2 test was used to evaluate between-group differences, using p<0.05 as the cut-off for statistically significant differences. Results the sample comprised 34 patients: IV, 10 cases; IA, 15 cases, and combined (IV + IA), 9 cases. The average age of the patients was 71.1±6.4 years. No significant differences in the outcome measures were found between the groups, with the exception of a significantly lower maximum hemoglobin loss in the combined versus the IV group (p=0.02). There were no differences between the groups in the amount of blood in the drain or the rate of transfusions. Conclusions the data from this preliminary study, as well as data from the literature, confirm that TXA administration is safe and effective in reducing total blood loss in TKA, and no administration protocol seems to be superior to the others. Level of evidence Level II, prospective comparative study. PMID:28217656

  9. Comparison of five methods for determination of total plasma protein concentration.

    PubMed

    Okutucu, Burcu; Dinçer, Ayşşe; Habib, Omer; Zihnioglu, Figen

    2007-08-01

    Quantitation of exact total protein content is often a key step and is common to many applications in general biochemistry research and routine clinical laboratory practice. Before embarking on any type of protein analysis, particularly comparative techniques, it is important to accurately quantitate the amount of protein in the sample. In order to assess the quality of total protein estimation results, five methods were tested and were applied to the same pooled plasma sample. For this aim, Bradford (Coomassie Brilliant Blue), Lowry (Folin-Ciocalteau), Biüret, Pesce and Strande (Ponceau-S/TCA), and modified method of Schaffner-Weismann (Amido Black 10B) were used. The last two methods employ simultaneous precipitation of proteins with the acid containing dye solutions followed by dissolution of precipitate in a NaOH solution. It is shown that each assay has advantages and disadvantages relative to sensitivity, ease of performance, acceptance in literature, accuracy and reproducibility/coefficient of variation. All of the methods tested show a CV %<6. Besides pooled plasma, a known concentration of human serum albumin was also analyzed and discussed by means of standardization of plasma total protein content.

  10. Intravenous versus topical tranexamic acid in primary total hip replacement

    PubMed Central

    Zhang, Pei; Liang, Yuan; Chen, Pengtao; Fang, Yongchao; He, Jinshan; Wang, Jingcheng

    2016-01-01

    Abstract Background: As the prevalence of total hip arthroplasty (THA) is increasing, it is usually associated with considerable blood loss. Tranexamic acid (TXA) has been reported to reduce perioperative blood loss in hip joint arthroplasty. But the best route of TXA administration continues to be controversial. So, we conducted a meta-analysis that integrated all data from the 7 included trials to compare the effectiveness and safety of topical and intravenous TXA administration in primary THA. The endpoints assessed in this meta-analysis include the comparisons of total blood loss, postoperative hemoglobin decline, transfusion rates, the incidence rate of deep vein thrombosis (DVT), pulmonary embolisms (PE), and wound infection. Methods: Literature searches of PubMed, EMBASE, the Cochrane Library, the Chinese Biomedical Literature database, the CNKI database, and Wan Fang Data were performed up to August 30, 2016. Randomized controlled trials (RCTs) were included in our meta-analysis if they compared the efficiency and safety of intravenous versus topical administration of TXA in patients who underwent primary THA. The endpoints included the comparisons of total blood loss, postoperative hemoglobin decline, transfusion rates, the incidence rate of DVT, PE, and wound infection. A meta-analysis was performed following the guidelines of the Cochrane Reviewer's Handbook and the PRISMA statement. The pooling of data was carried out by using RevMan 5.3, Denmark. Results: Seven RCTs involving 964 patients met the inclusion criteria. Our meta-analysis indicated that there were no significant differences in the 2 groups in terms of total blood loss ([mean difference (MD) = −14.74, 95% confidence interval (CI): −89.21 to 59.74, P = 0.7], transfusion rates [RD = −0.02, 95% CI: −0.05 to 0.02, P = 0.39]; no significant differences were found regarding the incidence of adverse effects such as deep venous thrombosis [DVT] [RD = 0.00, 95% CI: −0

  11. 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.

  12. Nucleic acids, proteins, and chirality

    NASA Technical Reports Server (NTRS)

    Usher, D. A.; Profy, A. T.; Walstrum, S. A.; Needels, M. C.; Bulack, S. C.; Lo, K. M.

    1984-01-01

    The present investigation is concerned with experimental results related, in one case, to the chirality of nucleotides, and, in another case, to the possibility of a link between the chirality of nucleic acids, and that of peptides. It has been found that aminoacylation of the 'internal' hydroxyl group of a dinucleoside monophosphate can occur stereoselectively. However, this reaction has not yet been made a part of a working peptide synthesis scheme. The formation and cleavage of oligonucleotides is considered. In the event of the formation of a helical complex between the oligonucleotide and the polymer, 1-prime,5-prime-bonds in the oligomer are found to become more resistant towards cleavage. The conditions required for peptide bond formation are examined, taking into account the known structures of RNA and possible mechanisms for prebiotic peptide bond formation. The possibility is considered that the 2-prime,5-prime-internucleotide linkage could have played an important part in the early days of biological peptide synthesis.

  13. Changes in total nitrogen and amino acid composition of New Zealand Undaria pinnatifida with growth, location and plant parts.

    PubMed

    Zhou, April Yongdong; Robertson, John; Hamid, Nazimah; Ma, Qianli; Lu, Jun

    2015-11-01

    Undaria pinnatifida is known as unwanted organism in New Zealand. However, Wakame is a traditional food made of U. pinnatifida, which is now cultured extensively in East Asia. Therefore, it is important to examine this introduced alga as a potential source of dietary protein for human consumption in New Zealand. This study determined total nitrogen content and amino acid profile of New Zealand U. pinnatifida harvested from the Marlborough Sounds on a monthly basis from June to November 2011. Total average nitrogen content and crude protein content was 21.02 mg/g dry weight and 13.1% of dry weight, respectively. The three most abundant amino acids that contributed to flavour (glutamic acid, aspartic acid and alanine) were present and the most abundant essential amino acids were arginine, leucine, lysine and valine. The results showed that the amino acid content in blades from the exposed farm was significantly higher (P < 0.05) than the others. Sporophyll maturation of U. pinnatifida in New Zealand influenced protein content and amino acid composition. Sporophyll, considered as a waste product by many, was found to be a potentially good source of protein.

  14. Electrocatalysis in proteins, nucleic acids and carbohydrates.

    PubMed

    Paleček, Emil; Bartošík, Martin; Ostatná, Veronika; Trefulka, Mojmír

    2012-02-01

    The ability of proteins to catalyze hydrogen evolution has been known for more than 80 years, but the poorly developed d.c. polarographic "pre-sodium wave" was of little analytical use. Recently, we have shown that by using constant current chronopotentiometric stripping analysis, proteins produce a well-developed peak H at hanging mercury drop and solid amalgam electrodes. Peak H sensitively reflects changes in protein structures due to protein denaturation, single amino acid exchange, etc. at the picomole level. Unmodified DNA and RNA do not yield such a peak, but they produce electrocatalytic voltammetric signals after modification with osmium tetroxide complexes with nitrogen ligands [Os(VIII)L], binding covalently to pyrimidine bases in nucleic acids. Recently, it has been shown that six-valent [Os(VI)L] complexes bind to 1,2-diols in polysaccharides and oligosaccharides, producing voltammetric responses similar to those of DNA-Os(VIII)L adducts. Electrocatalytic peaks produced by Os-modified nucleic acids, proteins (reaction with tryptophan residues) and carbohydrates are due to the catalytic hydrogen evolution, allowing determination of oligomers at the picomolar level.

  15. Essential amino acid supplementation in patients following total knee arthroplasty

    PubMed Central

    Dreyer, Hans C.; Strycker, Lisa A.; Senesac, Hilary A.; Hocker, Austin D.; Smolkowski, Keith; Shah, Steven N.; Jewett, Brian A.

    2013-01-01

    Background. By the year 2030, 3.48 million older U.S. adults are projected to undergo total knee arthroplasty (TKA). Following this surgery, considerable muscle atrophy occurs, resulting in decreased strength and impaired functional mobility. Essential amino acids (EAAs) have been shown to attenuate muscle loss during periods of reduced activity and may be beneficial for TKA patients. Methods. We used a double-blind, placebo-controlled, randomized clinical trial with 28 older adults undergoing TKA. Patients were randomized to ingest either 20 g of EAAs (n = 16) or placebo (n = 12) twice daily between meals for 1 week before and 2 weeks after TKA. At baseline, 2 weeks, and 6 weeks after TKA, an MRI was performed to determine mid-thigh muscle and adipose tissue volume. Muscle strength and functional mobility were also measured at these times. Results. TKA patients receiving placebo exhibited greater quadriceps muscle atrophy, with a –14.3 ± 3.6% change from baseline to 2 weeks after surgery compared with –3.4 ± 3.1% for the EAA group (F = 5.16, P = 0.036) and a –18.4 ± 2.3% change from baseline to 6 weeks after surgery for placebo versus –6.2 ± 2.2% for the EAA group (F = 14.14, P = 0.001). EAAs also attenuated atrophy in the nonoperated quadriceps and in the hamstring and adductor muscles of both extremities. The EAA group performed better at 2 and 6 weeks after surgery on functional mobility tests (all P < 0.05). Change in quadriceps muscle atrophy was significantly associated with change in functional mobility (F = 5.78, P = 0.021). Conclusion. EAA treatment attenuated muscle atrophy and accelerated the return of functional mobility in older adults following TKA. Trial registration. Clinicaltrials.gov NCT00760383. Funding. Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), Office of the Director (OD), and the National Institutes of Health Office of Dietary Supplements (ODS), NIH grant K01HD057332, and the Medical

  16. The colorimetric detection and quantitation of total protein.

    PubMed

    Krohn, Randall I

    2011-09-01

    Protein quantification is an important step for handling protein samples for isolation and characterization; it is a prerequisite step before submitting proteins for chromatographic, electrophoretic, or immunochemical analysis and separation. Colorimetric methods are fast, simple, and not laborious. This unit describes a number of assays able to detect protein concentrations in the low microgram to milligram per milliliter ranges in a variety of formats.

  17. Conformations of amino acids in proteins.

    PubMed

    Hovmöller, Sven; Zhou, Tuping; Ohlson, Tomas

    2002-05-01

    The main-chain conformations of 237 384 amino acids in 1042 protein subunits from the PDB were analyzed with Ramachandran plots. The populated areas of the empirical Ramachandran plot differed markedly from the classical plot in all regions. All amino acids in alpha-helices are found within a very narrow range of phi, psi angles. As many as 40% of all amino acids are found in this most populated region, covering only 2% of the Ramachandran plot. The beta-sheet region is clearly subdivided into two distinct regions. These do not arise from the parallel and antiparallel beta-strands, which have quite similar conformations. One beta region is mainly from amino acids in random coil. The third and smallest populated area of the Ramachandran plot, often denoted left-handed alpha-helix, has a different position than that originally suggested by Ramachandran. Each of the 20 amino acids has its own very characteristic Ramachandran plot. Most of the glycines have conformations that were considered to be less favoured. These results may be useful for checking secondary-structure assignments in the PDB and for predicting protein folding.

  18. Measurement of protein using bicinchoninic acid.

    PubMed

    Smith, P K; Krohn, R I; Hermanson, G T; Mallia, A K; Gartner, F H; Provenzano, M D; Fujimoto, E K; Goeke, N M; Olson, B J; Klenk, D C

    1985-10-01

    Bicinchoninic acid, sodium salt, is a stable, water-soluble compound capable of forming an intense purple complex with cuprous ion (Cu1+) in an alkaline environment. This reagent forms the basis of an analytical method capable of monitoring cuprous ion produced in the reaction of protein with alkaline Cu2+ (biuret reaction). The color produced from this reaction is stable and increases in a proportional fashion over a broad range of increasing protein concentrations. When compared to the method of Lowry et al., the results reported here demonstrate a greater tolerance of the bicinchoninate reagent toward such commonly encountered interferences as nonionic detergents and simple buffer salts. The stability of the reagent and resulting chromophore also allows for a simplified, one-step analysis and an enhanced flexibility in protocol selection. This new method maintains the high sensitivity and low protein-to-protein variation associated with the Lowry technique.

  19. Total and lipid-bound serum sialic acid in benign and malignant breast disease.

    PubMed

    Romppanen, J; Eskelinen, M; Tikanoja, S; Mononen, I

    1997-01-01

    Elevation in the total sialic acid (TSA), TSA/total protein (TSA/TP) and lipid-bound sialic acid (LASA) concentration in serum occurs in breast cancer and we have studied the applicability of the assays in classification of undefined breast tumors. Sialic acid was determined by HPLC and the statistical evaluation included the receiver operating characteristic (ROC) and Youden's index analyses. In cancer patients, the serum LASA and TSA concentration was significantly higher (p < 0.05) than in patients with benign breast disease and all the markers were significantly higher (p < 0.0001) than in normal controls. All the markers had a low accuracy (AUCs < 0.75) in differentiating between breast cancer and benign breast disease and at the specificity level of 0.95 the corresponding sensitivities were 0.32 (TSA), 0.14 (TSA/TP) and 0.23 (LASA). The results indicate that both breast cancer and benign breast disease cause elevation of TSA, TSA/TP and LASA values in serum and do not provide reliable classification of undefined breast tumors.

  20. 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

  1. Interference of N-hydroxysuccinimide with bicinchoninic acid protein assay.

    PubMed

    Vashist, Sandeep Kumar; Dixit, Chandra Kumar

    2011-07-29

    We report here substantial interference from N-hydroxysuccinimide (NHS) in the bicinchoninic acid (BCA) protein assay. NHS is one of the most commonly used crosslinking agents in bioanalytical sciences, which can lead to serious potential errors in the BCA protein assay based protein estimation if it is present in the protein analyte solution. It was identified to be a reducing substance, which interferes with the BCA protein assay by reducing Cu(2+) in the BCA working reagent. The absorbance peak and absorbance signal of NHS were very similar to those of bovine serum albumin (BSA), thereby indicating a similar BCA reaction mechanism for NHS and protein. However, the combined absorbance of NHS and BSA was not additive. The time-response measurements of the BCA protein assay showed consistent single-phase kinetics for NHS and gradually decreasing kinetics for BSA. The error in protein estimation due to the presence of NHS was counteracted effectively by plotting additional BCA standard curve for BSA with a fixed concentration of NHS. The difference between the absorbance values of BSA and BSA with a fixed NHS concentration provided the absorbance contributed by NHS, which was then subtracted from the total absorbance of analyte sample to determine the actual absorbance of protein in the analyte sample.

  2. The measurement of total serum proteins by the Biuret method.

    PubMed

    Lubran, M M

    1978-01-01

    The biuret reaction for proteins provides a simple and precise method for measuring serum proteins; Beer's law is obeyed to at least 10 g per dl. Several stable biuret reagents are available. Hemoglobin is the only important cause of interference which cannot be minimized by use of a sample blank. The mechanism of the biuret reaction is described and attention is drawn to the heterogeneity of the serum proteins and to the use of a certified albumin standard.

  3. Inadequacy of prebiotic synthesis as origin of proteinous amino acids.

    PubMed

    Wong, J T; Bronskill, P M

    1979-07-18

    The production of some nonproteinous, and lack of production of other proteinous, amino acids in model prebiotic synthesis, along with the instability of glutamine and asparagine, suggest that not all of the 20 present day proteinous amino acids gained entry into proteins directly from the primordial soup. Instead, a process of active co-evolution of the genetic code and its constituent amino acids would have to precede the final selection of these proteinous amono acids.

  4. Effect of Growth on Fatty Acid Composition of Total Intramuscular Lipid and Phospholipids in Ira Rabbits.

    PubMed

    Xue, Shan; He, Zhifei; Lu, Jingzhi; Tao, Xiaoqi; Zheng, Li; Xie, Yuejie; Xiao, Xia; Peng, Rong; Li, Hongjun

    2015-01-01

    The changes in fatty acid composition of total intramuscular lipid and phospholipids were investigated in the longissimus dorsi, left-hind leg muscle, and abdominal muscle of male Ira rabbits. Changes were monitored at 35, 45, 60, 75, and 90 d. Analysis using gas chromatography identified 21 types of fatty acids. Results showed that the intramuscular lipid increased and the intramuscular phospholipids (total intramuscular lipid %) decreased in all muscles with increasing age (p<0.05). An abundant amount of unsaturated fatty acids, especially polyunsaturated fatty acids, was distributed in male Ira rabbits at different ages and muscles. Palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), and arachidonic acid (C20:4) were the major fatty acids, which account to the dynamic changes of the n-6/n-3 value in Ira rabbit meat.

  5. Salivary total sialic acid levels increase in breast cancer patients: a preliminary study.

    PubMed

    Oztürk, Leyla Koç; Emekli-Alturfan, Ebru; Kaşikci, Emel; Demir, Gokhan; Yarat, Aysen

    2011-09-01

    Breast cancer is the most common cancer in women living in the Western world, even though it occurs worldwide. Cancer and cancer therapy induce multiple oral complications including dental and periodontal disease. Saliva is a complex and dynamic biologic fluid, which reflects both oral and systemic changes. While saliva is easily accessible body fluid, there has been little effort to study its value in cancer diagnosis. Sialic acids (SA), the end moieties of the carbohydrate chains, are biologically important and essential for functions of glycoconjugates that are reported to be altered in both blood and saliva of various cancer patients. Increased sialylation has been shown to be a characteristic feature in cancer tissue and blood in breast cancer patients. However, there is no data about salivary SA in breast cancer. The aim of this study was to evaluate salivary total sialic acid (TSA) levels in breast cancer patients who were under chemotheraphy. The study included 15 breast cancer patients in different stages and 10 healthy individuals as age-matched controls. Unstimulated whole saliva was collected. Salivary total protein and SA levels were determined. Flow rate was calculated from salivary volume by the time of secretion. Salivary SA was significantly higher and total protein was lower in breast cancer patients compared to controls. It is concluded that sialylation may be increased in saliva of patients with breast cancer as the same way for cancer tissue and for blood . Increased salivary SA may therefore be useful as a non-invasive predictive marker for breast cancer patients and for the prevention and management of oral complications of cancer and cancer therapy to improve oral function and quality-of-life. The effects of different types of chemotherapies and different stages of the disease on salivary SA levels and salivary sialo-glycomic are worthy of being further investigated in breast cancer patients.

  6. The natural non-protein amino acid N-β-methylamino-L-alanine (BMAA) is incorporated into protein during synthesis.

    PubMed

    Glover, W Broc; Mash, Deborah C; Murch, Susan J

    2014-11-01

    N-β-methylamino-L-alanine (BMAA) is an amino acid produced by cyanobacteria and accumulated through trophic levels in the environment and natural food webs. Human exposure to BMAA has been linked to progressive neurodegenerative diseases, potentially due to incorporation of BMAA into protein. The insertion of BMAA and other non-protein amino acids into proteins may trigger protein misfunction, misfolding and/or aggregation. However, the specific mechanism by which BMAA is associated with proteins remained unidentified. Such studies are challenging because of the complexity of biological systems and samples. A cell-free in vitro protein synthesis system offers an excellent approach for investigation of changing amino acid composition in protein. In this study, we report that BMAA incorporates into protein as an error in synthesis when a template DNA sequence is used. Bicinchoninic acid assay of total protein synthesis determined that BMAA effectively substituted for alanine and serine in protein product. LC-MS/MS confirmed that BMAA was selectively inserted into proteins in place of other amino acids, but isomers N-(2-aminoethyl)glycine (AEG) and 2,4-diaminobutyric acid (DAB) did not share this characteristic. Incorporation of BMAA into proteins was significantly higher when genomic DNA from post-mortem brain was the template. About half of BMAA in the synthetic proteins was released with denaturation with sodium dodecylsulfonate and dithiothreitol, but the remaining BMAA could only be released by acid hydrolysis. Together these data demonstrate that BMAA is incorporated into the amino acid backbone of proteins during synthesis and also associated with proteins through non-covalent bonding.

  7. Fast and selective determination of total protein in milk powder via titration of moving reaction boundary electrophoresis.

    PubMed

    Guo, Cheng-ye; Wang, Hou-yu; Liu, Xiao-ping; Fan, Liu-yin; Zhang, Lei; Cao, Cheng-xi

    2013-05-01

    In this paper, moving reaction boundary titration (MRBT) was developed for rapid and accurate quantification of total protein in infant milk powder, from the concept of moving reaction boundary (MRB) electrophoresis. In the method, the MRB was formed by the hydroxide ions and the acidic residues of milk proteins immobilized via cross-linked polyacrylamide gel (PAG), an acid-base indicator was used to denote the boundary motion. As a proof of concept, we chose five brands of infant milk powders to study the feasibility of MRBT method. The calibration curve of MRB velocity versus logarithmic total protein content of infant milk powder sample was established based on the visual signal of MRB motion as a function of logarithmic milk protein content. Weak influence of nonprotein nitrogen (NPN) reagents (e.g., melamine and urea) on MRBT method was observed, due to the fact that MRB was formed with hydroxide ions and the acidic residues of captured milk proteins, rather than the alkaline residues or the NPN reagents added. The total protein contents in infant milk powder samples detected via the MRBT method were in good agreement with those achieved by the classic Kjeldahl method. In addition, the developed method had much faster measuring speed compared with the Kjeldahl method.

  8. [Determination of total protein content in soya-bean milk via visual moving reaction boundary titration].

    PubMed

    Guo, Chengye; Wang, Houyu; Zhang, Lei; Fan, Liuyin; Cao, Chengxi

    2013-11-01

    A visual, rapid and accurate moving reaction boundary titration (MRBT) method was used for the determination of the total protein in soya-bean milk. During the process, moving reaction boundary (MRB) was formed by hydroxyl ions in the catholyte and soya-bean milk proteins immobilized in polyacrylamide gel (PAG), and an acid-base indicator was used to denote the boundary motion. The velocity of MRB has a relationship with protein concentration, which was used to obtain a standard curve. By paired t-test, there was no significant difference of the protein content between MRBT and Kjeldahl method at 95% confidence interval. The procedure of MRBT method required about 10 min, and it had linearity in the range of 2.0-14.0 g/L, low limit of detection (0.05 g/L), good precision (RSD of intra-day < 1.90% and inter-day < 4.39%), and high recoveries (97.41%-99.91%). In addition, non-protein nitrogen (NPN) such as melamine added into the soya-bean milk had weak influence on MRBT results.

  9. Difference in amounts between titratable acid and total carboxylic acids produced by oral streptococci during sugar metabolism.

    PubMed

    Iwami, Y; Hata, S; Takahashi, N; Yamada, T

    1989-01-01

    The acid produced by the resting cells of Streptococcus mutants NCTC 10449 and HS 6 and S. sanguis ATCC 10556 during sugar metabolism was estimated with a pH-stat and a carboxylic acid analyzer. Lactic, formic, acetic, pyruvic, and carbonic acids were detected in the reaction mixtures, but propionic, citric, succinic, iso-butyric, butyric, iso-valeric, and valeric acids were not detected. The amount of titratable acid estimated by alkaline titration with the pH-stat was larger than the amount of total carboxylic acids estimated with the carboxylic acid analyzer. The difference in quantity between the titratable and the total carboxylic acids increased significantly with an increase in the period of incubation with sugar. Moreover, the value of the alkaline titration of standard lactic, formic, acetic, and pyruvic acids was equal to the amount analyzed with the carboxylic acid analyzer. The results indicated that these two streptococci produced not only these carboxylic acids but also other acid(s), possibly non-carboxylic acid(s), during their sugar metabolism.

  10. Truly Absorbed Microbial Protein Synthesis, Rumen Bypass Protein, Endogenous Protein, and Total Metabolizable Protein from Starchy and Protein-Rich Raw Materials: Model Comparison and Predictions.

    PubMed

    Parand, Ehsan; Vakili, Alireza; Mesgaran, Mohsen Danesh; van Duinkerken, Gert; Yu, Peiqiang

    2015-07-29

    This study was carried out to measure truly absorbed microbial protein synthesis, rumen bypass protein, and endogenous protein loss, as well as total metabolizable protein, from starchy and protein-rich raw feed materials with model comparisons. Predictions by the DVE2010 system as a more mechanistic model were compared with those of two other models, DVE1994 and NRC-2001, that are frequently used in common international feeding practice. DVE1994 predictions for intestinally digestible rumen undegradable protein (ARUP) for starchy concentrates were higher (27 vs 18 g/kg DM, p < 0.05, SEM = 1.2) than predictions by the NRC-2001, whereas there was no difference in predictions for ARUP from protein concentrates among the three models. DVE2010 and NRC-2001 had highest estimations of intestinally digestible microbial protein for starchy (92 g/kg DM in DVE2010 vs 46 g/kg DM in NRC-2001 and 67 g/kg DM in DVE1994, p < 0.05 SEM = 4) and protein concentrates (69 g/kg DM in NRC-2001 vs 31 g/kg DM in DVE1994 and 49 g/kg DM in DVE2010, p < 0.05 SEM = 4), respectively. Potential protein supplies predicted by tested models from starchy and protein concentrates are widely different, and comparable direct measurements are needed to evaluate the actual ability of different models to predict the potential protein supply to dairy cows from different feedstuffs.

  11. Phytic acid reduction in soy protein improves zinc bioavailability

    SciTech Connect

    Zhou, J.R.; Wong, M.S.; Burns, R.A.; Erdman, J.W. Jr. Mead Johnson Research Center, Evansville, IN )

    1991-03-15

    The objective of this study was to confirm previous studies that have suggested that reduction of phytic acid in soy improved zinc bioavailability (BV). Two commercially-produced soybean isolates containing either a normal phytic acid level or a reduced level were formulated into diets so as to provide 6 or 9 ppm zinc. Control diets were egg white protein-based and contained 3, 6 or 9 ppm zinc from zinc carbonate. Weanling male rats were fed these diets for 21 days and food intake and weight gain monitored. Slope ratio analysis of total tibia zinc content compared to total zinc intake revealed that zinc BV from reduced phytic acid soy isolate-containing diets was indistinguishable from control egg white diets. In contrast, zinc BV from normal soy isolate diets was significantly reduced compared to reduced phytic acid and control diets. These results coupled with other results indicate that phytic acid is the inhibitory factor in soybean products that results in reduced zinc BV.

  12. Effects of total body irradiation on fatty acid and total lipid content of rats.

    PubMed

    Chukwuemeka, Nwokocha; Philippe, Mounmbegna; Magdalene, Nwokocha; Onyezuligbo, Onyekachi

    2012-01-01

    We examined time-dependent changes in plasma lipids of rats given total body irradiation (TBI) with X-rays at 3 Gy. for consecutive periods. Animals were exposed to x ray radiations consecutively for 20 days at 5 day interval thereafter five animals were picked at random and sacrificed (5, 10, 15 and 20 days after beginning the exposure). The triacylglycerols and total cholesterol serum levels were significant differences between control and experimental groups after the first exposure (5 days), values for the triacylglcerols were significantly higher with the second (day 10) and third (day 15) radiation exposures but not with the fourth radiation exposures (day 20) (p<0.05). However, the serum cholesterol values were not found to be significant with the second and third exposures but with the fourth exposure (day 20) (p<0.05). The serum HDL-C concentrations were not significantly different between control and experimental groups at any time analyzed. But the LDL cholesterol was found to decrease on days 5 and 20 of the experimental period. Our results indicate that the applied long term exposure to x rays ionization radiations exposure may induce slight but statistically significant alterations in some serum lipids profile of rats, within the physiological range. The mechanisms for the effects of these ionizing radiations on serum lipid profile are not well understand yet, we suggest that the changes could be due to some non-specific stress reactions. The consequences of our observation are not known yet, but could point to some possible clinical intervention.

  13. Comparison of the Effects of Eicosapentaenoic Acid and Docosahexaenoic Acid on the Eradication of Helicobacter pylori Infection, Serum Inflammatory Factors and Total Antioxidant Capacity.

    PubMed

    Khandouzi, Nafiseh; Shidfar, Farzad; Agah, Shahram; Hosseini, Agha Fatemeh; Dehnad, Afsaneh

    2015-01-01

    Helicobacter pylori infection, the most common chronic bacterial infection in the world, and an important cause of gastrointestinal disorders, may be involved in the pathogenesis of some extra-gastrointestinal disturbances, as well as an increase in blood levels of certain inflammatory markers. Anti-bacterial activity against Helicobacter pylori and anti-inflammatory properties of omega-3 fatty acids have been studied in several research studies. The purpose of the present study was the comparison of the effects of Eicosapentaenoic Acid and Docosahexaenoic Acid supplementation on Helicobacter pylori eradication, serum levels of some inflammatory markers and total antioxidant capacity. In a randomized, double-blind, placebo-controlled clinical trial, 97 Helicobacter pylori positive patients (64 patients in the two intervention groups and 33 in the control group), received 2 grams daily of Eicosapentaenoic Acid, Docosahexaenoic Acid or Medium Chain Triglyceride oil as placebo, along with conventional tetra-drug Helicobacter pylori eradication regimen, for 12 weeks. Helicobacter pylori eradication test and measurement of concentration of interleukine-6, interleukine-8, high-sensitivity C-reactive protein and total antioxidant capacity were performed after the intervention. There was no significant difference in eradication rate of the infection, levels of interleukine-6 and total antioxidant capacity among the three groups, while the levels of interleukine-8 and high-sensitivity C-reactive protein were statistically different. Eicosapentaenoic Acid or Docosahexaenoic Acid supplementation had no significant differential impact on the eradication of Helicobacter pylori infection, and serum levels of interleukine-6 and total antioxidant capacity. However, it had a desirable effect on the levels of interleukine-8 and high-sensitivity C-reactive protein in Helicobacter pylori positive patients.

  14. Comparison of the Effects of Eicosapentaenoic Acid and Docosahexaenoic Acid on the Eradication of Helicobacter pylori Infection, Serum Inflammatory Factors and Total Antioxidant Capacity

    PubMed Central

    Khandouzi, Nafiseh; Shidfar, Farzad; Agah, Shahram; Hosseini, Agha Fatemeh; Dehnad, Afsaneh

    2015-01-01

    Helicobacter pylori infection, the most common chronic bacterial infection in the world, and an important cause of gastrointestinal disorders, may be involved in the pathogenesis of some extra-gastrointestinal disturbances, as well as an increase in blood levels of certain inflammatory markers. Anti-bacterial activity against Helicobacter pylori and anti-inflammatory properties of omega-3 fatty acids have been studied in several research studies. The purpose of the present study was the comparison of the effects of Eicosapentaenoic Acid and Docosahexaenoic Acid supplementation on Helicobacter pylori eradication, serum levels of some inflammatory markers and total antioxidant capacity. In a randomized, double-blind, placebo-controlled clinical trial, 97 Helicobacter pylori positive patients (64 patients in the two intervention groups and 33 in the control group), received 2 grams daily of Eicosapentaenoic Acid, Docosahexaenoic Acid or Medium Chain Triglyceride oil as placebo, along with conventional tetra-drug Helicobacter pylori eradication regimen, for 12 weeks. Helicobacter pylori eradication test and measurement of concentration of interleukine-6, interleukine-8, high-sensitivity C-reactive protein and total antioxidant capacity were performed after the intervention. There was no significant difference in eradication rate of the infection, levels of interleukine-6 and total antioxidant capacity among the three groups, while the levels of interleukine-8 and high-sensitivity C-reactive protein were statistically different. Eicosapentaenoic Acid or Docosahexaenoic Acid supplementation had no significant differential impact on the eradication of Helicobacter pylori infection, and serum levels of interleukine-6 and total antioxidant capacity. However, it had a desirable effect on the levels of interleukine-8 and high-sensitivity C-reactive protein in Helicobacter pylori positive patients. PMID:25561921

  15. Automated Simultaneous Determination of Serum Total Protein and Globulin.

    DTIC Science & Technology

    biuret reaction while the serum globulin is determined by the reaction of globulins with copper in the presence of sulfuric and acetic acids to form...colored metalloprotein complexes. A major advantage of the proposed combined procedure is that the latter reaction is not influenced by the tryptophan

  16. Determination of total proteins: a study of reaction between quinones and proteins.

    PubMed

    Zaia, D A; Verri, W A; Zaia, C T

    1999-06-14

    A previous study was undertaken to test the reaction of several quinones (p-benzoquinone; 2,5-dichloro and 2,6-dichloro p-benzoquinone; tetrachloro-p-benzoquinone; tetrachloro-o-benzoquinone; 2,5-dichloro-3,6-dihydroxy-p-benzoquinone; benz[a]anthracene-7,12-dione) with bovine serum albumin (BSA). From this study, we have devised a spectrophotometric method for determination of total proteins. The quinone, tetrachloro-p-benzoquinone (p-chloranil), showed the best result. The product of reaction between proteins and p-chloranil absorbed at 360 nm and Beer's law was followed up to 200 mug ml(-1) of BSA. The product of reaction of BSA/p-chloranil was stable for 30 min, after that the absorbance increased 16% and kept stable for 24 h. The p-chloranil method showed a limit of detection (1.25 mug ml(-1)) lower than the biuret method (52.0 mug ml(-1)) or p-benzoquinone (PBQ) method (2.6-4.0 mug ml(-1)). The method was applied to spectrophotometric determination of total proteins in blood plasma; the results were compared with the biuret method that is widely used in clinical analysis.

  17. 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.

  18. Amino acid composition of Lagenaria siceraria seed flour and protein fractions.

    PubMed

    Ogunbusola, Moriyike Esther; Fagbemi, Tayo Nathaniel; Osundahunsi, Oluwatooyin Faramade

    2010-12-01

    Defatted seed flours of Lagenaria siceraria (calabash and bottle gourd) were fractionated into their major protein fractions. The amino acid composition of seed flours and their protein fractions were determined and the protein quality was evaluated. Glutamic acid (139-168 mg/g protein) was the most abundant amino acid followed by aspartic acid (89.0-116 mg/g protein) in both the seed flours and their protein fractions. The total essential amino acid ranged from 45.8 to 51.5%. The predicted protein efficiency ratio and the predicted biological value ranged from 2.4 to 2.9 and 8.7 to 44.0, respectively. Lysine and sulphur amino acids were mostly concentrated in the globulin fractions. The first and second limiting amino acids in seed flours and protein fractions were methionine and valine or threonine. The seed flours contained adequate essential amino acids required by growing school children and adults. The seed has potential as protein supplement in cereal based complementary diets or in the replacement of animal proteins in conventional foods.

  19. Determination of the amount of protein and amino acids extracted from the microbial protein (SCP) of lignocellulosic wastes.

    PubMed

    Ahmadi, A R; Ghoorchian, H; Hajihosaini, R; Khanifar, J

    2010-04-15

    With the increasing world population, the use of lignocellulosic wastes for production of microbial protein as animal feed becomes a necessity of our time. In order to verify the most productive protein, the amount of protein and amino acid extracted from Single Cell Protein (SCP) needs to be determined by an effective method. In this study Microbial protein was produced by treatment of wheat straw with Pleurotus florida; with heat at 100 degrees C and NaOH 2% as substrate by solid state fermentation. Concentration of protein was 62.8% per 100 g of dried microbial protein. Then the extracted protein hydrolyzed with HCl 6 Normal for 48 h under 110 degrees C temperature condition. Then the amino acids analyzed by using A-200 Amino Nova analyzer. The results of this study indicated that the ratio of essential amino acids to total amino acids was 65.6%. The concentration of essnyial amino acids were: Lysine = 9.5, histidine = 19.8, threonine = 0.6, valine = 6.6, methionine = 2.1, isoleucine = 7.3, leucine = 6.8, phenylalanine = 4.3 and arginine = 8.3 g/100 g of extracted protein that indicated the obtained microbial protein can be a good or suitable substitute in the food program of animal feed.

  20. Nucleic acids encoding human trithorax protein

    DOEpatents

    Evans, Glen A.; Djabali, Malek; Selleri, Licia; Parry, Pauline

    2001-01-01

    In accordance with the present invention, there is provided an isolated peptide having the characteristics of human trithorax protein (as well as DNA encoding same, antisense DNA derived therefrom and antagonists therefor). The invention peptide is characterized by having a DNA binding domain comprising multiple zinc fingers and at least 40% amino acid identity with respect to the DNA binding domain of Drosophila trithorax protein and at least 70% conserved sequence with respect to the DNA binding domain of Drosophila trithorax protein, and wherein said peptide is encoded by a gene located at chromosome 11 of the human genome at q23. Also provided are methods for the treatment of subject(s) suffering from immunodeficiency, developmental abnormality, inherited disease, or cancer by administering to said subject a therapeutically effective amount of one of the above-described agents (i.e., peptide, antagonist therefor, DNA encoding said peptide or antisense DNA derived therefrom). Also provided is a method for the diagnosis, in a subject, of immunodeficiency, developmental abnormality, inherited disease, or cancer associated with disruption of chromosome 11 at q23.

  1. Degradation of Cry1Ab protein from genetically modified maize (MON810) in relation to total dietary feed proteins in dairy cow digestion.

    PubMed

    Paul, Vijay; Guertler, Patrick; Wiedemann, Steffi; Meyer, Heinrich H D

    2010-08-01

    To investigate the relative degradation and fragmentation pattern of the recombinant Cry1Ab protein from genetically modified (GM) maize MON810 throughout the gastrointestinal tract (GIT) of dairy cows, a 25 months GM maize feeding study was conducted on 36 lactating Bavarian Fleckvieh cows allocated into two groups (18 cows per group) fed diets containing either GM maize MON810 or nearly isogenic non-GM maize as the respective diet components. All cows were fed a partial total mixed ration (pTMR). During the feeding trial, 8 feed (4 transgenic (T) and 4 non-transgenic (NT) pTMR) and 42 feces (26 T and 18 NT) samples from the subset of cows fed T and NT diets, and at the end of the feeding trial, digesta contents of rumen, abomasum, small intestine, large intestine and cecum were collected after the slaughter of six cows of each feeding group. Samples were analyzed for Cry1Ab protein and total protein using Cry1Ab specific ELISA and bicinchoninic acid assay, respectively. Immunoblot analyses were performed to evaluate the integrity of Cry1Ab protein in feed, digesta and feces samples. A decrease to 44% in Cry1Ab protein concentration from T pTMR to the voided feces (9.40 versus 4.18 mug/g of total proteins) was recorded. Concentrations of Cry1Ab protein in GIT digesta of cows fed T diets varied between the lowest 0.38 mug/g of total proteins in abomasum to the highest 3.84 mug/g of total proteins in rumen. Immunoblot analysis revealed the extensive degradation of recombinant Cry1Ab protein into a smaller fragment of around 34 kDa in GIT. The results of the present study indicate that the recombinant Cry1Ab protein from MON810 is increasingly degraded into a small fragment during dairy cow digestion.

  2. Denatured mammalian protein mixtures exhibit unusually high solubility in nucleic acid-free pure water.

    PubMed

    Futami, Junichiro; Fujiyama, Haruna; Kinoshita, Rie; Nonomura, Hidenori; Honjo, Tomoko; Tada, Hiroko; Matsushita, Hirokazu; Abe, Yoshito; Kakimi, Kazuhiro

    2014-01-01

    Preventing protein aggregation is a major goal of biotechnology. Since protein aggregates are mainly comprised of unfolded proteins, protecting against denaturation is likely to assist solubility in an aqueous medium. Contrary to this concept, we found denatured total cellular protein mixture from mammalian cell kept high solubility in pure water when the mixture was nucleic acids free. The lysates were prepared from total cellular protein pellet extracted by using guanidinium thiocyanate-phenol-chloroform mixture of TRIzol, denatured and reduced total protein mixtures remained soluble after extensive dialysis against pure water. The total cell protein lysates contained fully disordered proteins that readily formed large aggregates upon contact with nucleic acids or salts. These findings suggested that the highly flexible mixtures of disordered proteins, which have fully ionized side chains, are protected against aggregation. Interestingly, this unusual solubility is characteristic of protein mixtures from higher eukaryotes, whereas most prokaryotic protein mixtures were aggregated under identical conditions. This unusual solubility of unfolded protein mixtures could have implications for the study of intrinsically disordered proteins in a variety of cells.

  3. 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.

  4. 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.

  5. 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.

  6. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G.; Wang, Lei

    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.

  7. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G.; Wang, Lei

    2009-04-28

    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.

  8. Spectrophotometric total protein assay with copper(II)-neocuproine reagent in alkaline medium.

    PubMed

    Sözgen, Kevser; Cekic, Sema Demirci; Tütem, Esma; Apak, Resat

    2006-02-28

    Total protein assay was made using copper(II)-neocuproine (Nc) reagent in alkaline medium (with the help of a hydroxide-carbonate-tartarate solution) after 30min incubation at 40 degrees C. The absorbance of the reduction product, Cu(I)-Nc complex, was recorded at 450nm against a reagent blank. The absorptivity of the developed method for bovine serum albumin (BSA) was 0.023lmg(-1)cm(-1), greater than that of Lowry assay (0.0098), and much greater than that of Cu(II)-bicinchoninic acid (BCA) assay (0.00077). The linear range of the developed method (8-100mgl(-1) BSA) was as wide as that of Lowry, and much wider than that of BCA (200-1000mgl(-1) BSA) assay. The sensitivity of the method was greater than those of Cu-based assays (biuret, Lowry, and BCA) with a LOD of 1mgl(-1) BSA. The within-run and between-run precisions as RSD were 0.73 and 1.01%, respectively. The selectivity of the proposed method for protein was much higher than those of dye-binding and Lowry assays: Most common interferents to other protein assays such as tris, ethanolamine, deoxycholate, CsCl, citrate, and triton X-100 were tolerated at 100-fold concentrations in the analysis of 10mgl(-1) BSA, while the tolerance limits for other interferents, e.g., (NH(4))(2)SO(4) and acetylsalicylic acid (50-fold), SDS (25-fold), and glycerol (20-fold) were at acceptable levels. The redox reaction of Cu(II)-Nc as an outer-sphere electron transfer agent with the peptide bond and with four amino acid residues (cystine, cysteine, tryptophan, and tyrosine) was kinetically more favourable than that of Cu(II) alone in the biuret assay. Since the reduction product of Cu(II) with protein, i.e., Cu(I), was coordinatively saturated with Nc in the stable Cu(Nc)(2)(+) chelate, re-oxidation of the formed Cu(I) with Fenton-like reactions was not possible, thereby preventing a loss of chromophore. After conventional protein extraction, precipitation, and redissolution procedures, the protein contents of the minced meat

  9. Cellular Retinoic Acid Binding Protein and Breast Cancer

    DTIC Science & Technology

    2006-05-01

    fatty acid probe anilinonaphtalene-8- sulphonic acid (ANS) was measured. ANS readily associates with various FABPs and its fluorescence is highly...DAMD17-03-1-0249 TITLE: Cellular Retinoic Acid Binding Protein and Breast Cancer PRINCIPAL INVESTIGATOR: Leslie J. (Willmert) Donato...DATES COVERED (From - To) 14 Apr 03 – 13 Apr 06 5a. CONTRACT NUMBER Cellular Retinoic Acid Binding Protein and Breast Cancer 5b. GRANT NUMBER

  10. Association of fatty acids in serum phospholipids with hay fever, specific and total immunoglobulin E.

    PubMed

    Kompauer, Iris; Demmelmair, Hans; Koletzko, Berthold; Bolte, Gabriele; Linseisen, Jakob; Heinrich, Joachim

    2005-04-01

    The dietary intake of certain fatty acids might contribute to the development of allergic diseases such as hay fever and asthma. We investigated the association between the concentrations of fifteen fatty acids in serum phospholipids, as a marker of dietary intake and metabolism, and hay fever, allergic sensitisation and total IgE in adults. Data from a population-based cross-sectional study on respiratory health, including the measurement of fatty acids in the serum phospholipids of 740 adults between 20 and 64 years of age, were analysed. Positive associations were found between hay fever and arachidonic acid, and allergic sensitisation and oleic acid. No other fatty acids showed any association with hay fever or allergic sensitisation. Elevated levels of total IgE were not related to fatty acids. Concentrations of long-chain n-3 fatty acids, trans fatty acids or saturated fatty acids in serum phospholipids were not associated with allergic diseases in adults in this study. The present result on the association between hay fever and arachidonic acid is consistent with current hypotheses but warrants further research.

  11. Enteral obeticholic acid promotes intestinal growth in total parenteral nutrition fed neonatal pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Intestinal atrophy is an adverse outcome associated with prolonged total parenteral nutrition (PN) partly due to disruption of normal enterohepatic circulation of bile acids. Previously we showed that enteral treatment with chenodeoxycholic acid (CDCA), a dual agonist for the nuclear receptor, farne...

  12. Effect of Tranexamic Acid on Blood Loss and Blood Transfusion Reduction after Total Knee Arthroplasty

    PubMed Central

    Seol, Young-Jun; Seon, Jong-Keun; Lee, Seung-Hun; Jin, Cheng; Prakash, Jatin; Park, Yong-Jin

    2016-01-01

    Purpose Total knee arthroplasty (TKA) accompanies the risk of bleeding and need for transfusion. There are several methods to reduce postoperative blood loss and blood transfusion. One such method is using tranexamic acid during TKA. The purpose of this study was to confirm whether tranexamic acid reduces postoperative blood loss and blood transfusion after TKA. Materials and Methods A total of 100 TKA patients were included in the study. The tranexamic acid group consisted of 50 patients who received an intravenous injection of tranexamic acid. The control included 50 patients who received a placebo injection. The amounts of drainage, postoperative hemoglobin, and transfusion were compared between the groups. Results The mean amount of drainage was lower in the tranexamic acid group (580.6±355.0 mL) than the control group (886.0±375.5 mL). There was a reduction in the transfusion rate in the tranexamic acid group (48%) compared with the control group (64%). The hemoglobin level was higher in the tranexamic acid group than in the control group at 24 hours postoperatively. The mean units of transfusion were smaller in the tranexamic acid group (0.76 units) than in the control group (1.28 units). Conclusions Our data suggest that intravenous injection of tranexamic acid decreases the total blood loss and transfusion after TKA. PMID:27595071

  13. Addition of Amino Acids to Further Stabilize Lyophilized Sucrose-Based Protein Formulations: I. Screening of 15 Amino Acids in Two Model Proteins.

    PubMed

    Forney-Stevens, Kelly M; Bogner, Robin H; Pikal, Michael J

    2016-02-01

    In small amounts, the low molecular weight excipients-sorbitol and glycerol-have been shown to stabilize lyophilized sucrose-based protein formulations. The purpose of this study was to explore the use of amino acids as low molecular weight excipients to similarly enhance stability. Model proteins, recombinant human serum albumin and α-chymotrypsin, were formulated with sucrose in combination with one of 15 amino acid additives. Each formulation was lyophilized at 1:1:0.3 (w/w) protein-sucrose-amino acid. Percent total soluble aggregate was measured by size-exclusion chromatography before and after storage at 50 °C for 2 months. Classical thought might suggest that the addition of the amino acids to the sucrose-protein formulations would be destabilizing because of a decrease in the system's glass transition temperature. However, significant improvement in storage stability was observed for almost all formulations at the ratio of amino acid used. Weak correlations were found between the extent of stabilization and both amino acid molar volume and side-chain charge. The addition of amino acids at a modest level generally improves storage stability, often by more than a 50% increase, for lyophilized sucrose-based protein formulations.

  14. Estimation of lactose interference in vaccines and a proposal of methodological adjustment of total protein determination by the lowry method.

    PubMed

    Kusunoki, Hideki; Okuma, Kazu; Hamaguchi, Isao

    2012-01-01

    For national regulatory testing in Japan, the Lowry method is used for the determination of total protein content in vaccines. However, many substances are known to interfere with the Lowry method, rendering accurate estimation of protein content difficult. To accurately determine the total protein content in vaccines, it is necessary to identify the major interfering substances and improve the methodology for removing such substances. This study examined the effects of high levels of lactose with low levels of protein in freeze-dried, cell culture-derived Japanese encephalitis vaccine (inactivated). Lactose was selected because it is a reducing sugar that is expected to interfere with the Lowry method. Our results revealed that concentrations of ≥ 0.1 mg/mL lactose interfered with the Lowry assays and resulted in overestimation of the protein content in a lactose concentration-dependent manner. On the other hand, our results demonstrated that it is important for the residual volume to be ≤ 0.05 mL after trichloroacetic acid precipitation in order to avoid the effects of lactose. Thus, the method presented here is useful for accurate protein determination by the Lowry method, even when it is used for determining low levels of protein in vaccines containing interfering substances. In this study, we have reported a methodological adjustment that allows accurate estimation of protein content for national regulatory testing, when the vaccine contains interfering substances.

  15. 7 CFR 51.1178 - Maximum anhydrous citric acid permissible for corresponding total soluble solids.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... corresponding total soluble solids. 51.1178 Section 51.1178 Agriculture Regulations of the Department of... solids. For determining the grade of juice, the maximum permissible anhydrous citric acid content in relation to corresponding total soluble solids in the fruit is set forth in the following Table II...

  16. 7 CFR 51.1178 - Maximum anhydrous citric acid permissible for corresponding total soluble solids.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... with the minimum ratio of total soluble solids to anhydrous citric acid: Table II Total soluble solids (average pct) Maximum anhydrous citric acid (average pct) Minimum ratio of total soluble solids to... 1.027 9.25-1 9.6 1.043 9.20-1 9.7 1.060 9.15-1 9.8 1.077 9.10-1 9.9 1.094 9.05-1 10.0 1.111...

  17. Indole diterpene synthetic studies. Total synthesis of (+)-nodulisporic acid F and construction of the heptacyclic cores of (+)-nodulisporic acids A and B and (-)-nodulisporic acid D.

    PubMed

    Smith, Amos B; Davulcu, Akin H; Cho, Young Shin; Ohmoto, Kazuyuki; Kürti, László; Ishiyama, Haruaki

    2007-06-22

    A first-generation strategy for construction of (+)-nodulisporic acids A (1) and B (2) is described. The strategy entails union of the eastern and western hemisphere subtargets via the indole synthesis protocol developed in our laboratory. Subsequent elaboration of rings E and F, however, revealed the considerable acid instability of the C(24) hydroxyl, thereby preventing further advancement. Nonetheless, preparation of the heptacyclic core of (+)-nodulisporic acids A and B, the total synthesis of (+)-nodulisporic acid F, the simplest member of the nodulisporic acid family, and elaboration of the heptacyclic core of (-)-nodulisporic acid D were achieved.

  18. Multisite clickable modification of proteins using lipoic acid ligase.

    PubMed

    Plaks, Joseph G; Falatach, Rebecca; Kastantin, Mark; Berberich, Jason A; Kaar, Joel L

    2015-06-17

    Approaches that allow bioorthogonal and, in turn, site-specific chemical modification of proteins present considerable opportunities for modulating protein activity and stability. However, the development of such approaches that enable site-selective modification of proteins at multiple positions, including internal sites within a protein, has remained elusive. To overcome this void, we have developed an enzymatic approach for multisite clickable modification based on the incorporation of azide moieties in proteins using lipoic acid ligase (LplA). The ligation of azide moieties to the model protein, green fluorescent protein (GFP), at the N-terminus and two internal sites using lipoic acid ligase was shown to proceed efficiently with near-complete conversion. Modification of the ligated azide groups with poly(ethylene glycol) (PEG), α-d-mannopyranoside, and palmitic acid resulted in highly homogeneous populations of protein-polymer, protein-sugar, and protein-fatty acid conjugates. The homogeneity of the conjugates was confirmed by mass spectrometry (MALDI-TOF) and SDS-PAGE electrophoresis. In the case of PEG attachment, which involved the use of strain-promoted azide-alkyne click chemistry, the conjugation reaction resulted in highly homogeneous PEG-GFP conjugates in less than 30 min. As further demonstration of the utility of this approach, ligated GFP was also covalently immobilized on alkyne-terminated self-assembled monolayers. These results underscore the potential of this approach for, among other applications, site-specific multipoint protein PEGylation, glycosylation, fatty acid modification, and protein immobilization.

  19. Retardation signal for fluorescent determination of total protein content via rapid and sensitive chip moving reaction boundary electrophoretic titration.

    PubMed

    Wang, Houyu; Shi, Yongting; Yan, Jian; Dong, Jingyu; Li, Si; Xiao, Hua; Xie, Haiyang; Fan, Liu-Yin; Cao, Cheng-Xi

    2014-03-18

    A novel concept and theory of moving reaction boundary (MRB) retardation signal (RMRB) was advanced for determination of total protein content via MRB electrophoretic titration (MRBET). The theoretical results revealed that the retardation extent of boundary displacment, viz., the RMRB value, was as a function of protein content. Thus, the RMRB value of a sample could be used to determine its total protein content according to the relevant calibration curve. To demonstrate the concept and theoretical results, a novel microdevice was designed for the relevant experiments of MRBET. The microdevice has 30 identical work cells, each of which is composed of five ultrashort single microchannels (5 mm). In the microdevice, fluorescein isothiocyanate (FITC) was used to denote MRB motion and RMRB value for the first time, the polyacrylamide gel (PAG) containing protein sample was photopolymerized in microchannels, and the MRB was created with acid or alkali and target protein sample. As compared to the classic Kjeldahl method and conventional MRBET performed in glass tube, the developed titration chip has the following merits: good sensitivity (0.3-0.4 μg/mL vs 150-200 μg/mL of protein concentration, 0.6-0.8 ng vs 30-2000 μg of absolute protein content), rapid analysis (20-60 s vs 15-200 min), and portable low-power (15 V vs 200 V).

  20. Nitrogen effects on proteins, chlorophylls and fatty acids during the growth of Arthrospira platensis.

    PubMed

    Ayachi, Samah; El Abed, Amor; Dhifi, Wissal; Marzouk, Brahim

    2007-06-01

    Spirulina platensis (=Arthrospira platensis) is a tunisian strain which has been isolated for the first time in Oued Essed (Sousse, Sidi Bou Ali). Biomass evolution, proteins, chlorophylls and fatty acids composition of this alga were monitored by varying nitrogen concentrations in the culture medium. Nitrogen stress was provoked by adding sodium nitrate (NaNO3) in the culture medium with concentrations varying from 0 to 5 g/l. Results obtained showed that nitrogen depletion increased total proteins and total chlorophylls. The addition of NaNO3 (5g/l) led to an increase of total fatty acids amounts and modify fatty acids composition. Optimal quantities of palmitic, gamma -linolenic and oleic acids were obtained with NaNO3 free-cultures. Thus, the tunisian strain has valuable biological substances, worthy to determine the optimal conditions for its propagation.

  1. [Determination of total aminothiols and neuroactive amino acids in plasma by high performance liquid chromatography with fluorescence detection].

    PubMed

    Pozdeev, V K; Pozdeev, N V

    2010-01-01

    This paper describes a simple and sensitive reversed-phase HPLC method for the determination of total homocysteine, total cysteine, total glutathione (GSH+GSSG), and neuroactive amino acids (Asp, Glu, Tau, GABA) using precolumn derivatization with ortho-phtaldialdehyde and fluorimetric detection at 360 and 470 nm for emission and excitation, respectively. Derivatization was performed with ortho-phthaldialdehyde in the presence of 2-mercaptoethanol after alkylation of the free sulfhydryl groups with iodoacetic acid. For determination of total aminothiols, the disulfide bonds were reduced and protein-bound thiols were released by addition of dithiothreitol to the plasma sample. The advantage of this method is the simultaneous determination of both homocysteine/cysteine/glutathione and neuroactive amino acids in the sample. The plasma levels of studied compounds were determined in 14 healthy volunteers (20-45 years old) and 55 patients with chronic hepatitis C (20-49 years old) and the resulting numbers were in a good agreement the studies published earlier. The calibration curves were linear over a concentration range of 5-100 microM in plasma (r2 = 0.985-0.996). The intraday and interday coefficients of variation were 3-6% and 4-7%, respectively. The recovery of the standards added to the plasma samples ranged from 94 to 102%. The limits of detection (LOD) were 0.2-0.5 ng per 10 microl injection volume (signal-to-noise ratio of 3).

  2. Comparison of total protein and phospholipase A(2) levels in individual coralsnake venoms.

    PubMed

    Kopper, Randall A; Harper, George R; Zimmerman, Sloane; Hook, Jessica

    2013-12-15

    Studies of differences or changes in venom protein levels or enzymatic activities have significance only if contrasted to the normal variations between individual snakes. This study involves the analysis and comparison of venom from 13 individual Texas coralsnakes (Micrurus tener tener) in order to detect differences in the volume, total protein concentration, electrophoretic profile, and PLA2 enzyme activity. A significant inverse correlation between venom volume and total protein concentration was found. Although the 13 venoms were indistinguishable from their electrophoretic protein profiles, phospholipase A2 enzymatic activities varied considerably.

  3. Evaluation, Validation, and Demonstration of a Total Protein Assay for Application to Biotoxin Fate Studies

    DTIC Science & Technology

    2007-10-01

    Laboratories, Burlingame, CA at 5.0 mg/mL in 10 mM phosphate, 0.15 M NaCI, 0.08% sodium azide solution, pH 7.8. Lysozyme from chicken egg white was purchased...mg/L of total protein. 15. SUBJECT TERMS Total Protein Ricin SEB BSA Lysozyme Biotoxin Fate COTS Micro BCA Assay Coomassieg Plus Protein Assay...protein response for bovine serum albumin (BSA), staphylococcal enterotoxin B (SEB), and lysozyme in the desired concentration range of 1 to 25 mg/L

  4. Los Alamos sequence analysis package for nucleic acids and proteins.

    PubMed Central

    Kanehisa, M I

    1982-01-01

    An interactive system for computer analysis of nucleic acid and protein sequences has been developed for the Los Alamos DNA Sequence Database. It provides a convenient way to search or verify various sequence features, e.g., restriction enzyme sites, protein coding frames, and properties of coded proteins. Further, the comprehensive analysis package on a large-scale database can be used for comparative studies on sequence and structural homologies in order to find unnoted information stored in nucleic acid sequences. PMID:6174934

  5. Effects of fatty acid supplements on ruminal and total tract nutrient digestion in lactating dairy cows.

    PubMed

    Harvatine, K J; Allen, M S

    2006-03-01

    Saturated and unsaturated fatty acid supplements (FS) were evaluated for effects on ruminal digestion kinetics, and ruminal and postruminal nutrient digestion. Eight early lactation ruminally and duodenally cannulated cows (77 +/- 12 days in milk, mean +/- SD) were used in a replicated 4 x 4 Latin square design experiment with 21-d periods. Treatments were control and a linear substitution of 2.5% fatty acids from supplemented saturated FS (SAT; prilled, hydrogenated free fatty acids) for partially unsaturated FS (UNS; calcium soaps of long-chain fatty acids). All rations contained identical forage and concentrate components including 37.2% forage and 13.5% cottonseed. Saturated FS linearly decreased ruminal digestibility of dry matter and organic matter and linearly decreased ruminal neutral detergent fiber (NDF) digestibility. The reduction in ruminal NDF digestibility was because of a linear decrease in digestion rate and a linear increase in passage rate of potentially digestible NDF with increasing saturated FS. Total tract digestibility of NDF was not different between treatments because of compensatory postruminal digestion. Ruminal fatty acid and C18 fatty acid digestibility tended to increase linearly with increasing unsaturated FS, and postruminal C18 fatty acid digestibility decreased with increasing saturated FS. Saturated FS linearly decreased ruminal organic matter digestibility and decreased intestinal long-chain fatty acid digestibility, although differences in fatty acid digestibility may be partially explained by fatty acid intake.

  6. Amino acid sequences of proteins from Leptospira serovar pomona.

    PubMed

    Alves, S F; Lefebvre, R B; Probert, W

    2000-01-01

    This report describes a partial amino acid sequences from three putative outer envelope proteins from Leptospira serovar pomona. In order to obtain internal fragments for protein sequencing, enzymatic and chemical digestion was performed. The enzyme clostripain was used to digest the proteins 32 and 45 kDa. In situ digestion of 40 kDa molecular weight protein was accomplished using cyanogen bromide. The 32 kDa protein generated two fragments, one of 21 kDa and another of 10 kDa that yielded five residues. A fragment of 24 kDa that yielded nineteen residues of amino acids was obtained from 45 kDa protein. A fragment with a molecular weight of 20 kDa, yielding a twenty amino acids sequence from the 40 kDa protein.

  7. Dietary exposure estimates for the food preservatives benzoic acid and sorbic acid in the total diet in Taiwan.

    PubMed

    Ling, Min-Pei; Lien, Keng-Wen; Wu, Chiu-Hua; Ni, Shih-Pei; Huang, Hui-Ying; Hsieh, Dennis P H

    2015-02-25

    The purpose was to assess the health risk to general consumers in Taiwan associated with dietary intake of benzoic acid and sorbic acid by conducting a total diet study (TDS). The hazard index (HI) in percent acceptable daily intake (%ADI) of benzoic acid and sorbic acid for eight exposure groups classified by age were calculated. In high-intake consumers, the highest HI of benzoic acid was 54.1%ADI for males aged 1-2 years old at the 95th percentile, whereas for females, the HI was 61.7%ADI for aged over 66 years old. The highest HI of sorbic acid for male and female consumers aged 3-6 years old at the 95th percentile were 14.0%ADI and 12.2%ADI, respectively. These results indicate that the use of benzoic acid and sorbic acid as preservatives at the current level of use in the Taiwanese diet does not constitute a public health and safety concern.

  8. Serum amino acid concentrations in patients receiving total parenteral nutrition with an amino acid plus dextrose mixture.

    PubMed

    Philcox, J C; Hartley, T F; Worthley, L I; Thomas, D W

    1984-01-01

    The results of monitoring the serum amino acid concentrations during three infusion regimens using a 5:4 mixture of 70% glucose and the synthetic L-amino acid solution, Synthamin 17 (Travasol) are reported. Twelve stabilized patients received continuous total parenteral nutrition (TPN), eight of whom were subsequently placed on a second regimen of cyclical feeding. A separate group of five patients was infused with amino acids, both with and without simultaneous glucose. The serum amino acid concentrations indicated that the supply of valine, leucine, isoleucine, lysine, and histidine, and the synthesis of taurine from the infused methionine was suboptimal, particularly if the period of TPN was prolonged. The synthesis of tyrosine from phenylalanine appeared to be inversely proportional to the infusion rate of the TPN mixture, in particular the glucose component, resulting in depressed tyrosine and increased phenylalanine concentrations in serum during continuous iv nutrition. Cyclical infusions, on the other hand, permitted the tyrosine and phenylalanine concentrations to return to normal during the noninfusion stage of the cycle. Amino acid measurements enabled us to design an amino acids additive mixture which normalized the serum concentrations in three long-term home parenteral nutrition patients. As a result of these investigations serum amino acid measurements are used routinely to monitor the efficacy of TPN and accommodate any specific amino acid requirements of individual patients.

  9. Comparison of Free Total Amino Acid Compositions and Their Functional Classifications in 13 Wild Edible Mushrooms.

    PubMed

    Sun, Liping; Liu, Qiuming; Bao, Changjun; Fan, Jian

    2017-02-24

    Thirteen popular wild edible mushroom species in Yunnan Province, Boletus bicolor, Boletus speciosus, Boletus sinicus, Boletus craspedius, Boletus griseus, Boletus ornatipes, Xerocomus, Suillus placidus, Boletinus pinetorus, Tricholoma terreum, Tricholomopsis lividipileata, Termitomyces microcarpus, and Amanita hemibapha, were analyzed for their free amino acid compositions by online pre-column derivazation reversed phase high-performance liquid chromatography (RP-HPLC) analysis. Twenty free amino acids, aspartic acid, glutamic acid, serine, glycine, alanine, praline, cysteine, valine, methionine, phenylalanine, isoleucine, leucine, lysine, histidine, threonine, asparagines, glutamine, arginine, tyrosine, and tryptophan, were determined. The total free amino acid (TAA) contents ranged from 1462.6 mg/100 g in B. craspedius to 13,106.2 mg/100 g in T. microcarpus. The different species showed distinct free amino acid profiles. The ratio of total essential amino acids (EAA) to TAA was 0.13-0.41. All of the analyzed species showed high contents of hydrophobic amino acids, at 33%-54% of TAA. Alanine, cysteine, glutamine, and glutamic acid were among the most abundant amino acids present in all species. The results showed that the analyzed mushrooms possessed significant free amino acid contents, which may be important compounds contributing to the typical mushroom taste, nutritional value, and potent antioxidant properties of these wild edible mushrooms. Furthermore, the principal component analysis (PCA) showed that the accumulative variance contribution rate of the first four principal components reached 94.39%. Cluster analysis revealed EAA composition and content might be an important parameter to separate the mushroom species, and T. microcarpus and A. hemibapha showed remarkable EAA content among the 13 species.

  10. Kainic acid inhibits protein amino acid incorporation in select rat brain regions.

    PubMed

    Planas, A M; Soriano, M A; Ferrer, I; Rodríguez-Farré, E

    1994-11-21

    Regional incorporation of labelled methionine into proteins was studied with quantitative autoradiography in different regions of the rat brain 2.5 h following systemic kainic acid administration. Labelled protein concentration was found reduced to approximately 40% of control values in the pyramidal cell layer of hippocampus, piriform, entorhinal and perirhinal cortices, ventral lateral septum and mediodorsal thalamic nucleus. These regions showed increased levels of label not incorporated into proteins, indicating that free labelled methionine was available for protein synthesis. Reduction of protein amino acid incorporation in those brain regions selectively affected by kainic acid may be involved in subsequent tissue damage.

  11. Amino acid metabolism and protein synthesis in malarial parasites*

    PubMed Central

    Sherman, I. W.

    1977-01-01

    Malaria-infected red cells and free parasites have limited capabilities for the biosynthesis of amino acids. Therefore, the principal amino acid sources for parasite protein synthesis are the plasma free amino acids and host cell haemoglobin. Infected cells and plasmodia incorporate exogenously supplied amino acids into protein. However, the hypothesis that amino acid utilization (from an external source) is related to availability of that amino acid in haemoglobin is without universal support: it is true for isoleucine and for Plasmodium knowlesi and P. falciparum, but not for methionine, cysteine, and other amino acids, and it does not apply to P. lophurae. More by default than by direct evidence, haemoglobin is believed to be the main amino acid reservoir available to the intraerythrocytic plasmodium. Haemoglobin, ingested via the cytostome, is held in food vacuoles where auto-oxidation takes place. As a consequence, haem is released and accumulates in the vacuole as particulate haemozoin (= malaria pigment). Current evidence favours the view that haemozoin is mainly haematin. Acid and alkaline proteases (identified in crude extracts from mammalian and avian malarias) are presumably secreted directly into the food vacuole. They then digest the denatured globin and the resulting amino acids are incorporated into parasite protein. Cell-free protein synthesizing systems have been developed using P. knowlesi and P. lophurae ribosomes. In the main these systems are typically eukaryotic. Studies of amino acid metabolism are exceedingly limited. Arginine, lysine, methionine, and proline are incorporated into protein, whereas glutamic acid is metabolized via an NADP-specific glutamic dehydrogenase. Glutamate oxidation generates NADPH and auxiliary energy (in the form of α-ketoglutarate). The role of red cell glutathione in the economy of the parasite remains obscure. Important goals for future research should be: quantitative assessment of the relative importance of

  12. [Amino acid composition and biologic value of the proteins of several sorts of buckwheat].

    PubMed

    Sarkisova, N E; Kirilenko, S K

    1976-01-01

    The amino acids composition of summary proteins in unground buckwheat of four common and promising varieties grown in the Ukraine was investigated by using ion-exchange chromatography with an automatic analyzor Hd-1200 E. Between individual varieties of buckweheat no essential differences in the amino acids content were in evidence. The total proteins of the buckwheat grit contain high quantities of lysine, treonine, leucine, glutamic acid and arginine. The amino acids score was instrumental in determining the biological value and in eliciting amino acids limiting this value in different grits. These data may be made use of in the practice of public catering for estimating formulae of meals prepared with grits differently combined with other products securing an improved amino acids composition of ready-to-eat meals.

  13. Total synthesis of racemic and (R) and (S)-4-methoxyalkanoic acids and their antifungal activity.

    PubMed

    Das, Biswanath; Shinde, Digambar Balaji; Kanth, Boddu Shashi; Kamle, Avijeet; Kumar, C Ganesh

    2011-07-01

    The total synthesis of 4-methoxydecanoic acid and 4-methoxyundecanoic acid in racemic and stereoselective [(R) and (S)] forms has been accomplished. For stereoselective synthesis of the compounds (S) and (R)-BINOL complexes have been used to generate the required chiral centres. The antifungal activity of these compounds has been studied against different organisms and the results were found to be impressive. The activity of the compounds in racemic and in stereoselective forms was compared. (R)-4-Methoxydecanoic acid was found to be most potent (MIC: 0.019 mg/mL against Candida albicans MTCC 227, C. albicans MTCC 4748, Aspergillus brasiliensis (niger) MTCC 281 and Issatchenkia orientalis MTCC 3020).

  14. Fluorimetric determination of total ascorbic acid by a stopped-flow mixing technique.

    PubMed

    Pérez-Ruiz, T; Martínez-Lozano, C; Tomás, V; Fenoll, J; Fenol, J

    2001-08-01

    A simple, rapid and automatic fluorimetric method for the determination of total ascorbic acid is described. The method makes use of the stopped-flow mixing technique in order to achieve the rapid oxidation of ascorbic acid by dissolved oxygen to dehydroascorbic acid, which then reacts with o-phenylenediamine to form a fluorescent quinoxaline. The initial rate and fluorescence signal of this system are directly proportional to the ascorbic acid concentration. The calibration graph was linear over the range 0.1-30 microg ml(-1) (kinetic method) and 0.25-34 microg ml(-1) (equilibrium method). The precision (% RSD) was close to 0.5%. The method has been used for the determination of ascorbic acid in pharmaceutical formulations, fruit juices, soft drinks and blood serum.

  15. KINETICS OF AMINO ACID INCORPORATION INTO SERUM PROTEINS

    PubMed Central

    Green, H.; Anker, H. S.

    1955-01-01

    1. The effect of varying body temperature on the rate of amino acid incorporation into serum protein does not give support to the idea that the rate of this process is adjusted in vivo to restore those protein molecules destroyed by thermal denaturation. The experimentally observed Q10 was about 3.9. 2. When amino acids are injected into the blood of animals in a steady state of serum protein turnover, a period of time elapses before these amino acids can be found in the serum proteins. This has been called transit time. At a given temperature (31°) it is the same in rabbits, turtles, and Limulus (1 hour). In rabbits and turtles it has a Q10 of 3.2. It appears to be specifically related to the process of synthesis (or release) of serum proteins. 3. It was not possible to affect the transit time or the incorporation rate by the administration of amino acid analogues. PMID:13221773

  16. 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.

  17. Effects of gamma irradiation on chickpea seeds vis-a-vis total seed storage proteins, antioxidant activity and protein profiling.

    PubMed

    Bhagyawant, S S; Gupta, N; Shrivastava, N

    2015-10-23

    The present work describes radiation—induced effects on seed composition vis—à—vis total seed proteins, antioxidant levels and protein profiling employing two dimensional gel electrophoresis (2D—GE) in kabuli and desi chickpea varities. Seeds were exposed to the radiation doses of 1,2,3,4 and 5 kGy. The total protein concentrations decreased and antioxidant levels were increased with increasing dose compared to control seed samples. Radiation induced effects were dose dependent to these seed parameters while it showed tolerance to 1 kGy dose. Increase in the dose was complimented with increase in antioxidant levels, like 5 kGy enhanced % scavenging activities in all the seed extracts. Precisely, the investigations reflected that the dose range from 2 to 5 kGy was effective for total seed storage proteins, as depicted quantitatively and qualitative 2D—GE means enhance antioxidant activities in vitro.

  18. Review of methods for determination of total protein and peptide concentration in biological samples.

    PubMed

    Sapan, Christine V; Lundblad, Roger L

    2015-04-01

    Clinical proteomics can be defined as the use of proteomic technologies to identify and measure biomarkers in fluids and tissues. The current work is intended to review various methods used for the determination of the total concentration of protein or peptide in fluids and tissues and the application of such methods to clinical proteomics. Specifically, this article considers the approaches to the measurement of total protein concentration, not the measurement of the concentration of a specific protein or group of proteins in a larger mixture of proteins. The necessity of understanding various concepts such as fit-for-use, quality-by-design, and other regulatory elements is discussed, as is the significance of using suitable standards for the protein quality of various samples.

  19. Comparison study between fasting total serum bile acid and post prandial bile acid in hepatic diseases: a preliminary study.

    PubMed

    Boonyapisit, S; Lekhakula, S; Amornkittichareon, B; Shumnumsirivath, D

    1994-01-01

    Fasting bile acid, two-hour post prandial bile acid and other liver function tests (Bili, AST, ALT, ALB, Glob, ALP) were measured in 22 normal and 28 liver diseased patients. In normal volunteers, the mean value of fasting total serum bile acid (FTBA) and postprandial serum bile acid (PTBA) were 3.08 mumole/L (S.D. 1.65) range 0.21-6.26 mumol/L, and 8.07 mumole/L (S.D. 2.99) range 4.06-15.65 mumole/L. Comparison between FTBA, PTBA and other liver function tests in various liver diseases from this study the PTBA was not statistically significant superior to FTBA. Therefore, it is not necessary to do the PTBA at this time until more data is available.

  20. Common housekeeping proteins are upregulated in colorectal adenocarcinoma and hepatocellular carcinoma, making the total protein a better "housekeeper"

    PubMed Central

    Yu, Jiekai; Yang, Xuhan; Yang, Chao; Zhou, Daizhan; Wang, Qingyu; Qin, Shengying; Yan, Xiaomei; He, Lin; Han, Dongmei; Wan, Chunling

    2016-01-01

    Housekeeping proteins are essential endogenous controls for normalization as they are expected to be stably expressed. However, the stability of the expression level of housekeeping proteins needs to be assessed considering various experimental conditions. Our study evaluated the degree of variability of 7 commonly used housekeeping proteins with regard to their potential utility as normalizers in 56 pairs of matched colorectal adenocarcinoma (CRC) tissue samples and 6 pairs of hepatocellular carcinoma (HCC) tissue samples using multiple reaction monitoring (MRM) and Western blot analyses. A comprehensive experimental design and strict statistical analysis revealed that the expression levels of these 7 housekeeping proteins were not as stable as expected and they all exhibited upregulations to varying degrees in both the CRC and the HCC tissue samples. Consequently, we verified that using the amount of total protein instead of that of an individual protein can serve as a preferable control for studies of protein expression that require normalization. PMID:27556505

  1. Effect of water salinity on total protein and electrophoretic pattern of serum proteins of grass carp, Ctenopharyngodon idella

    PubMed Central

    Peyghan, Rahim; Khadjeh, Gholam Hosain; Enayati, Ala

    2014-01-01

    In this study the effects of water salinity on serum total protein and its components in grass carp were investigated. The aim of this study was to determine the effect of salinity tolerance of fish on total serum protein level and its components as an indicator of liver and kidney activity. One hundred and twenty grass carp were divided into four groups, randomly. The first three groups were reared in concentration of 4, 8 and 12 g L-1 of salt solution, respectively, and the fourth group was reared in freshwater and served as control. After 3 weeks, blood samples were collected and after harvesting the blood serum, serum total protein and protein components were measured with Biuret and electrophoresis methods, respectively. Results showed that mean value of serum total protein in the control and three salinities groups were 2.75, 3.28, 2.90 and 3.13 g dL-1, respectively. Five fractions of serum protein were electrophoretically observed as: albumin (Alb), alpha-1 globulin (α1-glu), alpha-2 globulin (α2-glu), beta globulin (β-glu) and gamma globulin (γ-glu). There were not any significant differences between the average mean of serum total protein of experimental and control groups (p > 0.05). However, Alb, α1-glu and β-glu levels in the experimental groups were significantly higher than in the control group (p < 0.05). The average of α2-glu and γ-glu revealed no significant difference between the experimental groups (p > 0.05). In conclusion, our results showed that increasing water salinity could have a significant effect on Alb, α1-glu and β-glu levels but not on total serum protein in grass carp. PMID:25568723

  2. Economic impact of tranexamic acid in healthy patients undergoing primary total hip and knee arthroplasty.

    PubMed

    Gillette, Blake P; Maradit Kremers, Hilal; Duncan, Christopher M; Smith, Hugh M; Trousdale, Robert T; Pagnano, Mark W; Sierra, Rafael J

    2013-09-01

    Tranexamic acid (TA) has been shown to reduce perioperative blood loss and blood transfusion. While concern remains about the cost of antifibrinolytic medication, we hypothesized that routine use of tranexamic acid would result in lower direct hospital total cost by decreasing costs associated with blood transfusion, laboratory testing, and room & board. Patients with an American Society of Anesthesiologists (ASA) class II or less undergoing primary total hip or knee arthroplasty at a single institution during 2007-2008 were retrospectively reviewed. The estimated mean direct hospital total cost, operating room, blood/lab, room & board, and pharmacy costs were compared between patients who did and did not receive TA. The study population included 1018 patients, and 580 patients received TA. The mean direct total cost of hospitalization with and without TA was $15,099 and $15,978 (P<.0002) respectively, a difference of $879. The only increased cost associated with TA was the pharmacy cost which was $921 versus $781 (P<.0001). The routine use of tranexamic acid TA was associated with lower mean direct hospital total costs after primary total hip and knee arthroplasty as the increase in pharmacy costs was more than offset by cost savings in other categories.

  3. Amino acid composition of proteins reduces deleterious impact of mutations

    PubMed Central

    Hormoz, Sahand

    2013-01-01

    The evolutionary origin of amino acid occurrence frequencies in proteins (composition) is not yet fully understood. We suggest that protein composition works alongside the genetic code to minimize impact of mutations on protein structure. First, we propose a novel method for estimating thermodynamic stability of proteins whose sequence is constrained to a fixed composition. Second, we quantify the average deleterious impact of substituting one amino acid with another. Natural proteome compositions are special in at least two ways: 1) Natural compositions do not generate more stable proteins than the average random composition, however, they result in proteins that are less susceptible to damage from mutations. 2) Natural proteome compositions that result in more stable proteins (i.e. those of thermophiles) are also tuned to have a higher tolerance for mutations. This is consistent with the observation that environmental factors selecting for more stable proteins also enhance the deleterious impact of mutations. PMID:24108121

  4. Novel approaches using alkaline or acid/guanidine treatment to eliminate therapeutic antibody interference in the measurement of total target ligand.

    PubMed

    Salimi-Moosavi, Hossein; Lee, Jean; Desilva, Binodh; Doellgast, George

    2010-04-06

    Measurement of the total target ligand can help to provide pharmacokinetic (PK) and pharmacodynamic (PD) informations. However, the presence of monocloncal antibody therapeutics (ThAs) interferes with ELISA determinations of the total target proteins. The interferences can cause over- or under-estimation of the target protein analysis. The nature of interferences was dependent upon the ThA, target protein, antibody reagents and assay conditions of the ELISA. We have developed novel alkaline and acid/guanidine treatment approaches to dissociate the protein binding and preferentially denature the ThA. The neutralized target proteins can be determined by ELISA. These methods provide reproducible measurements of total target protein without ThA interference. Serum samples, standards and QCs containing target protein and ThA were treated with alkaline buffer (pH>13) containing casein or acid/guanidine buffer (pH<1). Total target proteins for two different ThA systems were successfully measured and interferences were completely eliminated by the treatments. These methods were successfully applied to analysis in pre-clinical serum samples.

  5. Serum protein concentration in low-dose total body irradiation of normal and malnourished rats.

    PubMed

    Viana, W C M; Lambertz, D; Borges, E S; Neto, A M O; Lambertz, K M F T; Amaral, A

    2016-12-01

    Among the radiotherapeutics' modalities, total body irradiation (TBI) is used as treatment for certain hematological, oncological and immunological diseases. The aim of this study was to evaluate the long-term effects of low-dose TBI on plasma concentration of total protein and albumin using prematurely and undernourished rats as animal model. For this, four groups with 9 animals each were formed: Normal nourished (N); Malnourished (M); Irradiated Normal nourished (IN); Irradiated Malnourished (IM). At the age of 28 days, rats of the IN and IM groups underwent total body gamma irradiation with a source of cobalt-60. Total protein and Albumin in the blood serum was quantified by colorimetry. This research indicates that procedures involving low-dose total body irradiation in children have repercussions in the reduction in body-mass as well as in the plasma levels of total protein and albumin. Our findings reinforce the periodic monitoring of total serum protein and albumin levels as an important tool in long-term follow-up of pediatric patients in treatments associated to total body irradiation.

  6. Variability in phytic acid content and protein digestibility of grain legumes.

    PubMed

    Chitra, U; Vimala, V; Singh, U; Geervani, P

    1995-02-01

    Several genotypes, number given within parenthesis, of chickpea, pigeonpea, urd bean, mung bean and soybean, differing in seed characteristics were analyzed for phytic acid, in vitro protein digestibility (IVPD), protein, total phosphorus, and seed size. Phytic acid contents and IVPD values differed significantly among and within these species. Phytic acid content (mg/g) was the highest in soybean (36.4) followed by urd bean (13.7), pigeonpea (12.7), mung bean (12.0) and chickpea (9.6). On an average, phytic acid constituted 78.2 percent of the total phosphorus content and this percentage figure was the highest in soybean and the lowest in mung bean. In vitro protein digestibility (IVPD) of pigeonpea and chickpea genotypes varied from 60.4 to 74.4 percent and 65.3 to 79.4 percent, respectively. The IVPD values of genotypes of mung bean, urd bean and soybean ranged from 67.2 to 72.2 percent, 55.7 to 63.3 percent and 62.7 to 71.6 percent, respectively. There was a significant negative correlation between phytic acid and IVPD of these genotypes. Phytic acid was significantly and positively correlated with protein but the magnitude of correlation was very low in chickpea and pigeonpea. Results indicate that the genotypes of pulses with low phytic acid content could be identified and used in breeding program to improve their nutritive value and utilization.

  7. The protein digestibility-corrected amino acid score.

    PubMed

    Schaafsma, G

    2000-07-01

    The protein digestibility-corrected amino acid score (PDCAAS) has been adopted by FAO/WHO as the preferred method for the measurement of the protein value in human nutrition. The method is based on comparison of the concentration of the first limiting essential amino acid in the test protein with the concentration of that amino acid in a reference (scoring) pattern. This scoring pattern is derived from the essential amino acid requirements of the preschool-age child. The chemical score obtained in this way is corrected for true fecal digestibility of the test protein. PDCAAS values higher than 100% are not accepted as such but are truncated to 100%. Although the principle of the PDCAAS method has been widely accepted, critical questions have been raised in the scientific community about a number of issues. These questions relate to 1) the validity of the preschool-age child amino acid requirement values, 2) the validity of correction for fecal instead of ileal digestibility and 3) the truncation of PDCAAS values to 100%. At the time of the adoption of the PDCAAS method, only a few studies had been performed on the amino acid requirements of the preschool-age child, and there is still a need for validation of the scoring pattern. Also, the scoring pattern does not include conditionally indispensable amino acids. These amino acids also contribute to the nutrition value of a protein. There is strong evidence that ileal, and not fecal, digestibility is the right parameter for correction of the amino acid score. The use of fecal digestibility overestimates the nutritional value of a protein, because amino acid nitrogen entering the colon is lost for protein synthesis in the body and is, at least in part, excreted in urine as ammonia. The truncation of PDCAAS values to 100% can be defended only for the limited number of situations in which the protein is to be used as the sole source of protein in the diet. For evaluation of the nutritional significance of proteins as

  8. Cassava interspecific hybrids with increased protein content and improved amino acid profiles.

    PubMed

    Gomes, P T C; Nassar, N M A

    2013-04-12

    Cassava (Manihot esculenta) is a principal food for large populations of poor people in the tropics and subtropics. Its edible roots are poor in protein and lack several essential amino acids. Interspecific hybrids may acquire high protein characteristics from wild species. We analyzed 19 hybrids of M. esculenta with its wild relative, M. oligantha, for crude protein, amino acid profile, and total cyanide. Some hybrids produced roots with high protein content of up to 5.7%, while the common cultivar that we examined had just 2.3% crude protein. The essential amino acids alanine, phenylalanine, and valine were detected in the hybrids. The sulfur-containing amino acids cysteine and methionine were found at relatively high concentrations in the roots of 4 hybrids. The proportion of lysine in one hybrid was 20 times higher than in the common cultivar. The levels of total cyanide ranged from 19.73 to 172.56 mg/kg and most of the roots analyzed were classified as "non-toxic" and "low toxic". Furthermore, 2 progenies showed reasonable levels of cyanide, but higher protein content and amino acid profile more advantageous than the common cassava.

  9. Four proteins synthesized in response to deoxyribonucleic acid damage in Micrococcus radiodurans.

    PubMed Central

    Hansen, M T

    1980-01-01

    Four proteins, alpha beta, gamma, and delta, preferentially synthesized in ultraviolet light-treated cells of Micrococcus radiodurans, were characterized in terms of their molecular weights and isoelectric points. Within the sublethal-dose range, the differential rate of synthesis for these proteins increased linearly with the inducing UV dose. The degree of induction reached 100-fold, and the most abundant protein beta, amounted to approximately 2% of the total newly synthesized protein after irradiation. Damage caused by ionizing radiation or by treatment with mitomycin C also provoked the synthesis of the four proteins. The proportions between the individual proteins, however, varied strikingly with the damaging agent. In contrast to treatments which introduced damage in the cellular deoxyribonucleic acid, the mere arrest of deoxyribonucleic acid replication, caused by nalidixic acid or by starvation for thymine, failed to elicit the synthesis of either protein. Repair of deoxyribonucleic acid damage requires that a number of versatile and efficient processes by employed. It is proposed that the induced proteins participate in deoxyribonucleic acid repair in M. radiodurans. Mechanisms are discussed which would allow a differentiated cellular response to damages of sufficiently distinctive nature. Images PMID:7354007

  10. [Materials for carrying out external quality control in the determination of total protein].

    PubMed

    Pishak, V P; Iarmol'chuk, G M

    1998-06-01

    Quaternary bis-ammonium compound No. 82 (QAC-82) is a highly effective antibacterial biochemical stabilizer of total protein. In concentrations of 3.4-13.6 mmol/liter, it preserves the content of protein in the blood serum for 40 days at 18-20 degrees C. For preparing reference material, 5 ml human or animal serum is added to 95 ml 5.25 mM aqueous solution of QAC-82, mixed, stored at room temperature, and used for assessing the quality of total protein measurements by the biuret method.

  11. Sex Steroid Modulation of Fatty Acid Utilization and Fatty Acid Binding Protein Concentration in Rat Liver

    PubMed Central

    Ockner, Robert K.; Lysenko, Nina; Manning, Joan A.; Monroe, Scott E.; Burnett, David A.

    1980-01-01

    The mechanism by which sex steroids influence very low density hepatic lipoprotein triglyceride production has not been fully elucidated. In previous studies we showed that [14C]oleate utilization and incorporation into triglycerides were greater in hepatocyte suspensions from adult female rats than from males. The sex differences were not related to activities of the enzymes of triglyceride biosynthesis, whereas fatty acid binding protein (FABP) concentration in liver cytosol was greater in females. These findings suggested that sex differences in lipoprotein could reflect a sex steroid influence on the availability of fatty acids for hepatocellular triglyceride biosynthesis. In the present studies, sex steroid effects on hepatocyte [14C]oleate utilization and FABP concentration were investigated directly. Hepatocytes from immature (30-d-old) rats exhibited no sex differences in [14C]oleate utilization. With maturation, total [14C]oleate utilization and triglyceride biosynthesis increased moderately in female cells and decreased markedly in male cells; the profound sex differences in adults were maximal by age 60 d. Fatty acid oxidation was little affected. Rats were castrated at age 30 d, and received estradiol, testosterone, or no hormone until age 60 d, when hepatocyte [14C]oleate utilization was studied. Castration virtually eliminated maturational changes and blunted the sex differences in adults. Estradiol or testosterone largely reproduced the appropriate adult pattern of [14C]oleate utilization regardless of the genotypic sex of the treated animal. In immature females and males, total cytosolic FABP concentrations were similar. In 60-d-old animals, there was a striking correlation among all groups (females, males, castrates, and hormone-treated) between mean cytosolic FABP concentration on the one hand, and mean total [14C]oleate utilization (r = 0.91) and incorporation into triglycerides (r = 0.94) on the other. In 30-d-old animals rates of [14C

  12. Contents of total fat, fatty acids, starch, sugars and dietary fibre in Swedish market basket diets.

    PubMed

    Becker, W; Eriksson, A; Haglund, M; Wretling, S

    2015-05-14

    The typical dietary supply of total fat, fatty acids, starch, sugars, polyols and dietary fibre in Sweden was assessed from analyses of market baskets (MB) purchased in 2005 and 2010. MB were based on food balance sheets, with each basket comprising about 130 foods, which represented more than 90% of annual dietary supply. Foods were divided into ten to twelve categories. In 2010, total fat contributed 34% of energy (E%), SFA 14.3 E%, MUFA 12.8 E%, PUFA 4.6 E%, n-6 fatty acids 3.6 E%, n-3 fatty acids 1.0 E% and trans-fatty acids (TFA) 0.5 E%. Glycaemic carbohydrates contributed 47 E%, monosaccharides 9 E%, sucrose 11 E%, disaccharides 15 E% and total sugars 24 E%. Added sugars contributed about 15 E%. Dietary fibre content was about 1.7 g/MJ in the 2010 MB. Compared with the 2005 MB, the dietary supply of TFA and dietary fibre was lower, otherwise differences were small. The present MB survey shows that the content of SFA and added sugars was higher than the current Nordic Nutrition Recommendations, while the content of PUFA and especially dietary fibre was lower. TFA levels decreased and dietary supply was well below the recommendations of the WHO. These results emphasise a focus on quality and food sources of fat and carbohydrates, limiting foods rich in SFA and added sugars and replacing them with foods rich in dietary fibre and cis-unsaturated fatty acids.

  13. Changes in total plasma content of electrolytes and proteins with maximal exercise.

    NASA Technical Reports Server (NTRS)

    Van Beaumont, W.; Strand, J. C.; Petrofsky, J. S.; Hipskind, S. G.; Greenleaf, J. E.

    1973-01-01

    To determine to what extent the increases in concentration of plasma proteins and electrolytes with short maximal work were a result of hemoconcentration, the changes in plasma volume and total content of the plasma constituents were simultaneously evaluated. The results obtained from six human subjects indicated that in comparison to preexercise values there was a net decrease in total content of plasma protein, sodium, and chloride in the first 2 min of the postexercise period, due primarily to a significant loss (13-15%) of plasma fluid. The total plasma potassium content was increased immediately after exercise but was significantly below the preexercise plasma content after 2 min of recovery.

  14. TOTAL PARTICLE, SULFATE, AND ACIDIC AEROSOL EMISSIONS FROM KEROSENE SPACE HEATERS

    EPA Science Inventory

    Chamber studies were conducted on four unvented kerosene space heaters to assess emissions of total particle, sulfate, and acidic aerosol. The heaters tested represented four burner designs currently in use by the public. Kerosene space heaters are a potential source of fine part...

  15. Total synthesis of (±)-epithuriferic acid methyl ester via Diels-Alder reaction.

    PubMed

    Koprowski, Marek; Bałczewski, Piotr; Owsianik, Krzysztof; Różycka-Sokołowska, Ewa; Marciniak, Bernard

    2016-02-07

    In this paper, we have described the first total synthesis of (±)-epithuriferic acid methyl ester from non-natural sources, in four steps (20% overall yield). The key step involves the Diels-Alder reaction of isobenzofuran with methyl 3-(dimethoxyphosphoryl)acrylate which is controlled by "ortho" regio- and endo stereoselectivities due to the COOMe group.

  16. Total synthesis of (−)-dihydroprotolichesterenic acid via diastereoselective conjugate addition to chiral fumarates

    PubMed Central

    Hethcox, J. Caleb; Shanahan, Charles S.; Martin, Stephen F.

    2013-01-01

    A diastereoselective conjugate addition of a variety of monoorganocuprates, Li[RCuI], to chiral fumarates to provide funtionalized succinates has been developed. The utility of this reaction is demonstrated in a concise total synthesis of (−)-dihydroprotolichesterenic acid that required only four steps and proceeded in an overall 31% yield. PMID:23539490

  17. 21 CFR 862.1020 - Acid phosphatase (total or prostatic) test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Acid phosphatase (total or prostatic) test system. 862.1020 Section 862.1020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  18. 21 CFR 862.1020 - Acid phosphatase (total or prostatic) test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Acid phosphatase (total or prostatic) test system. 862.1020 Section 862.1020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  19. 21 CFR 862.1020 - Acid phosphatase (total or prostatic) test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Acid phosphatase (total or prostatic) test system. 862.1020 Section 862.1020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  20. 21 CFR 862.1020 - Acid phosphatase (total or prostatic) test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Acid phosphatase (total or prostatic) test system. 862.1020 Section 862.1020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  1. 21 CFR 862.1020 - Acid phosphatase (total or prostatic) test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Acid phosphatase (total or prostatic) test system. 862.1020 Section 862.1020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  2. [Metabolic pathway and metabolites of total diterpene acid isolated from Pseudolarix kaempferi].

    PubMed

    Liu, Peng; Guo, Hong-Zhu; Sun, Jiang-Hao; Xu, Man; Guo, Hui; Sun, Shi-Feng; Guo, De-An

    2014-08-01

    The preliminary metabolic profile of total diterpene acid (TDA) isolated from Pseudolarix kaempferi was investigated by using in vivo and in vitro tests. Pseudolaric acid C2 (PC2) was identified as the predominant metabolite in plasma, urine, bile and feces after both oral and intravenous administrations to rats using HPLC-UV and HPLC-ESI/MS(n), and demethoxydeacetoxypseudolaric acid B (DDPB), a metabolite proposed to be the glucoside of PC2 (PC2G), as well as pseudolaric acid C (PC), pseudolaric acid A (PA), pseudolaric acid A O-beta-D glucopyranoside (PAG), pseudolaric acid B O-beta-D glucopyranoside (PBG) and deacetylpseudolaric acid A (DPA) originated from TDA could also be detected. It was demonstrated by tests that the metabolism of TDA is independent of intestinal microflora, and neither of pepsin and trypsin is in charge of metabolism of TDA, TDA is also stable in both pH environments of gastric tract and intestinal tract. The metabolites of TDA in whole blood in vitro incubation were found to be PC2, DDPB and PC2G, which demonstrated that the metabolic reaction of TDA in vivo is mainly occurred in blood and contributed to be the hydrolysis of plasma esterase to ester bond, as well as the glucosylation reaction. These results clarified the metabolic pathway of TDA for the first time, which is of great significance to the in vivo active form and acting mechanism research of P. kaempferi.

  3. Dietary protein and plasma total homocysteine, cysteine concentrations in coronary angiographic subjects

    PubMed Central

    2013-01-01

    Background Dietary patterns are associated with plasma total homocysteine (tHcy) concentrations in healthy populations, but the associations between dietary protein and tHcy, total cysteine (tCys) in high risk populations are unclear. We therefore examined the association between dietary protein and tHcy and tCys concentrations in coronary angiographic subjects. Methods We conducted a cross-sectional study of 1015 Chinese patients who underwent coronary angiography (40–85 y old). With the use of food-frequency questionnaires, we divided the total protein intakes into high animal-protein and high plant-protein diets. Circulating concentrations of tHcy and tCys were simultaneously measured by high-performance liquid chromatography with fluorescence detection. Results We found that high animal-protein diet was positively associated with hyperhomocysteinemia after adjustment for potential confounders, with the subjects in the highest quartile of intake having the greatest increase in risk (OR: 4.14, 95% CI: 2.67-6.43), whereas high plant-protein diet was inversely related to hyperhomocysteinemia, with a higher intake being protective. Compared with the first quartile of intake, the adjusted OR was 0.59 (95% CI: 0.38-0.91) for the fourth quartile. The total protein intake was positively associated with the risk of hypercysteinemia and the participants in highest quartile had significant OR of 1.69 (95% CI: 1.02-2.87) compared with those in lowest quartile. In multivariate linear regression analyses, high animal-protein and total-protein intakes were positively associated with plasma tHcy and tCys concentrations. The plant-protein intake was a negative determinant of plasma tHcy concentrations. Conclusions High animal-protein diet was positively associated with high tHcy concentrations, whereas high plant-protein diet was inversely associated with tHcy concentrations. Furthermore the total protein intake was strongly related to tCys concentrations. PMID:24195518

  4. Protein packing: dependence on protein size, secondary structure and amino acid composition.

    PubMed

    Fleming, P J; Richards, F M

    2000-06-02

    We have used the occluded surface algorithm to estimate the packing of both buried and exposed amino acid residues in protein structures. This method works equally well for buried residues and solvent-exposed residues in contrast to the commonly used Voronoi method that works directly only on buried residues. The atomic packing of individual globular proteins may vary significantly from the average packing of a large data set of globular proteins. Here, we demonstrate that these variations in protein packing are due to a complex combination of protein size, secondary structure composition and amino acid composition. Differences in protein packing are conserved in protein families of similar structure despite significant sequence differences. This conclusion indicates that quality assessments of packing in protein structures should include a consideration of various parameters including the packing of known homologous proteins. Also, modeling of protein structures based on homologous templates should take into account the packing of the template protein structure.

  5. Stable carbon isotopic compositions of organic acids in total suspended particles and dusts from Guangzhou, China

    NASA Astrophysics Data System (ADS)

    Ma, Shexia; Peng, Ping'an; Song, Jianzhong; Zhao, Jinping; He, Lulu; Sheng, Guoying; Fu, Jiamo

    2010-10-01

    Stable carbon isotopic compositions of individual organic acids were determined in total suspended particles and dusts from Guangzhou. The δ 13C values of high molecular weight n-alkanoic acids (C 20-C 28) varied from -34.1‰ to -32.4‰ and tended to be heavier in summer and lighter in winter. These δ 13C values indicate that high molecular weight n-alkanoic acids were derived mainly from emission by C 3 plants. Reduced biological synthesis of high molecular weight n-alkanoic acids in winter may be the reason for the light carbon isotopic composition. The δ 13C values of low molecular weight n-alkanoic acids (C 10-C 18) changed from -31.7‰ to -30.3‰ and exhibited a reverse seasonal trend, i.e., heavier in winter and lighter in summer. Slightly heavier δ 13C values of low molecular weight n-alkanoic acids than those of high molecular weight n-alkanoic acids suggested that they may be emitted from blended sources, e.g., anthropogenic sources and vegetation waxes. Lighter δ 13C values in summer may be attributed to relatively low anthropogenic sources and high botanic sources in summer. Dicarboxylic acids and aromatic acids have been proposed as secondary products from photochemical degradation. The average δ 13C values of dicarboxylic acids and aromatic acids were heavier, and ranged from -25.2‰ to -22.9‰ and from -30.0‰ to -27.6‰, respectively. Both dicarboxylic acids and aromatic acids displayed the same temporal variations in the δ 13C values, i.e., negative δ 13C in the summer samples and positive in the winter samples, which may be controlled by photochemical reactions; they are generally severe in winter in Guangzhou under the monsoon weather system. The heaviest δ 13C values were observed in dicarboxylic acids, indicating that dicarboxylic acids were formed by fast and more complete oxidation reactions. These results indicate that the stable carbon isotopic composition of organic acids may provide important information about sources and

  6. Re-evaluation of turbidimetry of proteins by use of aromatic sulfonic acids and chloroacetic acids.

    PubMed

    Ebina, S; Nagai, Y

    1979-02-01

    From studies on 11 different proteins (including native albumin and albumin with reduced disulfide-bridges) treated with sulfosalicylic, 2-naphthalenesulfonic, toluenesulfonic, dichloroacetic, or trichloroacetic acids, we elucidate the interactions determining the resulting turbidities and other factors affecting turbidities, and we discuss the clinical utility of such turbidimetry. At least three interactions are important in determining turbidity: reduction of positive charges on the protein, hydrogen bonding of the non-ionized chloroacetic acids with the protein, and hydrophobic interaction of the aromatic sulfonic acids with albumin. Turbidity varies appreciably with the species of acid and protein, concentrations of acid, temperature, and standing time after acid is added. We conclude that this technique should be restricted to confirming proteinuria.

  7. Fatty Acid-Binding Protein in Small Intestine IDENTIFICATION, ISOLATION, AND EVIDENCE FOR ITS ROLE IN CELLULAR FATTY ACID TRANSPORT

    PubMed Central

    Ockner, Robert K.; Manning, Joan A.

    1974-01-01

    A soluble fatty acid-binding protein (FABP), mol wt ∼ 12,000 is present in intestinal mucosa and other tissues that utilize fatty acids, including liver, myocardium, adipose, and kidney. This protein binds long chain fatty acids both in vivo and in vitro. FABP was isolated from rat intestine by gel filtration and isoelectric focusing. It showed a reaction of complete immunochemical identity with proteins in the 12,000 mol wt fatty acid-binding fractions of liver, myocardium, and adipose tissue supernates. (The presence of immunochemically nonidentical 12,000 mol wt FABP in these tissues is not excluded.) By quantitative radial immunodiffusion, supernatant FABP concentration in mucosa from proximal and middle thirds of jejuno-ileum significantly exceeded that in distal third, duodenum, and liver, expressed as micrograms per milligram soluble protein, micrograms per gram DNA, and micrograms per gram tissue. FABP concentration in villi was approximately three times greater than in crypts. Small quantities of FABP were present in washed nuclei-cell membrane, mitochondrial and microsomal fractions. However, the amount of FABP solubilized per milligram membrane protein was similar for all particulate fractions, and total membrane-associated FABP was only about 16% of supernatant FABP. Intestinal FABP concentration was significantly greater in animals maintained on high fat diets than on low fat; saturated and unsaturated fat diets did not differ greatly in this regard. The preponderance of FABP in villi from proximal and middle intestine, its ability to bind fatty acids in vivo as well as in vitro, and its response to changes in dietary fat intake support the concept that this protein participates in cellular fatty acid transport during fat absorption. Identical or closely related 12,000 mol wt proteins may serve similar functions in other tissues. Images PMID:4211161

  8. Comparison of total protein concentration in skeletal muscle as measured by the Bradford and Lowry assays.

    PubMed

    Seevaratnam, Rajini; Patel, Barkha P; Hamadeh, Mazen J

    2009-06-01

    The Lowry and Bradford assays are the most commonly used methods of total protein quantification, yet vary in several aspects. To date, no comparisons have been made in skeletal muscle. We compared total protein concentrations of mouse red and white gastrocnemius, reagent stability, protein stability and range of linearity using both assays. The Lowry averaged protein concentrations 15% higher than the Bradford with a moderate correlation (r = 0.36, P = 0.01). However, Bland-Altman analysis revealed considerable bias (15.8 +/- 29.7%). Both Lowry reagents and its protein-reagent interactions were less stable over time than the Bradford. The linear range of concentration was smaller for the Lowry (0.05-0.50 mg/ml) than the Bradford (0-2.0 mg/ml). We conclude that the Bradford and Lowry measures of total protein concentration in skeletal muscle are not interchangeable. The Bradford and Lowry assays have various strengths and weaknesses in terms of substance interference and protein size. However, the Bradford provides greater reagent stability, protein-reagent stability and range of linearity, and requires less time to analyse compared to the Lowry assay.

  9. Direct sensing of total acidity by chronopotentiometric flash titrations at polymer membrane ion-selective electrodes.

    PubMed

    Gemene, Kebede L; Bakker, Eric

    2008-05-15

    Polymer membrane ion-selective electrodes containing lipophilic ionophores are traditionally interrogated by zero current potentiometry, which, ideally, gives information on the sample activity of ionic species. It is shown here that a discrete cathodic current pulse across an H (+)-selective polymeric membrane doped with the ionophore ETH 5294 may be used for the chronopotentiometric detection of pH in well-buffered samples. However, a reduction in the buffer capacity leads to large deviations from the expected Nernstian response slope. This is explained by the local depletion of hydrogen ions at the sample-membrane interface as a result of the galvanostatically imposed ion flux in direction of the membrane. This depletion is found to be a function of the total acidity of the sample and can be directly monitored chronopotentiometrically in a flash titration experiment. The subsequent application of a baseline potential pulse reverses the extraction process of the current pulse, allowing one to interrogate the sample with minimal perturbation. In one protocol, total acidity is found to be proportional to the magnitude of applied current at the flash titration end point. More conveniently, the square root of the flash titration end point time observed at a fixed applied current is a linear function of the total acid concentration. This suggests that it is possible to perform rapid localized pH titrations at ion-selective electrodes without the need for volumetric titrimetry. The technique is explored here for acetic acid, MES and citric acid with promising results. Polymeric membrane electrodes based on poly(vinyl chloride) plasticized with o-nitrophenyl octyl ether in a 1:2 mass ratio may be used for the detection of acids of up to ca. 1 mM concentration, with flash titration times on the order of a few seconds. Possible limitations of the technique are discussed, including variations of the acid diffusion coefficients and influence of electrical migration.

  10. Amino acid composition and crude protein values of some Cyanobacteria from Çanakkale (Turkey).

    PubMed

    Akgül, Rıza; Kızılkaya, Bayram; Akgül, Füsun; Erduğan, Hüseyin

    2015-09-01

    Cyanobacteria (blue-green algae) form an important component of integrated nutrient managements in agriculture and are exploited in commercial biotechnological ventures. In this study, Rivularia bullata (Poir) Berkeley ex Bornet & Flahault, Nostocs pongiaeforme C. Agardh ex Bornet & Flahault were researched for their amino acid composition and crude protein values. R. bullata was collected from coastal zones of the Gulf of Saros and N. spongiaeforme from the Ayazma Stream. The levels of amino acids were measured in algae samples using EZ: fast kits (EZ: fast GC/FID Protein Hydrolysate Amino Acid Kit) by gas chromatography. The crude proteins of samples were determined by the Kjeldahl method and were calculated using a nitrogen conversion factor of 6.25. Thirty-two amino acids were investigated, for N. spongiaeforme eight free essential amino acids (EAA), eight free non-essential amino acids (NEAA) and eleven other amino acids (OAA); for R. bullata eight EAA, eight NEAA and eight OAA were detected. Aspartic acid is the major constituent for both species. The total protein percents were determined for N. spongiaeforme as % 19.83 and for R. bullata as % 6.15. When considering the increasing world population and reducing natural products; Cyanobacteria will benew feed sources for all living.

  11. BIOACTIVE PROTEINS, PEPTIDES, AND AMINO ACIDS FROM MACROALGAE(1).

    PubMed

    Harnedy, Pádraigín A; FitzGerald, Richard J

    2011-04-01

    Macroalgae are a diverse group of marine organisms that have developed complex and unique metabolic pathways to ensure survival in highly competitive marine environments. As a result, these organisms have been targeted for mining of natural biologically active components. The exploration of marine organisms has revealed numerous bioactive compounds that are proteinaceous in nature. These include proteins, linear peptides, cyclic peptides and depsipeptides, peptide derivatives, amino acids, and amino acid-like components. Furthermore, some species of macroalgae have been shown to contain significant levels of protein. While some protein-derived bioactive peptides have been characterized from macroalgae, macroalgal proteins currently still represent good candidate raw materials for biofunctional peptide mining. This review will provide an overview of the important bioactive amino-acid-containing compounds that have been identified in macroalgae. Moreover, the potential of macroalgal proteins as substrates for the generation of biofunctional peptides for utilization as functional foods to provide specific health benefits will be discussed.

  12. Free and total amino acid composition in blue-green algae.

    PubMed

    Campanella, Luigi; Russo, Mario Vincenzo; Avino, Pasquale

    2002-04-01

    A simple, accurate and reproducible analytical method is described for the extraction and the simultaneous determination of 18 amino acids in different for geographical origin Spirulina alga samples using phenylisothiocianate as derivatizating agent in natural feed. The best experimental hydrolysis conditions have been studied varying the temperature, the time and the hydrolyzing reagent. The separation and the quantitative analysis of the by-products have been carried out by HPLC analysis and UV detection. An amino acid pattern is compared with that proposed by the Food Agriculture Organization (FAO) for an ideal protein and with those of some traditional feed.

  13. 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

  14. Protein quality and digestibility of sorghum in preschool children: balance studies and plasma free amino acids.

    PubMed

    MacLeon, W C; Lopez de Romaña, G; Placko, R P; Graham, G G

    1981-11-01

    The protein quality and digestibility of two high lysine (2.9-3.0 g/100 g protein) and tow conventional varieties (lysine content 2.1-2.2 g/100 g protein) of whole grain sorghum milled as flour were assessed through balance studies in 13 children 6-30 months of age. Sorghum protein provided 6.4 or 8.0% of dietary energy. Control diets contained 64% kcal protein as casein. Children consumed 100-150 kcal/kg body weight/day. Sorghum consumption was associated with weight loss or poor weight gain. We found no difference by variety in apparent nitrogen absorption or retention. Mean absorption and retention of nitrogen (+/- SD) from 26 six-day sorghum dietary periods were 46 +/- 17% and 14 +/- 10% of intake, respectively (corresponding preceding casein control values: 81 +/- 5% and 38 +/- 3%). Stool weight and energy losses during sorghum periods averaged 2.5 to 3 times control values. Plasma amino acids were determined in eleven children after 16 days of sorghum consumption. Fasting concentration of total amino acids (TAA) was similar to values previously obtained with wheat protein at similar levels of intake. Total concentration of essential amino acids (TEAA) was low as were concentrations of lysine (Lys) and threonine (Thr). Analysis of postprandial changes of the Lys/TEAA and Thr/TEAA molar ratios confirmed that Lys was the first limiting amino acid.

  15. Protein turnover, amino acid profile and amino acid flux in juvenile shrimp Litopenaeus vannamei: effects of dietary protein source.

    PubMed

    Mente, Eleni; Coutteau, Peter; Houlihan, Dominic; Davidson, Ian; Sorgeloos, Patrick

    2002-10-01

    The effect of dietary protein on protein synthesis and growth of juvenile shrimps Litopenaeus vannamei was investigated using three different diets with equivalent protein content. Protein synthesis was investigated by a flooding dose of tritiated phenylalanine. Survival, specific growth and protein synthesis rates were higher, and protein degradation was lower, in shrimps fed a fish/squid/shrimp meal diet, or a 50% laboratory diet/50% soybean meal variant diet, than in those fed a casein-based diet. The efficiency of retention of synthesized protein as growth was 94% for shrimps fed the fish meal diet, suggesting a very low protein turnover rate; by contrast, the retention of synthesized protein was only 80% for shrimps fed the casein diet. The amino acid profile of the casein diet was poorly correlated with that of the shrimps. 4 h after a single meal the protein synthesis rates increased following an increase in RNA activity. A model was developed for amino acid flux, suggesting that high growth rates involve a reduction in the turnover of proteins, while amino acid loss appears to be high.

  16. Mitotic apparatus: the selective extraction of protein with mild acid.

    PubMed

    Bibring, T; Baxandall, J

    1968-07-26

    The treatment of isolated mitotic apparatus with mild (pH 3) hydrochloric acid results in the extraction of less than 10 percent of its protein, accompanied by the selective morphological disappearance of the microtubules. The same extraction can be shown to dissolve outer doublet microtubules from sperm flagella. A protein with points of similarity to the flagellar microtubule protein is the major component of the extract from mitotic apparatus.

  17. Manipulating Fatty Acid Biosynthesis in Microalgae for Biofuel through Protein-Protein Interactions

    PubMed Central

    Blatti, Jillian L.; Beld, Joris; Behnke, Craig A.; Mendez, Michael; Mayfield, Stephen P.; Burkart, Michael D.

    2012-01-01

    Microalgae are a promising feedstock for renewable fuels, and algal metabolic engineering can lead to crop improvement, thus accelerating the development of commercially viable biodiesel production from algae biomass. We demonstrate that protein-protein interactions between the fatty acid acyl carrier protein (ACP) and thioesterase (TE) govern fatty acid hydrolysis within the algal chloroplast. Using green microalga Chlamydomonas reinhardtii (Cr) as a model, a structural simulation of docking CrACP to CrTE identifies a protein-protein recognition surface between the two domains. A virtual screen reveals plant TEs with similar in silico binding to CrACP. Employing an activity-based crosslinking probe designed to selectively trap transient protein-protein interactions between the TE and ACP, we demonstrate in vitro that CrTE must functionally interact with CrACP to release fatty acids, while TEs of vascular plants show no mechanistic crosslinking to CrACP. This is recapitulated in vivo, where overproduction of the endogenous CrTE increased levels of short-chain fatty acids and engineering plant TEs into the C. reinhardtii chloroplast did not alter the fatty acid profile. These findings highlight the critical role of protein-protein interactions in manipulating fatty acid biosynthesis for algae biofuel engineering as illuminated by activity-based probes. PMID:23028438

  18. Manipulating fatty acid biosynthesis in microalgae for biofuel through protein-protein interactions.

    PubMed

    Blatti, Jillian L; Beld, Joris; Behnke, Craig A; Mendez, Michael; Mayfield, Stephen P; Burkart, Michael D

    2012-01-01

    Microalgae are a promising feedstock for renewable fuels, and algal metabolic engineering can lead to crop improvement, thus accelerating the development of commercially viable biodiesel production from algae biomass. We demonstrate that protein-protein interactions between the fatty acid acyl carrier protein (ACP) and thioesterase (TE) govern fatty acid hydrolysis within the algal chloroplast. Using green microalga Chlamydomonas reinhardtii (Cr) as a model, a structural simulation of docking CrACP to CrTE identifies a protein-protein recognition surface between the two domains. A virtual screen reveals plant TEs with similar in silico binding to CrACP. Employing an activity-based crosslinking probe designed to selectively trap transient protein-protein interactions between the TE and ACP, we demonstrate in vitro that CrTE must functionally interact with CrACP to release fatty acids, while TEs of vascular plants show no mechanistic crosslinking to CrACP. This is recapitulated in vivo, where overproduction of the endogenous CrTE increased levels of short-chain fatty acids and engineering plant TEs into the C. reinhardtii chloroplast did not alter the fatty acid profile. These findings highlight the critical role of protein-protein interactions in manipulating fatty acid biosynthesis for algae biofuel engineering as illuminated by activity-based probes.

  19. Modifications of proteins by polyunsaturated fatty acid peroxidation products

    NASA Astrophysics Data System (ADS)

    Refsgaard, Hanne H. F.; Tsai, Lin; Stadtman, Earl R.

    2000-01-01

    The ability of unsaturated fatty acid methyl esters to modify amino acid residues in bovine serum albumin (BSA), glutamine synthetase, and insulin in the presence of a metal-catalyzed oxidation system [ascorbate/Fe(III)/O2] depends on the degree of unsaturation of the fatty acid. The fatty acid-dependent generation of carbonyl groups and loss of lysine residues increased in the order methyl linoleate < methyl linolenate < methyl arachidonate. The amounts of alkyl hydroperoxides, malondialdehyde, and a number of other aldehydes that accumulated when polyunsaturated fatty acids were oxidized in the presence of BSA were significantly lower than that observed in the absence of BSA. Direct treatment of proteins with various lipid hydroperoxides led to a slight increase in the formation of protein carbonyl derivatives, whereas treatment with the hydroperoxides together with Fe(II) led to a substantial increase in the formation of protein carbonyls. These results are consistent with the proposition that metal-catalyzed oxidation of polyunsaturated fatty acids can contribute to the generation of protein carbonyls by direct interaction of lipid oxidation products (α,β-unsaturated aldehydes) with lysine residues (Michael addition reactions) and also by interactions with alkoxyl radicals obtained by Fe(II) cleavage of lipid hydroperoxides that are formed. In addition, saturated aldehydes derived from the polyunsaturated fatty acids likely react with lysine residues to form Schiff base adducts.

  20. Improved proteomic analysis following trichloroacetic acid extraction of Bacillus anthracis spore proteins.

    PubMed

    Deatherage Kaiser, Brooke L; Wunschel, David S; Sydor, Michael A; Warner, Marvin G; Wahl, Karen L; Hutchison, Janine R

    2015-11-01

    Proteomic analysis of bacterial samples provides valuable information about cellular responses and functions under different environmental pressures. Analysis of cellular proteins is dependent upon efficient extraction from bacterial samples, which can be challenging with increasing complexity and refractory characteristics. While no single method can recover 100% of the bacterial proteins, selected protocols can improve overall protein isolation, peptide recovery, or enrichment for certain classes of proteins. The method presented here is technically simple, does not require specialized equipment such as a mechanical disrupter, and is effective for protein extraction of the particularly challenging sample type of Bacillus anthracis Sterne spores. The ability of Trichloroacetic acid (TCA) extraction to isolate proteins from spores and enrich for spore-specific proteins was compared to the traditional mechanical disruption method of bead beating. TCA extraction improved the total average number of proteins identified within a sample as compared to bead beating (547 vs 495, respectively). Further, TCA extraction enriched for 270 spore proteins, including those typically identified by first isolating the spore coat and exosporium layers. Bead beating enriched for 156 spore proteins more typically identified from whole spore proteome analyses. The total average number of proteins identified was equal using TCA or bead beating for easily lysed samples, such as B. anthracis vegetative cells. As with all assays, supplemental methods such as implementation of an alternative preparation method may simplify sample preparation and provide additional insight to the protein biology of the organism being studied.

  1. Rapid online nonenzymatic protein digestion combining microwave heating acid hydrolysis and electrochemical oxidation.

    PubMed

    Basile, Franco; Hauser, Nicolas

    2011-01-01

    We report an online nonenzymatic method for site-specific digestion of proteins to yield peptides that are well suited for collision-induced dissociation tandem mass spectrometry. The method combines online microwave heating acid hydrolysis at aspartic acid and online electrochemical oxidation at tryptophan and tyrosine. The combined microwave/electrochemical digestion is reproducible and produces peptides with an average sequence length of 10 amino acids. This peptide length is similar to the average peptide length of 9 amino acids obtained by digestion of proteins with the enzyme trypsin. As a result, the peptides produced by this novel nonenzymatic digestion method, when analyzed by electrospray ionization mass spectrometry, produce protonated molecules with mostly +1 and +2 charge states. The combination of these two nonenzymatic methods overcomes shortcomings with each individual method in that (i) peptides generated by the microwave-hydrolysis method have an average amino acid length of 16 amino acids and (ii) the electrochemical-cleavage method is unable to reproducibly digest proteins with molecular masses above 4 kDa. Preliminary results are presented on the application and utility of this rapid online digestion (total of 6 min of digestion time) on a series of standard peptides and proteins as well as an Escherichia coli protein extract.

  2. Production of hydrophobic amino acids from biobased resources: wheat gluten and rubber seed proteins.

    PubMed

    Widyarani; Sari, Yessie W; Ratnaningsih, Enny; Sanders, Johan P M; Bruins, Marieke E

    2016-09-01

    Protein hydrolysis enables production of peptides and free amino acids that are suitable for usage in food and feed or can be used as precursors for bulk chemicals. Several essential amino acids for food and feed have hydrophobic side chains; this property may also be exploited for subsequent separation. Here, we present methods for selective production of hydrophobic amino acids from proteins. Selectivity can be achieved by selection of starting material, selection of hydrolysis conditions, and separation of achieved hydrolysate. Several protease combinations were applied for hydrolysis of rubber seed protein concentrate, wheat gluten, and bovine serum albumin (BSA). High degree of hydrolysis (>50 %) could be achieved. Hydrophobic selectivity was influenced by the combination of proteases and by the extent of hydrolysis. Combination of Pronase and Peptidase R showed the highest selectivity towards hydrophobic amino acids, roughly doubling the content of hydrophobic amino acids in the products compared to the original substrates. Hydrophobic selectivity of 0.6 mol-hydrophobic/mol-total free amino acids was observed after 6 h hydrolysis of wheat gluten and 24 h hydrolysis of rubber seed proteins and BSA. The results of experiments with rubber seed proteins and wheat gluten suggest that this process can be applied to agro-industrial residues.

  3. IR-UV photochemistry of protein-nucleic acid systems

    SciTech Connect

    Kozub, J.; Edwards, G.

    1995-12-31

    UV light has often been used to induce the formation of covalent bonds between DNA (or RNA) and tightly-bound protein molecules. However, the internal photoreactions of nucleic acids and proteins limit the yield and complicate the analysis of intermolecular crosslinks. In an ongoing search for improved reaction specificity or new photoreactions in these systems, we have employed UV photons from a Nd:YAG-pumped dye laser and mid-IR photons from the Vanderbilt FEL. Having crosslinked several protein-nucleic acid systems with nanosecond UV laser pulses, we are currently studying the effect of various IR wavelengths on a model system (gene 32 protein and poly[dT]). We have found that irradiation with sufficiently intense FEL macropulses creates an altered form of gene 32 protein which was not observed with UV-only irradiation. The electrophoretic nobility of the product is consistent with the formation of a specific protein-protein crosslink. No evidence of the non-specific protein damage typically induced by UV light is found. The yield of the new photoproduct is apparently enhanced by exposure to FEL macropulses which are synchronized with UV laser pulses. With ideal exposure parameters, the two-color reaction effectively competes with UV-only reactions. Experiments designed to determine the reaction mechanism and to demonstrate FEL-induced reactions in other protein-nucleic acid systems are currently underway.

  4. Reference intervals for total protein concentration, serum protein fractions, and albumin/globulin ratios in clinically healthy dairy cows.

    PubMed

    Alberghina, Daniela; Giannetto, Claudia; Vazzana, Irene; Ferrantelli, Vincenzo; Piccione, Giuseppe

    2011-01-01

    The aim of the current study was to evaluate total serum protein concentration measured by the biuret reaction as well as albumin and globulin protein fractions determined by agarose gel electrophoresis. These data were used to establish reference intervals in dairy cows of different ages. Blood was collected from 111 clinically healthy Modicana dairy cows by means of jugular venipuncture. Reference intervals (mean ± standard deviation) were determined for total protein (67.54 ± 11.53 g/l), albumin (31.86 ± 4.60 g/l), α(1)-globulin (5.77 ± 2.20 g/l), α(2)-globulin (5.84 ± 1.90 g/l), β-globulin (7.46 ± 1.94 g/l), and γ-globulin (16.73 ± 4.54 g/l) concentrations as well as for albumin/globulin (A/G) ratio (0.88 ± 0.43). Values from 2-, 3-, 4-, 5-, and 6-year-old cows were compared statistically. One-way analysis of variance showed age-related differences for α-globulin and β-globulin fractions only. The results of the current study provide reference intervals for total protein concentration as well as albumin and globulin protein fractions in 2- to 6-year-old dairy cows.

  5. Protein and Essential Amino Acids to Protect Musculoskeletal Health during Spaceflight: Evidence of a Paradox?

    PubMed Central

    Hackney, Kyle J.; English, Kirk L.

    2014-01-01

    Long-duration spaceflight results in muscle atrophy and a loss of bone mineral density. In skeletal muscle tissue, acute exercise and protein (e.g., essential amino acids) stimulate anabolic pathways (e.g., muscle protein synthesis) both independently and synergistically to maintain neutral or positive net muscle protein balance. Protein intake in space is recommended to be 12%–15% of total energy intake (≤1.4 g∙kg−1∙day−1) and spaceflight is associated with reduced energy intake (~20%), which enhances muscle catabolism. Increasing protein intake to 1.5–2.0 g∙kg−1∙day−1 may be beneficial for skeletal muscle tissue and could be accomplished with essential amino acid supplementation. However, increased consumption of sulfur-containing amino acids is associated with increased bone resorption, which creates a dilemma for musculoskeletal countermeasures, whereby optimizing skeletal muscle parameters via essential amino acid supplementation may worsen bone outcomes. To protect both muscle and bone health, future unloading studies should evaluate increased protein intake via non-sulfur containing essential amino acids or leucine in combination with exercise countermeasures and the concomitant influence of reduced energy intake. PMID:25370374

  6. A biotin enrichment strategy identifies novel carbonylated amino acids in proteins from human plasma.

    PubMed

    Havelund, Jesper F; Wojdyla, Katarzyna; Davies, Michael J; Jensen, Ole N; Møller, Ian Max; Rogowska-Wrzesinska, Adelina

    2017-03-06

    Protein carbonylation is an irreversible protein oxidation correlated with oxidative stress, various diseases and ageing. Here we describe a peptide-centric approach for identification and characterisation of up to 14 different types of carbonylated amino acids in proteins. The modified residues are derivatised with biotin-hydrazide, enriched and characterised by tandem mass spectrometry. The strength of the method lies in an improved elution of biotinylated peptides from monomeric avidin resin using hot water (95°C) and increased sensitivity achieved by reduction of analyte losses during sample preparation and chromatography. For the first time MS/MS data analysis utilising diagnostic biotin fragment ions is used to pinpoint sites of biotin labelling and improve the confidence of carbonyl peptide assignments. We identified a total of 125 carbonylated residues in bovine serum albumin after extensive in vitro metal ion-catalysed oxidation. Furthermore, we assigned 133 carbonylated sites in 36 proteins in native human plasma protein samples. The optimised workflow enabled detection of 10 hitherto undetected types of carbonylated amino acids in proteins: aldehyde and ketone modifications of leucine, valine, alanine, isoleucine, glutamine, lysine and glutamic acid (+14Da), an oxidised form of methionine - aspartate semialdehyde (-32Da) - and decarboxylated glutamic acid and aspartic acid (-30Da).

  7. Influence of various nitrogen applications on protein and amino acid profiles of amaranth and quinoa.

    PubMed

    Thanapornpoonpong, Sa-nguansak; Vearasilp, Suchada; Pawelzik, Elke; Gorinstein, Shela

    2008-12-10

    The effect of nitrogen application levels (0.16 and 0.24 g N kg(-1) soil) on seed proteins and their amino acid compositions of amaranth (Amaranthus spp.) and quinoa (Chenopodium quinoa Willd) was studied. Total proteins of amaranth and quinoa had high contents of lysine (6.3-8.2 g 100 g(-1) protein) but low contents of methionine (1.2-1.8 g 100 g(-1) protein). Seed proteins were fractionated on the basis of different solubility in water, saline, and buffer as albumin-1 (Albu-1), albumin-2 (Albu-2), globulin (Glob), and glutelin (Glu) and were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Albu-1 was high in lysine (5.4-8.6 g 100 g(-1) protein), while Albu-2, which is a part of storage proteins, had a high leucine content (7.2-8.9 g 100 g(-1) protein) as an effect of different nitrogen application levels. Glu fractions were well-balanced in their essential amino acids with the exception of methionine. In conclusion, nitrogen application can be used for the nutritional improvement in human diet by increasing and maintaining protein and essential amino acid contents.

  8. Production of total reducing sugar (TRS) from acid hydrolysed potato peels by sonication and its optimization.

    PubMed

    Bhattacharyya, Saurav; Chakraborty, Sudip; Datta, Siddhartha; Drioli, Enrico; Bhattacharjee, Chiranjib

    2013-01-01

    Potato peel is a waste biomass which can be a source of raw material for biofuel production. This biomass contains a sufficient amount of total reducing sugar (TRS), which can be extracted and further treated with microbial pathways to produce bioethanol. The extraction of TRS from potato peels by hydrolysis in dilute sulphuric acid was investigated at different acid concentrations (0.50%, 0.75% and 1% w/v) and sonication was carried out to improve the extent of sugar extraction after hydrolysis. Response surface methodology based on central composite design was used to verify the experimental data and later applied for the optimization of the main important reaction variables including amplitude (60%, 80% and 100%), cycle (0.6, 0.8 and 1.0) and treatment time (5, 10 and 15 min) for the responses of TRS extraction by acid hydrolysis and later compared with the experimental data.

  9. Nucleic acid compositions and the encoding proteins

    DOEpatents

    Preston, III, James F.; Chow, Virginia; Nong, Guang; Rice, John D.; St. John, Franz J.

    2014-09-02

    The subject invention provides at least one nucleic acid sequence encoding an aldouronate-utilization regulon isolated from Paenibacillus sp. strain JDR-2, a bacterium which efficiently utilizes xylan and metabolizes aldouronates (methylglucuronoxylosaccharides). The subject invention also provides a means for providing a coordinately regulated process in which xylan depolymerization and product assimilation are coupled in Paenibacillus sp. strain JDR-2 to provide a favorable system for the conversion of lignocellulosic biomass to biobased products. Additionally, the nucleic acid sequences encoding the aldouronate-utilization regulon can be used to transform other bacteria to form organisms capable of producing a desired product (e.g., ethanol, 1-butanol, acetoin, 2,3-butanediol, 1,3-propanediol, succinate, lactate, acetate, malate or alanine) from lignocellulosic biomass.

  10. Antioxidant activity, ascorbic acid and total phenol of exotic fruits occurring in Brazil.

    PubMed

    de Assis, Sandra Aparecida; Vellosa, José Carlos Rebuglio; Brunetti, Iguatemy Lourenço; Khalil, Najeh Maissar; Leite, Kátia Maria da Silva Cerqueira; Martins, Antonio Baldo Geraldo; Oliveira, Olga Maria Mascarenhas de Faria

    2009-08-01

    The antioxidant activity, ascorbic acid and phenolic content were studied in 10 exotic fruits from Brazil: abiu, acerola, wax jambu, cashew, mamey sapote, carambola or star fruit, Surinam cherry, longan, sapodilla and jaboticaba. The ascorbic acid was determined by 2,6-dichloroindophenol titrimetic methods and total phenols were measured colorimetrically using the Folin-Ciocalteu reagent. The antioxidant activity was investigated with three different methods: hypochlorous acid scavenging activity, 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation decolorization assay, and 2,2-diphenyl-1-picrylhydrazyl radical scavenging method. The highest content of vitamin C (1,525.00 mg/100 g pulp) occurred in acerola. The total phenol content was higher in abiu, acerola, Surinam cherry and sapodilla. In relation to antioxidant activity, acerola has showed the great values in all three different methods tested. It was found that the fruits have a significant antioxidant effect when tested by each method, respectively, and these antioxidant capacities are promising. The sample concentration also influenced its antioxidant power.

  11. Effect of acute bile acid pool depletion on total and ionized calcium concentrations in human bile.

    PubMed

    Gleeson, D; Murphy, G M; Dowling, R H

    1995-04-01

    Although calcium salts are important components of gallstones, there are few data on the total and ionized calcium content of human bile. Therefore, in 14 fasting T-tube patients studied 7-11 days after cholecystectomy, we measured bile flow, bile acid [BA], total [CaTOT] and free ionized [Ca++] calcium concentrations, in 20-30 min bile collections during acute BA pool depletion induced by 6-8 h of continuous bile drainage. During washout of the BA pool there were parallel falls in bile flow, BA output and total calcium output (correlation coefficients ranging from 0.59 to 0.99; P < 0.02-0.001). In 12 of the 14 patients, [CaTOT] also fell (from 1.84 +/- 0.29 to 1.32 +/- 0.34 mmol L-1) in parallel with [BA] (from 34.0 +/- 14.0 to 8.2 +/- 8.0 mmol L-1; r = 0.75-0.98; P < 0.005). In contrast, biliary [Ca++] remained virtually unchanged. These data suggest that the BAs are linked to the bound, rather than to the free, ionized, fraction of biliary calcium, which is consistent with in vivo calcium binding by BAs. A model is proposed in which BA-induced biliary calcium secretion results from (i) bile acid-induced water flow via solvent drag; and (ii) calcium binding in the bile canaliculus by bile acids, which induces paracellular diffusion of Ca++, thereby maintaining [Ca++] independent of [BA].

  12. Evidence that the ZNT3 protein controls the total amount of elemental zinc in synaptic vesicles

    USGS Publications Warehouse

    Linkous, D.H.; Flinn, J.M.; Koh, J.Y.; Lanzirotti, A.; Bertsch, P.M.; Jones, B.F.; Giblin, L.J.; Frederickson, C.J.

    2008-01-01

    The ZNT3 protein decorates the presynaptic vesicles of central neurons harboring vesicular zinc, and deletion of this protein removes staining for zinc. However, it has been unclear whether only histochemically reactive zinc is lacking or if, indeed, total elemental zinc is missing from neurons lacking the Slc30a3 gene, which encodes the ZNT3 protein. The limitations of conventional histochemical procedures have contributed to this enigma. However, a novel technique, microprobe synchrotron X-ray fluorescence, reveals that the normal 2- to 3-fold elevation of zinc concentration normally present in the hippocampal mossy fibers is absent in Slc30a3 knockout (ZNT3) mice. Thus, the ZNT3 protein evidently controls not only the "stainability" but also the actual mass of zinc in mossy-fiber synaptic vesicles. This work thus confirms the metal-transporting role of the ZNT3 protein in the brain. ?? The Histochemical Society, Inc.

  13. [Total protein analysis by two-dimensional electrophoresis in cysticerci of Taenia solium and Taenia asiatica].

    PubMed

    Fang, Wen; Xiao, Liang-Liang; Bao, Huai-En; Mu, Rong

    2011-06-01

    Two 20-day-old three-way crossed hybrid pigs were infected with 80000 Taenia solium or T. asiatica eggs, respectively. Immature cysticerci of the two species in liver were collected at 40 days after infection. The total proteins were separated by two-dimensional electrophoresis, and differentially expressed proteins were analyzed by Image-Master 2D Platinum 6.0 software. The results showed that there were (236 +/- 12) and (231 +/- 14) protein spots in 2D electrophoresis gel images of T. solium and T. asiatica, respectively, with 3 proteins up-regulated and 7 proteins down-regulated in T. solium cysticercus by 2-fold or more compared with those in T. asiatica cysticercus.

  14. Evidence That the ZNT3 Protein Controls the Total Amount of Elemental Zinc in Synaptic Vesicles

    SciTech Connect

    Linkous,D.; Flinn, J.; Koh, J.; Lanzirotti, A.; Bertsch, P.; Jones, B.; Giblin, L.; Fredrickson, C.

    2008-01-01

    The ZNT3 protein decorates the presynaptic vesicles of central neurons harboring vesicular zinc, and deletion of this protein removes staining for zinc. However, it has been unclear whether only histochemically reactive zinc is lacking or if, indeed, total elemental zinc is missing from neurons lacking the Slc30a3 gene, which encodes the ZNT3 protein. The limitations of conventional histochemical procedures have contributed to this enigma. However, a novel technique, microprobe synchrotron X-ray fluorescence, reveals that the normal 2- to 3-fold elevation of zinc concentration normally present in the hippocampal mossy fibers is absent in Slc30a3 knockout (ZNT3) mice. Thus, the ZNT3 protein evidently controls not only the 'stainability' but also the actual mass of zinc in mossy-fiber synaptic vesicles. This work thus confirms the metal-transporting role of the ZNT3 protein in the brain.

  15. Real-time measurements of amino acid and protein hydroperoxides using coumarin boronic acid.

    PubMed

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

    2014-08-08

    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 × 10(3) 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.

  16. Overexpression of a Gene Involved in Phytic Acid Biosynthesis Substantially Increases Phytic Acid and Total Phosphorus in Rice Seeds.

    PubMed

    Tagashira, Yusuke; Shimizu, Tomoe; Miyamoto, Masanobu; Nishida, Sho; Yoshida, Kaoru T

    2015-04-24

    The manipulation of seed phosphorus is important for seedling growth and environmental P sustainability in agriculture. The mechanism of regulating P content in seed, however, is poorly understood. To study regulation of total P, we focused on phytic acid (inositol hexakisphosphate; InsP₆) biosynthesis-related genes, as InsP₆ is a major storage form of P in seeds. The rice (Oryza sativa L.) low phytic acid mutant lpa1-1 has been identified as a homolog of archael 2-phosphoglycerate kinase. The homolog might act as an inositol monophosphate kinase, which catalyzes a key step in InsP₆ biosynthesis. Overexpression of the homolog in transgenic rice resulted in a significant increase in total P content in seed, due to increases in InsP₆ and inorganic phosphates. On the other hand, overexpression of genes that catalyze the first and last steps of InsP₆ biosynthesis could not increase total P levels. From the experiments using developing seeds, it is suggested that the activation of InsP₆ biosynthesis in both very early and very late periods of seed development increases the influx of P from vegetative organs into seeds. This is the first report from a study attempting to elevate the P levels of seed through a transgenic approach.

  17. Functional domains of the fatty acid transport proteins: studies using protein chimeras.

    PubMed

    DiRusso, Concetta C; Darwis, Dina; Obermeyer, Thomas; Black, Paul N

    2008-03-01

    Fatty acid transport proteins (FATP) function in fatty acid trafficking pathways, several of which have been shown to participate in the transport of exogenous fatty acids into the cell. Members of this protein family also function as acyl CoA synthetases with specificity towards very long chain fatty acids or bile acids. These proteins have two identifying sequence motifs: The ATP/AMP motif, an approximately 100 amino acid segment required for ATP binding and common to members of the adenylate-forming super family of proteins, and the FATP/VLACS motif that consists of approximately 50 amino acid residues and is restricted to members of the FATP family. This latter motif has been implicated in fatty acid transport in the yeast FATP orthologue Fat1p. In the present studies using a yeast strain containing deletions in FAT1 (encoding Fat1p) and FAA1 (encoding the major acyl CoA synthetase (Acsl) Faa1p) as an experimental platform, the phenotypic and functional properties of specific murine FATP1-FATP4 and FATP6-FATP4 protein chimeras were evaluated in order to define elements within these proteins that further distinguish the fatty acid transport and activation functions. As expected from previous work FATP1 and FATP4 were functional in the fatty acid transport pathway, while and FATP6 was not. All three isoforms were able to activate the very long chain fatty acids arachidonate (C(20:4)) and lignocerate (C(24:0)), but with distinguishing activities between saturated and highly unsaturated ligands. A 73 amino acid segment common to FATP1 and FATP4 and between the ATP/AMP and FATP/VLACS motifs was identified by studying the chimeras, which is hypothesized to contribute to the transport function.

  18. Plasma amino acid response to graded levels of escape protein.

    PubMed

    Gibb, D J; Klopfenstein, T J; Britton, R A; Lewis, A J

    1992-09-01

    A trial was conducted to examine the potential of using plasma amino acid responses to graded levels of escape protein to determine limiting amino acids in cattle. Growing calves (n = 120; mean BW = 220 +/- 21 kg) were fed a basal diet of corncob:sorghum silage (61:39) and were individually supplemented with distillers' dried grains (DDG), heat-damaged DDG (H-DDG), feather meal (FTH), or urea. The urea supplement was mixed with DDG and H-DDG to allow 0, 20, 35, 50, 65, or 80% of the supplemental CP to come from distillers' protein and maintain an 11.5% CP diet. Urea supplement was mixed with FTH to allow 0, 22, 39, 56, 73, or 90% of the supplemental CP to come from FTH. Dietary CP ranged from 11.5% at the 0% level to 17.3% at the 90% level. Plasma concentration of most essential plasma amino acids responded (P less than .10) linearly and(or) quadratically to increased escape protein. The broken-line response of plasma methionine at low DDG intake suggested that methionine was limiting at low levels of escape protein. An initial decrease followed by a plateau fit by a broken line indicated that histidine became limiting in FTH diets, and lysine eventually became limiting for DDG, H-DDG, and FTH diets before maximum BW gain was reached. Results indicate that plasma amino acid responses may identify amino acids that become limiting with increasing escape protein.

  19. Total Protein of Whole Saliva as a Biomarker of Anaerobic Threshold

    ERIC Educational Resources Information Center

    Bortolini, Miguel Junior Sordi; De Agostini, Guilherme Gularte; Reis, Ismair Teodoro; Lamounier, Romeu Paulo Martins Silva; Blumberg, Jeffrey B.; Espindola, Foued Salmen

    2009-01-01

    Saliva provides a convenient and noninvasive matrix for assessing specific physiological parameters, including some biomarkers of exercise. We investigated whether the total protein concentration of whole saliva (TPWS) would reflect the anaerobic threshold during an incremental exercise test. After a warm-up period, 13 nonsmoking men performed a…

  20. Stimulation of nonselective amino acid export by glutamine dumper proteins.

    PubMed

    Pratelli, Réjane; Voll, Lars M; Horst, Robin J; Frommer, Wolf B; Pilot, Guillaume

    2010-02-01

    Phloem and xylem transport of amino acids involves two steps: export from one cell type to the apoplasm, and subsequent import into adjacent cells. High-affinity import is mediated by proton/amino acid cotransporters, while the mechanism of export remains unclear. Enhanced expression of the plant-specific type I membrane protein Glutamine Dumper1 (GDU1) has previously been shown to induce the secretion of glutamine from hydathodes and increased amino acid content in leaf apoplasm and xylem sap. In this work, tolerance to low concentrations of amino acids and transport analyses using radiolabeled amino acids demonstrate that net amino acid uptake is reduced in the glutamine-secreting GDU1 overexpressor gdu1-1D. The net uptake rate of phenylalanine decreased over time, and amino acid net efflux was increased in gdu1-1D compared with the wild type, indicating increased amino acid export from cells. Independence of the export from proton gradients and ATP suggests that overexpression of GDU1 affects a passive export system. Each of the seven Arabidopsis (Arabidopsis thaliana) GDU genes led to similar phenotypes, including increased efflux of a wide spectrum of amino acids. Differences in expression profiles and functional properties suggested that the GDU genes fulfill different roles in roots, vasculature, and reproductive organs. Taken together, the GDUs appear to stimulate amino acid export by activating nonselective amino acid facilitators.

  1. Development of a new method for determination of total haem protein in fish muscle.

    PubMed

    Chaijan, Manat; Undeland, Ingrid

    2015-04-15

    Using classic haem protein quantification methods, the extraction step in buffer or acid acetone often becomes limiting if muscle is oxidised and/or stored; haem-proteins then tend to bind to muscle components like myofibrils and/or biomembranes. The objective of this study was to develop a new haem protein determination method for fish muscle overcoming such extractability problems. The principle was to homogenise and heat samples in an SDS-containing phosphate buffer to dissolve major muscle components and convert ferrous/ferric haem proteins to hemichromes with a unique absorption peak at 535 nm. Hb-recovery tests with the new and classic methods showed that the new method and Hornsey's method performed significantly better on fresh Hb-enriched cod mince than Brown's and Drabkin's methods; recovery was ⩾98%. However, in highly oxidised samples and in cod protein isolates made with acid pH-shift processing, the new method performed better than Hornsey's method (63% and 87% vs. 50% and 68% recovery). Further, the new method performed well in fish muscle with ⩽30% lipid, <5% NaCl and pH 5.5-7.0; it was also unaffected by freezing/frozen storage.

  2. Selective precipitation of ribonucleic acid from a mixture of total cellular nucleic acids extracted from cultured mammalian cells

    PubMed Central

    Harrison, P. R.

    1971-01-01

    A simple and reproducible method is described for precipitating RNA selectively from total mammalian-cell nucleic acids extracted by the phenol–sodium dodecyl sulphate procedure at pH8.0. Under specified conditions bulk RNA is precipitated almost quantitatively whereas bulk DNA remains in solution. Minor components of RNA (detected by pulse-labelling and chromatography on methylated albumin–kieselguhr) and rapidly labelled components of DNA containing single-stranded regions are also precipitated. The usefulness of the method is discussed in the context of isolating separately both RNA and DNA from cultured cells that are difficult to obtain in quantity. PMID:5165620

  3. The multiple roles of fatty acid handling proteins in brain

    PubMed Central

    Moullé, Valentine S. F.; Cansell, Céline; Luquet, Serge; Cruciani-Guglielmacci, Céline

    2012-01-01

    Lipids are essential components of a living organism as energy source but also as constituent of the membrane lipid bilayer. In addition fatty acid (FA) derivatives interact with many signaling pathways. FAs have amphipathic properties and therefore require being associated to protein for both transport and intracellular trafficking. Here we will focus on several FA handling proteins, among which the fatty acid translocase/CD36 (FAT/CD36), members of fatty acid transport proteins (FATPs), and lipid chaperones fatty acid-binding proteins (FABPs). A decade of extensive studies has helped decipher the mechanism of action of these proteins in peripheral tissue with high lipid metabolism. However, considerably less information is available regarding their role in the brain, despite the high lipid content of this tissue. This review will primarily focus on the recent studies that have highlighted the crucial role of lipid handling proteins in brain FA transport, neuronal differentiation and development, cognitive processes and brain diseases. Finally a special focus will be made on the recent studies that have revealed the role of FAT/CD36 in brain lipid sensing and nervous control of energy balance. PMID:23060810

  4. Okadaic acid: the archetypal serine/threonine protein phosphatase inhibitor.

    PubMed

    Dounay, A B; Forsyth, C J

    2002-11-01

    As the first recognized member of the "okadaic acid class" of phosphatase inhibitors, the marine natural product okadaic acid is perhaps the most well-known member of a diverse array of secondary metabolites that have emerged as valuable probes for studying the roles of various cellular protein serine/threonine phosphatases. This review provides a historical perspective on the role that okadaic acid has played in stimulating a broad spectrum of modern scientific research as a result of the natural product's ability to bind to and inhibit important classes of protein serine / threonine phosphatases. The relationships between the structure and biological activities of okadaic acid are briefly reviewed, as well as the structural information regarding the particular cellular receptors protein phosphatases 1 (PP1) and 2A. Laboratory syntheses of okadaic acid and its analogs are thoroughly reviewed. Finally, an interpretation of the critical contacts observed between okadaic acid and PP1 by X-ray crystallography is provided, and specific molecular recognition hypotheses that are testable via the synthesis and assay of non-natural analogs of okadaic acid are suggested.

  5. Improved biuret procedure for routine determination of urinary total proteins in clinical proteinuria.

    PubMed

    Rice, E W

    1975-03-01

    This communication describes and evaluates an improved routine methodology for quantitating clinical proteinuria. Based on investigations of Piscator and of Savory et al., a modified Tsuchiya's reagent (ethanolic HCI-phosphotungstic acid) is used to precipitate proteins at 56 degrees C, followed by biuret spectrophotometry at 540 nm. The accuracy of the proposed procedure was assessed by comparisons with results obtained by using an ultrafiltration membrane that retains solutes with an average molecular weight in excess of 10 000 for separating of urinary proteins before they are measured with the biuret reaction. Precision of the method (coefficient of variation) is typically 2-3%.

  6. Study on furundu, a traditional Sudanese fermented roselle (Hibiscus sabdariffa L.) seed: effect on in vitro protein digestibility, chemical composition, and functional properties of the total proteins.

    PubMed

    Yagoub, Abu El-Gasim A; Mohamed, Babiker E; Ahmed, Abdel Halim R; El Tinay, Abdullahi H

    2004-10-06

    Furundu, a meat substitute, is traditionally prepared by cooking the karkade (Hibiscus sabdariffa L.) seed and then fermenting it for 9 days. Physicochemical and functional properties of raw and cooked seed and of furundu ferments were analyzed. Furundu preparation resulted in significant changes in karkade seed major nutrients. Total polyphenols and phytic acid were also reduced. The increase in total acidity and fat acidity coupled with a decrease in pH indicates microbial hydrolysis of the major nutrients; proteins, carbohydrates, and fats. In vitro digestibility of the seed proteins reached the maximum value (82.7%) at the sixth day of fermentation, but thereafter it significantly decreased. The effect of furundu preparation on N solubility profiles and functional properties, such as emulsification and foaming properties and other related parameters, is investigated in water and in 1 M NaCl extracts from defatted flour samples. The results show that cooking followed by fermentation affects proteins solubility in water and 1 M NaCl. The foaming capacity (FC) from the flour of raw seed decreased as a result of cooking. Fermentation for 9 days significantly increased the FC of the cooked seed, restoring the inherent value. Foam from fermented samples collapsed more rapidly during a period of 120 min as compared to the foam from raw and cooked karkade seeds; stability in 1 M NaCl was lower as compared to those in water. In water, the emulsion stability (ES) from the fermented samples was significantly higher than that of the raw seed flour. Addition of 1 M NaCl significantly decreased the ES of the fermented samples.

  7. Quantitative colorimetric assay for total protein applied to the red wine Pinot noir.

    PubMed

    Smith, Mark R; Penner, Mike H; Bennett, Samuel E; Bakalinsky, Alan T

    2011-07-13

    A standard method for assaying protein in red wine is currently lacking. The method described here is based on protein precipitation followed by dye binding quantification. Improvements over existing approaches include minimal sample processing prior to protein precipitation with cold trichloroacetic acid/acetone and quantification based on absorbance relative to a commercially available standard representative of proteins likely to be found in wine, the yeast mannoprotein invertase. The precipitation method shortened preparation time relative to currently published methods and the mannoprotein standard yielded values comparable to those obtained by micro-Kjeldahl analysis. The assay was used to measure protein in 48 Pinot noir wines from 6 to 32 years old. The protein content of these wines was found to range from 50 to 102 mg/L with a mean value of 70 mg/L. The availability of a simple and relatively rapid procedure for assaying protein provides a practical tool to quantify a wine component that has been overlooked in routine analyses of red wines.

  8. Hydrodynamic size-based separation and characterization of protein aggregates from total cell lysates

    PubMed Central

    Tanase, Maya; Zolla, Valerio; Clement, Cristina C; Borghi, Francesco; Urbanska, Aleksandra M; Rodriguez-Navarro, Jose Antonio; Roda, Barbara; Zattoni, Andrea; Reschiglian, Pierluigi; Cuervo, Ana Maria; Santambrogio, Laura

    2016-01-01

    Herein we describe a protocol that uses hollow-fiber flow field-flow fractionation (FFF) coupled with multiangle light scattering (MALS) for hydrodynamic size-based separation and characterization of complex protein aggregates. The fractionation method, which requires 1.5 h to run, was successfully modified from the analysis of protein aggregates, as found in simple protein mixtures, to complex aggregates, as found in total cell lysates. In contrast to other related methods (filter assay, analytical ultracentrifugation, gel electrophoresis and size-exclusion chromatography), hollow-fiber flow FFF coupled with MALS allows a flow-based fractionation of highly purified protein aggregates and simultaneous measurement of their molecular weight, r.m.s. radius and molecular conformation (e.g., round, rod-shaped, compact or relaxed). The polyethersulfone hollow fibers used, which have a 0.8-mm inner diameter, allow separation of as little as 20 μg of total cell lysates. In addition, the ability to run the samples in different denaturing and nondenaturing buffer allows defining true aggregates from artifacts, which can form during sample preparation. The protocol was set up using Paraquat-induced carbonylation, a model that induces protein aggregation in cultured cells. This technique will advance the biochemical, proteomic and biophysical characterization of molecular-weight aggregates associated with protein mutations, as found in many CNS degenerative diseases, or chronic oxidative stress, as found in aging, and chronic metabolic and inflammatory conditions. PMID:25521790

  9. Total Protein Extraction for Metaproteomics Analysis of Methane Producing Biofilm: The Effects of Detergents

    PubMed Central

    Huang, Hung-Jen; Chen, Wei-Yu; Wu, Jer-Horng

    2014-01-01

    Protein recovery is crucial for shotgun metaproteomics to study the in situ functionality of microbial populations from complex biofilms but still poorly addressed by far. To fill this knowledge gap, we systematically evaluated the sample preparation with extraction buffers comprising four detergents for the metaproteomics analysis of a terephthalate-degrading methanogenic biofilm using an on-line two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS) system. Totally, 1018 non-repeated proteins were identified with the four treatments. On the whole, each treatment could recover the biofilm proteins with specific distributions of molecular weight, hydrophobicity, and isoelectric point. The extraction buffers containing zwitterionic and anionic detergents were found to harvest the proteins with better efficiency and quality, allowing identification up to 76.2% of total identified proteins with the LC-MS/MS analysis. According to the annotation with a relevant metagenomic database, we further observed different taxonomic profiles of bacterial and archaeal members and discriminable patterns of the functional expression among the extraction buffers used. Overall, the finding of the present study provides first insight to the effect of the detergents on the characteristics of extractable proteins from biofilm and the developed protocol combined with nano 2D-LC/MS/MS analysis can improve the metaproteomics studies on microbial functionality of biofilms in the wastewater treatment systems. PMID:24914765

  10. Total protein extraction for metaproteomics analysis of methane producing biofilm: the effects of detergents.

    PubMed

    Huang, Hung-Jen; Chen, Wei-Yu; Wu, Jer-Horng

    2014-06-06

    Protein recovery is crucial for shotgun metaproteomics to study the in situ functionality of microbial populations from complex biofilms but still poorly addressed by far. To fill this knowledge gap, we systematically evaluated the sample preparation with extraction buffers comprising four detergents for the metaproteomics analysis of a terephthalate-degrading methanogenic biofilm using an on-line two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS) system. Totally, 1018 non-repeated proteins were identified with the four treatments. On the whole, each treatment could recover the biofilm proteins with specific distributions of molecular weight, hydrophobicity, and isoelectric point. The extraction buffers containing zwitterionic and anionic detergents were found to harvest the proteins with better efficiency and quality, allowing identification up to 76.2% of total identified proteins with the LC-MS/MS analysis. According to the annotation with a relevant metagenomic database, we further observed different taxonomic profiles of bacterial and archaeal members and discriminable patterns of the functional expression among the extraction buffers used. Overall, the finding of the present study provides first insight to the effect of the detergents on the characteristics of extractable proteins from biofilm and the developed protocol combined with nano 2D-LC/MS/MS analysis can improve the metaproteomics studies on microbial functionality of biofilms in the wastewater treatment systems.

  11. Total Soluble Protein Extraction for Improved Proteomic Analysis of Transgenic Rice Plant Roots.

    PubMed

    Raorane, Manish L; Narciso, Joan O; Kohli, Ajay

    2016-01-01

    With the advent of high-throughput platforms, proteomics has become a powerful tool to search for plant gene products of agronomic relevance. Protein extractions using multistep protocols have been shown to be effective to achieve better proteome profiles than simple, single-step extractions. These protocols are generally efficient for above ground tissues such as leaves. However, each step leads to loss of some amount of proteins. Additionally, compounds such as proteases in the plant tissues lead to protein degradation. While protease inhibitor cocktails are available, these alone do not seem to suffice when roots are included in the plant sample. This is obvious given the lack of high molecular weight (HMW) proteins obtained from samples that include root tissue. For protein/proteome analysis of transgenic plant roots or of seedlings, which include root tissue, such pronounced protein degradation is especially undesirable. A facile protein extraction protocol is presented, which ensures that despite the inclusion of root tissues there is minimal loss in total protein components.

  12. Defining meal requirements for protein to optimize metabolic roles of amino acids12345

    PubMed Central

    Anthony, Tracy G; Rasmussen, Blake B; Adams, Sean H; Lynch, Christopher J; Brinkworth, Grant D; Davis, Teresa A

    2015-01-01

    Dietary protein provides essential amino acids (EAAs) for the synthesis of new proteins plus an array of other metabolic functions; many of these functions are sensitive to postprandial plasma and intracellular amino acid concentrations. Recent research has focused on amino acids as metabolic signals that influence the rate of protein synthesis, inflammation responses, mitochondrial activity, and satiety, exerting their influence through signaling systems including mammalian/mechanistic target of rapamycin complex 1 (mTORC1), general control nonrepressed 2 (GCN2), glucagon-like peptide 1 (GLP-1), peptide YY (PYY), serotonin, and insulin. These signals represent meal-based responses to dietary protein. The best characterized of these signals is the leucine-induced activation of mTORC1, which leads to the stimulation of skeletal muscle protein synthesis after ingestion of a meal that contains protein. The response of this metabolic pathway to dietary protein (i.e., meal threshold) declines with advancing age or reduced physical activity. Current dietary recommendations for protein are focused on total daily intake of 0.8 g/kg body weight, but new research suggests daily needs for older adults of ≥1.0 g/kg and identifies anabolic and metabolic benefits to consuming at least 20–30 g protein at a given meal. Resistance exercise appears to increase the efficiency of EAA use for muscle anabolism and to lower the meal threshold for stimulation of protein synthesis. Applying this information to a typical 3-meal-a-day dietary plan results in protein intakes that are well within the guidelines of the Dietary Reference Intakes for acceptable macronutrient intakes. The meal threshold concept for dietary protein emphasizes a need for redistribution of dietary protein for optimum metabolic health. PMID:25926513

  13. Defining meal requirements for protein to optimize metabolic roles of amino acids.

    PubMed

    Layman, Donald K; Anthony, Tracy G; Rasmussen, Blake B; Adams, Sean H; Lynch, Christopher J; Brinkworth, Grant D; Davis, Teresa A

    2015-04-29

    Dietary protein provides essential amino acids (EAAs) for the synthesis of new proteins plus an array of other metabolic functions; many of these functions are sensitive to postprandial plasma and intracellular amino acid concentrations. Recent research has focused on amino acids as metabolic signals that influence the rate of protein synthesis, inflammation responses, mitochondrial activity, and satiety, exerting their influence through signaling systems including mammalian/mechanistic target of rapamycin complex 1 (mTORC1), general control nonrepressed 2 (GCN2), glucagon-like peptide 1 (GLP-1), peptide YY (PYY), serotonin, and insulin. These signals represent meal-based responses to dietary protein. The best characterized of these signals is the leucine-induced activation of mTORC1, which leads to the stimulation of skeletal muscle protein synthesis after ingestion of a meal that contains protein. The response of this metabolic pathway to dietary protein (i.e., meal threshold) declines with advancing age or reduced physical activity. Current dietary recommendations for protein are focused on total daily intake of 0.8 g/kg body weight, but new research suggests daily needs for older adults of ≥1.0 g/kg and identifies anabolic and metabolic benefits to consuming at least 20-30 g protein at a given meal. Resistance exercise appears to increase the efficiency of EAA use for muscle anabolism and to lower the meal threshold for stimulation of protein synthesis. Applying this information to a typical 3-meal-a-day dietary plan results in protein intakes that are well within the guidelines of the Dietary Reference Intakes for acceptable macronutrient intakes. The meal threshold concept for dietary protein emphasizes a need for redistribution of dietary protein for optimum metabolic health.

  14. Assessment of the protein quality of 15 new northern adapted cultivars of quality protein maize using amino acid analysis.

    PubMed

    Zarkadas, C G; Hamilton, R I; Yu, Z R; Choi, V K; Khanizadeh, S; Rose, N G; Pattison, P L

    2000-11-01

    Amino acid determinations were carried out on 15 new northern adapted cultivars of quality protein maize (QPM) containing opaque-2 modifier genes to ascertain whether their amino acid scoring patterns could be used to select high-lysine QPM genotypes and to assess their protein quality. Total protein in these cultivars ranged from 8.0 to 10.2% compared to two commercial maize varieties, Dekalb DK435 (7.9%) and Pioneer 3925 (10.3%). Four of these QPM genotypes, QPM-C26, QPM-C21, QPM-C79, and QPM-C59, contained high levels of lysine (4.43-4.58 g of lysine/100 g of protein), whereas the remaining varied from 3.43 to 4.21 g of lysine/100 g of protein, compared to Dekalb DK435 and Pioneer 3925, which contained 2.9 and 3. 1 g of lysine/100 g of protein, respectively. Although lysine is the first limiting amino acid in QPM inbreds, the high-lysine QPM genotypes may supply approximately 70.2-72.6% of human protein requirements, compared to 46.2% for Dekalb DK435 and 50.1% for Pioneer 3925, 55-63% for oats, and 59-60.3% for barley. Northern adapted QPM genotypes may have the potential to increase their lysine content even further, either by an increase in specific high-lysine-containing nonzein proteins, such as the synthesis of factor EF-1a, or by a further reduction in the 19 and 22 kDa alpha-zein in the endosperm or both. This knowledge could assist maize breeders in the selection of new high-performance QPM genotypes with improved protein quality and quantity.

  15. Roles of intrinsic disorder in protein-nucleic acid interactions.

    PubMed

    Dyson, H Jane

    2012-01-01

    Interactions between proteins and nucleic acids typify the role of disordered segments, linkers, tails and other entities in the function of complexes that must form with high affinity and specificity but which must be capable of dissociating when no longer needed. While much of the emphasis in the literature has been on the interactions of disordered proteins with other proteins, disorder is also frequently observed in nucleic acids (particularly RNA) and in the proteins that interact with them. The interactions of disordered proteins with DNA most often manifest as molding of the protein onto the B-form DNA structure, although some well-known instances involve remodeling of the DNA structure that seems to require that the interacting proteins be disordered to various extents in the free state. By contrast, induced fit in RNA-protein interactions has been recognized for many years-the existence and prevalence of this phenomenon provides the clearest possible evidence that RNA and its interactions with proteins must be considered as highly dynamic, and the dynamic nature of RNA and its multiplicity of folded and unfolded states is an integral part of its nature and function.

  16. Pyridoxic acid excretion during low vitamin B-6 intake, total fasting, and bed rest

    NASA Technical Reports Server (NTRS)

    Coburn, S. P.; Thampy, K. G.; Lane, H. W.; Conn, P. S.; Ziegler, P. J.; Costill, D. L.; Mahuren, J. D.; Fink, W. J.; Pearson, D. R.; Schaltenbrand, W. E.

    1995-01-01

    Vitamin B-6 metabolism in 10 volunteers during 21 d of total fasting was compared with results from 10 men consuming a diet low only in vitamin B-6 (1.76 mumol/d) and with men consuming a normal diet during bed rest. At the end of the fast mean plasma concentrations of vitamin B-6 metabolites and urinary excretion of 4-pyridoxic acid tended to be higher in the fasting subjects than in the low-vitamin B-6 group. The fasting subjects lost approximately 10% of their total vitamin B-6 pool and approximately 13% of their body weight. The low-vitamin B-6 group lost only approximately 4% of their vitamin B-6 pool. Compared with baseline, urinary excretion of pyridoxic acid was significantly increased during 17 wk of bed rest. There was no increase in pyridoxic acid excretion during a second 15-d bed rest study. These data suggest the possibility of complex interactions between diet and muscle metabolism that may influence indexes that are frequently used to assess vitamin B-6 status.

  17. Acidic and total primary sulfates: development of emission factors for major stationary combustion sources

    SciTech Connect

    Goklany, I.M.; Hoffnagle, G.F.; Brackbill, E.A.

    1984-01-01

    ''Best estimates'' of emission factors for major sources of acidic and total primary sulfates are developed for use in the compilation of emission inventories for the eastern U.S. These may, in turn, be used for modeling of acidic or sulfate deposition. The factors are based upon a critical evaluation of the generic measurement methods used to quantify total and acidic primary sulfate emissions, and an exhaustive review and critique of individual papers and studies available in the open literature which present measurement data on primary sulfate emissions. It develops a qualitative rating scheme which specifies the level of confidence that should be attached to the emission factor determinations. The paper concludes that much of the existing data on primary sulfates from stationary combustion sources are, probably, significantly biased upward and, therefore, inappropriate for the derivation of emission factors. Therefore, existing estimates of primary sulfate emissions for these source categories are, probably, substanitally inflated. It also concludes that, for most source categories, very little confidence can be attached to the best estimates because of the paucity of data obtained from measurement techniques which are likely to be free of systematic bias. 68 references.

  18. A new microwave acid digestion bomb method for the determination of total fluorine.

    PubMed

    Grobler, S R; Louw, A J

    1998-01-01

    A new microwave acid digestion method for total fluorine analysis was compared to the reliable reverse-extraction technique. The commercially available Parr bombs which are compatible with microwave heating were modified for this purpose. The Mann-Whitney statistical test did not show any significant differences (p > 0.05) in the determinations of total fluorine in various samples between the two above-mentioned methods. The microwave method also gave high fluorine recoveries (> 97%) when fluoride was added to different samples. The great advantage of the microwave acid digestion bomb method is that the digestion under pressure is so aggressive that only a few minutes is needed for complete digestion (also of covalently bonded fluorine), which reduces the time for fluorine analysis dramatically, while no loss of fluorine or contamination from extraneous sources could take place during the ashing procedure. The digestion solution was made up of 300 microliter of concentrated nitric acid plus 537 microliter of water. After digestion 675 microliter of approximately 8.5 M sodium hydroxide plus 643 microliter of citrate/TISAB buffer was added resulting in an alkaline solution (pH approximately 12) which was finally adjusted to a pH of approximately 5.3 for fluoride determination.

  19. Effects of total gastrectomy on plasma silicon and amino acid concentrations in men.

    PubMed

    Tatara, Marcin R; Krupski, Witold; Szpetnar, Maria; Dąbrowski, Andrzej; Bury, Paweł; Szabelska, Anna; Charuta, Anna; Boguszewska-Czubara, Anna; Maciejewski, Ryszard; Wallner, Grzegorz

    2015-12-01

    The aim of the study was to determine one-year effects of total gastrectomy on plasma silicon and free amino acid concentrations in patients and evaluate changes of volumetric bone mineral density (vBMD) in lumbar spine. Eight patients were enrolled to the control (CTR) group. Six patients subjected to total gastrectomy (GX group) were included to the experimental group. vBMD in trabecular and cortical bone was measured in lumbar vertebrae at baseline (before surgery) and one year later using quantitative computed tomography. Plasma concentrations of silicon and free amino acids were determined at baseline and one year later using photometric method and ion-exchange chromatography. Body weights within CTR and GX groups were not different after one-year follow-up when compared to the baseline values (P > 0.05). An average annual decrease of vBMD in the trabecular bone in the gastrectomized patients reached 15.0% in lumbar spine and was significantly different in comparison to the percentage changes observed in CTR group (P = 0.02). One-year percentage change of vBMD in the cortical bone in L1 and L2 has shown significantly decreased values by 10.5 and 9.1% in the GX group when compared to the percentage change observed in the controls (P < 0.05). Plasma concentration of adipic acid was significantly higher by 101.6% one year after total gastrectomy procedure in the patients when compared to the baseline value (P = 0.01). Plasma concentration of silicon was significantly lowered by 26.7% one year after the total gastrectomy when compared to the baseline value (P = 0.009). Total gastrectomy in patients has induced severe osteoporotic changes in lumbar spine within one-year period. The observed osteoporotic changes were associated with decreased plasma concentration of silicon indicating importance of exocrine and endocrine functions of stomach for silicon homeostasis maintenance. Gastrectomy-induced bone loss was not related to decreased amino acid

  20. Proteomic Analysis of Responsive Proteins Induced in Japanese Birch Plantlet Treated with Salicylic Acid

    PubMed Central

    Suzuki, Hiromu; Takashima, Yuya; Ishiguri, Futoshi; Yoshizawa, Nobuo; Yokota, Shinso

    2014-01-01

    The present study was performed to unravel the mechanisms of systemic acquired resistance (SAR) establishment and resistance signaling pathways against the canker-rot fungus (Inonotus obliquus strain IO-U1) infection in Japanese birch plantlet No.8. Modulation of protein-profile induced by salicylic acid (SA)-administration was analyzed, and SA-responsive proteins were identified. In total, 5 specifically expressed, 3 significantly increased, and 3 significantly decreased protein spots were identified using liquid chromatography/tandem mass spectrometry (LC/MS/MS) and the sequence tag method. These proteins were malate dehydrogenase, succinate dehydrogenase, phosphoglycerate kinase, diaminopimalate decarboxylase, arginase, chorismate mutase, cyclophilin, aminopeptidase, and unknown function proteins. These proteins are considered to be involved in SAR-establishment mechanisms in the Japanese birch plantlet No 8. PMID:28250384

  1. A general method of protein purification for recombinant unstructured non-acidic proteins.

    PubMed

    Campos, Francisco; Guillén, Gabriel; Reyes, José L; Covarrubias, Alejandra A

    2011-11-01

    Typical late embryogenesis abundant (LEA) proteins accumulate in response to water deficit imposed by the environment or by plant developmental programs. Because of their physicochemical properties, they can be considered as hydrophilins and as a paradigm of intrinsically unstructured proteins (IUPs) in plants. To study their biophysical and biochemical characteristics large quantities of highly purified protein are required. In this work, we report a fast and simple purification method for non-acidic recombinant LEA proteins that does not need the addition of tags and that preserves their in vitro protective activity. The method is based on the enrichment of the protein of interest by boiling the bacterial protein extract, followed by a differential precipitation with trichloroacetic acid (TCA). Using this procedure we have obtained highly pure recombinant LEA proteins of groups 1, 3, and 4 and one recombinant bacterial hydrophilin. This protocol will facilitate the purification of this type of IUPs, and could be particularly useful in proteomic projects/analyses.

  2. Substitution of aspartic acid with glutamic acid increases the unfolding transition temperature of a protein.

    PubMed

    Lee, Duck Yeon; Kim, Kyeong-Ae; Yu, Yeon Gyu; Kim, Key-Sun

    2004-07-30

    Proteins from thermophiles are more stable than those from mesophiles. Several factors have been suggested as causes for this greater stability, but no general rule has been found. The amino acid composition of thermophile proteins indicates that the content of polar amino acids such as Asn, Gln, Ser, and Thr is lower, and that of charged amino acids such as Arg, Glu, and Lys is higher than in mesophile proteins. Among charged amino acids, however, the content of Asp is even lower in thermophile proteins than in mesophile proteins. To investigate the reasons for the lower occurrence of Asp compared to Glu in thermophile proteins, Glu was substituted with Asp in a hyperthermophile protein, MjTRX, and Asp was substituted with Glu in a mesophile protein, ETRX. Each substitution of Glu with Asp decreased the Tm of MjTRX by about 2 degrees C, while each substitution of Asp with Glu increased the Tm of ETRX by about 1.5 degrees C. The change of Tm destabilizes the MjTRX by 0.55 kcal/mol and stabilizes the ETRX by 0.45 kcal/mol in free energy.

  3. Total dietary fat and omega-3 fatty acids have modest effects on urinary sex hormones in postmenopausal women

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Total fat and omega-3 fatty acids in the diet may affect breast cancer risk by altering estrogen metabolism. The purpose of this study was to elucidate the effects of differing total fat and omega-3 fatty acid content of diets on a panel of urinary estrogens and metabolites. A controlled, cross-ove...

  4. Spectrophotometric study of total protein-albumin methods applied to cerebrospinal fluid.

    PubMed

    Artiss, J D; Thibert, R J; Zak, B

    1981-02-01

    A spectrophotometric study was carried out for three proteins assays when modification of their serum procedures using bromcresol green, bromcresol purple and biuret reagents were applied to the determinations of total proteins and albumin in cerebrospinal fluids. A novel concentration device wherein the sample itself was used as the primary diluent for the three reagents concentrated to contain the proper amounts of chemicals in smaller volumes than suggested in their serum procedures allowed reasonable absorbance signals to be obtained. Low molecular weight molecules were separated from the albumin and globulins of the fluids by centrifugal ultrafiltration using a 25K cutoff and spectra were obtained for both high and low molecular weight fractions. Some materials were obtained in the separated ultrafiltrates which gave reactions with all three reagents, reactions which either overlapped the spectra of the albumin reactions or superimposed the spectra obtained with the total protein reaction. A screening procedure for cerebrospinal fluid total proteins or centrifugally ultrafiltered albumin appears reasonable as an inference from studies made, although further elucidation of the low molecular weight fractions in needed as a confirmation device.

  5. A High-throughput Screening Assay for Determining Cellular Levels of Total Tau Protein

    PubMed Central

    Dehdashti, Seameen J.; Zheng, Wei; Gever, Joel R.; Wilhelm, Robert; Nguyen, Dac-Trung; Sittampalam, Gurusingham; McKew, John C.; Austin, Christopher P.; Prusiner, Stanley B.

    2014-01-01

    The microtubule-associated protein (MAP) tau has been implicated in the pathology of numerous neurodegenerative diseases. In the past decade, the hyperphosphorylated and aggregated states of tau protein have been important targets in the drug discovery field for the potential treatment of Alzheimer’s disease. Although several compounds have been reported to reduce the hyperphosphorylated state of tau or impact the stabilization of tau, their therapeutic activities are still to be validated. Recently, reduction of total cellular tau protein has emerged as an alternate intervention point for drug development and a potential treatment of tauopathies. We have developed and optimized a homogenous assay, using the AlphaLISA and HTRF assay technologies, for the quantification of total cellular tau protein levels in the SH-SY5Y neuroblastoma cell line. The signal-to-basal ratios were 375 and 5.3, and the Z’ factors were 0.67 and 0.60 for the AlphaLISA and HTRF tau assays, respectively. The clear advantages of this homogeneous tau assay over conventional total tau assays, such as ELISA and Western blot, are the elimination of plate wash steps and miniaturization of the assay into 1536-well plate format for the ultra–high-throughput screening of large compound libraries. PMID:23905996

  6. 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.

  7. Attenuated total reflectance Fourier-transform infrared spectroscopy of carboxylic acids adsorbed onto mineral surfaces

    NASA Astrophysics Data System (ADS)

    Kubicki, J. D.; Schroeter, L. M.; Itoh, M. J.; Nguyen, B. N.; Apitz, S. E.

    1999-09-01

    A suite of naturally-occurring carboxylic acids (acetic, oxalic, citric, benzoic, salicylic and phthalic) and their corresponding sodium salts were adsorbed onto a set of common mineral substrates (quartz, albite, illite, kaolinite and montmorillonite) in batch slurry experiments. Solution pH's of approximately 3 and 6 were used to examine the effects of pH on sorption mechanisms. Attenuated total reflectance Fourier-transform infrared (ATR FTIR) spectroscopy was employed to obtain vibrational frequencies of the organic ligands on the mineral surfaces and in solution. UV/visible spectroscopy on supernatant solutions was also employed to confirm that adsorption from solution had taken place for benzoic, salicylic and phthalic acids. Molecular orbital calculations were used to model possible surface complexes and interpret the experimental spectra. In general, the tectosilicates, quartz and albite feldspar, did not chemisorb (i.e., strong, inner-sphere adsorption) the carboxylate anions in sufficient amounts to produce infrared spectra of the organics after rinsing in distilled water. The clays (illite, kaolinite and montmorillonite) each exhibited similar ATR FTIR spectra. However, the illite sample used in this study reacted to form strong surface and aqueous complexes with salicylic acid before being treated to remove free Fe-hydroxides. Chemisorption of carboxylic acids onto clays is shown to be limited without the presence of Fe-hydroxides within the clay matrix.

  8. Rapid intravenous administration of amino acids prevents biliary sludge induced by total parenteral nutrition in humans.

    PubMed

    Wu, Z S; Yu, L; Lin, Y J; Jun, Z J; Min, W S; Jun, Y; Hua, Z B

    2000-01-01

    The aim of this study was to evaluate whether daily rapid intravenous administration of amino acids (IVAA) prevented the formation of biliary sludge in humans receiving long-term total parenteral nutrition (TPN). Thirty adult patients receiving TPN for more than 28 consecutive days were studied. They were randomized to receive either saline solution (placebo) intravenously (15 patients) or 6.9% branched chain amino acid (BCAA)-enriched amino acid (15 synthetic amino acids; Freamine HBC) solution given by administration rapid intravenous (15 patients). The groups were similar with respect to age, sex, diagnosis, liver function test results, amylase levels, TPN time, and time of study. All patients underwent weekly ultrasound studies. Volume and emptying studies of the gallbladder in response to the study drug were performed after 1 week. As a result, none of the patients receiving rapid IVAA had sludge, whereas 11 of the 15 patients receiving placebo had sludge (P < 0.01). Results of emptying studies showed significant contraction of the gallbladder in those in the rapid IVAA group, but not in the placebo group. Consequently, the data suggest that rapid IVAA given daily prevents TPN-induced stasis and sludge in the gallbladder. We conclude that rapid IVAA should be used as routine prophylaxis against biliary sludge and formation of gallstones in patients receiving long-term TPN.

  9. Quantitative Analysis of Alcohol, Sugar, and Tartaric Acid in Alcoholic Beverages Using Attenuated Total Reflectance Spectroscopy

    PubMed Central

    Nagarajan, R.; Gupta, A.; Bajaj, M. M.

    2006-01-01

    Mid-infrared (MIR) spectroscopy in attenuated total reflectance (ATR) mode was used for quantifying ethanol, sucrose, and tartaric acid in alcoholic beverages. One hundred synthetic samples were prepared with different ethanol, sucrose, and tartaric acid concentrations. Experiments were carried out on Bio-Rad 175 C FTS using an ATR accessory. Spectra were recorded in the wavelength region 600–4000 cm −1 . Calibration was performed using partial least squares (PLS) algorithm. Commercially available alcoholic beverages (gin, rum, vodka, etc.) were experimented and concentration of ethanol in these samples was predicted using the developed calibration model. Chemical analysis of these commercial samples was carried out in order to compare the results. The agreement between ATR results with those of chemical analysis revealed good reliability and repeatability of the technique used. PMID:17671618

  10. Acidic and total primary sulfates: development of emission factors for major stationary combustion sources

    SciTech Connect

    Goklamy, I.M.

    1984-02-01

    This paper develops 'best estimates' of emission factors for major sources of acidic and total primary sulphates for use in the compilation of emission inventories for the eastern US, which may, in turn, be used for modelling acidic or sulphate deposition. The authors conclude that much of the existing data on primary sulphates from stationary combustion sources are probably significantly biased upward and, therefore, inappropriate for the derivation of emission factors. Existing estimates of primary sulphate emissions for these source categories are probably substantially inflated. It also concludes that for most source categories, very little confidence can be attached to the best estimates because of the paucity of data obtained from measurement techniques which are likely to be free of systematic bias.

  11. Total and lipid-bound serum sialic acid as markers for carcinoma of the uterine cervix.

    PubMed

    Vivas, I; Spagnuolo, L; Palacios, P

    1992-08-01

    Concentrations of total sialic acid (TSA) and lipid-bound sialic acid (LSA) were determined in serum samples from 88 patients with squamous cell carcinoma of the cervix, 26 normal subjects, and 44 patients with benign uterine or ovarian disease. TSA concentrations in patients with cervical cancer were found to correlate with advanced-stage disease. LSA concentrations were only increased in stage IV of the disease. Sensitivity of the test for stage IB was zero for TSA and 27% for LSA. The specificity of both markers was about 80% due to a low incidence of false-positive values in the pathologic control group. Measurements of TSA or LSA appear to be of no value for the early detection of cervical cancer or to complement the clinical staging of this tumor.

  12. [Studies on hydroxyapatite applicatied in coprecipitate of total salvianolic acids phospholipid complex].

    PubMed

    Chen, Xiao-Yun; Zhang, Zhen-Hai; Liu, Dan; Sun, E; Jia, Xiao-Bin

    2014-03-01

    The purpose of this research was to prepare total salvianolic acids-phytosome-HA coprecipitate to improve drug dissolution and its micromeritic properties. Firstly, the coprecipitate was prepared by solvent method and in vitro dissolution of tripterine was performed with the salvianolic acid B and danshensu as criteria. At the same time, the micromeritic properties was characterizated, the structure of samples was characterized by TEM, DSC, XRD and FTIR. Results showed that when the ratio of drug to HA was 1:2, it had a better dissolution, the accumulative drug-release percent in vitro at 60 min was over 90%. At the same time, it has good liquidity and low moisture absorption. Its micromeritic properties have improved. It is proved that the drug still existed amorphously by microstructure analysis. The preparation process is simple and feasible, it has practical value.

  13. Total, free, and protein-bound thiols in plasma of peritoneal dialysis and predialysis patients.

    PubMed

    Przemysław, Włodek; Piotr, Książek; Grażyna, Chwatko; Danuta, Kowalczyk-Pachel; Małgorzata, Iciek; Bernadeta, Marcykiewicz; Małgorzata, Suliga; Witold, Smoleński

    2011-12-01

    Thiol compounds such as glutathione, homocysteine, and cysteinyl-glycine are the natural reservoir of reductive capacity of the cells. Chronic renal failure is accompanied by disturbances in redox status of plasma thiols. The aim of the present study was to compare the changes in concentrations of different forms of thiols in plasma of terminal renal failure patients, nondialyzed and on peritoneal dialysis. Total concentrations of different redox forms of thiols were determined by high performance liquid chromatography. We observed that total concentration of glutathione in terminal renal failure patients decreased and total concentration of the remaining thiols in these patients significantly increased. Continuous ambulatory peritoneal dialysis had the following features in comparison with nondialyzed patients: (1) glutathione and cysteine concentration was restored and (2) free fraction of thiols rose, while protein-bound fraction dropped (except for homocysteine). Continuous ambulatory peritoneal dialysis corrects total concentration of glutathione and cysteine, in comparison with nondialyzed patients.

  14. Total trans fatty acid analysis in spreadable cheese by capillary zone electrophoresis.

    PubMed

    De Castro, Patrícia Mendonça; Barra, Marcelo Macedo; Costa Ribeiro, Mauro Carlos; Aued-Pimentel, Sabria; Da Silva, Simone Alves; De Oliveira, Marcone Augusto Leal

    2010-02-10

    An alternative method for determination of total trans fatty acids expressed as elaidic acid by capillary zone electrophoresis (CZE) under indirect UV detection at 224 nm within an analysis time of 7.5 min was developed. The optimized running electrolyte includes 15.0 mmol L(-1) KH(2)PO(4)/Na(2)HPO(4) buffer (pH approximately 7.0), 4.0 mmol L(-1) SDBS, 8.0 mmol L(-1) Brij35, 45%v/v ACN, 8% methanol, and 1.5% v/v n-octanol. Baseline separation of the critical pair C18-9cis/C18:1-9t with a resolution higher than 1.5 was achieved using C15:0 as the internal standard. The optimum capillary electrophoresis (CE) conditions for the background electrolyte were established with the aid of Raman spectroscopy and experiments of a 3(2) factorial design. After response factor (R(F)) calculations, the CE method was applied to total trans fatty acid (TTFA) analysis in a hydrogenated vegetable fat (HVF) sample, and compared with the American Oil Chemists' Society (AOCS) official method by gas chromatography (GC). The methods were compared with an independent sample t test, and no significant difference was found between CE and GC methods within the 95% confidence interval for six genuine replicates of TTFA analysis (p-value > 0.05). The CE method was applied to TTFA analysis in a spreadable cheese sample. Satisfactory results were obtained, indicating that the optimized methodology can be used for trans fatty acid determination for these samples.

  15. Comparison between intravenous and intra-articular regimens of tranexamic acid in reducing blood loss during total knee arthroplasty.

    PubMed

    Soni, Ashwani; Saini, Raghav; Gulati, Anmol; Paul, Rajesh; Bhatty, Shiraj; Rajoli, Sreekanth Reddy

    2014-08-01

    Tranexamic acid is an antifibrinolytic drug used widely to prevent bleeding. Its use in reducing bleeding during total knee arthroplasty surgery is well proven but there is no final consensus regarding the regimen. The purpose of our study was to compare the effectiveness of intravenous and intra-articular regimen of tranexamic acid during the total knee arthroplasty surgery. A total of 40 patients were received three doses of intravenous tranexamic acid during total knee arthroplasty surgery. Intra-articular tranexamic acid was used in 40 patients during the surgery. We concluded that intra-articular tranexamic acid is equally effective as three dose intravenous regimen in reducing blood loss during total knee arthroplasty surgery.

  16. Application of infrared portable sensor technology for predicting perceived astringency of acidic whey protein beverages.

    PubMed

    Wang, Ting; Tan, Siow-Ying; Mutilangi, William; Plans, Marcal; Rodriguez-Saona, Luis

    2016-12-01

    Formulating whey protein beverages at acidic pH provides better clarity but the beverages typically develop an unpleasant and astringent flavor. Our aim was to evaluate the application of infrared spectroscopy and chemometrics in predicting astringency of acidic whey protein beverages. Whey protein isolate (WPI), whey protein concentrate (WPC), and whey protein hydrolysate (WPH) from different manufacturers were used to formulate beverages at pH ranging from 2.2 to 3.9. Trained panelists using the spectrum method of descriptive analysis tested the beverages providing astringency scores. A portable Fourier transform infrared spectroscopy attenuated total reflectance spectrometer was used for spectra collection that was analyzed by multivariate regression analysis (partial least squares regression) to build calibration models with the sensory astringency scores. Beverage astringency scores fluctuated from 1.9 to 5.2 units and were explained by pH, protein type (WPC, WPI, or WPH), source (manufacturer), and their interactions, revealing the complexity of astringency development in acidic whey protein beverages. The WPC and WPH beverages showed an increase in astringency as the pH of the solution was lowered, but no relationship was found for WPI beverages. The partial least squares regression analysis showed strong relationship between the reference astringency scores and the infrared predicted values (correlation coefficient >0.94), giving standard error of cross-validation ranging from 0.08 to 0.12 units, depending on whey protein type. Major absorption bands explaining astringency scores were associated with carboxylic groups and amide regions of proteins. The portable infrared technique allowed rapid prediction of astringency of acidic whey protein beverages, providing the industry a novel tool for monitoring sensory characteristics of whey-containing beverages.

  17. Fish protein hydrolysate reduces plasma total cholesterol, increases the proportion of HDL cholesterol, and lowers acyl-CoA:cholesterol acyltransferase activity in liver of Zucker rats.

    PubMed

    Wergedahl, Hege; Liaset, Bjørn; Gudbrandsen, Oddrun Anita; Lied, Einar; Espe, Marit; Muna, Ziad; Mørk, Sverre; Berge, Rolf K

    2004-06-01

    There is growing evidence that soy protein improves the blood lipid profiles of animals and humans. We compared the effects of fish protein hydrolysate (FPH), soy protein, and casein (control) on lipid metabolism in Wistar rats and genetically obese Zucker (fa/fa) rats. In Zucker rats, FPH treatment affected the fatty acid composition in liver, plasma, and triacylglycerol-rich lipoproteins. The mRNA levels of Delta 5 and Delta 6 desaturases were reduced by FPH and soy protein feeding compared with casein feeding. In Zucker rats both FPH and soy protein treatment reduced the plasma cholesterol level. Furthermore, the HDL cholesterol:total cholesterol ratio was greater in these rats and in the Wistar rats fed FPH and soy protein compared with those fed casein. Although fecal total bile acids were greater in soy protein-fed Zucker rats than in casein-fed controls, those fed FPH did not differ from the controls. However, the acyl-CoA:cholesterol acyltransferase activity was reduced in Zucker rats fed FPH and tended to be lower (P = 0.13) in those fed soy protein compared with those fed casein. Low ratios of methionine to glycine and lysine to arginine in the FPH and soy protein diets, compared with the casein diet, may be involved in lowering the plasma cholesterol concentration. Our results indicate that the effects of FPH and soy protein on fatty acid metabolism are similar in many respects, but the hypocholesterolemic effects of FPH and soy protein appear to be due to different mechanisms. FPH may have a role as a cardioprotective nutrient.

  18. Nucleic Acid Programmable Protein Array: A Just-In-Time Multiplexed Protein Expression and Purification Platform

    PubMed Central

    Qiu, Ji; LaBaer, Joshua

    2012-01-01

    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. PMID:21943897

  19. 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.

  20. 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.

  1. Development of radiation sterilized dip slides for enumerating lactic acid bacteria and total count in foodstuffs

    NASA Astrophysics Data System (ADS)

    Eisenberg, E.; Padova, R.; Kirsch, E.; Weissman, Sh.; Hirshfeld, T.; Shenfeld, A.

    APT agar (APT) used for enumeration of lactic acid bacteria and Plate Count agar (PCA) applied for total count were sterilized by gamma radiation using radiation dose of 10-15 kGy. Radiosterilized PCA and APT modified by adding catalase prior to irradiation, or APT with increased content of yeast extract performed, as well as, the heat sterilized commercial media. Growth performance was evaluated on several strains of microorganisms, as well as, by enumeration of bacteria in food products. Radiosterilization of culture media in final packaging, can be applied to produce dip slide kits containing PCA or APT.

  2. Synthesis of hydrochloric acid solution for total mercury determination in natural waters.

    PubMed

    Patel-Sorrentino, Nathalie; Benaim, Jean-Yves; Cossa, Daniel; Lucas, Yves

    2011-01-01

    Total mercury (Hg(T)) determination requires the addition of concentrated hydrochloric acid solution (≥10 mol L(-1) HCl) in relatively high amounts to preserve the samples and to prepare reagent solutions. A method for the preparation of concentrated HCl with Hg(T) concentration of lower than 5 ng L(-1) is described in this article. It is based on the well-known chemical reaction: 2 NH(4)Cl + H(2)SO(4) → (NH(4))(2)SO(4) + 2 HCl. This method is validated thanks to the US Environmental Protection Agency method 1631 and standard reference materials BCR-579 (mercury in coastal seawater).

  3. Human soleus and vastus lateralis muscle protein metabolism with an amino acid infusion.

    PubMed

    Carroll, Chad C; Fluckey, James D; Williams, Rick H; Sullivan, Dennis H; Trappe, Todd A

    2005-03-01

    The calf muscles, compared with the thigh, are less responsive to resistance exercise in ambulatory and bed-rested individuals, apparently due to muscle-specific differences in protein metabolism. We chose to evaluate the efficacy of using amino acids to elevate protein synthesis in the soleus, because amino acids have been shown to have a potent anabolic effect in the vastus lateralis. Mixed muscle protein synthesis in the soleus and vastus lateralis was measured before and after infusion of mixed amino acids in 10 individuals (28 +/- 1 yr). Phosphorylation of ribosomal protein p70 S6 kinase (p70S6K; Thr389) and eukaryotic initiation factor 4E-binding protein-1 (4E-BP1; Thr37/46) was also evaluated at rest and after 3 h of amino acid infusion. Basal protein synthesis was similar (P = 0.126), and amino acids stimulated protein synthesis to a similar extent (P = 0.004) in the vastus lateralis (0.043 +/- 0.011%/h) and soleus (0.032 +/- 0.017%/h). Phosphorylation of p70S6K (P = 0.443) and 4E-BP1 (P = 0.192) was not increased in either muscle; however, the soleus contained more total (P = 0.002) and phosphorylated (P = 0.013) 4E-BP1 than the vastus lateralis. These data support the need for further study of amino acid supplementation as a means to compensate for the reduced effectiveness of calf resistance exercise in ambulatory individuals and those exposed to extended periods of unloading. The greater 4E-BP1 in the soleus suggests that there is a muscle-specific distribution of general translational initiation machinery in human skeletal muscle.

  4. Design of a Protein-Targeting System for Lactic Acid Bacteria

    PubMed Central

    Dieye, Y.; Usai, S.; Clier, F.; Gruss, A.; Piard, J.-C.

    2001-01-01

    We designed an expression and export system that enabled the targeting of a reporter protein (the staphylococcal nuclease Nuc) to specific locations in Lactococcus lactis cells, i.e., cytoplasm, cell wall, or medium. Optimization of protein secretion and of protein cell wall anchoring was performed with L. lactis cells by modifying the signals located at the N and C termini, respectively, of the reporter protein. Efficient translocation of precursor (∼95%) is obtained using the signal peptide from the lactococcal Usp45 protein and provided that the mature protein is fused to overall anionic amino acids at its N terminus; those residues prevented interactions of Nuc with the cell envelope. Nuc could be covalently anchored to the peptidoglycan by using the cell wall anchor motif of the Streptococcus pyogenes M6 protein. However, the anchoring step proved to not be totally efficient in L. lactis, as considerable amounts of protein remained membrane associated. Our results may suggest that the defect is due to limiting sortase in the cell. The optimized expression and export vectors also allowed secretion and cell wall anchoring of Nuc in food-fermenting and commensal strains of Lactobacillus. In all strains tested, both secreted and cell wall-anchored Nuc was enzymatically active, suggesting proper enzyme folding in the different locations. These results provide the first report of a targeting system in lactic acid bacteria in which the final location of a protein is controlled and biological activity is maintained. PMID:11418555

  5. A prospective double-blind placebo controlled trial of topical tranexamic acid in total knee arthroplasty.

    PubMed

    Georgiadis, Andrew G; Muh, Stephanie J; Silverton, Craig D; Weir, Robb M; Laker, Michael W

    2013-09-01

    Tranexamic acid (TNA) reduces postoperative blood loss in general and obstetrical surgery but there is limited orthopaedic literature regarding its use in the topical setting. To study the effect of topical TNA after primary total knee arthroplasty (TKA), 101 patients were randomized to topical administration of 2.0g TNA in 75mL of normal saline (50 patients) or placebo (51 patients). Operative technique, drug administration, and venous thromboembolism prophylaxis were standardized. All patients underwent screening ultrasound of the operative extremity. Total blood loss was lower in the TNA group (940.2±327.1mL) than the placebo group (1293.1±532.7mL)(P<0.001), and four patients in the placebo group and none in the TNA group received postoperative transfusion (P=0.118). We recommend administration of topical TNA in primary TKA in healthy patients to decrease perioperative blood loss.

  6. Blood Sparing Efficacy of Oral Tranexamic Acid in Primary Total Knee Arthroplasty: A Randomized Controlled Trial

    PubMed Central

    Lee, Qunn Jid; Ching, Wai Yee; Wong, Yiu Chung

    2017-01-01

    Purpose Tranexamic acid (TXA) is effective in reducing blood loss in primary total knee replacement. Almost all studies used an intravenous form or a topical form. The aim of this study was to assess the blood sparing efficacy and the safety of oral TXA. Materials and Methods All patients with primary total knee replacement performed in our institute from January 2015 to October 2015 were eligible. Oral TXA group was given 1 g oral TXA 2 hours before induction of anesthesia and 6 hours and 12 hours postoperatively. The control group was not given TXA. Results There were 94 cases in the oral TXA group and 95 cases in the control group. There was no difference in the baseline characteristics. The oral TXA group had a significantly lower hemoglobin drop (1.7 g/dL vs. 2.5 g/dL), lower drain output (154 mL vs. 203 mL), lower hidden blood loss (244 mL vs. 423 mL) and lower total blood loss (398 mL vs. 626 mL). There was no difference in transfusion rate (1.1% vs. 3.2%) and thromboembolic complication. There was no infection or mortality in both groups. Conclusions Oral TXA is effective in reducing blood loss in primary total knee replacement. It is a safe alternative to the intravenous or topical form. PMID:28231650

  7. Increased hepatocellular carcinoma risk in chronic hepatitis B patients with persistently elevated serum total bile acid: a retrospective cohort study

    PubMed Central

    Wang, Haoliang; Shang, Xiaoyun; Wan, Xing; Xiang, Xiaomei; Mao, Qing; Deng, Guohong; Wu, Yuzhang

    2016-01-01

    To investigate the association between long-term changes of serum total bile acid and hepatocellular carcinoma in chronic hepatitis B patients, we did a retrospective cohort study of 2262 chronic hepatitis B patients with regular antiviral treatment using data from the Hepatitis Biobank at Southwest Hospital Program from 2004 to 2014. Patients in the study were classified into 3 groups according to persistence of elevated serum total bile acid during follow-up: none-low, medium, and high persistence of elevated serum total bile acid. The association between persistence of elevated serum total bile acid and hepatocellular carcinoma was estimated using Cox proportional hazard models and Kaplan-Meier analysis including information about patients’ demographic and clinical characteristics. There were 62 hepatocellular carcinoma cases during a total follow-up of 14756.5 person-years in the retrospective study. Compared to patients with none-low persistence of elevated total bile acid, the multivariate adjusted hazard ratios (95% confidence interval) were 2.37 (1.16–4.84), and 2.57 (1.28–5.16) for patients with medium, and high persistence of elevated total bile acid. Our findings identified persistence of elevated serum total bile acid as an independent risk factor of hepatocellular carcinoma in chronic hepatitis B patients. PMID:27905528

  8. Total serum bile acid as a potential marker for the diagnosis of cholangiocarcinoma without jaundice.

    PubMed

    Sombattheera, Sutthikan; Proungvitaya, Tanakorn; Limpaiboon, Temduang; Wongkham, Sopit; Wongkham, Chaisiri; Luvira, Vor; Proungvitaya, Siriporn

    2015-01-01

    Diagnosis of cholangiocarcinoma (CCA) is difficult when patients do not show jaundice. The aim of this study was to examine the feasibility of using the total serum bile acid (TSBA) level as an aid for the diagnosis of CCA in patients without jaundice. For this purpose, TSBA of the following groups were measured using a Beckman Synchron CX4 clinical chemistry analyzer: 60 cases of CCA with total serum bilirubin ≤2 mg/dL (low total bilirubin group, LTB); 32 cases of CCA with total serum bilirubin >2 mg/dL (high total bilirubin group, HTB); and 115 healthy controls. Liver function parameters such as serum cholesterol, albumin, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were also examined. The results showed that the TSBA of both LTB and HTB groups of the CCA patients were significantly higher than that of the healthy controls. Also, significant correlation was observed between TSBA and total bilirubin levels in the HTB group of CCA patients. However, no such correlation was seen in the LTB group. The cut-off value of TSBA was determined for the LTB group of CCA patients using the receiver operating characteristic curve analysis, and it was 6.05 μmol/L with the sensitivity and specificity of 46.7% and 84.4%, respectively. In addition, the ALP level was correlated well with the TSBA level and ALP in HTB group was significantly higher than that of LTB group. Moreover, the combination of high TSBA and high ALP levels gave higher specificity up to 97.4%. TSBA might be useful for the diagnosis of CCA patients without jaundice.

  9. Estimation of salivary flow rate, pH, buffer capacity, calcium, total protein content and total antioxidant capacity in relation to dental caries severity, age and gender

    PubMed Central

    Pandey, Pallavi; Reddy, N. Venugopal; Rao, V. Arun Prasad; Saxena, Aditya; Chaudhary, C. P.

    2015-01-01

    Purpose: The aim of the study was to evaluate salivary flow rate, pH, buffering capacity, calcium, total protein content and total antioxidant capacity in relation to dental caries, age and gender. Materials and Methods: The study population consisted of 120 healthy children aged 7–15 years that was further divided into two groups: 7–10 years and 11–15 years. In this 60 children with DMFS/dfs = 0 and 60 children with DMFS/dfs ≥5 were included. The subjects were divided into two groups; Group A: Children with DMFS/dfs = 0 (caries-free) Group B: Children with DMFS/dfs ≥5 (caries active). Unstimulated saliva samples were collected from all groups. Flow rates were determined, and samples analyzed for pH, buffer capacity, calcium, total protein and total antioxidant status. Salivary antioxidant activity is measured with spectrophotometer by an adaptation of 2,2’-azino-di-(3-ethylbenzthiazoline-6-sulphonate) assays. Results: The mean difference of the two groups; caries-free and caries active were proved to be statistically significant (P < 0.05) for salivary calcium, total protein and total antioxidant level for both the sexes in the age group 7–10 years and for the age 11–15 years the mean difference of the two groups were proved to be statistically significant (P < 0.05) for salivary calcium level for both the sexes. Salivary total protein and total antioxidant level were proved to be statistically significant for male children only. Conclusions: In general, total protein and total antioxidants in saliva were increased with caries activity. Calcium content of saliva was found to be more in caries-free group and increased with age. PMID:25821379

  10. Thiol-based Redox Proteins in Brassica napus Guard Cell Abscisic Acid and Methyl Jasmonate Signaling

    PubMed Central

    Zhu, Mengmeng; Zhu, Ning; Song, Wen-yuan; Harmon, Alice C.; Assmann, Sarah M.; Chen, Sixue

    2014-01-01

    SUMMARY Reversibly oxidized cysteine sulfhydryl groups serve as redox sensors or targets of redox sensing that are important in different physiological processes. Little is known, however, about redox sensitive proteins in guard cells and how they function in stomatal signaling. In this study, Brassica napus guard cell proteins altered by redox in response to abscisic acid (ABA) or methyl jasmonate (MeJA) were identified by complementary proteomics approaches, saturation differential in-gel electrophoresis (DIGE) and isotope-coded affinity tag (ICAT). In total, 65 and 118 potential redox responsive proteins were identified in ABA and MeJA treated guard cells, respectively. All the proteins contain at least one cysteine, and over half of them are predicted to form intra-molecular disulfide bonds. Most of the proteins fall into the functional groups of energy, stress and defense, and metabolism. Based on the peptide sequences identified by mass spectrometry, 30 proteins were common to ABA and MeJA treated samples. A total of 44 cysteines was mapped in all the identified proteins, and their levels of redox sensitivity were quantified. Two of the proteins, a SNRK2 kinase and an isopropylmalate dehydrogenase were confirmed to be redox regulated and involved in stomatal movement. This study creates an inventory of potential redox switches, and highlights a protein redox regulatory mechanism in guard cell ABA and MeJA signal transduction. PMID:24580573

  11. Malic acid or orthophosphoric acid-heat treatments for protecting sunflower (Helianthus annuus) meal proteins against ruminal degradation and increasing intestinal amino acid supply.

    PubMed

    Arroyo, J M; González, J; Ouarti, M; Silván, J M; Ruiz del Castillo, M L; de la Peña Moreno, F

    2013-02-01

    increases for those AA showing a high resistance to degradation in the untreated meal were also observed. The total supply of metabolisable AA was increased by 3.87 times for sulphur-containing AA, whereas that of lysine was increased by 2.5 times, mainly because of lysine losses with heat treatments. These treatments and especially that with malic acid would be useful to increase the protein value of these meals but their combined use with lysine-rich protein concentrates would improve the metabolisable protein profile.

  12. Urinary intestinal fatty acid binding protein predicts necrotizing enterocolitis.

    PubMed

    Gregory, Katherine E; Winston, Abigail B; Yamamoto, Hidemi S; Dawood, Hassan Y; Fashemi, Titilayo; Fichorova, Raina N; Van Marter, Linda J

    2014-06-01

    Necrotizing enterocolitis, characterized by sudden onset and rapid progression, remains the most significant gastrointestinal disorder among premature infants. In seeking a predictive biomarker, we found intestinal fatty acid binding protein, an indicator of enterocyte damage, was substantially increased within three and seven days before the diagnosis of necrotizing enterocolitis.

  13. [Photochemistry and UV Spectroscopy of Proteins and Nucleic Acids].

    PubMed

    Wierzchowski, Kazimierz Lech

    2015-01-01

    The article presents a short history of David Shugar studies in the field of photochemistry and UV spectroscopy of proteins and nucleic acids, carried out since the late 1940s. to the beginning of the 1970s. of the 20th century, with some references to the state of related research in those days.

  14. Amino acid nutrition beyond methionine and lysine for milk protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Amino acids are involved in many important physiological processes affecting the production, health, and reproduction of high-producing dairy cows. Most research and recommendations for lactating dairy cows has focused on methionine and lysine for increasing milk protein yield. This is because these...

  15. Stimulation of protein synthesis by phosphatidic acid in rat cardiomyocytes.

    PubMed

    Xu, Y J; Yau, L; Yu, L P; Elimban, V; Zahradka, P; Dhalla, N S

    1996-12-13

    Phosphatidic acid (PA) was observed to stimulate protein synthesis in adult cardiomyocytes in a time- and concentration-dependent manner. The maximal stimulation in protein synthesis (142 +/- 12% vs 100% as the control) was achieved at 10 microM PA within 60 min and was inhibited by actinomycin D (107 +/- 4% of the control) or cycloheximide (105 +/- 6% of the control). The increase in protein synthesis due to PA was attenuated or abolished by preincubation of cardiomyocytes with a tyrosine kinase inhibitor, genistein (94 +/- 9% of the control), phospholipase C inhibitors 2-nitro-4-carboxyphenyl N,N-diphenyl carbamate or carbon-odithioic acid O-(octahydro-4,7-methanol-1H-inden-5-yl (101 +/- 6 and 95 +/- 5% of the control, respectively), protein kinase C inhibitors staurosporine or polymyxin B (109 +/- 3 and 93 +/- 3% of the control), and chelators of extracellular and intracellular free Ca2+ EGTA or BAPTA/AM (103 +/- 6 and 95 +/- 6% of the control, respectively). PA at different concentrations (0.1 to 100 microM) also caused phosphorylation of a cell surface protein of approximately 24 kDa. In addition, mitogen-activated protein kinase was stimulated by PA in a concentration-dependent manner; maximal stimulation (217 +/- 6% of the control) was seen at 10 microM PA. These data suggest that PA increases protein synthesis in adult rat cardiomyocytes and thus may play an important role in the development of cardiac hypertrophy.

  16. Automated multiple flow-injection analysis in clinical chemistry: determination of total protein with Biuret reagent.

    PubMed

    Shideler, C E; Stewart, K K; Crump, J; Wills, M R; Savory, J; Renoe, B W

    1980-09-01

    We have examined the feasibility of the automated multiple flow-injection technique for application to clinical chemistry by adapting to this system the biuret method for the determination of total protein. Samples were discretely and rapidly introduced into a continuously flowing, nonsegmented reagent stream by means of an automatic sampler and high-pressure injection valve. Pumps operating at 1380-2070 kPa (200-300 psi) were utilized to introduce the biuret reagent and saline diluent into the system separately at flow rates of 72 and 47 microL/s, respectively. Use of 20-microL sample and a 3.0-s reaction-delay coil was adequately sensitive for analysis for total protein by this method. Samples were analyzed at a rate of 150/h with no detectable between-sample carryover. Within-run precision studies yielded relative standard deviations of 2.5% and less. Total protein values obtained by this method correlated well with those obtained by centrifugal analyzer and bubble-segmented continuous-flow biuret methods.

  17. [Abnormal Serum Total Protein Measurement by Lipoprotein-X in an Infant with Biliary Atresia].

    PubMed

    Futatsugi, Akiko; Hidaka, Eiko; Kubota, Noriko; Nishijima, Fumie; Yoshizawa, Katsumi; Ishimine, Nau; Sugano, Mitsutoshi; Hori, Atsushi; Hidaka, Hiroya

    2015-11-01

    Lipoprotein-X (LP-X) in cholestatic jaundice causes abnormal reaction in assays for low-density lipoprotein-cholesterol, but the effects on other test items are unknown. Here, we report an infant with biliary atresia showing abnormal reaction in total serum protein assay using the biuret method, and lipoprotein-X (LP-X) was then detected. In this 11-month-old female infant, jaundice was observed at 2 months old, and a diagnosis of biliary atresia was made. On biochemical tests at 12 months old, the total serum protein concentrations detected by the biuret method were very high, and the response curve and linearity of dilution were abnormal. LP-X was detected by agar electrophoresis. In addition and recovery experiments with normal serum fractionation of the patient's LP-X-rich lipoprotein fraction prepared by ultracentrifugation, normal γ-globulin fractionation showed an abnormal reaction by the biuret method. In infants with biliary atresia, we showed that the total serum protein assay by the biuret method was influenced by LP-X-rich lipoprotein, which may be caused by abnormal reaction of LP-X and γ-globulin. [Case Report].

  18. Amino Acid Composition of an Organic Brown Rice Protein Concentrate and Isolate Compared to Soy and Whey Concentrates and Isolates.

    PubMed

    Kalman, Douglas S

    2014-06-30

    A protein concentrate (Oryzatein-80™) and a protein isolate (Oryzatein-90™) from organic whole-grain brown rice were analyzed for their amino acid composition. Two samples from different batches of Oryzatein-90™ and one sample of Oryzatein-80™ were provided by Axiom Foods (Los Angeles, CA, USA). Preparation and analysis was carried out by Covance Laboratories (Madison, WI, USA). After hydrolysis in 6-N hydrochloric acid for 24 h at approximately 110 °C and further chemical stabilization, samples were analyzed by HPLC after pre-injection derivitization. Total amino acid content of both the isolate and the concentrate was approximately 78% by weight with 36% essential amino acids and 18% branched-chain amino acids. These results are similar to the profiles of raw and cooked brown rice except in the case of glutamic acid which was 3% lower in the isolate and concentrate. The amino acid content and profile of the Oryzatein-90™ isolate was similar to published values for soy protein isolate but the total, essential, and branched-chain amino acid content of whey protein isolate was 20%, 39% and 33% greater, respectively, than that of Oryzatein-90™. These results provide a valuable addition to the nutrient database of protein isolates and concentrates from cereal grains.

  19. Simultaneous determination of amino acid nitrogen and total acid in soy sauce using near infrared spectroscopy combined with characteristic variables selection.

    PubMed

    Zhao, Jiewen; Ouyang, Qin; Chen, Quansheng; Lin, Hao

    2013-08-01

    Amino acid nitrogen and total acid are two most important quality indices to assess the quality of soy sauce in China. This work employed near infrared spectroscopy combined with synergy interval partial least square and genetic algorithm to detect amino acid nitrogen and total acid content in soy sauce. First, synergy interval partial least square was used to select efficient spectral regions from the full spectrum region; and then, genetic algorithm was used to selected variables from the efficient spectral regions, to build partial least square model. The optimal genetic algorithm synergy interval partial least square models were obtained as follows: Rc  = 0.9988 and Rp = 0.9988 for amino acid nitrogen content model using 64 variables; Rc = 0.9917 and Rp = 0.9902 for total acid content model using 81 variables. Genetic algorithm synergy interval partial least square models showed superiority over the partial least square and synergy interval partial least square models. The results indicated that amino acid nitrogen and total acid content in soy sauce could be rapidly determined by near infrared spectroscopy technique. Also, the results indicated that genetic algorithm synergy interval partial least square can improve the performance in measurement of amino acid nitrogen and total acid content by near infrared spectroscopy.

  20. Changes in total soluble proteins and Ca2+ upon cryopreservation of Prunus mume pollen.

    PubMed

    Zhang, Y L; Li, B L; Wang, H; Liu, Y

    2012-01-01

    Mei Flowers (Prunus mume) are traditional Chinese ornamental plants. Fifty-one Mei cultivars have been conserved in a pollen cryobank since 2003. We used two-dimensional electrophoresis (2-DE) of total soluble proteins and flow cytometric detection of Ca2+ fluorescence to probe changes in pollen grains before and after cryopreservation. Results indicated that: (1) electrophoresis maps of total soluble proteins before and after cryostorage of pollen from three cultivars were different, even though 70 percent of the protein spots among these three cultivars were matched after cryopreservation. We found some protein spots that changed in all three cultivars; their molecular weights and pI were between 12.6-72.8 kDa and 5.6-7.3, respectively; (2): the geometric mean of Ca2+ fluorescence intensity (GMFI) value of cryopreserved pollen was significantly higher compared with that of fresh pollen in cultivar 'Beijing Yudie'. GMFI increased during pollen germination in our studied cultivars, especially after 0.5 and 1.0 h of culturing. In addition, no positive correlation was found between pollen germination rate and GMFI in the present study.

  1. Enantiomeric deoxycholic acid: total synthesis, characterization, and preliminary toxicity toward colon cancer cell lines.

    PubMed

    Katona, Bryson W; Rath, Nigam P; Anant, Shrikant; Stenson, William F; Covey, Douglas F

    2007-11-23

    Deoxycholic acid (DCA) is an endogenous secondary bile acid implicated in numerous pathological conditions including colon cancer formation and progression and cholestatic liver disease. DCA involvement in these disease processes results partly from its ability to modulate signaling cascades within the cell, presumably through both direct receptor activation and general detergent mediated membrane changes. To further explore DCA induced changes in cell signaling, we completed a total synthesis of enantiomeric deoxycholic acid (ent-DCA) from achiral 2-methyl-1,3-cyclopentanedione. Using a modified method of the synthesis of ent-testosterone that proceeds through the (R)-(-)-Hajos-Parrish ketone, we have completed the successful synthesis of ent-DCA in 25 steps with a yield of 0.3% with all stereochemical assignments of the product confirmed by X-ray crystallography. Our studies toward this synthesis also uncovered the methodology for the development of a novel A,B-cis steroidal skeleton system containing a C3-C9 single bond as well as conditions to selectively ketalize the typically less reactive 12-carbonyl in poly-keto A,B-cis androgens. The critical micelle concentration (cmc) of ent-DCA, determined by a dye solubilization method, was identical to the cmc of natural DCA. Toxicity studies toward HT-29 and HCT-116 human colon cancer cell lines demonstrated that ent-DCA had similar effects on proliferation, yet showed a markedly decreased ability to induce apoptosis as compared to natural DCA.

  2. Leukocyte protease binding to nucleic acids promotes nuclear localization and cleavage of nucleic acid binding proteins.

    PubMed

    Thomas, Marshall P; Whangbo, Jennifer; McCrossan, Geoffrey; Deutsch, Aaron J; Martinod, Kimberly; Walch, Michael; Lieberman, Judy

    2014-06-01

    Killer lymphocyte granzyme (Gzm) serine proteases induce apoptosis of pathogen-infected cells and tumor cells. Many known Gzm substrates are nucleic acid binding proteins, and the Gzms accumulate in the target cell nucleus by an unknown mechanism. In this study, we show that human Gzms bind to DNA and RNA with nanomolar affinity. Gzms cleave their substrates most efficiently when both are bound to nucleic acids. RNase treatment of cell lysates reduces Gzm cleavage of RNA binding protein targets, whereas adding RNA to recombinant RNA binding protein substrates increases in vitro cleavage. Binding to nucleic acids also influences Gzm trafficking within target cells. Preincubation with competitor DNA and DNase treatment both reduce Gzm nuclear localization. The Gzms are closely related to neutrophil proteases, including neutrophil elastase (NE) and cathepsin G. During neutrophil activation, NE translocates to the nucleus to initiate DNA extrusion into neutrophil extracellular traps, which bind NE and cathepsin G. These myeloid cell proteases, but not digestive serine proteases, also bind DNA strongly and localize to nuclei and neutrophil extracellular traps in a DNA-dependent manner. Thus, high-affinity nucleic acid binding is a conserved and functionally important property specific to leukocyte serine proteases. Furthermore, nucleic acid binding provides an elegant and simple mechanism to confer specificity of these proteases for cleavage of nucleic acid binding protein substrates that play essential roles in cellular gene expression and cell proliferation.

  3. Kinetic study of sulphuric acid hydrolysis of protein feathers.

    PubMed

    Ben Hamad Bouhamed, Sana; Kechaou, Nabil

    2017-02-28

    Poultry feather keratin is the most important by-product from the poultry industry due to its abundance. Different methods have been still applied to process this by-product such as enzymatic hydrolysis which is expensive and inapplicable at the industrial level. This paper presents a study of acid hydrolysis of poultry feathers using different types of acids, sulphuric acid concentration, different temperatures and solid to liquid ratio to obtain a liquid product rich in peptides. The feathers analysis revealed a crude protein content of 88.83%. A maximum peptides production of 676 mg/g was reached using sulphuric acid, 1 molar acid concentration and 50 g/l solid to liquid ratio at a temperature of 90 °C after 300 min. A reaction scheme for protein aggregation and decomposition to polypeptides and amino acids was proposed and a kinetic model for peptides production was developed. The proposed kinetic model proved to be well adapted to the experimental data with R (2) = 0.99.

  4. Seasonal and spatial changes of free and bound organic acids in total suspended particles in Guangzhou, China

    NASA Astrophysics Data System (ADS)

    Ma, Shexia; Peng, Ping'an; Song, Jianzhong; Bi, Xinhui; Zhao, Jinping; He, Lulu; Sheng, Guoying; Fu, Jiamo

    2010-12-01

    The concentrations and compositions of free and bound organic acids in total suspended particles from typical urban, suburban and forest park sites of Guangzhou were determined in this study. The free form of organic acids (solvent extractable) in aerosols in Guangzhou varied with site and season. The suburban samples contained the highest contents of alkanoic, alkenoic and dicarboxylic acids. These findings were consistent with a higher supply of hydrocarbons and NOx in the suburban area. However, concentrations of aromatic acids were similar in the urban, suburban and forest park sites. Generally, winter season samples of the acids from anthropogenic sources contained more organic acids than summer season samples due to stronger removal by wet deposition in the summer. For the acids from botanic sources, the summer season samples were higher. In addition to the free acids, bound acids (solvent non-extractable) mainly formed by esterification of free acids were also found in the samples. In general, bound acids were higher than free acids. Esterification is mainly controlled by the pKa of organic acids and the atmospheric pH value. This explains why aromatic and dicarboxylic acids occur mainly as bound forms and why the samples from urban sites contained high levels of bound acids as the pH of rain water can reach 4.53. Concentrations of alkanoic and alkenoic acids in the aerosols of Guangzhou were much higher than those in the other areas studied.

  5. A Platform for Combined DNA and Protein Microarrays Based on Total Internal Reflection Fluorescence

    PubMed Central

    Asanov, Alexander; Zepeda, Angélica; Vaca, Luis

    2012-01-01

    We have developed a novel microarray technology based on total internal reflection fluorescence (TIRF) in combination with DNA and protein bioassays immobilized at the TIRF surface. Unlike conventional microarrays that exhibit reduced signal-to-background ratio, require several stages of incubation, rinsing and stringency control, and measure only end-point results, our TIRF microarray technology provides several orders of magnitude better signal-to-background ratio, performs analysis rapidly in one step, and measures the entire course of association and dissociation kinetics between target DNA and protein molecules and the bioassays. In many practical cases detection of only DNA or protein markers alone does not provide the necessary accuracy for diagnosing a disease or detecting a pathogen. Here we describe TIRF microarrays that detect DNA and protein markers simultaneously, which reduces the probabilities of false responses. Supersensitive and multiplexed TIRF DNA and protein microarray technology may provide a platform for accurate diagnosis or enhanced research studies. Our TIRF microarray system can be mounted on upright or inverted microscopes or interfaced directly with CCD cameras equipped with a single objective, facilitating the development of portable devices. As proof-of-concept we applied TIRF microarrays for detecting molecular markers from Bacillus anthracis, the pathogen responsible for anthrax. PMID:22438738

  6. Evaluation of oxidative stress via total antioxidant status, sialic acid, malondialdehyde and RT-PCR findings in sheep affected with bluetongue

    PubMed Central

    Aytekin, I.; Aksit, H.; Sait, A.; Kaya, F.; Aksit, D.; Gokmen, M.; Baca, A. Unsal

    2015-01-01

    Introduction Bluetongue (BT) is a non-contagious infectious disease of ruminants. The disease agent bluetongue virus (BTV) is classified in the Reoviridae family Orbivirus. Aims and objectives The aim of this study was to determine serum malondialdehyde (MDA), total antioxidative stres (TAS), total sialic acid (TSA), ceruloplasmin, triglyceride, alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyltransferase (GGT), cholesterol, creatinine, albumin, and total protein levels in sheep with and without bluetongue (BT). Materials and Methods The study included 13 Sakiz crossbreed sheep, aged 1–4 years and usually in the last stage of pregnancy, as the BT group and a control group consisting of 10 healthy sheep. All sheep were clinically examined before collecting blood samples. Serum ALT, AST, cholesterol, triglyceride, albumin, GGT, total protein, creatinine and TAS levels were measured using commercially available kits as per manufacturer's recommendations using a Biochemistry Auto Analyzer (Sinnowa D280, China). Serum lipid peroxidation was estimated through a previously described method in which MDA reacts with thiobarbituric acid (TBA) to form a coloured complex at a maximum absorbance of 535 nm. The TSA value was measured at 549 nm using the method described by Warren (1959): sialic acid was oxidised to formyl-pyruvic acid, which reacts with TBA to form a pink product. The ceruloplasmin concentration was measured according to Sunderman and Nomoto (1970): ceruloplasmin and p-phenylenediamine formed a coloured oxidation product that was proportional to the concentration of serum ceruloplasmin. Real time RT-PCR and conventional RT-PCR were performed as described by Shaw and others (2007). Results Biochemistry analysis of serum showed that in the BT group, TSA, MDA, triglyceride and ALT and AST were higher and that ceruloplasmin and TAS were lower than in the control group. Serum albumin, cholesterol, creatinine, total protein and GGT did

  7. Production of Fatty Acids and Protein by Nannochloropsis in Flat-Plate Photobioreactors

    PubMed Central

    Wijffels, René H.; Bolla, Sylvie; Kiron, Viswanath

    2017-01-01

    Nannochloropsis is an industrially-promising microalga that may be cultivated for alternative sources of nutrition due to its high productivity, protein content and lipid composition. We studied the growth and biochemical profile of Nannochloropsis 211/78 (CCAP) in optimized flat-plate photobioreactors. Eighteen cultivations were performed at two nutrient concentrations. The fatty acid, protein content and calorific values were analyzed after 8, 12 and 16 days. Neutral lipids were separated and the changes in fatty acids in triglycerides (TAGs) during nutrient depletion were recorded. The maximum cell density reached 4.7 g∙L-1 and the maximum productivity was 0.51 g∙L-1∙d-1. During nutrient-replete conditions, eicosapentaneoic acid (EPA) and total protein concentrations measured 4.2–4.9% and 50–55% of the dry mass, respectively. Nutrient starvation induced the accumulation of fatty acids up to 28.3% of the cell dry weight, largely due to the incorporation of C16:0 and C16:1n-7 fatty acyl chains into neutral lipids. During nutrient starvation the total EPA content did not detectibly change, but up to 37% was transferred from polar membrane lipids to the neutral lipid fraction. PMID:28103296

  8. Production of Fatty Acids and Protein by Nannochloropsis in Flat-Plate Photobioreactors.

    PubMed

    Hulatt, Chris J; Wijffels, René H; Bolla, Sylvie; Kiron, Viswanath

    2017-01-01

    Nannochloropsis is an industrially-promising microalga that may be cultivated for alternative sources of nutrition due to its high productivity, protein content and lipid composition. We studied the growth and biochemical profile of Nannochloropsis 211/78 (CCAP) in optimized flat-plate photobioreactors. Eighteen cultivations were performed at two nutrient concentrations. The fatty acid, protein content and calorific values were analyzed after 8, 12 and 16 days. Neutral lipids were separated and the changes in fatty acids in triglycerides (TAGs) during nutrient depletion were recorded. The maximum cell density reached 4.7 g∙L-1 and the maximum productivity was 0.51 g∙L-1∙d-1. During nutrient-replete conditions, eicosapentaneoic acid (EPA) and total protein concentrations measured 4.2-4.9% and 50-55% of the dry mass, respectively. Nutrient starvation induced the accumulation of fatty acids up to 28.3% of the cell dry weight, largely due to the incorporation of C16:0 and C16:1n-7 fatty acyl chains into neutral lipids. During nutrient starvation the total EPA content did not detectibly change, but up to 37% was transferred from polar membrane lipids to the neutral lipid fraction.

  9. In situ fluorescent protein imaging with metal film-enhanced total internal reflection microscopy.

    PubMed

    Burghardt, Thomas P; Charlesworth, Jon E; Halstead, Miriam F; Tarara, James E; Ajtai, Katalin

    2006-06-15

    Fluorescence detection of single molecules provides a means to investigate protein dynamics minus ambiguities introduced by ensemble averages of unsynchronized protein movement or of protein movement mimicking a local symmetry. For proteins in a biological assembly, taking advantage of the single molecule approach could require single protein isolation from within a high protein concentration milieu. Myosin cross-bridges in a muscle fiber are proteins attaining concentrations of approximately 120 muM, implying single myosin detection volume for this biological assembly is approximately 1 attoL (10(-18) L) provided that just 2% of the cross-bridges are fluorescently labeled. With total internal reflection microscopy (TIRM) an exponentially decaying electromagnetic field established on the surface of a glass-substrate/aqueous-sample interface defines a subdiffraction limit penetration depth into the sample that, when combined with confocal microscopy, permits image formation from approximately 3 attoL volumes. Demonstrated here is a variation of TIRM incorporating a nanometer scale metal film into the substrate/glass interface. Comparison of TIRM images from rhodamine-labeled cross-bridges in muscle fibers contacting simultaneously the bare glass and metal-coated interface show the metal film noticeably reduces both background fluorescence and the depth into the sample from which fluorescence is detected. High contrast metal film-enhanced TIRM images allow secondary label visualization in the muscle fibers, facilitating elucidation of Z-disk structure. Reduction of both background fluorescence and detection depth will enhance TIRM's usefulness for single molecule isolation within biological assemblies.

  10. A conserved patch of hydrophobic amino acids modulates Myb activity by mediating protein-protein interactions.

    PubMed

    Dukare, Sandeep; Klempnauer, Karl-Heinz

    2016-07-01

    The transcription factor c-Myb plays a key role in the control of proliferation and differentiation in hematopoietic progenitor cells and has been implicated in the development of leukemia and certain non-hematopoietic tumors. c-Myb activity is highly dependent on the interaction with the coactivator p300 which is mediated by the transactivation domain of c-Myb and the KIX domain of p300. We have previously observed that conservative valine-to-isoleucine amino acid substitutions in a conserved stretch of hydrophobic amino acids have a profound effect on Myb activity. Here, we have explored the function of the hydrophobic region as a mediator of protein-protein interactions. We show that the hydrophobic region facilitates Myb self-interaction and binding of the histone acetyl transferase Tip60, a previously identified Myb interacting protein. We show that these interactions are affected by the valine-to-isoleucine amino acid substitutions and suppress Myb activity by interfering with the interaction of Myb and the KIX domain of p300. Taken together, our work identifies the hydrophobic region in the Myb transactivation domain as a binding site for homo- and heteromeric protein interactions and leads to a picture of the c-Myb transactivation domain as a composite protein binding region that facilitates interdependent protein-protein interactions of Myb with regulatory proteins.

  11. Further evaluation of the interrelationship between the hepatocellular transport of bile acids and endocytosed proteins.

    PubMed Central

    Herrera, M. C.; el-Mir, M. Y.; Monte, M. J.; Perez-Barriocanal, F.; Marin, J. J.

    1992-01-01

    Experiments on the relationship between the hepatocellular transport of endogenous or exogenously loaded bile acids (sodium taurocholate, TC, 0.5 mumol/min/100 g body wt) and horseradish peroxidase (HRP) or immunoglobulin A (IgA) (0.5 mg/100 g body wt) were carried out on anaesthetized Wistar rats. The time course of HRP excretion into bile (acceleration in the secretory peak), but not the total amount of HRP output, was affected by TC infusion. Administration of HRP was found to have no stimulatory effect on either spontaneous or TC-induced bile flow, bile acid, lecithin or cholesterol output. Spontaneous bile acid output was increased (25 and 67%, respectively) in rats that were treated for 12-h fasting or by oral administration of TC (45 mg/100 g body wt, every 12 h, for 2 days). These manoeuvres did not change the inability of HRP and IgA to increase bile acid output. Exogenous TC load had no stimulatory effect on the hepatocellular transport of endogenous bile acid pool, that was labelled by a combination of fasting and oral administration of 14C-glycocholic acid 12 h before the experiments. Therefore, exogenous bile acid load-induced stimulation of transcytosis had no effect on endogenous bile acid output. Moreover, bile secretion of both endogenous and exogenously loaded bile acids is unaffected by the administration of proteins, irrespective of whether they are endocytosed by a receptor or nonreceptor mediated process. PMID:1571280

  12. Expression of liver fatty acid binding protein in hepatocellular carcinoma☆

    PubMed Central

    Cho, Soo-Jin; Ferrell, Linda D.; Gill, Ryan M.

    2017-01-01

    Summary Loss of expression of liver fatty acid binding protein (LFABP) by immunohistochemistry has been shown to be characteristic of a subset of hepatocellular adenomas (HCAs) in which HNF1A is inactivated. Transformation to hepatocellular carcinoma is thought to be a very rare phenomenon in the HNF1A-inactivated variant of HCA. However, we recently observed 2 cases at our institution, 1 definite hepatocellular carcinoma and 1 possible hepatocellular carcinoma, with loss of LFABP staining, raising the possibility that LFABP down-regulation may be associated with hepatocellular carcinogenesis. Our aim was to evaluate hepatocellular carcinomas arising in various backgrounds and with varying degrees of differentiation for loss of LFABP staining. Twenty total cases of hepatocellular carcinoma were examined. Thirteen cases arose in a background of cirrhosis due to hepatitis C (n = 8) or steatohepatitis (n = 5); 7 cases arose in a noncirrhotic background, with 2 cases arising within HNF1A-inactivated variant HCA and 2 cases arising within inflammatory variant HCA. Complete loss of expression of LFABP was seen in 6 of 20 cases, including 2 cases of hepatocellular carcinoma arising within HNF1A-inactivated variant HCA. Thus, loss of staining for LFABP appears to be common in hepatocellular carcinoma and may be seen in well-differentiated hepatocellular carcinoma. Therefore, LFABP loss should not be interpreted as evidence for hepatocellular adenoma over carcinoma, when other features support a diagnosis of hepatocellular carcinoma. The findings raise consideration for a role of HNF1A inactivation in hepatocellular carcinogenesis, particularly in less differentiated tumors. PMID:26997447

  13. A rapid method for detecting protein-nucleic acid interactions by protein induced fluorescence enhancement

    PubMed Central

    Valuchova, Sona; Fulnecek, Jaroslav; Petrov, Alexander P.; Tripsianes, Konstantinos; Riha, Karel

    2016-01-01

    Many fundamental biological processes depend on intricate networks of interactions between proteins and nucleic acids and a quantitative description of these interactions is important for understanding cellular mechanisms governing DNA replication, transcription, or translation. Here we present a versatile method for rapid and quantitative assessment of protein/nucleic acid (NA) interactions. This method is based on protein induced fluorescence enhancement (PIFE), a phenomenon whereby protein binding increases the fluorescence of Cy3-like dyes. PIFE has mainly been used in single molecule studies to detect protein association with DNA or RNA. Here we applied PIFE for steady state quantification of protein/NA interactions by using microwell plate fluorescence readers (mwPIFE). We demonstrate the general applicability of mwPIFE for examining various aspects of protein/DNA interactions with examples from the restriction enzyme BamHI, and the DNA repair complexes Ku and XPF/ERCC1. These include determination of sequence and structure binding specificities, dissociation constants, detection of weak interactions, and the ability of a protein to translocate along DNA. mwPIFE represents an easy and high throughput method that does not require protein labeling and can be applied to a wide range of applications involving protein/NA interactions. PMID:28008962

  14. Dietary protein's and dietary acid load's influence on bone health.

    PubMed

    Remer, Thomas; Krupp, Danika; Shi, Lijie

    2014-01-01

    A variety of genetic, mechano-response-related, endocrine-metabolic, and nutritional determinants impact bone health. Among the nutritional influences, protein intake and dietary acid load are two of the factors most controversially discussed. Although in the past high protein intake was often assumed to exert a primarily detrimental impact on bone mass and skeletal health, the majority of recent studies indicates the opposite and suggests a bone-anabolic influence. Studies examining the influence of alkalizing diets or alkalizing supplement provision on skeletal outcomes are less consistent, which raises doubts about the role of acid-base status in bone health. The present review critically evaluates relevant key issues such as acid-base terminology, influencing factors of intestinal calcium absorption, calcium balance, the endocrine-metabolic milieu related to metabolic acidosis, and some methodological aspects of dietary exposure and bone outcome examinations. It becomes apparent that for an adequate identification and characterization of either dietary acid load's or protein's impact on bone, the combined assessment of both nutritional influences is necessary.

  15. Effect of total mixed ration composition on fermentation and efficiency of ruminal microbial crude protein synthesis in vitro.

    PubMed

    Boguhn, J; Kluth, H; Rodehutscord, M

    2006-05-01

    The goal of this study was to identify dietary factors that affect fermentation and efficiency of microbial crude protein (CP(M)) synthesis in the rumen in vitro. We used 16 total mixed, dairy cow rations with known digestibilities that varied in ingredient composition and nutrient content. Each ration was incubated in a Rusitec (n = 3) for 15 d, and fermentation of different fractions was assessed. Observed extents of fermentation in 24 h were 35 to 47% for organic matter, 25 to 60% for crude protein, 3 to 28% for neutral detergent fiber, and 31 to 45% for gross energy. Organic matter fermentation depended on the content of crude protein and neutral detergent fiber in the ration. We studied net synthesis of CP(M) using an 15N dilution technique and found that 7 d of continuous 15N application are needed to achieve an 15N enrichment plateau in the N of isolated microbes in this type of study. The efficiency of CP(M) synthesis was 141 to 286 g/kg of fermented organic matter or 4.9 to 11.1 g/MJ of metabolizable energy, and these ranges agree with those found in the literature. Multiple regressions to predict the efficiency of CP(M) synthesis by diet data showed that crude protein was the only dietary chemical fraction that had a significant effect. Fat content and the inclusion rate of corn silage in the ration also tended to improve efficiency. We suggest that microbial need for preformed amino acids may explain the crude protein effect. A large part of the variation in efficiency of microbial activity still remains unexplained.

  16. Erythrocyte fatty acid composition does not influence levels of free, bioavailable, and total 25-hydroxy vitamin D.

    PubMed

    Carlsson, Martin; Nilsson, Ingela; Brudin, Lars; Von, Siv-Ping; Wanby, Pär

    2017-02-01

    In vitro, mono- and polyunsaturated fatty acids (FAs) may decrease the binding affinity of vitamin D metabolites for vitamin D-binding protein, which in turn may influence their bioavailability. FAs incorporated as phospholipids in erythrocyte (ery-) cell membranes reflect dietary intake. The purpose of this study was to investigate ery-FA composition in relation to markers for vitamin D. In healthy females (age 22.6 ± 2.0 years) total 25(OH)D was measured by LC-MS/MS (n = 78), free 25(OH)D with ELISA (n = 64 of 78), and bioavailable 25(OH)D was calculated. Analysis of ery-FA composition was by gas chromatography (n = 56 of 78). A strong correlation between total 25(OH)D and free 25(OH)D was seen (r = .66, p < .001), and between total-25(OH)D and bioavailable 25(OH)D (r = .68, p < .001). No correlations between 25(OH)D fractions and specific fatty acids were found, and in particular, no associations with mono- and poly-unsaturated FA compositions. All 25(OH)D fractions were correlated with leptin (total 25(OH)D (r = -.33, p < .003); bioavailable 25(OH)D (r = -.47, p < .001); free 25(OH)D (r = -.44, p < .001). Associations were found between PTH and total 25(OH)D (r = -.35, p = .002) and weaker between bioavailable 25(OH)D (r = -.35, p = .040) and free 25(OH)D (r = -.28, p = .079). All fractions of 25(OH)D appear to correlate in a similar way to PTH, BMI and body fat (leptin). No association was found between ery-FA composition and free/bioavailable 25(OH)D. It is unlikely that FAs are a strong uncoupling factor of DBP-bound 25(OH)D.

  17. The effectiveness and safety of tranexamic acid in bilateral total knee arthroplasty

    PubMed Central

    Weng, Kedi; Zhang, Xingen; Bi, Qing; Zhao, Chen

    2016-01-01

    Abstract Objective: A meta-analysis was performed to investigate the effectiveness and safety of tranexamic acid (TXA) for the treatment of blood loss after a bilateral total knee arthroplasty (TKA). Methods: Patients prepared for bilateral TKA and intervention including TXA versus placebo were comprehensively retrieved from MEDLINE (PubMed), Embase, Cochrane Central Register of Controlled Trials (CENTRAL), and Web of Science from the time of the establishment of these databases to January 2016. The outcomes were all calculated by Stata 12.0 software. The continuous endpoints (total blood loss and blood loss in drainage) were calculated as mean difference (MD) and 95% confidence intervals (CIs). Binary variables (the need for transfusion, and the occurrence of deep venous thrombosis [DVT]) were calculated as relative risk (RR) with 95% CIs. Results: Pooled results revealed that treatment with TXA associated with less need for transfusion (P = 0.000) and the value of Hb drop postoperatively (P = 0.290) after bilateral TKA. The results also indicated that TXA can decrease the total blood loss and blood loss in drainage after bilateral TKA (P < 0.05). Meanwhile, TXA can decrease the blood units transfused per patient by 1.23 U (P = 0.001). There is no statistically significant difference in terms of the occurrence of DVT between the 2 groups (P = 0.461). Conclusion: Based on the current evidence, TXA can decrease the need for transfusion and the total blood loss without increasing the occurrence of DVT, and its administration is recommended routinely in bilateral TKA. PMID:27684841

  18. Secreted Protein Acidic and Rich in Cysteine in Ocular Tissue

    PubMed Central

    Scavelli, Kurt; Chatterjee, Ayan

    2015-01-01

    Abstract Secreted protein acidic and rich in cysteine (SPARC), also known as osteonectin or BM-40, is the prototypical matricellular protein. Matricellular proteins are nonstructural secreted proteins that provide an integration between cells and their surrounding extracellular matrix (ECM). Regulation of the ECM is important in maintaining the physiologic function of tissues. Elevated levels of SPARC have been identified in a variety of diseases involving pathologic tissue remodeling, such as hepatic fibrosis, systemic sclerosis, and certain carcinomas. Within the eye, SPARC has been identified in the trabecular meshwork, lens, and retina. Studies have begun to show the role of SPARC in these tissues and its possible role, specifically in primary open-angle glaucoma, cataracts, and proliferative vitreoretinopathy. SPARC may, therefore, be a therapeutic target in the treatment of certain ocular diseases. Further investigation into the mechanism of action of SPARC will be necessary in the development of SPARC-targeted therapy. PMID:26167673

  19. Saturated fatty acids modulate autophagy's proteins in the hypothalamus.

    PubMed

    Portovedo, Mariana; Ignacio-Souza, Letícia M; Bombassaro, Bruna; Coope, Andressa; Reginato, Andressa; Razolli, Daniela S; Torsoni, Márcio A; Torsoni, Adriana S; Leal, Raquel F; Velloso, Licio A; Milanski, Marciane

    2015-01-01

    Autophagy is an important process that regulates cellular homeostasis by degrading dysfunctional proteins, organelles and lipids. In this study, the hypothesis that obesity could lead to impairment in hypothalamic autophagy in mice was evaluated by examining the hypothalamic distribution and content of autophagic proteins in animal with obesity induced by 8 or 16 weeks high fat diet to induce obesity and in response to intracerebroventricular injections of palmitic acid. The results showed that chronic exposure to a high fat diet leads to an increased expression of inflammatory markers and downregulation of autophagic proteins. In obese mice, autophagic induction leads to the downregulation of proteins, such as JNK and Bax, which are involved in the stress pathways. In neuron cell-line, palmitate has a direct effect on autophagy even without inflammatory activity. Understanding the cellular and molecular bases of overnutrition is essential for identifying new diagnostic and therapeutic targets for obesity.

  20. Augmentation of protein-derived acetic acid production by heat-alkaline-induced changes in protein structure and conformation.

    PubMed

    Wang, Xu; Li, Yanbo; Liu, Junxin; Ren, Nan-Qi; Qu, Jiuhui

    2016-01-01

    Waste-derived acetic acid (HAc) is an attractive feedstock for microbe-mediated biofuel production. However, fermentative conversion of HAc from waste-activated sludge (WAS) has low yield because of the high concentration of proteins not readily utilizable by microorganisms without prior hydrolysis. We investigated a combined technology for HAc augmentation during sludge protein fermentation. The maximal HAc yield increased over two-fold, reaching 0.502 ± 0.021 g/g protein (0.36 ± 0.01 g COD/g COD, ∼52% of the total volatile fatty acids) when synthetic sludge protein was heated at 120 °C for 30 min, treated at pH 12 for 24 h, and fermented at pH 9 for 72 h. Comprehensive analysis illustrated that the heat-alkaline pretreatment significantly induced protein fragmentation, simultaneously increasing the efficiency of protein biohydrolysis (from 35.5% to 85.9%) by inducing conformational changes indicative of protein unfolding. Consequently, the native α-helix content was decreased from 67.3% to 32.5% by conversion to an unordered shape, whose content increased from 27.5% to 45.5%; disulfide bonds were cleaved, whereas the main S-S stretching pattern was altered from gauche-gauche-gauche to gauche-gauche-trans, consequently causing increased protein susceptibility to proteolytic hydrolysis (76.3% vs. 47.0%). Economic analysis indicated that anaerobic fermentation with appropriate heat-alkaline pretreatment is a cost-effective approach for waste conversion to energy sources such as HAc.

  1. 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

  2. Total RNA and protein content, Cyclin B1 expression and developmental competence of prepubertal goat oocytes.

    PubMed

    Anguita, Begoña; Paramio, Maria-Teresa; Jiménez-Macedo, Ana R; Morató, Roser; Mogas, Teresa; Izquierdo, Dolors

    2008-01-30

    The aim of this study was to examine the relationship between the developmental competence of oocytes and their total RNA and protein contents, and the level of Cyclin B1 transcription. Ovaries from prepubertal goats were collected from a slaughterhouse. Oocytes were recovered by slicing and those with two or more layers of cumulus cells and homogenous cytoplasm were matured in vitro (20-25 oocytes per drop) for 27 h. Both before and after IVM, samples of oocytes were denuded and categorised into four group treatments by diameter (<110 microm, 110-125 microm, 125-135 microm; >135 microm), separated into sub-groups of 10 oocytes per treatment-replicate and stored in liquid nitrogen until total RNA content analysis by spectophotometry, total protein content analysis by a colorimetric assay and Cyclin B1 transcription analysis by RT-PCR. For the study of developmental competence, the rest of the matured oocytes were fertilised in vitro in groups of 20-25 for 24 h. Presumptive zygotes were denuded, sorted into the four categories of diameter noted above, and placed into culture drops in groups of 18-25 for in vitro culture. Cleavage rate was evaluated at 48 hpi and embryo development at 8 d post-insemination. There were four replicates of each treatment for each assay or evaluation point of the experiment. There were no significant differences between the size categories of oocytes at collection in total RNA content, total protein content and Cyclin B1 mRNA. There were significant differences (P<0.05) in the expression of Cyclin B1 before IVM with oocytes in the >135 mm diameter category having the highest value for this variant. There were no significant differences in these characteristics between the categories of oocyte diameter after IVM except in respect of total RNA content, which was lower for the largest size of oocytes (>135 microm; mean+/-S.D.=12.3+/-1.84 ng/oocyte) than the other three size groups (19.2+/-1.38-22.1+/-4.44 ng/oocyte; P<0.05). Significant

  3. Relationship between nine haloacetic acids with total organic halogens in different experimental conditions.

    PubMed

    Pourmoghadas, Hossein; Kinman, Riley N

    2013-04-03

    The effects of pH and bromide ion concentration on the formation of nine haloacetic acids (HAAs) and total organic halogens (TOX) in chlorinated drinking water have been evaluated. In an extensive study, the relationships of nine HAAs with TOX have been investigated. Honesty Significant Differences test (HSD) and ANOVA tests were used for the statistical analyses. The study determined the concentration range of nine HAAs as of a percentage of TOX at varying experimental conditions. Statistical analyses showed that the parameters pH and Br had significant effects on the formation of nine HAAs and TOX. This study also showed that brominated and mixed species of HAAs would be dominant in the presence of high bromide ion concentration which contributes a high percentage of the TOX. The results of this study could be used to set up a maximum contaminant level of TOX as a water quality standard for chlorination by-products.

  4. Can tranexamic acid change preoperative anemia management during total joint arthroplasty?

    PubMed Central

    Phan, Duy L; Rinehart, Joseph B; Schwarzkopf, Ran

    2015-01-01

    AIM: To investigate the postoperative transfusion and complication rates of anemic and nonanemic total joint arthroplasty patients given tranexamic acid (TXA). METHODS: A cross-sectional prospective study was conducted of primary hip and knee arthroplasty cases performed from 11/2012 to 6/2014. Exclusion criteria included revision arthroplasty, bilateral arthroplasty, acute arthroplasty after fracture, and contraindication to TXA. Patients were screened prior to surgery, with anemia was defined as hemoglobin of less than 12 g/dL for females and of less than 13 g/dL for males. Patients were divided into four different groups, based on the type of arthroplasty (total hip or total knee) and hemoglobin status (anemic or nonanemic). Intraoperatively, all patients received 2 g of intravenous TXA during surgery. Postoperatively, allogeneic blood transfusion (ABT) was directed by both clinical symptoms and relative hemoglobin change. Complications were recorded within the first two weeks after surgery and included thromboembolism, infection, and wound breakdown. The differences in transfusion and complication rates, as well as the relative hemoglobin change, were compared between anemic and nonanemic groups. RESULTS: A total of 232 patients undergoing primary joint arthroplasty were included in the study. For the total hip arthroplasty cohort, 21% (18/84) of patients presented with preoperative anemia. Two patients in the anemic group and two patients in the nonanemic group needed ABTs; this was not significantly different (P = 0.20). One patient in the anemic group presented with a deep venous thromboembolism while no patients in the nonanemic group had an acute complication; this was not significantly different (P = 0.21). For nonanemic patients, the average change in hemoglobin was 2.73 ± 1.17 g/dL. For anemic patients, the average change in hemoglobin was 2.28 ± 0.96 g/dL. Between the two groups, the hemoglobin difference of 0.45 g/dL was not significant (P = 0

  5. A high-performance direct transmethylation method for total fatty acids assessment in biological and foodstuff samples.

    PubMed

    Castro-Gómez, Pilar; Fontecha, Javier; Rodríguez-Alcalá, Luis M

    2014-10-01

    Isolation is the main bottleneck in the analysis of fatty acids in biological samples and foods. In the last few decades some methods described direct derivatization procedures bypassing these steps. They involve the utilization of methanolic HCL or BF3 as catalysts, but several evidences from previous works suggest these reagents are unstable, lead to the formation of artifacts and alter the distribution of specific compounds as hydroxy fatty acids or CLA. However, the main issue is that they are excellent esterification reagents but poor in transterification, being not suitable for the analysis of all lipid classes and leading to erroneous composition quantitations. The present research work is a comprehensive comparison of six general methylation protocols using base, acid or base/acid catalysts plus a proposed method in the analysis of total fatty acids in lipid standards mixtures, foodstuff and biological samples. The addition of aprotic solvents to the reaction mixture to avoid alterations was also tested. Results confirmed that procedures solely involving acid catalyst resulted in incomplete derivatizations and alteration of the fatty acid profile, partially corrected by addition of the aprotic solvent. The proposed method combining sodium methoxyde and sulfuric acid showed absence of alteration of the FAME profile and the best values for response factors (short chain fatty acids to PUFA), accuracy in the determination of total cholesterol and derivatization performance, thus showing a high reliability in the determination of the total fatty acid composition in biological samples and foods.

  6. Fermentation characteristics and lactic Acid bacteria succession of total mixed ration silages formulated with peach pomace.

    PubMed

    Hu, Xiaodong; Hao, Wei; Wang, Huili; Ning, Tingting; Zheng, Mingli; Xu, Chuncheng

    2015-04-01

    The objective of this study was to assess the use of peach pomace in total mixed ration (TMR) silages and clarify the differences in aerobic stability between TMR and TMR silages caused by lactic acid bacteria (LAB). The TMR were prepared using peach pomace, alfalfa hay or Leymus chinensis hay, maize meal, soybean meal, cotton meal, limestone, a vitamin-mineral supplement, and salt in a ratio of 6.0:34.0:44.4:7.0:5.0:2.5:1.0:0.1 on a dry matter (DM) basis. Fermentation quality, microbial composition, and the predominant LAB were examined during ensiling and aerobic deterioration. The results indicated that the TMR silages with peach pomace were well fermented, with low pH and high lactic acid concentrations. The aerobic stability of TMR silages were significantly higher than that of TMR. Compared with TMR silages with alfalfa hay, TMR silage with Leymus chinensis hay was much more prone to deterioration. Although the dominant LAB were not identical in TMR, the same dominant species, Lactobacillus buchneri and Pediococcus acidilactici, were found in both types of TMR silages after 56 d of ensiling, and they may play an important role in the aerobic stability of TMR silages.

  7. Fermentation Characteristics and Lactic Acid Bacteria Succession of Total Mixed Ration Silages Formulated with Peach Pomace

    PubMed Central

    Hu, Xiaodong; Hao, Wei; Wang, Huili; Ning, Tingting; Zheng, Mingli; Xu, Chuncheng

    2015-01-01

    The objective of this study was to assess the use of peach pomace in total mixed ration (TMR) silages and clarify the differences in aerobic stability between TMR and TMR silages caused by lactic acid bacteria (LAB). The TMR were prepared using peach pomace, alfalfa hay or Leymus chinensis hay, maize meal, soybean meal, cotton meal, limestone, a vitamin-mineral supplement, and salt in a ratio of 6.0:34.0:44.4:7.0:5.0:2.5:1.0:0.1 on a dry matter (DM) basis. Fermentation quality, microbial composition, and the predominant LAB were examined during ensiling and aerobic deterioration. The results indicated that the TMR silages with peach pomace were well fermented, with low pH and high lactic acid concentrations. The aerobic stability of TMR silages were significantly higher than that of TMR. Compared with TMR silages with alfalfa hay, TMR silage with Leymus chinensis hay was much more prone to deterioration. Although the dominant LAB were not identical in TMR, the same dominant species, Lactobacillus buchneri and Pediococcus acidilactici, were found in both types of TMR silages after 56 d of ensiling, and they may play an important role in the aerobic stability of TMR silages. PMID:25656205

  8. Total synthesis and structure-activity relationship studies of a series of selective G protein inhibitors

    NASA Astrophysics Data System (ADS)

    Xiong, Xiao-Feng; Zhang, Hang; Underwood, Christina R.; Harpsøe, Kasper; Gardella, Thomas J.; Wöldike, Mie F.; Mannstadt, Michael; Gloriam, David E.; Bräuner-Osborne, Hans; Strømgaard, Kristian

    2016-11-01

    G proteins are key mediators of G protein-coupled receptor signalling, which facilitates a plethora of important physiological processes. The cyclic depsipeptides YM-254890 and FR900359 are the only known specific inhibitors of the Gq subfamily of G proteins; however, no synthetic route has been reported previously for these complex natural products and they are not easily isolated from natural sources. Here we report the first total synthesis of YM-254890 and FR900359, as well as of two known analogues, YM-385780 and YM-385781. The versatility of the synthetic approach also enabled the design and synthesis of ten analogues, which provided the first structure-activity relationship study for this class of compounds. Pharmacological characterization of all the compounds at Gq-, Gi- and Gs-mediated signalling provided succinct information on the structural requirements for inhibition, and demonstrated that both YM-254890 and FR900359 are highly potent inhibitors of Gq signalling, with FR900359 being the most potent. These natural products and their analogues represent unique tools for explorative studies of G protein inhibition.

  9. Brillouin spectroscopy as a new method of screening for increased CSF total protein during bacterial meningitis.

    PubMed

    Steelman, Zachary; Meng, Zhaokai; Traverso, Andrew J; Yakovlev, Vladislav V

    2015-05-01

    Bacterial meningitis is a disease of pronounced clinical significance, especially in the developing world. Immediate treatment with antibiotics is essential, and no single test can provide a conclusive diagnosis. It is well established that elevated total protein in cerebrospinal fluid (CSF) is associated with bacterial meningitis. Brillouin spectroscopy is a widely used optical technique for noninvasive determination of the elastic moduli of materials. We found that elevated protein levels in CSF alter the fluid elasticity sufficiently to be measurable by Brillouin spectroscopy, with model healthy and diseased fluids distinguishable to marked significance (P = 0.014), which increases with sample concentration by dialysis. Typical raw output of a 2-stage VIPA Brillouin spectrometer: inelastically scattered Brillouin peaks (arrows) and elastically scattered incident radiation (center cross).

  10. Sulfo-N-hydroxysuccinimide interferes with bicinchoninic acid protein assay.

    PubMed

    Vashist, Sandeep Kumar; Zhang, BinBin; Zheng, Dan; Al-Rubeaan, Khalid; Luong, John H T; Sheu, Fwu-Shan

    2011-10-01

    This study revealed a major interference from sulfo-N-hydroxysuccinimide (sulfo-NHS) in the bicinchoninic acid (BCA) protein assay. Sulfo-NHS, a common reagent used in bioconjugation and analytical biochemistry, exhibited absorbance signals and absorbance peaks at 562 nm, comparable to bovine serum albumin (BSA). However, the combined absorbance of sulfo-NHS and BSA was not strictly additive. The sulfo-NHS interference was suggested to be caused by the reduction of Cu(2+) in the BCA Kit's reagent B (4% cupric sulfate) in a manner similar to that of the protein.

  11. Hypochlorous acid-mediated protein oxidation: how important are chloramine transfer reactions and protein tertiary structure?

    PubMed

    Pattison, David I; Hawkins, Clare L; Davies, Michael J

    2007-08-28

    Hypochlorous acid (HOCl) is a powerful oxidant generated from H2O2 and Cl- by the heme enzyme myeloperoxidase, which is released from activated leukocytes. HOCl possesses potent antibacterial properties, but excessive production can lead to host tissue damage that occurs in numerous human pathologies. As proteins and amino acids are highly abundant in vivo and react rapidly with HOCl, they are likely to be major targets for HOCl. In this study, two small globular proteins, lysozyme and insulin, have been oxidized with increasing excesses of HOCl to determine whether the pattern of HOCl-mediated amino acid consumption is consistent with reported kinetic data for isolated amino acids and model compounds. Identical experiments have been carried out with mixtures of N-acetyl amino acids (to prevent reaction at the alpha-amino groups) that mimic the protein composition to examine the role of protein structure on reactivity. The results indicate that tertiary structure facilitates secondary chlorine transfer reactions of chloramines formed on His and Lys side chains. In light of these data, second-order rate constants for reactions of Lys side chain and Gly chloramines with Trp side chains and disulfide bonds have been determined, together with those for further oxidation of Met sulfoxide by HOCl and His side chain chloramines. Computational kinetic models incorporating these additional rate constants closely predict the experimentally observed amino acid consumption. These studies provide insight into the roles of chloramine formation and three-dimensional structure on the reactions of HOCl with isolated proteins and demonstrate that kinetic models can predict the outcome of HOCl-mediated protein oxidation.

  12. Polyunsaturated Branched-Chain Fatty Acid Geranylgeranoic Acid Induces Unfolded Protein Response in Human Hepatoma Cells

    PubMed Central

    Iwao, Chieko; Shidoji, Yoshihiro

    2015-01-01

    The acyclic diterpenoid acid geranylgeranoic acid (GGA) has been reported to induce autophagic cell death in several human hepatoma-derived cell lines; however, the molecular mechanism for this remains unknown. In the present study, several diterpenoids were examined for ability to induce XBP1 splicing and/or lipotoxicity for human hepatoma cell lines. Here we show that three groups of diterpenoids emerged: 1) GGA, 2,3-dihydro GGA and 9-cis retinoic acid induce cell death and XBP1 splicing; 2) all-trans retinoic acid induces XBP1 splicing but little cell death; and 3) phytanic acid, phytenic acid and geranylgeraniol induce neither cell death nor XBP1 splicing. GGA-induced ER stress/ unfolded protein response (UPR) and its lipotoxicity were both blocked by co-treatment with oleic acid. The blocking activity of oleic acid for GGA-induced XBP1 splicing was not attenuated by methylation of oleic acid. These findings strongly suggest that GGA at micromolar concentrations induces the so-called lipid-induced ER stress response/UPR, which is oleate-suppressive, and shows its lipotoxicity in human hepatoma cells. PMID:26186544

  13. Diagnosis of drowning: Electrolytes and total protein in sphenoid sinus liquid.

    PubMed

    Hayakawa, Akira; Terazawa, Koichi; Matoba, Kotaro; Horioka, Kie; Fukunaga, Tatsushige

    2017-04-01

    In this study, electrolyte (sodium (Na), chlorine (Cl), and magnesium (Mg)) and total protein (TP) concentrations and volume of liquid in the sphenoid sinus were examined to determine their usefulness to elucidate whether drowning occurred in freshwater or seawater. We examined 68 cases (seawater drowning group: 27 cases, freshwater drowning group: 21 cases, non-drowning group: 20 cases). There was a significant difference in Na, Cl, Mg, and TP concentrations of liquid in the sphenoid sinus among the three groups (seawater drowning, freshwater drowning, and non-drowning groups). To distinguish freshwater drowning from seawater drowning, Na, Cl, and Mg concentrations of liquid in the sphenoid sinus might serve as useful indicators.

  14. Association between Hyaluronic Acid Injections and Time-to-Total Knee Replacement Surgery.

    PubMed

    Altman, Roy; Fredericson, Michael; Bhattacharyya, Samir K; Bisson, Brad; Abbott, Thomas; Yadalam, Sashidhar; Kim, Myoung

    2016-10-01

    This study assessed the association between hyaluronic acid (HA) injections and time-to-total knee replacement (TKR) surgery for patients with knee osteoarthritis (OA). Patients 18 to 64 years of age who had TKR surgery between January 1, 2006 and December 31, 2011 were identified from the MarketScan Commercial claims database. All patients had 6 years or more of continuous enrollment prior to TKR surgery. There were two cohorts (1) patients with HA injections prior to TKR surgery and (2) patients who did not have HA injections prior to TKR surgery. Time-to-TKR was defined as the total days from the date of diagnosis of knee OA on the patient's first visit to an orthopedic surgeon to the date of TKR surgery. Results included 22,555 patients who had TKR surgery: 14,132 in the non-HA and 8,423 in the HA cohort. In this retrospective analysis of patients undergoing TKR, the median Time-to-TKR surgery was 326 days for the non-HA and 908 days for the HA cohort, a difference of 582 days. Those receiving HA injections had a median 1.6-year longer Time-to-TKR surgery versus those who did not receive HA injections. These results have both clinical and economic implications.

  15. EFFECT OF TRANEXAMIC ACID ON BLEEDING CONTROL IN TOTAL KNEE ARTHROPLASTY

    PubMed Central

    SADIGURSKY, DAVID; ANDION, DANIEL; BOUREAU, PÉRICLES; FERREIRA, MARIA CORDULINA; CARNEIRO, ROGÉRIO JAMIL FERNANDES; COLAVOLPE, PAULO OLIVEIRA

    2016-01-01

    ABSTRACT Objectives: To analyze the effectiveness of intravenous (IV) tranexamic acid (TA) in reducing blood loss in total knee arthroplasty (TKA). Method: The population sample was composed of patients with a diagnosis of primary knee osteoarthritis. The patients undergoing TKA were divided in two groups. Group A: comprised patients who used IV TA and B group, formed by patients who did not use TA in the intra or post-operative period. For descriptive analysis, quantitative variables were represented by mean and standard deviations when their distribution was normal and interquartile ranges and medians for non-normal variables. Results: The mean age of patients was 68 years old, most of them were female and with involvement of the left knee. Postoperatively patients who had used IV TA showed less bleeding rate and less hemoglobin rate reduction. Conclusion: The use of IV TA in TKA reduces blood loss in peri- and postoperative periods. Regarding total blood loss reduction, hemoglobin rate and need for blood transfusions, IV TA should be used routinely during TKA since it has been shown to be safe with no increase in side effects as thromboembolic events. Level of Evidence III. Retrospective Comparative Study. PMID:27217813

  16. Amino acid sequence of band-3 protein from rainbow trout erythrocytes derived from cDNA.

    PubMed Central

    Hübner, S; Michel, F; Rudloff, V; Appelhans, H

    1992-01-01

    In this report we present the first complete band-3 cDNA sequence of a poikilothermic lower vertebrate. The primary structure of the anion-exchange protein band 3 (AE1) from rainbow trout erythrocytes was determined by nucleotide sequencing of cDNA clones. The overlapping clones have a total length of 3827 bp with a 5'-terminal untranslated region of 150 bp, a 2754 bp open reading frame and a 3'-untranslated region of 924 bp. Band-3 protein from trout erythrocytes consists of 918 amino acid residues with a calculated molecular mass of 101 827 Da. Comparison of its amino acid sequence revealed a 60-65% identity within the transmembrane spanning sequence of band-3 proteins published so far. An additional insertion of 24 amino acid residues within the membrane-associated domain of trout band-3 protein was identified, which until now was thought to be a general feature only of mammalian band-3-related proteins. PMID:1637296

  17. Boric acid gel enrichment of glycosylated proteins in human wound fluids.

    PubMed

    Krisp, Christoph; Kubutat, Caroline; Kyas, Andreas; Steinsträsser, Lars; Jacobsen, Frank; Wolters, Dirk

    2011-04-01

    The enrichment of glycosylated proteins by glycocapturing materials plays a pivotal role for the investigation of polysaccharide containing proteins in disease pathogenesis. Hence, we investigated a boric acid gel as a binding material for glycoprotein enrichment. The bovine proteins alpha-1-acid-glycoprotein (A1AG) and alpha-2-HS-glycoprotein (fetuin A) were spiked in human chronic wound fluids and were subsequently enriched by a boric acid gel affinity chromatography (BAGAC). The enrichment efficiency was evaluated by western blot analysis and mass spectrometry. Additionally, glycoproteins of human wound fluids from diabetes mellitus patients with chronic foot ulcers were analyzed after BAGAC enrichments. In total 104 glycoproteins were identified, with reported glycosylation sites. 60 proteins were detected in at least 2 out of 3 biological replicates and were used for quantitative analysis between the bound and unbound fractions. Almost 80% of these glycoproteins were more prominent in the bound fraction. Only 2 glycoproteins revealed higher spectral counts in the flow through fraction compared to the bound fraction. These findings demonstrate the capability of the BAGAC material to enrich glycosylated proteins from complex human wound fluids.

  18. Dietary total fat and fatty acids intake, serum fatty acids and risk of breast cancer: A meta-analysis of prospective cohort studies.

    PubMed

    Cao, Yi; Hou, Lin; Wang, Weijing

    2016-04-15

    Results from prospective cohort studies on the association between dietary total fat and fatty acids intake and risk of breast cancer remain controversial. Pertinent prospective cohort studies were identified by a search of Embase and PubMed from inception to September 2015. Study-specific relative risks (RRs) with 95% confidence intervals were pooled using a random-effect model. Between-study heterogeneity and publication bias were assessed, and sensitivity analysis was conducted. Twenty-four independent studies on dietary total fat and fatty acids intake and seven studies on serum fatty acids were included. The pooled RR of breast cancer for the highest vs. lowest category of dietary total fat intake was 1.10 (1.02-1.19); however, no association was observed in studies adjusting for traditional risk factors of breast cancer. No association was observed between animal fat, vegetable fat, saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), n-3 PUFA, n-6 PUFA, eicosapentaenoic acid, docosahexaenoic acid, alpha-linolenic acid, oleic acid, linoleic acid and arachidonic acid and risk of breast cancer. The pooled RRs of breast cancer for the highest vs. lowest category of serum SFA, MUFA, PUFA, n-3 PUFA and n-6 PUFA were 1.00 (0.78-1.28), 1.41 (0.99-2.03), 0.59 (0.27-1.30), 0.81 (0.60-1.10) and 0.84 (0.60-1.18), respectively. Results from this meta-analysis suggested that dietary total fat and fatty acids might be not associated with risk of breast cancer.

  19. Advanced oxidation protein products and total antioxidant activity in colorectal carcinoma.

    PubMed

    Avinash, S S; Anitha, M; Vinodchandran; Rao, Gayathri M; Sudha, K; Shetty, Beena V

    2009-01-01

    The present study was designed to assess the levels of advanced oxidation protein products (AOPP) and percent hemolysis (that indirectly indicates the degree of membrane damage secondary to lipid peroxidation) in colorectal carcinoma. Glutathione (GSH), total thiols and albumin were measured to determine the antioxidant status. Considering the dynamic interaction between various antioxidants in the body, we measured the total antioxidant activity (AOA). Globulin was measured to assess the inflammatory response secondary to oxidative stress. Investigations were conducted in 45 cases of recently diagnosed primary colorectal adenocarcinoma. As control, 45 age and sex matched healthy persons were chosen. GSH was estimated in whole blood, percent hemolysis in RBC suspension and other parameters in plasma. We observed a very high significant increase (P<0.001) in AOPP, percent hemolysis and a highly significant increase (P<0.01) in globulin in colorectal carcinoma. We observed a very high significant decrease (P<0.001) in whole blood GSH, total thiols, albumin, AOA and a significant decrease (P<0.05) in plasma GSH in colorectal carcinoma. A very high significant negative correlation between percent hemolysis and AOA and an apparent negative correlation between total thiols and AOPP was seen in colorectal carcinoma. This demonstrated oxidative stress, decreased antioxidant status and secondary inflammatory response in colorectal carcinoma.

  20. 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.

  1. Low Dose Gamma Irradiation Does Not Affect the Quality or Total Ascorbic Acid Concentration of "Sweetheart" Passionfruit (Passiflora edulis).

    PubMed

    Golding, John B; Blades, Barbara L; Satyan, Shashirekha; Spohr, Lorraine J; Harris, Anne; Jessup, Andrew J; Archer, John R; Davies, Justin B; Banos, Connie

    2015-08-26

    Passionfruit (Passiflora edulis, Sims, cultivar "Sweetheart") were subject to gamma irradiation at levels suitable for phytosanitary purposes (0, 150, 400 and 1000 Gy) then stored at 8 °C and assessed for fruit quality and total ascorbic acid concentration after one and fourteen days. Irradiation at any dose (≤1000 Gy) did not affect passionfruit quality (overall fruit quality, colour, firmness, fruit shrivel, stem condition, weight loss, total soluble solids level (TSS), titratable acidity (TA) level, TSS/TA ratio, juice pH and rot development), nor the total ascorbic acid concentration. The length of time in storage affected some fruit quality parameters and total ascorbic acid concentration, with longer storage periods resulting in lower quality fruit and lower total ascorbic acid concentration, irrespective of irradiation. There was no interaction between irradiation treatment and storage time, indicating that irradiation did not influence the effect of storage on passionfruit quality. The results showed that the application of 150, 400 and 1000 Gy gamma irradiation to "Sweetheart" purple passionfruit did not produce any deleterious effects on fruit quality or total ascorbic acid concentration during cold storage, thus supporting the use of low dose irradiation as a phytosanitary treatment against quarantine pests in purple passionfruit.

  2. Nucleic acid (cDNA) and amino acid sequences of the maize endosperm protein glutelin-2.

    PubMed Central

    Prat, S; Cortadas, J; Puigdomènech, P; Palau, J

    1985-01-01

    The cDNA coding for a glutelin-2 protein from maize endosperm has been cloned and the complete amino acid sequence of the protein derived for the first time. An immature maize endosperm cDNA bank was screened for the expression of a beta-lactamase:glutelin-2 (G2) fusion polypeptide by using antibodies against the purified 28 kd G2 protein. A clone corresponding to the 28 kd G2 protein was sequenced and the primary structure of this protein was derived. Five regions can be defined in the protein sequence: an 11 residue N-terminal part, a repeated region formed by eight units of the sequence Pro-Pro-Pro-Val-His-Leu, an alternating Pro-X stretch 21 residues long, a Cys rich domain and a C-terminal part rich in Gln. The protein sequence is preceded by 19 residues which have the characteristics of the signal peptide found in secreted proteins. Unlike zeins, the main maize storage proteins, 28 kd glutelin-2 has several homologous sequences in common with other cereal storage proteins. Images PMID:3839076

  3. Light enhanced the accumulation of total fatty acids (TFA) and docosahexaenoic acid (DHA) in a newly isolated heterotrophic microalga Crypthecodinium sp. SUN.

    PubMed

    Sun, Dongzhe; Zhang, Zhao; Mao, Xuemei; Wu, Tao; Jiang, Yue; Liu, Jin; Chen, Feng

    2017-03-01

    In the present study, light illumination was found to be efficient in elevating the total fatty acid content in a newly isolated heterotrophic microalga, Crypthecodinium sp. SUN. Under light illumination, the highest total fatty acid and DHA contents were achieved at 96h as 24.9% of dry weight and 82.8mgg(-1) dry weight, respectively, which were equivalent to 1.46-fold and 1.68-fold of those under the dark conditions. The elevation of total fatty acid content was mainly contributed by an increase of neutral lipids at the expense of starches. Moreover, light was found to alter the cell metabolism and led to a higher specific growth rate, higher glucose consumption rate and lower non-motile cell percentage. This is the first report that light can promote the total fatty acids accumulation in Crypthecodinium without growth inhibition.

  4. Deep ultraviolet mapping of intracellular protein and nucleic acid in femtograms per pixel.

    PubMed

    Cheung, Man C; Evans, James G; McKenna, Brian; Ehrlich, Daniel J

    2011-11-01

    By using imaging spectrophotometry with paired images in the 200- to 280-nm wavelength range, we have directly mapped intracellular nucleic acid and protein distributions across a population of Chinese hamster ovary (CHO-K1) cells. A broadband 100× objective with a numerical aperture of 1.2 NA (glycerin immersion) and a novel laser-induced-plasma point source generated high-contrast images with short (∼100 ms) exposures and a lateral resolution nearing 200 nm that easily resolves internal organelles. In a population of 420 CHO-K1 cells and 477 nuclei, we found a G1 whole-cell nucleic acid peak at 26.6 pg, a nuclear-isolated total nucleic acid peak at 11.4 pg, and, as inferred by RNase treatment, a G1 total DNA mass of 7.4 pg. At the G1 peak, we found a whole-cell protein mass of 95.6 pg, and a nuclear-isolated protein mass of 39.3 pg. An algorithm for protein quantification that senses peptide-bond (220-nm) absorbance was found to have a higher signal-to-noise ratio and to provide more reliable nucleic acid and protein determinations when compared to more classical 280/260-nm algorithms when used for intracellular mass mapping. Using simultaneous imaging with common nuclear stains (Hoechst 33342, Syto-14, and Sytox Orange), we have compared staining patterns to deep-UV images of condensed chromatin and have confirmed bias of these common nuclear stains related to nuclear packaging. The approach allows absolute mass measurements with no special sample preparation or staining. It can be used in conjunction with normal fluorescence microscopy and with relatively modest modification of the microscope.

  5. Hyperdimensional Analysis of Amino Acid Pair Distributions in Proteins

    PubMed Central

    Henriksen, Svend B.; Arnason, Omar; Söring, Jón; Petersen, Steffen B.

    2011-01-01

    Our manuscript presents a novel approach to protein structure analyses. We have organized an 8-dimensional data cube with protein 3D-structural information from 8706 high-resolution non-redundant protein-chains with the aim of identifying packing rules at the amino acid pair level. The cube contains information about amino acid type, solvent accessibility, spatial and sequence distance, secondary structure and sequence length. We are able to pose structural queries to the data cube using program ProPack. The response is a 1, 2 or 3D graph. Whereas the response is of a statistical nature, the user can obtain an instant list of all PDB-structures where such pair is found. The user may select a particular structure, which is displayed highlighting the pair in question. The user may pose millions of different queries and for each one he will receive the answer in a few seconds. In order to demonstrate the capabilities of the data cube as well as the programs, we have selected well known structural features, disulphide bridges and salt bridges, where we illustrate how the queries are posed, and how answers are given. Motifs involving cysteines such as disulphide bridges, zinc-fingers and iron-sulfur clusters are clearly identified and differentiated. ProPack also reveals that whereas pairs of Lys residues virtually never appear in close spatial proximity, pairs of Arg are abundant and appear at close spatial distance, contrasting the belief that electrostatic repulsion would prevent this juxtaposition and that Arg-Lys is perceived as a conservative mutation. The presented programs can find and visualize novel packing preferences in proteins structures allowing the user to unravel correlations between pairs of amino acids. The new tools allow the user to view statistical information and visualize instantly the structures that underpin the statistical information, which is far from trivial with most other SW tools for protein structure analysis. PMID:22174733

  6. Prepartum maternal diets supplemented with oilseeds alter the fatty acid profile in bovine neonatal plasma possibly through reduced placental expression of fatty acid transporter protein 4 and fatty acid translocase.

    PubMed

    Salehi, Reza; Ambrose, Divakar J

    2016-12-12

    In the present study, we determined the effects of maternal dietary fat and the type of fat on plasma fatty acids and the expression of placental fatty acid transporter genes. In Experiment 1, Holstein cows in the last 35 days of gestation received diets containing sunflower seed (n=8; high in linoleic acid (LA)), canola seed (n=7; high in oleic acid (OLA)) or no oilseed (n=7; control). Fatty acids were quantified in dam and neonate plasma at calving. In Experiment 2, placental cotyledons were collected (LA: n=4; OLA: n=4; control: n=5) to quantify gene expression. Maternal long-chain polyunsaturated fatty acids, neonatal total n-3 fatty acids and eicosapentaenoic acid (EPA) declined, whereas docosahexaenoic acid (DHA) and total fat tended to decline following fat supplementation prepartum. Feeding of LA versus OLA prepartum tended to increase peroxisome proliferator-activated receptor α (PPARA) expression, whereas peroxisome proliferator-activated receptor δ (PPARD) and peroxisome proliferator-activated receptor γ (PPARG) expression tended to be higher in OLA- than LA-fed cows. Expression of fatty acid transporter protein 4 (FATP4) and fatty acid translocase (FAT/CD36) expression was lower in placental tissue of cows fed fat compared with control cows. Reduced total n-3 fatty acids, EPA and DHA in neonates born of dams fed fat prepartum is likely due to changes in PPARs and reduced expression of placental FATP4 and FAT/CD36.

  7. Screening for non-protein amino acids in seeds of the Guam cycad, Cycas circinalis, by an improved GC-MS method.

    PubMed

    Oh, C H; Brownson, D M; Mabry, T J

    1995-02-01

    The non-protein amino acid, beta-N-methyl-amino-L-alanine (L-BMAA) from Cycas circinalis seeds, has been implicated as a causative agent of amyotrophic lateral sclerosis-Parkinsonism dementia complex (ALS-PDC), a disease known from Guam and other areas in the western Pacific. We analyzed C. circinalis seeds for additional free non-protein amino acids by a recently developed GC-MS method. The samples were prepared by water extraction of seed flour. The amino acids present in the extract were derivatized by N(O,S)-isobutyloxycarbonylation of the amine functional groups and then tert-butyldimethylsilylation of the carboxyl functional groups. Peaks for a total of seventeen derivatives of non-protein amino acids were detected by GC-MS. In addition to L-BMAA, four other non-protein amino acids were identified as beta-alanine, gamma-amino-butyric acid, pyroglutamic acid, and alpha-aminoadipic acid.

  8. [Refractometric measurement of total serum protein, comparison of refractometry and biuret test (author's transl)].

    PubMed

    Liappis, N; Jäkel, A

    1978-07-01

    Study on the Refractometric Determination of Total Protein in Serum, Comparison of the Refractometric and Biuret Method. Total protein concentration in serum was determined by the aid of the Abbé-refractometer and the biuret method. Both methods showed a good precision and accuracy. The investigation was carried out in 241 sera with normal bilirubin (up to 1 mg/100 ml), cholesterol (up to 200 mg/100 ml) and urea (up to 23,0 mg/100 ml) concentration, in 43 sera with increased (10,6-26,6 mg/100 ml) bilirubin concentration, in 129 sera with increased (200-520 mg/100 ml) cholesterol concentration and in 43 sera with increased (23,0-155,3 mg/100 ml) urea concentration. The comparison of the refractometric values with the values obtained by the biuret method in the 241 sera with normal bilirubin, cholesterol and urea concentration (correlation coefficient = 0,971) showed a close correlation and in the 43 sera with increased bilirubin concentration (correlation coefficient = 0,958) an acceptable correlation. However no close correlations were observed in the 129 sera with increased cholesterol concentration and in the 43 sera with increased urea concentration. The correlation lines diverged proportional with the increase of cholesterol and urea concentration from the expected correlation lines.

  9. The biological activities of protein/oleic acid complexes reside in the fatty acid.

    PubMed

    Fontana, Angelo; Spolaore, Barbara; Polverino de Laureto, Patrizia

    2013-06-01

    A complex formed by human α-lactalbumin (α-LA) and oleic acid (OA), named HAMLET, has been shown to have an apoptotic activity leading to the selective death of tumor cells. In numerous publications it has been reported that in the complex α-LA is monomeric and adopts a partly folded or "molten globule" state, leading to the idea that partly folded proteins can have "beneficial effects". The protein/OA molar ratio initially has been reported to be 1:1, while recent data have indicated that the OA-complex is given by an oligomeric protein capable of binding numerous OA molecules per protein monomer. Proteolytic fragments of α-LA, as well as other proteins unrelated to α-LA, can form OA-complexes with biological activities similar to those of HAMLET, thus indicating that a generic protein can form a cytotoxic complex under suitable experimental conditions. Moreover, even the selective tumoricidal activity of HAMLET-like complexes has been questioned. There is recent evidence that the biological activity of long chain unsaturated fatty acids, including OA, can be ascribed to their effect of perturbing the structure of biological membranes and consequently the function of membrane-bound proteins. In general, it has been observed that the cytotoxic effects exerted by HAMLET-like complexes are similar to those reported for OA alone. Overall, these findings can be interpreted by considering that the protein moiety does not have a toxic effect on its own, but merely acts as a solubilising agent for the inherently toxic fatty acid.

  10. Role of Ingested Amino Acids and Protein in the Promotion of Resistance Exercise–Induced Muscle Protein Anabolism123

    PubMed Central

    Rasmussen, Blake B

    2016-01-01

    The goal of this critical review is to comprehensively assess the evidence for the molecular, physiologic, and phenotypic skeletal muscle responses to resistance exercise (RE) combined with the nutritional intervention of protein and/or amino acid (AA) ingestion in young adults. We gathered the literature regarding the translational response in human skeletal muscle to acute exposure to RE and protein/AA supplements and the literature describing the phenotypic skeletal muscle adaptation to RE and nutritional interventions. Supplementation of protein/AAs with RE exhibited clear protein dose–dependent effects on translational regulation (protein synthesis) through mammalian target of rapamycin complex 1 (mTORC1) signaling, which was most apparent through increases in p70 ribosomal protein S6 kinase 1 (S6K1) phosphorylation, compared with postexercise recovery in the fasted or carbohydrate-fed state. These acute findings were critically tested via long-term exposure to RE training (RET) and protein/AA supplementation, and it was determined that a diminishing protein/AA supplement effect occurs over a prolonged exposure stimulus after exercise training. Furthermore, we found that protein/AA supplements, combined with RET, produced a positive, albeit minor, effect on the promotion of lean mass growth (when assessed in >20 participants/treatment); a negligible effect on muscle mass; and a negligible to no additional effect on strength. A potential concern we discovered was that the majority of the exercise training studies were underpowered in their ability to discern effects of protein/AA supplementation. Regardless, even when using optimal methodology and large sample sizes, it is clear that the effect size for protein/AA supplementation is low and likely limited to a subset of individuals because the individual variability is high. With regard to nutritional intakes, total protein intake per day, rather than protein timing or quality, appears to be more of a factor

  11. A Novel and Rapid Colorimetric Method for Measuring Total Phosphorus and Phytic Acid in Foods and Animal Feeds.

    PubMed

    2016-04-08

    Phytic acid, or myo-inositol hexakisphosphate, is the primary source of inositol and storage phosphorus in plant seeds and has considerable nutritional importance. In this form, phosphorus is unavailable for absorption by monogastric animals, and the strong chelating characteristic of phytic acid reduces the bioavailability of multivalent minerals such as iron, zinc, and calcium. Currently, there is no simple quantitative method for phytic acid; existing methods are complex, and the most commonly accepted method, AOAC Official Method (SM) 986.11, has limitations. The aim of this work was to develop and validate a simple, high-throughput method for the measurement of total phosphorus and phytic acid in foods and animal feeds. The method described here involves acid extraction of phytic acid, followed by dephosphorylation with phytase and alkaline phosphatase. The phosphate released from phytic acid is measured using a modified colorimetric molybdenum blue assay and calculated as total phosphorus or phytic acid content of the original sample. The method was validated to a maximum linearity of 3.0 g phytic acid/100 g sample. Accuracy ranged from 98 to 105% using pure phytic acid and from 97 to 115% for spiked samples. Repeatability ranged from 0.81 to 2.32%, and intermediate precision was 2.27%.

  12. Modulation of Fourier transform infrared spectra and total sialic acid levels by selenium during 1,2 dimethylhydrazine-induced colon carcinogenesis in rats.

    PubMed

    Ghadi, Fereshteh Ezzati; Malhotra, Anshoo; Ghara, Abdollah Ramzani; Dhawan, D K

    2013-01-01

    The present study investigated the modulatory potential of selenium supplementation, if any, on Fourier transform infrared (FTIR) spectra in brush border membranes (BBM) of colons and on serum total sialic acid as well as lipid bound sialic acid during 1,2 dimethyl hydrazine (DMH)-induced colorectal carcinogenesis in rats. The FTIR spectra of BBM from the colons of DMH-treated rats revealed a significant increase in the lipid contents but showed a significant decline in the protein contents. Further, decrease in the collagen as well as creatine contents was also noticed in the colons of DMH-treated rats. Supplementation with selenium appreciably restored protein as well as collagen contents and resulted in decreased lipids levels in the colons of DMH-treated rats. Interestingly, a significant increase in the levels of total sialic acid in serum of DMH-treated rats was observed which, however, got moderated significantly upon selenium supplementation. Moreover, no significant changes were observed in the levels of lipid bound sialic acid in all the treated groups as compared to controls. In conclusion, the present study suggested that supplementation of selenium act as a chemopreventive agent and delays considerably the process of colon carcinogenesis.

  13. Bacteriophage and impurity carryover and total organic carbon release during extended protein A chromatography.

    PubMed

    Lute, Scott; Brorson, Kurt

    2009-05-01

    In the biopharmaceutical industry, column chromatography residuals are routinely assessed by the direct measurement of mock eluates. In this study, we evaluated virus and other impurity carryover between protein A cycles and the feasibility of using a total organic carbon (TOC) analyzer to monitor for column impurity leakage as a correlate for actual measured carryover in mock eluates. Commercial process intermediates were used in scaled down studies of two protein A media, ProSep A (Millipore, Bedford, MA, USA) and MabSelect SuRe (GE Healthcare, Uppsala, Sweden). The chromatography system was programmed to run up to 200 normal load/elution cycles with periodic blank cycles to measure protein and phage carryover, and water flush cycles to measure TOC release. Sustained phage carryover was evident in each study. Carryover and TOC release was lowest in the case where cleaning was most stringent (50 mM NaOH/0.5 M Na(2)SO(4) with MabSelect SuRe). The TOC analysis at this time does not appear to be a viable practical means of measuring impurity carryover; direct measurements in mock eluates appears to be more predictive of column performance.

  14. Effect of degree of hydrolysis of whey protein on in vivo plasma amino acid appearance in humans.

    PubMed

    Farup, Jean; Rahbek, Stine Klejs; Storm, Adam C; Klitgaard, Søren; Jørgensen, Henry; Bibby, Bo M; Serena, Anja; Vissing, Kristian

    2016-01-01

    Whey protein is generally found to be faster digested and to promote faster and higher increases in plasma amino acid concentrations during the immediate ~60 min following protein ingestion compared to casein. The aim of the present study was to compare three different whey protein hydrolysates with varying degrees of hydrolysis (DH, % cleaved peptide bonds) to evaluate if the degree of whey protein hydrolysis influences the rate of amino acid plasma appearance in humans. A casein protein was included as reference. The three differentially hydrolysed whey proteins investigated were: High degree of hydrolysis (DH, DH = 48 %), Medium DH (DH = 27 %), and Low DH (DH = 23 %). The casein protein was intact. Additionally, since manufacturing of protein products may render some amino acids unavailable for utilisation in the body the digestibility and the biological value of all four protein fractions were evaluated in a rat study. A two-compartment model for the description of the postprandial plasma amino acid kinetics was applied to investigate the rate of postprandial total amino acid plasma appearance of the four protein products. The plasma amino acid appearance rates of the three whey protein hydrolysates (WPH) were all significantly higher than for the casein protein, however, the degree of hydrolysis of the WPH products did not influence plasma total amino acid appearance rate (estimates of DH and 95 % confidence intervals [CI] (mol L(-1) min(-1)): High DH 0.0585 [0.0454, 0.0754], Medium DH 0.0594 [0.0495, 0.0768], Low DH 0.0560 [0.0429, 0.0732], Casein 0.0194 [0.0129, 0.0291]). The four protein products were all highly digestible, while the biological value decreased with increasing degree of hydrolysis. In conclusion, the current study does not provide evidence that the degree of whey protein hydrolysis is a strong determinant for plasma amino acid appearance rate within the studied range of hydrolysis and protein dose.

  15. Polysaccharide-free nucleic acids and proteins of Abelmoschus esculentus for versatile molecular studies.

    PubMed

    Manoj-Kumar, A; Reddy, K N; Manjulatha, M; Blanco, L

    2012-01-01

    Abelmoschus esculentus (okra) is one of the polysaccharide rich crop plants. The polysaccharides interfere with nucleic acids and protein isolation thereby affecting the downstream molecular analysis. So, to understand the molecular systematics of okra, high quality DNA, RNA and proteins are essential. In this study we present a method for extracting genomic DNA, RNA and proteins from polysaccharide rich okra tissues. The conventional extraction procedures were integrated with purification treatments with pectinase, RNase and proteinase K, which improved the quality and quantity of DNA as well. Using SDS, additional washes with CIA and NaCl precipitation improved the RNA isolation both quantitatively and qualitatively. Finally, ammonium acetate mediated protein precipitation and re-solubilization increased the quality of total protein extracts from the okra leaves. All of the methods above not only eliminated the impurities but also improved the quality and quantity of nucleic acids and proteins. Further, we subjected these samples to versatile downstream molecular analyses such as restriction endonuclease digestion, RAPD, Southern, reverse transcription-PCR and Western analysis and were proved to be successful.

  16. Longitudinal evolution of true protein, amino acids and bioactive proteins in breast milk: a developmental perspective.

    PubMed

    Lönnerdal, Bo; Erdmann, Peter; Thakkar, Sagar K; Sauser, Julien; Destaillats, Frédéric

    2017-03-01

    The protein content of breast milk provides a foundation for estimating protein requirements of infants. Because it serves as a guideline for regulatory agencies issuing regulations for infant formula composition, it is critical that information on the protein content of breast milk is reliable. We have therefore carried out a meta-analysis of the protein and amino acid contents of breast milk and how they evolve during lactation. As several bioactive proteins are not completely digested in the infant and therefore represent "non-utilizable" protein, we evaluated the quantity, mechanism of action and digestive fate of several major breast milk proteins. A better knowledge of the development of the protein contents of breast milk and to what extent protein utilization changes with age of the infant will help improve understanding of protein needs in infancy. It is also essential when designing the composition of infant formulas, particularly when the formula uses a "staging" approach in which the composition of the formula is modified in stages to reflect changes in breast milk and changing requirements as the infant ages.

  17. Protein turnover, amino acid requirements and recommendations for athletes and active populations

    PubMed Central

    Poortmans, J.R.; Carpentier, A.; Pereira-Lancha, L.O.; Lancha, A.

    2012-01-01

    Skeletal muscle is the major deposit of protein molecules. As for any cell or tissue, total muscle protein reflects a dynamic turnover between net protein synthesis and degradation. Noninvasive and invasive techniques have been applied to determine amino acid catabolism and muscle protein building at rest, during exercise and during the recovery period after a single experiment or training sessions. Stable isotopic tracers (13C-lysine, 15N-glycine, 2H5-phenylalanine) and arteriovenous differences have been used in studies of skeletal muscle and collagen tissues under resting and exercise conditions. There are different fractional synthesis rates in skeletal muscle and tendon tissues, but there is no major difference between collagen and myofibrillar protein synthesis. Strenuous exercise provokes increased proteolysis and decreased protein synthesis, the opposite occurring during the recovery period. Individuals who exercise respond differently when resistance and endurance types of contractions are compared. Endurance exercise induces a greater oxidative capacity (enzymes) compared to resistance exercise, which induces fiber hypertrophy (myofibrils). Nitrogen balance (difference between protein intake and protein degradation) for athletes is usually balanced when the intake of protein reaches 1.2 g·kg−1·day−1 compared to 0.8 g·kg−1·day−1 in resting individuals. Muscular activities promote a cascade of signals leading to the stimulation of eukaryotic initiation of myofibrillar protein synthesis. As suggested in several publications, a bolus of 15-20 g protein (from skimmed milk or whey proteins) and carbohydrate (± 30 g maltodextrine) drinks is needed immediately after stopping exercise to stimulate muscle protein and tendon collagen turnover within 1 h. PMID:22666780

  18. [Evaluation on contribution rate of each component total salvianolic acids and characterization of apparent oil/water partition coefficient].

    PubMed

    Yan, Hong-mei; Chen, Xiao-yun; Xia, Hai-jian; Liu, Dan; Jia, Xiao-bin; Zhang, Zhen-hai

    2015-02-01

    The difference between three representative components of total salvianolic acids in pharmacodynamic activity were compared by three different pharmacological experiments: HUVECs oxidative damage experiment, 4 items of blood coagulation in vitro experiment in rabbits and experimental myocardial ischemia in rats. And the effects of contribution rate of each component were calculated by multi index comprehensive evaluation method based on CRITIC weights. The contribution rates of salvianolic acid B, rosmarinic acid and Danshensu were 28.85%, 30.11%, 41.04%. Apparent oil/water partition coefficient of each representative components of total salvianolic acids in n-octyl alcohol-buffer was tested and the total salvianolic acid components were characterized based on a combination of the approach of self-defined weighting coefficient with effects of contribution rate. Apparent oil/water partition coefficient of total salvianolic acids was 0.32, 1.06, 0.89, 0.98, 0.90, 0.13, 0.02, 0.20, 0.56 when in octanol-water/pH 1.2 dilute hydrochloric acid solution/ pH 2.0, 2.5, 5.0, 5.8, 6.8, 7.4, 7.8 phosphate buffer solution. It provides a certain reference for the characterization of components.

  19. Estimates of Total Dietary Folic Acid Intake in the Australian Population Following Mandatory Folic Acid Fortification of Bread

    PubMed Central

    Dugbaza, Jacinta; Cunningham, Judy

    2012-01-01

    Mandatory folic acid fortification of wheat flour for making bread was implemented in Australia in September 2009, to improve the dietary folate status of women of child-bearing age, and help reduce the incidence of neural tube defects in the population. This paper presents estimates of folic acid intake in the target population and other subgroups of the Australian population following implementation of the mandatory folic acid fortification standard. In June/July 2010 one hundred samples from seven bread categories were purchased from around the country and individually analysed for the amount of folic acid they contained. A modification to the triple enzyme microbiological method was used to measure folic acid in the individual bread samples. The folic acid analytical values together with national food consumption data were used to generate estimates of the population's folic acid intake from fortified foods. Food Standards Australia New Zealand's (FSANZ) custom-built dietary modelling program (DIAMOND) was used for the estimates. The mean amount of folic acid found in white bread was 200 μg/100 g which demonstrated that folic-acid-fortified wheat flour was used to bake the bread. The intake estimates indicated an increase in mean folic acid intake of 159 μg per day for the target group. Other sub-groups of the population also showed increases in estimated mean daily intake of folic acid. PMID:22957218

  20. Coagulant properties of Moringa oleifera protein preparations: application to humic acid removal.

    PubMed

    Santos, Andréa F S; Paiva, Patrícia M G; Teixeira, José A C; Brito, António G; Coelho, Luana C B B; Nogueira, Regina

    2012-01-01

    This work aimed to characterize the coagulant properties of protein preparations from Moringa oleifera seeds in the removal of humic acids from water. Three distinct preparations were assayed, namely extract (seeds homogenized with 0.15 M NaCl), fraction (extract precipitated with 60% w/v ammonium sulphate) and cMoL (protein purified with guar gel column chromatography). The extract showed the highest coagulant activity in a protein concentration between 1 mg/L and 180 mg/L at pH 7.0. The zeta potential of the extract (-10 mV to -15 mV) was less negative than that of the humic acid (-41 mV to -42 mV) in a pH range between 5.0 and 8.0; thus, the mechanism that might be involved in this coagulation activity is adsorption and neutralization of charges. Reduction of total organic carbon (TOC) and dissolved organic carbon (DOC) was observed in water samples containing 9 mg/L carbon as humic acid when treated with 1 mg/L of the extract. A decrease in colour and in the aromatic content of the treated water was also observed. These results suggested that the extract from M. oleifera seeds in a low concentration (1 mg/L) can be an interesting natural alternative for removing humic acid from water in developing countries. The extract dose determined in the present study does not impart odour or colour to the treated water.

  1. vProtein: Identifying Optimal Amino Acid Complements from Plant-Based Foods

    PubMed Central

    Woolf, Peter J.; Fu, Leeann L.; Basu, Avik

    2011-01-01

    Background Indispensible amino acids (IAAs) are used by the body in different proportions. Most animal-based foods provide these IAAs in roughly the needed proportions, but many plant-based foods provide different proportions of IAAs. To explore how these plant-based foods can be better used in human nutrition, we have created the computational tool vProtein to identify optimal food complements to satisfy human protein needs. Methods vProtein uses 1251 plant-based foods listed in the United States Department of Agriculture standard release 22 database to determine the quantity of each food or pair of foods required to satisfy human IAA needs as determined by the 2005 daily recommended intake. The quantity of food in a pair is found using a linear programming approach that minimizes total calories, total excess IAAs, or the total weight of the combination. Results For single foods, vProtein identifies foods with particularly balanced IAA patterns such as wheat germ, quinoa, and cauliflower. vProtein also identifies foods with particularly unbalanced IAA patterns such as macadamia nuts, degermed corn products, and wakame seaweed. Although less useful alone, some unbalanced foods provide unusually good complements, such as Brazil nuts to legumes. Interestingly, vProtein finds no statistically significant bias toward grain/legume pairings for protein complementation. These analyses suggest that pairings of plant-based foods should be based on the individual foods themselves instead of based on broader food group-food group pairings. Overall, the most efficient pairings include sweet corn/tomatoes, apple/coconut, and sweet corn/cherry. The top pairings also highlight the utility of less common protein sources such as the seaweeds laver and spirulina, pumpkin leaves, and lambsquarters. From a public health perspective, many of the food pairings represent novel, low cost food sources to combat malnutrition. Full analysis results are available online at http

  2. Mechanisms of enhanced total organic carbon elimination from oxalic acid solutions by electro-peroxone process.

    PubMed

    Wang, Huijiao; Yuan, Shi; Zhan, Juhong; Wang, Yujue; Yu, Gang; Deng, Shubo; Huang, Jun; Wang, Bin

    2015-09-01

    Electro-peroxone (E-peroxone) is a novel electrocatalytic ozonation process that combines ozonation and electrolysis process to enhance pollutant degradation during water and wastewater treatment. This enhancement has been mainly attributed to several mechanisms that increase O3 transformation to ·OH in the E-peroxone system, e.g., electro-generation of H2O2 from O2 at a carbon-based cathode and its subsequent peroxone reaction with O3 to ·OH, electro-reduction of O3 to ·OH at the cathode, and O3 decomposition to ·OH at high local pH near the cathode. To get more insight how these mechanisms contribute respectively to the enhancement, this study investigated total organic carbon (TOC) elimination from oxalic acid (OA) solutions by the E-peroxone process. Results show that the E-peroxone process significantly increased TOC elimination rate by 10.2-12.5 times compared with the linear addition of the individual rates of corresponding ozonation and electrolysis process. Kinetic analyses reveal that the electrochemically-driven peroxone reaction is the most important mechanism for the enhanced TOC elimination rate, while the other mechanisms contribute minor to the enhancement by a factor of 1.6-2.5. The results indicate that proper selection of electrodes that can effectively produce H2O2 at the cathode is critical to maximize TOC elimination in the E-peroxone process.

  3. Elevated First-Trimester Total Bile Acid is Associated with the Risk of Subsequent Gestational Diabetes

    PubMed Central

    Hou, Wolin; Meng, Xiyan; Zhao, Weijing; Pan, Jiemin; Tang, Junling; Huang, Yajuan; Tao, Minfang; Liu, Fang; Jia, Weiping

    2016-01-01

    The aim of the current study is to assess whether total bile acid (TBA) level in first trimester pregnancy is associated with gestational diabetes mellitus (GDM). Biochemical parameters including serum TBA of 742 pregnant women were collected within 12 weeks of gestation and compared. At 24–28th weeks of gestation, 75 g oral glucose tolerance test (OGTT) was performed. The perinatal data of 330 women were collected. The results demonstrated women with GDM (n = 268) had higher first-trimester serum levels of TBA compared with healthy subjects (n = 474) (2.3 ± 1.4 μmol/L vs. 1.9 ± 1.0 μmol/L, P < 0.001). TBA was independently associated with GDM [adjusted odds ratio (AOR), 1.38; 95% confidence interval (CI), 1.18–1.61, P < 0.001]. Compared to the first category of TBA, women in the highest category had a marked increase in risk for GDM (AOR, 7.72; 95% CI, 3.22–18.50, P < 0.001). In conclusion, higher first-trimester TBA levels, even within normal range, may help indicate increased risk of GDM. PMID:27667090

  4. Total Syntheses of (−)-Pyrimidoblamic Acid and P-3A

    PubMed Central

    2015-01-01

    Total syntheses of (−)-pyrimidoblamic acid and P-3A are disclosed. Central to the convergent approach is a powerful inverse electron demand Diels–Alder reaction between substituted electron-deficient 1,2,3-triazines and a highly functionalized and chiral primary amidine, which forms the pyrimidine cores and introduces all necessary stereochemistry in a single step. Intrinsic in the convergent approach is the potential it provides for the late stage divergent synthesis of modified analogs bearing deep-seated changes in either the pyrimidine cores or the highly functionalized C2 side chain common to both natural products. The examination of the key cycloaddition reaction revealed that the inherent 1,2,3-triazine mode of cycloaddition (C4/N1 vs C5/N2) as well as the amidine regioselectivity were unaffected by introduction of two electron-withdrawing groups (−CO2R) at C4 and C6 of the 1,2,3-triazine even if C5 is unsubstituted (Me or H), highlighting the synthetic potential of the powerful pyrimidine synthesis. PMID:24410439

  5. Fatty acid acylation of rat brain myelin proteolipid protein in vitro: identification of the lipid donor.

    PubMed

    Bizzozero, O A; Lees, M B

    1986-02-01

    The immediate acyl chain donor for fatty acid esterification of proteolipid protein (PLP) was identified in an in vitro system. Rat brain total membranes, after removal of crude nuclear and mitochondrial fractions, were incubated with radioactive acyl donors, extracted with chloroform/methanol, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of [3H]palmitic acid, CoA, ATP, and Mg2+, acylation of endogenous PLP occurred at a linear rate for at least 2 h. The radioactivity was associated with the protein via an ester linkage, mainly as palmitic acid. Omission of ATP, CoA, Mg2+, or all three reduced fatty acid incorporation into PLP to 44, 27, 8, and 4%, respectively, of the values in the complete system. Incubation of the membrane fraction with [3H]palmitoyl-CoA in the absence of CoA and ATP led to highly labeled PLP. These data demonstrate that activation of free fatty acid is required for acylation. Phospholipids and glycolipids were not able to acylate the PLP directly. Finally, when isolated myelin was incubated with [3H]palmitoyl-CoA in the absence of cofactors, only PLP was labeled, thus confirming the identity of palmitoyl-CoA as the direct acyl chain donor and suggesting that the acylating activity and the PLP pool available for acylation are both in the myelin.

  6. Retinoic acid binding protein in normal and neopolastic rat prostate.

    PubMed

    Gesell, M S; Brandes, M J; Arnold, E A; Isaacs, J T; Ueda, H; Millan, J C; Brandes, D

    1982-01-01

    Sucrose density gradient analysis of cytosol from normal and neoplastic rat prostatic tissues exhibited a peak of (3H) retinoic acid binding in the 2S region, corresponding to the cytoplasmic retinoic acid binding protein (cRABP). In the Fisher-Copenhagen F1 rat, cRABP was present in the lateral lobe, but could not be detected in the ventral nor in the dorsal prostatic lobes. Four sublines of the R-3327 rat prostatic tumor contained similar levels of this binding protein. The absence of cRABP in the normal tissue of origin of the R-3327 tumor, the rat dorsal prostate, and reappearance in the neoplastic tissues follows a pattern described in other human and animal tumors. The occurrence of cRABP in the well-differentiated as well as in the anaplastic R-3327 tumors in which markers which reflect a state of differentiation and hormonal regulation, such as androgen receptor, 5 alpha reductase, and secretory acid phosphatase are either markedly reduced or absent, points to cRABP as a marker of malignant transformation.

  7. Dynamics of palmitic acid complexed with rat intestinal fatty acid binding protein.

    PubMed

    Zhu, L; Kurian, E; Prendergast, F G; Kemple, M D

    1999-02-02

    Dynamics of palmitic acid (PA), isotopically enriched with 13C at the second, seventh, or terminal methyl position, were investigated by 13C NMR. Relaxation measurements were made on PA bound to recombinant rat intestinal fatty acid binding protein (I-FABP) at pH 5.5 and 23 degreesC, and, for comparison, on PA incorporated into 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (MPPC) micelles, and dissolved in methanol. The 13C relaxation data, T1, and steady-state nuclear Overhauser effect (NOE) obtained at two different magnetic fields were interpreted using the model-free approach [Lipari, G., and Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559]. The overall rotational correlation time of the fatty acid.protein complex was 2.5 +/- 0.4 ns, which is substantially less than the value expected for the protein itself (>6 ns). Order parameters (S2), which are a measure of the amplitude of the internal motion of individual C-H vectors with respect to the PA molecule, while largest for C-2 and smallest for the methyl carbon, were relatively small (<0.4) in the protein complex. S2 values for given C-H vectors also were smaller for PA in the MPPC micelles and in methanol than in the protein complex. Correlation times reflective of the time scale of the internal motion of the C-H vectors were in all cases <60 ps. These results support the view that the fatty acid is not rigidly anchored within the I-FABP binding pocket, but rather has considerable freedom to move within the pocket.

  8. Effect of Acidic pH on Expression of Surface-Associated Proteins of Streptococcus oralis

    PubMed Central

    Wilkins, Joanna C.; Beighton, David; Homer, Karen A.

    2003-01-01

    Streptococcus oralis, a member of the mitis group of oral streptococci, is implicated in the pathogenesis of infective endocarditis and is the predominant aciduric non-mutans-group streptococcus in dental plaque. We undertook to identify the most abundant surface-associated proteins of S. oralis and to investigate changes in protein expression when the organism was grown under acidic culture conditions. Surface-associated proteins were extracted from cells grown in batch culture, separated by two-dimensional gel electrophoresis, excised, digested with trypsin, and analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry. Putative functions were assigned by homology to a translated genomic database of Streptococcus pneumoniae. A total of 27 proteins were identified; these included a lipoprotein, a ribosome recycling factor, and the glycolytic enzymes phosphoglycerate kinase, fructose bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, and enolase. The most abundant protein, phosphocarrier protein HPr, was present as three isoforms. Neither lactate dehydrogenase nor pyruvate oxidase, dominant intracellular proteins, were present among the proteins on the gels, demonstrating that proteins in the surface-associated pool did not arise as a result of cell lysis. Eleven of the proteins identified were differentially expressed when cells were grown at pH 5.2 versus pH 7.0, and these included superoxide dismutase, a homologue of dipeptidase V from Lactococcus lactis, and the protein translation elongation factors G, Tu, and Ts. This study has extended the range of streptococcal proteins known to be expressed at the cell surface. Further investigations are required to ascertain their functions at this extracellular location and determine how their expression is influenced by other environmental conditions. PMID:12957916

  9. Minimal genome encoding proteins with constrained amino acid repertoire

    PubMed Central

    Tsoy, Olga; Yurieva, Marina; Kucharavy, Andrey; O'Reilly, Mary; Mushegian, Arcady

    2013-01-01

    Minimal bacterial gene set comprises the genetic elements needed for survival of engineered bacterium on a rich medium. This set is estimated to include 300–350 protein-coding genes. One way of simplifying an organism with such a minimal genome even further is to constrain the amino acid content of its proteins. In this study, comparative genomics approaches and the results of gene knockout experiments were used to extrapolate the minimal gene set of mollicutes, and bioinformatics combined with the knowledge-based analysis of the structure-function relationships in these proteins and their orthologs, paralogs and analogs was applied to examine the challenges of completely replacing the rarest residue, cysteine. Among several known functions of cysteine residues, their roles in the active centers of the enzymes responsible for deoxyribonucleoside synthesis and transfer RNA modification appear to be crucial, as no alternative chemistry is known for these reactions. Thus, drastic reduction of the content of the rarest amino acid in a minimal proteome appears to be possible, but its complete elimination is challenging. PMID:23873957

  10. Retroviral nucleocapsid proteins possess potent nucleic acid strand renaturation activity.

    PubMed Central

    Dib-Hajj, F.; Khan, R.; Giedroc, D. P.

    1993-01-01

    The nucleocapsid protein (NC) is the major genomic RNA binding protein that plays integral roles in the structure and replication of all animal retroviruses. In this report, select biochemical properties of recombinant Mason-Pfizer monkey virus (MPMV) and HIV-1 NCs are compared. Evidence is presented that two types of saturated Zn2 NC-polynucleotide complexes can be formed under conditions of low [NaCl] that differ in apparent site-size (n = 8 vs. n = 14). The formation of one or the other complex appears dependent on the molar ratio of NC to RNA nucleotide with the putative low site-size mode apparently predominating under conditions of protein excess. Both MPMV and HIV-1 NCs kinetically facilitate the renaturation of two complementary DNA strands, suggesting that this is a general property of retroviral NCs. NC proteins increase the second-order rate constant for renaturation of a 149-bp DNA fragment by more than four orders of magnitude over that obtained in the absence of protein at 37 degrees C. The protein-assisted rate is 100-200-fold faster than that obtained at 68 degrees C, 1 M NaCl, solution conditions considered to be optimal for strand renaturation. Provided that sufficient NC is present to coat all strands, the presence of 400-1,000-fold excess nonhomologous DNA does not greatly affect the reaction rate. The HIV-1 NC-mediated renaturation reaction functions stoichiometrically, requiring a saturated strand of DNA nucleotide:NC ratio of about 7-8, rather than 14. Under conditions of less protein, the rate acceleration is not realized. The finding of significant nucleic acid strand renaturation activity may have important implications for various events of reverse transcription particularly in initiation and cDNA strand transfer. PMID:8443601

  11. G-protein coupled receptor solubilization and purification for biophysical analysis and functional studies, in the total absence of detergent

    PubMed Central

    Jamshad, Mohammed; Charlton, Jack; Lin, Yu-Pin; Routledge, Sarah J.; Bawa, Zharain; Knowles, Timothy J.; Overduin, Michael; Dekker, Niek; Dafforn, Tim R.; Bill, Roslyn M.; Poyner, David R.; Wheatley, Mark

    2015-01-01

    G-protein coupled receptors (GPCRs) constitute the largest class of membrane proteins and are a major drug target. A serious obstacle to studying GPCR structure/function characteristics is the requirement to extract the receptors from their native environment in the plasma membrane, coupled with the inherent instability of GPCRs in the detergents required for their solubilization. In the present study, we report the first solubilization and purification of a functional GPCR [human adenosine A2A receptor (A2AR)], in the total absence of detergent at any stage, by exploiting spontaneous encapsulation by styrene maleic acid (SMA) co-polymer direct from the membrane into a nanoscale SMA lipid particle (SMALP). Furthermore, the A2AR–SMALP, generated from yeast (Pichia pastoris) or mammalian cells, exhibited increased thermostability (∼5°C) compared with detergent [DDM (n-dodecyl-β-D-maltopyranoside)]-solubilized A2AR controls. The A2AR–SMALP was also stable when stored for prolonged periods at 4°C and was resistant to multiple freeze-thaw cycles, in marked contrast with the detergent-solubilized receptor. These properties establish the potential for using GPCR–SMALP in receptor-based drug discovery assays. Moreover, in contrast with nanodiscs stabilized by scaffold proteins, the non-proteinaceous nature of the SMA polymer allowed unobscured biophysical characterization of the embedded receptor. Consequently, CD spectroscopy was used to relate changes in secondary structure to loss of ligand binding ([3H]ZM241385) capability. SMALP-solubilization of GPCRs, retaining the annular lipid environment, will enable a wide range of therapeutic targets to be prepared in native-like state to aid drug discovery and understanding of GPCR molecular mechanisms. PMID:25720391

  12. Major phenolic acids and total antioxidant activity in mamaki leaves, Pipturus albidus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Three phenolic acids, (+) catechins, chlorogenic acid, and rutin, were identified and quantified in mamaki leaves (Pipturus albidus) using a liquid chromatograph-mass spectrometer technique. Concentrations of (+) catechins, chlorogenic acid, and rutin varied from 1.1 mg to 5.0 mg per gram of mamaki...

  13. Amino acid function relates to its embedded protein microenvironment: A study on disulfide-bridged cystine.

    PubMed

    Bhatnagar, Akshay; Apostol, Marcin I; Bandyopadhyay, Debashree

    2016-11-01

    In our previous study, we have shown that the microenvironments around conserved amino acids are also conserved in protein families (Bandyopadhyay and Mehler, Proteins 2008; 72:646-659). In this study, we have hypothesized that amino acids perform similar functions when embedded in a certain type of protein microenvironment. We have tested this hypothesis on the microenvironments around disulfide-bridged cysteines from high-resolution protein crystal structures. Although such cystines mainly play structural role in proteins, in certain enzymes they participate in catalysis and redox reactions. We have performed and report a functional annotation of enzymatically active cystines to their respective microenvironments. Three protein microenvironment clusters were identified: (i) buried-hydrophobic, (ii) exposed-hydrophilic, and (iii) buried-hydrophilic. The buried-hydrophobic cluster encompasses a small group of 22 redox-active cystines, mostly in alpha-helical conformations in a -C-x-x-C- motif from the Oxido-reductase enzyme class. All these cystines have high strain energy and near identical microenvironments. Most of the active cystines in hydrolase enzyme class belong to buried hydrophilic microenvironment cluster. In total there are 34 half-cystines detected in buried hydrophilic cluster from hydrolases, as a part of enzyme active site. Even within the buried hydrophilic cluster, there is clear separation of active half-cystines between surface exposed part of the protein and protein interior. Half-cystines toward the surface exposed region are higher in number compared to those in protein interior. Apart from cystines at the active sites of the enzymes, many more half-cystines were detected in buried hydrophilic cluster those are part of the microenvironment of enzyme active sites. However, no active half-cystines were detected in extremely hydrophilic microenvironment cluster, that is, exposed hydrophilic cluster, indicating that total exposure of cystine

  14. Total Acid Value Titration of Hydrotreated Biomass Fast Pyrolysis Oil: Determination of Carboxylic Acids and Phenolics with Multiple End-Point Detection

    SciTech Connect

    Christensen, E.; Alleman, T. L.; McCormick, R. L.

    2013-01-01

    Total acid value titration has long been used to estimate corrosive potential of petroleum crude oil and fuel oil products. The method commonly used for this measurement, ASTM D664, utilizes KOH in isopropanol as the titrant with potentiometric end point determination by pH sensing electrode and Ag/AgCl reference electrode with LiCl electrolyte. A natural application of the D664 method is titration of pyrolysis-derived bio-oil, which is a candidate for refinery upgrading to produce drop in fuels. Determining the total acid value of pyrolysis derived bio-oil has proven challenging and not necessarily amenable to the methodology employed for petroleum products due to the different nature of acids present. We presented an acid value titration for bio-oil products in our previous publication which also utilizes potentiometry using tetrabutylammonium hydroxide in place of KOH as the titrant and tetraethylammonium bromide in place of LiCl as the reference electrolyte to improve the detection of these types of acids. This method was shown to detect numerous end points in samples of bio-oil that were not detected by D664. These end points were attributed to carboxylic acids and phenolics based on the results of HPLC and GC-MS studies. Additional work has led to refinement of the method and it has been established that both carboxylic acids and phenolics can be determined accurately. Use of pH buffer calibration to determine half-neutralization potentials of acids in conjunction with the analysis of model compounds has allowed us to conclude that this titration method is suitable for the determination of total acid value of pyrolysis oil and can be used to differentiate and quantify weak acid species. The measurement of phenolics in bio-oil is subject to a relatively high limit of detection, which may limit the utility of titrimetric methodology for characterizing the acidic potential of pyrolysis oil and products.

  15. Determination of total mercury in seafood and other protein-rich products

    SciTech Connect

    Landi, S.; Fagioli, F.; Locatelli, C.

    1992-11-01

    A previously developed wet-digestion method for the determination of total mercury in plants by cold vapor atomic absorption spectroscopy (CVAAS) was extended to the analysis of seafood and other products rich in proteins. Oxidation of matrixes is accomplished by K{sub 2}Cr{sub 2}O{sub 7} in the presence of diluted H{sub 2}SO{sub 4}; a simple air condenser is used to reflux vapors released from the boiling mixture. The original procedure (A) and 2 modifications (B and C), which differ with respect to the mode of acidification and/or digestion time and the types of condensers used, were compared for precision and accuracy by means of National Institute of Standards and Technology Research Material 50 Albacore Tuna and proved to be reliable (Hg present, 0.95{plus_minus}0.1 {mu}g/g; Hg found, 0.97 {plus_minus} 0.029 {mu}g/g [A], 0.98 {plus_minus} 0.018 {mu}g/g [B], and 0.94 {plus_minus} 0.025 {mu}g/g [C]). The modified procedures were tested further in Hg recovery experiments on a variety of biological matrixes with different spiking substances and again showed good analytical characteristics (overall average recoveries = 98 {plus_minus} 5.1% for seafood and 100 {plus_minus} 3.6 for protein-rich baby foods). 22 refs., 1 fig., 5 tabs.

  16. Effects of copper on chlorophyll, proline, protein and abscisic acid level of sunflower (Helianthus annuus L.) seedlings.

    PubMed

    Zengin, Fikriye Kirbag; Kirbag, Sevda

    2007-07-01

    The effect of copperchloride (CuCl2) on the level of chlorophyll (a+b), proline, protein and abscisic acid in sunflower (Helianthus annuus L.) seedlings were investigated Control and copper treated (0.4, 0.5 and 0.6 mM) seedlings were grown for ten days in Hoagland solution. Abscisic acid content was determined in root, shoot and leaf tissues of seedlings by HPLC. Copper stress caused significant increase of the abscisic acid contents in roots, shoots and leaves of seedlings. The increase was dependent on the copper salt concentration. Enhanced accumulation of proline in the leaves of seedlings exposed to copper was determined, as well as a decrease of chlorophyll (a+b) and total protein (p < 0.05 or p < 0.01). It was observed that the level of chlorophyll (a+b) and total protein (p < 0.05 or p < 0.01) remarkably decreased as copper concentration increased to 0.6 mM, although the levels of proline and abscisic acid in the leaves of plants were increased--a dose-depended behavior The same trends were also observed with the level of abscisic acid of stems and roots. Copper has dose- depended effects on chlorophyll, proline, protein and abscisic acid level of sunflower (Helianthus annuus L.) seedlings. Thus, we assumed that copper levels increase above some critical points seedling growth get negative effects. This assumption is in line with previous findings.

  17. An amino acid code for irregular and mixed protein packing.

    PubMed

    Joo, Hyun; Chavan, Archana G; Fraga, Keith J; Tsai, Jerry

    2015-12-01

    To advance our understanding of protein tertiary structure, the development of the knob-socket model is completed in an analysis of the packing in irregular coil and turn secondary structure packing as well as between mixed secondary structure. The knob-socket model simplifies packing based on repeated patterns of two motifs: a three-residue socket for packing within secondary (2°) structure and a four-residue knob-socket for tertiary (3°) packing. For coil and turn secondary structure, knob-sockets allow identification of a correlation between amino acid composition and tertiary arrangements in space. Coil contributes almost as much as α-helices to tertiary packing. In irregular sockets, Gly, Pro, Asp, and Ser are favored, while in irregular knobs, the preference order is Arg, Asp, Pro, Asn, Thr, Leu, and Gly. Cys, His,Met, and Trp are not favored in either. In mixed packing, the knob amino acid preferences are a function of the socket that they are packing into, whereas the amino acid composition of the sockets does not depend on the secondary structure of the knob. A unique motif of a coil knob with an XYZ β-sheet socket may potentially function to inhibit β-sheet extension. In addition, analysis of the preferred crossing angles for strands within a β-sheet and mixed α-helice/β-sheet identifies canonical packing patterns useful in protein design. Lastly, the knob-socket model abstracts the complexity of protein tertiary structure into an intuitive packing surface topology map.

  18. An Amino Acid Code for Irregular and Mixed Protein Packing

    PubMed Central

    Joo, Hyun; Chavan, Archana; Fraga, Keith; Tsai, Jerry

    2015-01-01

    To advance our understanding of protein tertiary structure, the development of the knob-socket model is completed in an analysis of the packing in irregular coil and turn secondary structure packing as well as between mixed secondary structure. The knob-socket model simplifies packing based on repeated patterns of 2 motifs: a 3 residue socket for packing within 2° structure and a 4 residue knob-socket for 3° packing. For coil and turn secondary structure, knob-sockets allow identification of a correlation between amino acid composition and tertiary arrangements in space. Coil contributes almost as much as α-helices to tertiary packing. Irregular secondary structure involves 3 residue cliques of consecutive contacting residues or XYZ sockets. In irregular sockets, Gly, Pro, Asp and Ser are favored, while Cys, His, Met and Trp are not. For irregular knobs, the preference order is Arg, Asp, Pro, Asn, Thr, Leu, and Gly, while Cys, His, Met and Trp are not. In mixed packing, the knob amino acid preferences are a function of the socket that they are packing into, whereas the amino acid composition of the sockets does not depend on the secondary structure of the knob. A unique motif of a coil knob with an XYZ β-sheet socket may potentially function to inhibit β-sheet extension. In addition, analysis of the preferred crossing angles for strands within a β-sheet and mixed α-helices/β-sheets identifies canonical packing patterns useful in protein design. Lastly, the knob-socket model abstracts the complexity of protein tertiary structure into an intuitive packing surface topology map. PMID:26370334

  19. Chemical Functionalization of Germanium with Dextran Brushes for Immobilization of Proteins Revealed by Attenuated Total Reflection Fourier Transform Infrared Difference Spectroscopy.

    PubMed

    Schartner, Jonas; Hoeck, Nina; Güldenhaupt, Jörn; Mavarani, Laven; Nabers, Andreas; Gerwert, Klaus; Kötting, Carsten

    2015-07-21

    Protein immobilization studied by attenuated total reflection Fourier transform infrared (ATR-FT-IR) difference spectroscopy is an emerging field enabling the study of proteins at atomic detail. Gold or glass surfaces are frequently used for protein immobilization. Here, we present an alternative method for protein immobilization on germanium. Because of its high refractive index and broad spectral window germanium is the best material for ATR-FT-IR spectroscopy of thin layers. So far, this technique was mainly used for protein monolayers, which lead to a limited signal-to-noise ratio. Further, undesired protein-protein interactions can occur in a dense layer. Here, the germanium surface was functionalized with thiols and stepwise a dextran brush was generated. Each step was monitored by ATR-FT-IR spectroscopy. We compared a 70 kDa dextran with a 500 kDa dextran regarding the binding properties. All surfaces were characterized by atomic force microscopy, revealing thicknesses between 40 and 110 nm. To analyze the capability of our system we utilized N-Ras on mono-NTA (nitrilotriacetic acid) functionalized dextran, and the amount of immobilized Ras corresponded to several monolayers. The protein stability and loading capacity was further improved by means of tris-NTA for immobilization. Small-molecule-induced changes were revealed with an over 3 times higher signal-to-noise ratio compared to monolayers. This improvement may allow the observation of very small and so far hidden changes in proteins upon stimulus. Furthermore, we immobilized green fluorescent protein (GFP) and mCherry simultaneously enabling an analysis of the surface by fluorescence microscopy. The absence of a Förster resonance energy transfer (FRET) signal demonstrated a large protein-protein distance, indicating an even distribution of the protein within the dextran.

  20. Simplified protein design biased for prebiotic amino acids yields a foldable, halophilic protein

    PubMed Central

    Longo, Liam M.; Lee, Jihun; Blaber, Michael

    2013-01-01

    A compendium of different types of abiotic chemical syntheses identifies a consensus set of 10 “prebiotic” α-amino acids. Before the emergence of biosynthetic pathways, this set is the most plausible resource for protein formation (i.e., proteogenesis) within the overall process of abiogenesis. An essential unsolved question regarding this prebiotic set is whether it defines a “foldable set”—that is, does it contain sufficient chemical information to permit cooperatively folding polypeptides? If so, what (if any) characteristic properties might such polypeptides exhibit? To investigate these questions, two “primitive” versions of an extant protein fold (the β-trefoil) were produced by top-down symmetric deconstruction, resulting in a reduced alphabet size of 12 or 13 amino acids and a percentage of prebiotic amino acids approaching 80%. These proteins show a substantial acidification of pI and require high salt concentrations for cooperative folding. The results suggest that the prebiotic amino acids do comprise a foldable set within the halophile environment. PMID:23341608

  1. Blood count and C-reactive protein evolution in gastric cancer patients with total gastrectomy surgery

    PubMed Central

    CSENDES J., Attila; MUÑOZ Ch., Andrea; BURGOS L., Ana María

    2014-01-01

    Background The complete blood count (CBC) and C-reactive protein (CRP) are useful inflammatory parameters for ruling out acute postoperative inflammatory complications. Aim To determine their changes in gastric cancer patients submitted to total gastrectomy. Methods This is a prospective study, with 36 patients with gastric cancer who were submitted to elective total gastrectomy. On the first, third and fifth postoperative day (POD), blood count and CRP changes were assessed. Patients with postoperative complications were excluded. Results Twenty-one (58%) were men and 15 (42%) women. The mean age was 65 years. The leukocytes peaked on the 1st POD with a mean of 13,826 u/mm³, and decreased to 8,266 u/mm³ by the 5th POD. The bacilliforms peaked on the 1st POD with a maximum value of 1.48%. CRP reached its maximum level on the 3rd POD with a mean of 144.64 mg/l±44.84. Preoperative hematocrit (HCT) was 35% and 33.67% by the 5th POD. Hemoglobin, showed similar values. Conclusions Leukocytes increased during the 1st POD but reached normal values by the 5th POD. CRP peaked on the 3rd POD but did not reach normal values by the 5th POD. PMID:25626929

  2. Screening of immunomodulatory activity of total and protein extracts of some Moroccan medicinal plants.

    PubMed

    Daoudi, Abdeljlil; Aarab, Lotfi; Abdel-Sattar, Essam

    2013-04-01

    Herbal and traditional medicines are being widely used in practice in many countries for their benefits of treating different ailments. A large number of plants in Morocco were used in folk medicine to treat immune-related disorders. The objective of this study is to evaluate the immunomodulatory activity of protein extracts (PEs) of 14 Moroccan medicinal plants. This activity was tested on the proliferation of immune cells. The prepared total and PEs of the plant samples were tested using MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide) assay on the splenocytes with or without stimulation by concanavalin-A (Con-A), a mitogenic agent used as positive control. The results of this study indicated different activity spectra. Three groups of activities were observed. The first group represented by Citrullus colocynthis, Urtica dioica, Elettaria cardamomum, Capparis spinosa and Piper cubeba showed a significant immunosuppressive activity. The second group that showed a significant immunostimulatory activity was represented by Aristolochia longa, Datura stramonium, Marrubium vulgare, Sinapis nigra, Delphynium staphysagria, Lepidium sativum, Ammi visnaga and Tetraclinis articulata. The rest of the plant extracts did not alter the proliferation induced by Con-A. This result was more important for the PE than for the total extract. In conclusion, this study revealed an interesting immunomodulating action of certain PEs, which could explain their traditional use. The results of this study may also have implications in therapeutic treatment of infections, such as prophylactic and adjuvant with cancer chemotherapy.

  3. Reversible lysine modification on proteins by using functionalized boronic acids.

    PubMed

    Cal, Pedro M S D; Frade, Raquel F M; Cordeiro, Carlos; Gois, Pedro M P

    2015-05-26

    Iminoboronates have been utilized to successfully install azide and alkyne bioorthogonal functions on proteins, which may then be further reacted with their bioorthogonal counterparts. These constructs were also used to add polyethylene glycol (PEG) to insulin, a modification which has been shown to be reversible in the presence of fructose. Finally, iminoboronates were used to assemble a folic acid/paclitaxel small-molecule/drug conjugate in situ with an IC50  value of 20.7 nM against NCI-H460 cancer cells and negligible cytotoxicity against the CRL-1502 noncancer cells.

  4. Photolabeling of brain membrane proteins by lysergic acid diethylamide

    SciTech Connect

    Mahon, A.C.; Hartig, P.R.

    1982-04-05

    /sup 3/H-Lysergic acid diethylamide (/sup 3/H-LSD) is irreversibly incorporated into bovine caudate membranes during ultraviolet light illumination. The incorporated radioligand apparently forms a covalent bond with a sub-population of the membrane proteins. Although the photolabeling pattern differs significantly from the Coomassie blue staining pattern on SDS gels, the photolabeling is apparently not specific for LSD binding sites associated with neurotransmitter receptors. /sup 3/H-LSD photolabeling can occur during prolonged exposure of membrane samples to room lighting and thus may introduce artifacts into receptor binding assays.

  5. Acid-base chemistry of frustrated water at protein interfaces.

    PubMed

    Fernández, Ariel

    2016-01-01

    Water molecules at a protein interface are often frustrated in hydrogen-bonding opportunities due to subnanoscale confinement. As shown, this condition makes them behave as a general base that may titrate side-chain ammonium and guanidinium cations. Frustration-based chemistry is captured by a quantum mechanical treatment of proton transference and shown to remove same-charge uncompensated anticontacts at the interface found in the crystallographic record and in other spectroscopic information on the aqueous interface. Such observations are untenable within classical arguments, as hydronium is a stronger acid than ammonium or guanidinium. Frustration enables a directed Grotthuss mechanism for proton transference stabilizing same-charge anticontacts.

  6. Structure and function analysis of protein-nucleic acid complexes

    NASA Astrophysics Data System (ADS)

    Kuznetsova, S. A.; Oretskaya, T. S.

    2016-05-01

    The review summarizes published data on the results and achievements in the field of structure and function analysis of protein-nucleic acid complexes by means of main physical and biochemical methods, including X-ray diffraction, nuclear magnetic resonance spectroscopy, electron and atomic force microscopy, small-angle X-ray and neutron scattering, footprinting and cross-linking. Special attention is given to combined approaches. The advantages and limitations of each method are considered, and the prospects of their application for wide-scale structural studies in vivo are discussed. The bibliography includes 145 references.

  7. Dose response of whey protein isolate in addition to a typical mixed meal on blood amino acids and hormonal concentrations.

    PubMed

    Forbes, Scott C; McCargar, Linda; Jelen, Paul; Bell, Gordon J

    2014-04-01

    The purpose was to investigate the effects of a controlled typical 1-day diet supplemented with two different doses of whey protein isolate on blood amino acid profiles and hormonal concentrations following the final meal. Nine males (age: 29.6 ± 6.3 yrs) completed four conditions in random order: a control (C) condition of a typical mixed diet containing ~10% protein (0.8 g·kg1), 65% carbohydrate, and 25% fat; a placebo (P) condition calorically matched with carbohydrate to the whey protein conditions; a low-dose condition of 0.8 grams of whey protein isolate per kilogram body mass per day (g·kg1·d1; W1) in addition to the typical mixed diet; or a high-dose condition of 1.6 g·kg1·d1 (W2) of supplemental whey protein in addition to the typical mixed diet. Following the final meal, significant (p < .05) increases in total amino acids, essential amino acids (EAA), branch-chained amino acids (BCAA), and leucine were observed in plasma with whey protein supplementation while no changes were observed in the control and placebo conditions. There was no significant group difference for glucose, insulin, testosterone, cortisol, or growth hormone. In conclusion, supplementing a typical daily food intake consisting of 0.8 g of protein·kg1·d1 with a whey protein isolate (an additional 0.8 or 1.6 g·kg1·d1) significantly elevated total amino acids, EAA, BCAA, and leucine but had no effect on glucose, insulin, testosterone, cortisol, or growth hormone following the final meal. Future acute and chronic supplementation research examining the physiological and health outcomes associated with elevated amino acid profiles is warranted.

  8. Clamping drainage is unnecessary after minimally invasive total knee arthroplasty in patients with tranexamic acid

    PubMed Central

    Wu, Yuangang; Yang, Timin; Zeng, Yi; Li, Canfeng; Shen, Bin; Pei, Fuxing

    2017-01-01

    Abstract Background: Drainage and tranexamic acid (TXA) have been widely used in total knee arthroplasty (TKA). However, it remains unclear whether it is necessary to clamp the drain after minimally invasive TKA (MIS-TKA) when TXA is used. We therefore conducted a randomized controlled trial to compare the effects of clamping versus not clamping drainage following MIS-TKA in patients in whom TXA was used. Methods: From January 2015 to December 2015, 121 patients undergoing unilateral primary MIS-TKA were enrolled and randomly divided into 2 groups. In the clamping group (N = 60), drainage was clamped for the 1st 4 postoperative hours. In the nonclamping group (N = 61), drainage was not clamped. All patients underwent a minimidvastus approach and received 10 mg/kg TXA intravenously before tourniquet deflation. We recorded the total blood loss, drainage volume, and transfusion requirements in the postoperative period. We also measured the hemoglobin (Hb) and hematocrit (Hct) levels on postoperative days 1, 3, and 5. Other factors, including range of motion (ROM), visual analog scale (VAS), and occurrence of wound-related complications, deep vein thrombosis (DVT), and pulmonary embolism (PE) were recorded at the time of discharge and 1 and 6 months postoperatively. No statistically significant differences were found between the 2 groups with regard to age, gender, weight, BMI, preoperative Hb and Hct levels, preoperative ROM, VAS, duration of surgery, anesthesia method, and the American Society of Anesthesiologists classification. Results: The clamping group experienced better drainage volume results than the nonclamping group (P < 0.001). There were no statistically significant differences in TBL and transfusion requirements (P = 0.105 and 0.276, respectively); Hb and Hct levels on postoperative days 1, 3, and 5 were similar between the 2 groups. No significant differences were found for ROM, VAS, DVT, PE, wound-related complications, and hospital

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

    PubMed Central

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

    2014-01-01

    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 (OTSs) 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. PMID:24959531

  10. 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.

  11. Photo-CIDNP NMR spectroscopy of amino acids and proteins.

    PubMed

    Kuhn, Lars T

    2013-01-01

    Photo-chemically induced dynamic nuclear polarization (CIDNP) is a nuclear magnetic resonance (NMR) phenomenon which, among other things, is exploited to extract information on biomolecular structure via probing solvent-accessibilities of tryptophan (Trp), tyrosine (Tyr), and histidine (His) amino acid side chains both in polypeptides and proteins in solution. The effect, normally triggered by a (laser) light-induced photochemical reaction in situ, yields both positive and/or negative signal enhancements in the resulting NMR spectra which reflect the solvent exposure of these residues both in equilibrium and during structural transformations in "real time". As such, the method can offer - qualitatively and, to a certain extent, quantitatively - residue-specific structural and kinetic information on both the native and, in particular, the non-native states of proteins which, often, is not readily available from more routine NMR techniques. In this review, basic experimental procedures of the photo-CIDNP technique as applied to amino acids and proteins are discussed, recent improvements to the method highlighted, and future perspectives presented. First, the basic principles of the phenomenon based on the theory of the radical pair mechanism (RPM) are outlined. Second, a description of standard photo-CIDNP applications is given and it is shown how the effect can be exploited to extract residue-specific structural information on the conformational space sampled by unfolded or partially folded proteins on their "path" to the natively folded form. Last, recent methodological advances in the field are highlighted, modern applications of photo-CIDNP in the context of biological NMR evaluated, and an outlook into future perspectives of the method is given.

  12. Blood conservation with tranexamic acid in total hip arthroplasty: a randomized, double-blind study in 40 primary operations.

    PubMed

    Benoni, G; Fredin, H; Knebel, R; Nilsson, P

    2001-10-01

    We performed a randomized, double-blind study on the effect of tranexamic acid on blood loss and blood transfusions in 40 primary total hip arthroplasties. Tranexamic acid, 10 mg/kg body weight, or placebo, was given intravenously just before the operation. Blood loss during the operation and postoperatively into the drains was recorded, as also were blood hemoglobin concentrations. Ultrasound examination 1 week postoperatively was done to estimate the blood loss due to remaining hematomas. Total (operation + drain) blood loss was 0.76 (95 CI 0.63-0.89) L in the tranexamic acid group as compared to 1.0 (CI 0.81-1.2) L in the placebo group (p = 0.03). The number of blood transfusions during the day of operation was 2 vs. 10 (p = 0.07) and the total number during the hospital stay was 5 vs. 13 (p = 0.2). 1 patient in each group had a pulmonary embolism.

  13. Molecular evolution of monotreme and marsupial whey acidic protein genes.

    PubMed

    Sharp, Julie A; Lefèvre, Christophe; Nicholas, Kevin R

    2007-01-01

    Whey acidic protein (WAP), a major whey protein present in milk of a number of mammalian species has characteristic cysteine-rich domains known as four-disulfide cores (4-DSC). Eutherian WAP, expressed in the mammary gland throughout lactation, has two 4-DSC domains, (DI-DII) whereas marsupial WAP, expressed only during mid-late lactation, contains an additional 4-DSC (DIII), and has a DIII-D1-DII configuration. We report the expression and evolution of echidna (Tachyglossus aculeatus) and platypus (Onithorhynchus anatinus) WAP cDNAs. Predicted translation of monotreme cDNAs showed echidna WAP contains two 4-DSC domains corresponding to DIII-DII, whereas platypus WAP contains an additional domain at the C-terminus with homology to DII and has the configuration DIII-DII-DII. Both monotreme WAPs represent new WAP protein configurations. We propose models for evolution of the WAP gene in the mammalian lineage either through exon loss from an ancient ancestor or by rapid evolution via the process of exon shuffling. This evolutionary outcome may reflect differences in lactation strategy between marsupials, monotremes, and eutherians, and give insight to biological function of the gene products. WAP four-disulfide core domain 2 (WFDC2) proteins were also identified in echidna, platypus and tammar wallaby (Macropus eugenii) lactating mammary cells. WFDC2 proteins are secreted proteins not previously associated with lactation. Mammary gland expression of tammar WFDC2 during the course of lactation showed WFDC2 was elevated during pregnancy, reduced in early lactation and absent in mid-late lactation.

  14. Dependence of intestinal amino acid uptake on dietary protein or amino acid levels

    SciTech Connect

    Karasov, W.H.; Solberg, D.H.; Diamond, J.M.

    1987-05-01

    To understand how intestinal amino acid (AA) transport is regulated by dietary substrate levels, the authors measured uptake of seven radioactively-labelled AAs and glucose across the jejunal brush-border membrane of mice kept on one of three isocaloric rations differing in nitrogen content. In the high-protein ration, uptake increased by 77-81% for the nonessential, less toxic AAs, proline, and aspartate but only by 32-61% for the more toxic essential AAs tested. In the nitrogen-deficient ration, uptake decreased for the nonessential aspartate and proline but stayed constant or increased for essential AAs and for the nonessential alanine. These patterns imply independent regulation of the intestine's various AA transporters. With decreasing dietary AA (or protein), the imino acid and acidic AA private transporters are repressed, while activities of the basic AA transporter and the neutral AA public transporter decrease to an asymptote or else go through a minimum. These regulatory patterns can be understood as a compromise among conflicting constraints imposed by protein's multiple roles as a source of calories, nitrogen, and essential AAs and by the toxicity of essential AAs at high concentrations.

  15. Barley (Hordeum vulgare L.) low phytic acid 1-1: an endosperm-specific, filial determinant of seed total phosphorus.

    PubMed

    Raboy, Victor; Cichy, Karen; Peterson, Kevin; Reichman, Sarah; Sompong, Utumporn; Srinives, Peerasak; Saneoka, Hirofumi

    2014-01-01

    Inositol hexaphosphate (Ins P6 or "phytic acid") typically accounts for 75 (± 10%) of seed total phosphorus (P). In some cases, genetic blocks in seed Ins P6 accumulation can also alter the distribution or total amount of seed P. In nonmutant barley (Hordeum vulgare L.) caryopses, ~80% of Ins P6 and total P accumulate in the aleurone layer, the outer layer of the endosperm, with the remainder in the germ. In barley low phytic acid 1-1 (Hvlpa1-1) seed, both endosperm Ins P6 and total P are reduced (~45% and ~25%, respectively), but germs are phenotypically wild type. This translates into a net reduction in whole-seed total P of ~15%. Nutrient culture studies demonstrate that the reduction in endosperm total P is not due to a reduction in the uptake of P into the maternal plant. Genetic tests (analyses of testcross and F2 seed) reveal that the Hvlpa1-1 genotype of the filial seed conditions the seed total P reduction; sibling seed in the same head of barley that differ in their Hvlpa1-1 genotype (heterozygous vs. homozygous recessive) differ in their total P (normal vs. reduced, respectively). Therefore, Hvlpa1 functions as a seed-specific or filial determinant of barley endosperm total P.

  16. Effects of short-chain fatty acid-supplemented total parenteral nutrition on intestinal pro-inflammatory cytokine abundance.

    PubMed

    Milo, L A; Reardon, K A; Tappenden, K A

    2002-09-01

    We examined the effect of short-chain fatty acid-supplemented total parenteral nutrition on proinflammatory cytokine levels in piglets. Piglets (N = 22) received either standard total parenteral nutrition or total parenteral nutrition supplemented with short-chain fatty acids. After seven days of continuous nutrient infusion, proinflammatory cytokine (TNF-alpha, IL-1beta, IL-6) abundance in plasma, jejunal, and ileal samples and small intestinal myeloperoxidase was determined using western blotting. No differences were seen in TNF-alpha small intestinal abundance. IL-1beta was higher in the small intestine of the short-chain fatty acid group (P < 0.05). IL-6 was higher in intestinal samples of the short-chain fatty acid group (P = 0.05), with the ileum having a greater abundance of IL-6 than the jejunum (P < 0.005). No differences in proinflammatory cytokine abundance in the plasma or tissue myeloperoxidase were seen. These results indicate short-chain fatty acids beneficially increase small intestinal abundance of IL-1beta and IL-6 during total parenteral nutrition administration, while not affecting systemic production of these cytokines or intestinal inflammation.

  17. New data on the total lipid, cholesterol and fatty acid composition of raw and grilled beef longissimus dorsi.

    PubMed

    Bragagnolo, Neura; Rodriguez-Amaya, Délia B

    2003-09-01

    Simultaneous analyses of total lipids, cholesterol and fatty acids were carried out on raw and grilled beef longissimus dorsi trimmed of external fat. Cholesterol was determined by high performance liquid chromatography and the fatty acids by gas chromatography. Mean total lipid (g/100 g) ranged from 2.1 to 2.6 for raw beef and 3.5 to 4.0 for grilled beef steaks. Cholesterol levels (mg/100 g) ranged from 40 to 43 for raw beef and 67 to 70 for grilled beef steaks. The main intramuscular fatty acids of the raw and grilled meat were 14:0, 16:0, 16:1n7, 18:0, 18:1n9, 18:1n7 and 18:2n6. Grilled lean beef steaks had significantly higher contents of the principal fatty acids and most of the minor fatty acids. The higher values for the three components in the grilled meat were due to loss of moisture during grilling. There was no significant difference between the apparent and true retentions values, both indicating no significant loss or degradation of total lipids, cholesterol and fatty acids during grilling.

  18. Ribonucleic Acid Regulation in Permeabilized Cells of Escherichia coli Capable of Ribonucleic Acid and Protein Synthesis1

    PubMed Central

    Atherly, Alan G.

    1974-01-01

    A cell permeabilization procedure is described that reduces viability less than 10% and does not significantly reduce the rates of ribonucleic acid and protein synthesis when appropriately supplemented. Permeabilization abolishes the normal stringent coupling of protein and ribonucleic acid synthesis. PMID:4364330

  19. Conditions optimising on the yield of biomass, total lipid, and valuable fatty acids in two strains of Skeletonema menzelii.

    PubMed

    Jiang, Xiamin; Han, Qingxi; Gao, Xiuzhi; Gao, Guang

    2016-03-01

    Polyunsaturated fatty acids (PUFA) and eicosapentaenoic acid (EPA) are essential for the health of aquaculture organisms and human beings. A total of 9 species of diatoms were screened and two strains of Skeletonema menzelii were selected for further study due to their high growth rates, PUFA and EPA contents. The culture conditions for the yield of biomass, total lipid, EPA and PUFA were optimised. The quickest growth rates (0.28-0.41d(-1)) were achieved with moderate supplement of nitrogen, phosphorus, iron and silicon. The accumulation of total lipid (17.85-22.70% of dry weight) benefitted from deprivation of nitrogen, phosphorus and silicon, but a moderate iron supplement. Highest PUFA (38.26-50.48% of total fatty acids) or EPA (14.26-18.39% of total fatty acids) contents were observed under high nitrogen and phosphorus supplement, moderate available iron but deprivation of silicon. These findings make tangible contributions to culture S. menzelii for commercial production of PUFA or EPA.

  20. Competitive Binding to Cuprous Ions of Protein and BCA in the Bicinchoninic Acid Protein Assay

    PubMed Central

    Huang, Tao; Long, Mian; Huo, Bo

    2010-01-01

    Although Bicinchoninic acid (BCA) has been widely used to determine protein concentration, the mechanism of interaction between protein, copper ion and BCA in this assay is still not well known. Using the Micro BCA protein assay kit (Pierce Company), we measured the absorbance at 562 nm of BSA solutions with different concentrations of protein, and also varied the BCA concentration. When the concentration of protein was increased, the absorbance exhibited the known linear and nonlinear increase, and then reached an unexpected plateau followed by a gradual decrease. We introduced a model in which peptide chains competed with BCA for binding to cuprous ions. Formation of the well-known chromogenic complex of BCA-Cu1+-BCA was competed with the binding of two peptide bonds (NTPB) to cuprous ion, and there is the possibility of the existence of two new complexes. A simple equilibrium equation was established to describe the correlations between the substances in solution at equilibrium, and an empirical exponential function was introduced to describe the reduction reaction. Theoretical predictions of absorbance from the model were in good agreement with the measurements, which not only validated the competitive binding model, but also predicted a new complex of BCA-Cu1+-NTPB that might exist in the final solution. This work provides a new insight into understanding the chemical bases of the BCA protein assay and might extend the assay to higher protein concentration. PMID:21625379

  1. Competitive Binding to Cuprous Ions of Protein and BCA in the Bicinchoninic Acid Protein Assay.

    PubMed

    Huang, Tao; Long, Mian; Huo, Bo

    2010-01-01

    Although Bicinchoninic acid (BCA) has been widely used to determine protein concentration, the mechanism of interaction between protein, copper ion and BCA in this assay is still not well known. Using the Micro BCA protein assay kit (Pierce Company), we measured the absorbance at 562 nm of BSA solutions with different concentrations of protein, and also varied the BCA concentration. When the concentration of protein was increased, the absorbance exhibited the known linear and nonlinear increase, and then reached an unexpected plateau followed by a gradual decrease. We introduced a model in which peptide chains competed with BCA for binding to cuprous ions. Formation of the well-known chromogenic complex of BCA-Cu(1+)-BCA was competed with the binding of two peptide bonds (NTPB) to cuprous ion, and there is the possibility of the existence of two new complexes. A simple equilibrium equation was established to describe the correlations between the substances in solution at equilibrium, and an empirical exponential function was introduced to describe the reduction reaction. Theoretical predictions of absorbance from the model were in good agreement with the measurements, which not only validated the competitive binding model, but also predicted a new complex of BCA-Cu(1+)-NTPB that might exist in the final solution. This work provides a new insight into understanding the chemical bases of the BCA protein assay and might extend the assay to higher protein concentration.

  2. Shedding light on proteins, nucleic acids, cells, humans and fish

    NASA Technical Reports Server (NTRS)

    Setlow, Richard B.

    2002-01-01

    I was trained as a physicist in graduate school. Hence, when I decided to go into the field of biophysics, it was natural that I concentrated on the effects of light on relatively simple biological systems, such as proteins. The wavelengths absorbed by the amino acid subunits of proteins are in the ultraviolet (UV). The wavelengths that affect the biological activities, the action spectra, also are in the UV, but are not necessarily parallel to the absorption spectra. Understanding these differences led me to investigate the action spectra for affecting nucleic acids, and the effects of UV on viruses and cells. The latter studies led me to the discovery of the important molecular nature of the damages affecting DNA (cyclobutane pyrimidine dimers) and to the discovery of nucleotide excision repair. Individuals with the genetic disease xeroderma pigmentosum (XP) are extraordinarily sensitive to sunlight-induced skin cancer. The finding, by James Cleaver, that their skin cells were defective in DNA repair strongly suggested that DNA damage was a key step in carcinogenesis. Such information was important for estimating the wavelengths in sunlight responsible for human skin cancer and for predicting the effects of ozone depletion on the incidence of non-melanoma skin cancer. It took experiments with backcross hybrid fish to call attention to the probable role of the longer UV wavelengths not absorbed by DNA in the induction of melanoma. These reflections trace the biophysicist's path from molecules to melanoma.

  3. Topology of RNA-protein nucleobase-amino acid π-π interactions and comparison to analogous DNA-protein π-π contacts.

    PubMed

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

    2016-05-01

    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.

  4. Specific high-affinity binding of fatty acids to epidermal cytosolic proteins

    SciTech Connect

    Raza, H.; Chung, W.L.; Mukhtar, H. )

    1991-08-01

    Cytosol from rat, mouse, and human skin or rat epidermis was incubated with (3H)arachidonic acid, (14C)retinoic acid, (14C)oleic acid, (3H)leukotriene A4, (3H)prostaglandin E2 (PGE2) or (3H) 15-hydroxyeicosatetraenoic acid (15-HETE), and protein-bound ligands were separated using Lipidex-1000 at 4C to assess the binding specificity. The binding of oleic acid and arachidonic acid with rat epidermal cytosol was rapid, saturable, and reversible. Binding of oleic acid was competed out with the simultaneous addition of other ligands and found to be in the following order: arachidonic acid greater than oleic acid greater than linoleic acid greater than lauric acid greater than leukotriene A4 greater than 15-HETE = PGE1 greater than PGE2 = PGF2. Scatchard analysis of the binding with arachidonic acid, oleic acid, and retinoic acid revealed high-affinity binding sites with the dissociation constant in the nM range. SDS-PAGE analysis of the oleic acid-bound epidermal cytosolic protein(s) revealed maximum binding at the 14.5 kDa region. The presence of the fatty acid-binding protein in epidermal cytosol and its binding to fatty acids and retinoic acid may be of significance both in the trafficking and the metabolism of fatty acids and retinoids across the skin.

  5. Omega-3 fatty acids predict recurrent venous thromboembolism or total mortality in elderly patients with acute venous thromboembolism.

    PubMed

    Reiner, M F; Stivala, S; Limacher, A; Bonetti, N R; Méan, M; Egloff, M; Rodondi, N; Aujesky, D; von Schacky, C; Lüscher, T F; Camici, G G; Beer, J H

    2017-01-01

    Essentials The role of omega-3 fatty acids (n-3 FAs) in recurrent venous thromboembolism (VTE) is unknown. Association of n-3 FAs with recurrent VTE or total mortality was investigated in 826 patients. Whole blood n-3 FAs were inversely correlated with recurrent VTE or total mortality. Major and non-major bleeding was not increased in patients with higher levels of n-3 FAs.

  6. Detection of Protein-Protein Interaction Within an RNA-Protein Complex Via Unnatural-Amino-Acid-Mediated Photochemical Crosslinking.

    PubMed

    Yeh, Fu-Lung; Tung, Luh; Chang, Tien-Hsien

    2016-01-01

    Although DExD/H-box proteins are known to unwind RNA duplexes and modulate RNA structures in vitro, it is highly plausible that, in vivo, some may function to remodel RNA-protein complexes. Precisely how the latter is achieved remains a mystery. We investigated this critical issue by using yeast Prp28p, an evolutionarily conserved DExD/H-box splicing factor, as a model system. To probe how Prp28p interacts with spliceosome, we strategically placed p-benzoyl-phenylalanine (BPA), a photoactivatable unnatural amino acid, along the body of Prp28p in vivo. Extracts prepared from these engineered strains were then used to assemble in vitro splicing reactions for BPA-mediated protein-protein crosslinkings. This enabled us, for the first time, to "capture" Prp28p in action. This approach may be applicable to studying the roles of other DExD/H-box proteins functioning in diverse RNA-related pathways, as well as to investigating protein-protein contacts within an RNA-protein complex.

  7. Fatty Acids Found in Dairy, Protein, and Unsaturated Fatty Acids Are Associated with Risk of Pancreatic Cancer in a Case-Control Study

    PubMed Central

    Jansen, Rick J.; Robinson, Dennis P.; Frank, Ryan D.; Anderson, Kristin E.; Bamlet, William R.; Oberg, Ann L.; Rabe, Kari G.; Olson, Janet E.; Sinha, Rashmi; Petersen, Gloria M.; Stolzenberg-Solomon, Rachael Z.

    2013-01-01

    Although many studies have investigated meat and total fat in relation to pancreatic cancer risk, few have investigated dairy, fish and specific fatty acids. We evaluated the association between intake of meat, fish, dairy, specific fatty acids and related nutrients and pancreatic cancer. In our American-based Mayo Clinic case-control study 384 cases and 983 controls frequency matched on recruitment age, race, sex, and residence area (Minnesota, Wisconsin, or Iowa, United States) between 2004 and 2009. All subjects provided demographic information and completed 144-item food frequency questionnaire. Logistic regression calculated odds ratios (OR) and 95% confidence intervals (95% CI) were adjusted for age, sex, cigarette smoking, body mass index, and diabetes mellitus. Significant inverse association (trend p-value < 0.05) between pancreatic cancer and the groupings (highest vs. lowest consumption quintile OR [95% CI]): meat replacement (0.67 [0.43–1.02]), total protein (0.58 [0.39–0.86]), vitamin B12 (0.67 [0.44, 1.01]), zinc (0.48 [0.32, 0.71]), phosphorus (0.62 [0.41, 0.93]), vitamin E (0.51 [0.33, 0.78]), polyunsaturated fatty acids (0.64 [0.42, 0.98]), and Linoleic Acid (fatty acid 18:2) (0.62 [0.40–0.95]). Increased risk associations were observed for saturated fatty acids (1.48 [0.97–2.23]), Butyric Acid (fatty acid 4:0) (1.77 [1.19–2.64]), Caproic Acid (fatty acid 6:0) (2.15 [1.42–3.27]), Caprylic Acid (fatty acid 8:0) (1.87 [1.27–2.76]), and Capric Acid (fatty acid 10:0) (1.83 [1.23–2.74]). Our study suggests that eating a diet high in total protein and certain unsaturated fatty acids is associated with decreased risk of developing pancreatic cancer in a dose dependent manner whereas fats found in dairy increase risk. PMID:24590454

  8. Nucleic acid-binding specificity of human FUS protein

    PubMed Central

    Wang, Xueyin; Schwartz, Jacob C.; Cech, Thomas R.

    2015-01-01

    FUS, a nuclear RNA-binding protein, plays multiple roles in RNA processing. Five specific FUS-binding RNA sequence/structure motifs have been proposed, but their affinities for FUS have not been directly compared. Here we find that human FUS binds all these sequences with Kdapp values spanning a 10-fold range. Furthermore, some RNAs that do not contain any of these motifs bind FUS with similar affinity. FUS binds RNA in a length-dependent manner, consistent with a substantial non-specific component to binding. Finally, investigation of FUS binding to different nucleic acids shows that it binds single-stranded DNA with three-fold lower affinity than ssRNA of the same length and sequence, while binding to double-stranded nucleic acids is weaker. We conclude that FUS has quite general nucleic acid-binding activity, with the various proposed RNA motifs being neither necessary for FUS binding nor sufficient to explain its diverse binding partners. PMID:26150427

  9. Effects of linoleic and gamma-linolenic acids (efamol evening primrose oil) on fatty acid-binding proteins of rat liver.

    PubMed

    Dutta-Roy, A K; Demarco, A C; Raha, S K; Shay, J; Garvey, M; Horrobin, D F

    We have studied the effects of Efamol evening primrose oil (EPO) on fatty acid-binding proteins (L-FABP) of rat liver. EPO contains 72% cis-linoleic acid and 9% cis-gamma linolenic acid. EPO has been clinically used for treatment of a number of diseases in humans and animals. EPO is also known to lower cholesterol level in humans and animals. Feeding of an EPO supplemented diet to rats (n = 9) for 2 months decreases the oleate binding capacity of purified L-FABP of rat liver whereas the palmitate binding activity was increased by 38%. However, EPO feeding did not alter the L-FABP concentrations significantly as measured by using the fluorescence fatty acid probe, dansylamino undecanoic acid. Endogenous fatty acid analysis of L-FABPs revealed significant qualitative and quantitative changes in fatty acid pattern after EPO feeding. EPO feeding decreased the endogenous palmitate level by 53% and oleate level by 64% in L-FABPs and also EPO feeding decreased the total endogenous fatty acid content from 62 nanomole per mg of protein to 42 nanomole per mg of L-FABP (n = 3).

  10. 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.

  11. Quality Control of Gamma Irradiated Dwarf Mallow (Malva neglecta Wallr.) Based on Color, Organic Acids, Total Phenolics and Antioxidant Parameters.

    PubMed

    Pinela, José; Barros, Lillian; Antonio, Amilcar L; Carvalho, Ana Maria; Oliveira, M Beatriz P P; Ferreira, Isabel C F R

    2016-04-08

    This study addresses the effects of gamma irradiation (1, 5 and 8 kGy) on color, organic acids, total phenolics, total flavonoids, and antioxidant activity of dwarf mallow (Malva neglecta Wallr.). Organic acids were analyzed by ultra fast liquid chromatography (UFLC) coupled to a photodiode array (PDA) detector. Total phenolics and flavonoids were measured by the Folin-Ciocalteu and aluminium chloride colorimetric methods, respectively. The antioxidant activity was evaluated based on the DPPH(•) scavenging activity, reducing power, β-carotene bleaching inhibition and thiobarbituric acid reactive substances (TBARS) formation inhibition. Analyses were performed in the non-irradiated and irradiated plant material, as well as in decoctions obtained from the same samples. The total amounts of organic acids and phenolics recorded in decocted extracts were always higher than those found in the plant material or hydromethanolic extracts, respectively. The DPPH(•) scavenging activity and reducing power were also higher in decocted extracts. The assayed irradiation doses affected differently the organic acids profile. The levels of total phenolics and flavonoids were lower in the hydromethanolic extracts prepared from samples irradiated at 1 kGy (dose that induced color changes) and in decocted extracts prepared from those irradiated at 8 kGy. The last samples also showed a lower antioxidant activity. In turn, irradiation at 5 kGy favored the amounts of total phenolics and flavonoids. Overall, this study contributes to the understanding of the effects of irradiation in indicators of dwarf mallow quality, and highlighted the decoctions for its antioxidant properties.

  12. [Nutrition, acid-base metabolism, cation-anion difference and total base balance in humans].

    PubMed

    Mioni, R; Sala, P; Mioni, G

    2008-01-01

    The relationship between dietary intake and acid-base metabolism has been investigated in the past by means of the inorganic cation-anion difference (C(+)(nm)-A(-)(nm)) method based on dietary ash-acidity titration after the oxidative combustion of food samples. Besides the inorganic components of TA (A(-)(nm)-C(+)(nm)), which are under renal control, there are also metabolizable components (A(-)(nm)-C(+)(nm)) of TA, which are under the control of the intermediate metabolism. The whole body base balance, NBb(W), is obtained only by the application of C(+)(nm)-A(-)(nm) to food, feces and urine, while the metabolizable component (A(-)(nm)-C(+)(nm)) is disregarded. A novel method has been subsequently suggested to calculate the net balance of fixed acid, made up by the difference between the input of net endogenous acid production: NEAP = SO(4)(2-)+A(-)(m)-(C(+)(nm)-A(-)(nm)), and the output of net acid excretion: NAE = TA + NH(4)(+) - HCO(3)(-). This approach has been criticized because 1) it includes metabolizable acids, whose production cannot be measured independently; 2) the specific control of metabolizable acid and base has been incorrectly attributed to the kidney; 3) the inclusion of A-m in the balance input generates an acid overload; 4) the object of measurement in making up a balance has to be the same, a condition not fulfilled as NEAP is different from NAE. Lastly, by rearranging the net balance of the acid equation, the balance of nonmetabolizable acid equation is obtained. Therefore, any discrepancy between these two equations is due to the inaccuracy in the urine measurement of metabolizable cations and/or anions.

  13. Quantifying protein synthesis and degradation in Arabidopsis by dynamic 13CO2 labeling and analysis of enrichment in individual amino acids in their free pools and in protein.

    PubMed

    Ishihara, Hirofumi; Obata, Toshihiro; Sulpice, Ronan; Fernie, Alisdair R; Stitt, Mark

    2015-05-01

    Protein synthesis and degradation represent substantial costs during plant growth. To obtain a quantitative measure of the rate of protein synthesis and degradation, we supplied (13)CO2 to intact Arabidopsis (Arabidopsis thaliana) Columbia-0 plants and analyzed enrichment in free amino acids and in amino acid residues in protein during a 24-h pulse and 4-d chase. While many free amino acids labeled slowly and incompletely, alanine showed a rapid rise in enrichment in the pulse and a decrease in the chase. Enrichment in free alanine was used to correct enrichment in alanine residues in protein and calculate the rate of protein synthesis. The latter was compared with the relative growth rate to estimate the rate of protein degradation. The relative growth rate was estimated from sequential determination of fresh weight, sequential images of rosette area, and labeling of glucose in the cell wall. In an 8-h photoperiod, protein synthesis and cell wall synthesis were 3-fold faster in the day than at night, protein degradation was slow (3%-4% d(-1)), and flux to growth and degradation resulted in a protein half-life of 3.5 d. In the starchless phosphoglucomutase mutant at night, protein synthesis was further decreased and protein degradation increased, while cell wall synthesis was totally inhibited, quantitatively accounting for the inhibition of growth in this mutant. We also investigated the rates of protein synthesis and degradation during leaf development, during growth at high temperature, and compared synthesis rates of Rubisco large and small subunits of in the light and dark.

  14. Maternal folic acid supplementation to dams on marginal protein level alters brain fatty acid levels of their adult offspring.

    PubMed

    Rao, Shobha; Joshi, Sadhana; Kale, Anvita; Hegde, Mahabaleshwar; Mahadik, Sahebarao

    2006-05-01

    Studies on fetal programming of adult diseases have highlighted the importance of maternal nutrition during pregnancy. Folic acid and long-chain essential polyunsaturated fatty acids (LC-PUFAs) have independent effects on fetal growth. However, folic acid effects may also involve alteration of LC-PUFA metabolism. Because marginal deficiency of LC-PUFAs during critical periods of brain growth and development is associated with risks for adult diseases, it is highly relevant to investigate how maternal supplementation of such nutrients can alter brain fatty acid levels. We examined the impact of folic acid supplementation, conventionally used in maternal intervention, on brain essential fatty acid levels and plasma corticosterone concentrations in adult offspring at 11 months of age. Pregnant female rats from 4 groups (6 in each) were fed with casein diets either with 18 g protein/100 g diet (control diet) or treatment diets that were marginal in protein (MP), such as 12 g protein/100 g diet supplemented with 8 mg folic acid (FAS/MP), 12 g protein/100 g diet without folic acid (FAD/MP), or 12 g protein/100 g diet (MP) with 2 mg folic acid. Pups were weaned to a standard laboratory diet with 18 g protein/100 g diet. All male adult offspring in the FAS/MP group showed lower docosahexaenoic acid (P<.05) as compared with control adult offspring (6.04+/-2.28 vs 10.33+/-0.86 g/100 g fatty acids) and higher n-6/n-3 ratio (P<.05). Docosahexaenoic acid levels in FAS/MP adult offspring were also lower (P<.05) when compared with the MP group. Plasma corticosterone concentrations were higher (P<.05) in male adult offspring from the FAS/MP group compared with control as well as the MP adult offspring. Results suggest that maternal folic acid supplementation at MP intake decreased brain docosahexaenoic acid levels probably involving corticosterone increase.

  15. Sex Differences in Long Chain Fatty Acid Utilization and Fatty Acid Binding Protein Concentration in Rat Liver

    PubMed Central

    Ockner, Robert K.; Burnett, David A.; Lysenko, Nina; Manning, Joan A.

    1979-01-01

    Female sex and estrogen administration are associated with increased hepatic production of triglyceride-rich lipoproteins; the basis for this has not been fully elucidated. Inasmuch as hepatic lipoprotein production is also influenced by FFA availability and triglyceride biosynthesis, we investigated sex differences in FFA utilization in rat hepatocyte suspensions and in the components of the triglyceride biosynthetic pathway. Isolated adult rat hepatocyte suspensions were incubated with albumin-bound [14C]oleate for up to 15 min. At physiological and low oleate concentrations, cells from females incorporated significantly more 14C into glycerolipids, especially triglycerides, and into oxidation products than did male cells, per milligram cell protein. At 0.44 mM oleate, incorporation into triglycerides in female cells was approximately twice that in male cells. Comparable sex differences were observed in cells from fasted animals and when [14C]-glycerol incorporation was measured. At higher oleate concentrations, i.e., fatty acid:albumin mole ratios in excess of 2:1, these sex differences were no longer demonstrable, suggesting that maximal rates of fatty acid esterification and oxidation were similar in female and male cells. In female and male hepatic microsomes, specific activities of long chain acyl coenzyme A synthetase, phosphatidate phosphohydrolase, and diglyceride acyltransferase were similar, but glycerol-3-phosphate acyltransferase activity was slightly greater in females at certain substrate concentrations. Microsomal incorporation of [14C]oleate into total glycerolipids was not significantly greater in females. In further contrast to intact cells, microsomal incorporation of [14C]oleate into triglycerides, although significantly greater in female microsomes, accounted for only a small fraction of the fatty acid esterified. The binding affinity and stoichiometry of partially purified female hepatic fatty acid binding protein (FABP) were similar to

  16. Total salvianolic acid improves ischemia-reperfusion-induced microcirculatory disturbance in rat mesentery

    PubMed Central

    Wang, Ming-Xia; Liu, Yu-Ying; Hu, Bai-He; Wei, Xiao-Hong; Chang, Xin; Sun, Kai; Fan, Jing-Yu; Liao, Fu-Long; Wang, Chuan-She; Zheng, Jun; Han, Jing-Yan

    2010-01-01

    AIM: To investigate the effect of total salvianolic acid (TSA) on ischemia-reperfusion (I/R)-induced rat mesenteric microcirculatory dysfunctions. METHODS: Male Wistar rats were randomly distributed into 5 groups (n = 6 each): Sham group and I/R group (infused with saline), TSA group, TSA + I/R group and I/R + TSA group (infused with TSA, 5 mg/kg per hour). Mesenteric I/R were conducted by a ligation of the mesenteric artery and vein (10 min) and subsequent release of the occlusion. TSA was continuously infused either starting from 10 min before the ischemia or 10 min after reperfusion. Changes in mesenteric microcirculatory variables, including diameter of venule, velocity of red blood cells in venule, leukocyte adhesion, free radicals released from venule, albumin leakage and mast cell degranulation, were observed through an inverted intravital microscope. Meanwhile, the expression of adhesion molecules CD11b/CD18 on neutrophils was evaluated by flow cytometry. Ultrastructural evidence of mesenteric venules damage was assessed after microcirculation observation. RESULTS: I/R led to multiple responses in mesenteric post-capillary venules, including a significant increase in the adhesion of leukocytes, production of oxygen radicals in the venular wall, albumin efflux and enhanced mast cell degranulation in vivo. All the I/R-induced manifestations were significantly reduced by pre- or post-treatment with TSA, with the exception that the I/R-induced increase in mast cell degranulation was inhibited only by pre-treatment with TSA. Moreover, pre- or post-treatment with TSA significantly attenuated the expression of CD11b/CD18 on neutrophils, reducing the increase in the number of caveolae in the endothelial cells of mesentery post-capillary venules induced by I/R. CONCLUSION: The results demonstrated that TSA protects from and ameliorates the microcirculation disturbance induced by I/R, which was associated with TSA inhibiting the production of oxygen-free radicals in

  17. Assessing the Chemical Accuracy of Protein Structures via Peptide Acidity

    PubMed Central

    Anderson, Janet S.; Hernández, Griselda; LeMaster, David M.

    2012-01-01

    Although the protein native state is a Boltzmann conformational ensemble, practical applications often require a representative model from the most populated region of that distribution. The acidity of the backbone amides, as reflected in hydrogen exchange rates, is exquisitely sensitive to the surrounding charge and dielectric volume distribution. For each of four proteins, three independently determined X-ray structures of differing crystallographic resolution were used to predict exchange for the static solvent-exposed amide hydrogens. The average correlation coefficients range from 0.74 for ubiquitin to 0.93 for Pyrococcus furiosus rubredoxin, reflecting the larger range of experimental exchange rates exhibited by the latter protein. The exchange prediction errors modestly correlate with the crystallographic resolution. MODELLER 9v6-derived homology models at ~60% sequence identity (36% identity for chymotrypsin inhibitor CI2) yielded correlation coefficients that are ~0.1 smaller than for the cognate X-ray structures. The most recently deposited NOE-based ubiquitin structure and the original NMR structure of CI2 fail to provide statistically significant predictions of hydrogen exchange. However, the more recent RECOORD refinement study of CI2 yielded predictions comparable to the X-ray and homology model-based analyses. PMID:23182463

  18. Snake venom. The amino acid sequence of protein A from Dendroaspis polylepis polylepis (black mamba) venom.

    PubMed

    Joubert, F J; Strydom, D J

    1980-12-01

    Protein A from Dendroaspis polylepis polylepis venom comprises 81 amino acids, including ten half-cystine residues. The complete primary structures of protein A and its variant A' were elucidated. The sequences of proteins A and A', which differ in a single position, show no homology with various neurotoxins and non-neurotoxic proteins and represent a new type of elapid venom protein.

  19. Prediction of nucleic acid binding probability in proteins: a neighboring residue network based score.

    PubMed

    Miao, Zhichao; Westhof, Eric

    2015-06-23

    We describe a general binding score for predicting the nucleic acid binding probability in proteins. The score is directly derived from physicochemical and evolutionary features and integrates a residue neighboring network approach. Our process achieves stable and high accuracies on both DNA- and RNA-binding proteins and illustrates how the main driving forces for nucleic acid binding are common. Because of the effective integration of the synergetic effects of the network of neighboring residues and the fact that the prediction yields a hierarchical scoring on the protein surface, energy funnels for nucleic acid binding appear on protein surfaces, pointing to the dynamic process occurring in the binding of nucleic acids to proteins.

  20. Thermophysical properties of starch and whey protein composite prepared in presence of organic acid and esters

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previously, we prepared starch and protein composite by reactive mixing in presence of various organic acids and found that use of these acid esters resulted in composites with good mechanical properties. In this study, concentration (% w/w) of acid citrates in the starch-protein composites were var...

  1. Defining meal requirements for protein to optimize metabolic roles of amino acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Dietary protein provides essential amino acids (EAAs) for the synthesis of new proteins plus an array of other metabolic functions; many of these functions are sensitive to postprandial plasma and intracellular amino acid concentrations. Recent research has focused on amino acids as metabolic signal...

  2. Fatty Acid-Binding Protein 5 Facilitates the Blood-Brain Barrier Transport of Docosahexaenoic Acid.

    PubMed

    Pan, Yijun; Scanlon, Martin J; Owada, Yuji; Yamamoto, Yui; Porter, Christopher J H; Nicolazzo, Joseph A

    2015-12-07

    The brain has a limited ability to synthesize the essential polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) from its omega-3 fatty acid precursors. Therefore, to maintain brain concentrations of this PUFA at physiological levels, plasma-derived DHA must be transported across the blood-brain barrier (BBB). While DHA is able to partition into the luminal membrane of brain endothelial cells, its low aqueous solubility likely limits its cytosolic transfer to the abluminal membrane, necessitating the requirement of an intracellular carrier protein to facilitate trafficking of this PUFA across the BBB. As the intracellular carrier protein fatty acid-binding protein 5 (FABP5) is expressed at the human BBB, the current study assessed the putative role of FABP5 in the brain endothelial cell uptake and BBB transport of DHA in vitro and in vivo, respectively. hFAPB5 was recombinantly expressed and purified from Escherichia coli C41(DE3) cells and the binding affinity of DHA to hFABP5 assessed using isothermal titration calorimetry. The impact of FABP5 siRNA on uptake of (14)C-DHA into immortalized human brain microvascular endothelial (hCMEC/D3) cells was assessed. An in situ transcardiac perfusion method was optimized in C57BL/6 mice and subsequently used to compare the BBB influx rate (Kin) of (14)C-DHA between FABP5-deficient (FABP5(-/-)) and wild-type (FABP5(+/+)) C57BL/6 mice. DHA bound to hFABP5 with an equilibrium dissociation constant of 155 ± 8 nM (mean ± SEM). FABP5 siRNA transfection decreased hCMEC/D3 mRNA and protein expression of FABP5 by 53.2 ± 5.5% and 44.8 ± 13.7%, respectively, which was associated with a 14.1 ± 2.7% reduction in (14)C-DHA cellular uptake. By using optimized conditions for the in situ transcardiac perfusion (a 1 min preperfusion (10 mL/min) followed by perfusion of (14)C-DHA (1 min)), the Kin of (14)C-DHA was 0.04 ± 0.01 mL/g/s. Relative to FABP5(+/+) mice, the Kin of (14)C-DHA decreased 36.7 ± 12.4% in FABP5(-/-) mice

  3. Exogenous amino acids stimulate net muscle protein synthesis in the elderly.

    PubMed Central

    Volpi, E; Ferrando, A A; Yeckel, C W; Tipton, K D; Wolfe, R R

    1998-01-01

    We have investigated the response of amino acid transport and protein synthesis in healthy elderly individuals (age 71+/-2 yr) to the stimulatory effect of increased amino acid availability. Muscle protein synthesis and breakdown, and amino acid transport were measured in the postabsorptive state and during the intravenous infusion of an amino acid mixture. Muscle-free amino acid kinetics were calculated by means of a three compartment model using data obtained by femoral arterio-venous catheterization and muscle biopsies from the vastus lateralis during the infusion of stable isotope tracers of amino acids. In addition, muscle protein fractional synthetic rate (FSR) was measured. Peripheral amino acid infusion significantly increased amino acid delivery to the leg, amino acid transport, and muscle protein synthesis when measured either with the three compartment model (P < 0.05) or with the traditional precursor-product approach (FSR increased from 0. 0474+/-0.0054 to 0.0940+/-0.0143%/h, P < 0.05). Because protein breakdown did not change during amino acid infusion, a positive net balance of amino acids across the muscle was achieved. We conclude that, although muscle mass is decreased in the elderly, muscle protein anabolism can nonetheless be stimulated by increased amino acid availability. We thus hypothesize that muscle mass could be better maintained with an increased intake of protein or amino acids. PMID:9576765

  4. Total and individual carotenoids and phenolic acids content in fresh, refrigerated and processed spinach (Spinacia oleracea L.).

    PubMed

    Bunea, Andrea; Andjelkovic, Mirjana; Socaciu, Carmen; Bobis, Otilia; Neacsu, Madalina; Verhé, Roland; Camp, John Van

    2008-05-15

    The carotenoid and phenolic acid contents in fresh, stored and processed (blanched, frozen and boiled) spinach were comparatively determined by spectrophotometric and HPLC analyses. The major carotenoids identified after HPLC analysis in saponified samples were lutein (37-53μg/kg), β-carotene (18-31μg/kg), violaxanthin (9-23μg/kg) and neoxanthin (10-22μg/kg). These carotenoids were all affected by storage and/or heating. The content of carotenoids was best preserved after storage for one day at 4°C. The total phenolic content in the fresh spinach was 2088mg GAE/kg FW. After LC-MS analysis three phenolic acids were identified and quantified. These being ortho-coumaric acid (28-60mg/kg FW), ferulic acid (10-35mg/kg) and para-coumaric acid (1-30mg/kg) depending on the sample type. After storage of spinach at different temperatures (4°C or -18°C) the amount of total phenolic compounds decreased by around 20%, while the amount of individual phenolic acids increased by four times on average.

  5. Indirect and direct effects of salinity on the quantity and quality of total amino acids in Ulva ohnoi (Chlorophyta).

    PubMed

    Angell, Alex R; Mata, Leonardo; de Nys, Rocky; Paul, Nicholas A

    2015-06-01

    Salinity can affect the quantity and quality of total amino acids (TAAs) in seaweeds indirectly by altering growth rates and thereby diluting or concentrating the amino acid content of the biomass, or directly by altering the synthesis of specific amino acids and osmolytes. This study attempted to partition the indirect and direct effects of salinity on the quantity and quality of TAAs in the green seaweed Ulva ohnoi by culturing it under a range of salinities without nutrient limitation. Both the quantity and quality of TAAs varied across the salinity treatments. Quantity was most strongly related to the growth rate of the seaweed and was highest in the slowest growing seaweed. In contrast, the quality of TAAs (individual amino acids as a proportion of total content) was most strongly related to salinity for all amino acids, although this varied substantially among individual amino acids. Increases in salinity were positively correlated with the proportion of proline (46% increase), tyrosine (36% increase), and histidine (26% increase), whereas there was a negative correlation with alanine (29% decrease). The proportion of methionine, with strong links to the synthesis of the osmolyte dimethylsulfoniopropionate, did not correlate linearly with salinity and instead was moderately higher at the optimal salinities for growth. These results show that salinity simultaneously affects the quantity and quality of TAAs in seaweed through both indirect and direct mechanisms, with growth rates playing the overarching role in determining the quantity of TAAs.

  6. Administration of balanced or BCAA-enriched amino acid solution in septic rats. Effects on protein synthesis in the liver.

    PubMed Central

    Pedersen, P; Li, S J; Hasselgren, P O; LaFrance, R; Fischer, J E

    1988-01-01

    Total hepatic protein synthesis was measured in vivo with a flooding-dose technique, and the production of total secreted proteins, albumin, complement component C3, and seromucoid fraction was measured in perfused livers of septic rats that received one of three different solutions infused intravenously; Group 1 received 16.4% dextrose; Group 2 received Aminosyn (25% BCAA) in 10.6% dextrose, and Group 3 received Freamine HBC (45% BCAA) in 10.6% dextrose. All solutions were isocaloric, and the amino acid solutions were isonitrogenous. The solutions were administered for 18 or 48 hours after the induction of sepsis. There were no significant differences in mortality rates in the three treatment groups. The negative nitrogen balance seen in the dextrose-infused animals was reversed to the same degree by the two different amino acid solutions. There were no significant differences in hepatic protein synthesis rates in vivo between the three groups of rats. Synthesis rates of secreted proteins in perfused liver were similar in the different treatment groups in the 18-hour experiments, whereas in the 48-hour experiments, synthesis rates of total secreted proteins, C3, and the serumucoid fraction were higher in Group 1 than in Groups 2 and 3. The results suggest that administration of an amino acid solution improves nitrogen balance in sepsis, but that this effect is not caused by stimulated hepatic protein synthesis. The nitrogen-sparing effect during sepsis of a branched chain amino acid (BCAA)-enriched solution does not seem to be superior to that of a balanced amino acid solution. PMID:3143320

  7. Proteins, dietary acid load, and calcium and risk of postmenopausal fractures in the E3N French women prospective study

    PubMed Central

    Dargent-Molina, Patricia; Sabia, Sèverine; Touvier, Mathilde; Kesse, Emmanuelle; Bréart, Gérard; Clavel-Chapelon, Françoise; Boutron-Ruault, Marie-Christine

    2008-01-01

    Introduction Excess dietary proteins and “acid ash” diets have been suspected to increase the risk of osteoporosis, but experimental and epidemiological evidence is mixed. We aimed to determine whether the association between protein intake and the overall acid-base equilibrium of the diet (as renal net acid excretion (RNAE) estimate) and fracture risk vary according to calcium intake. Materials and Methods During an average of 8.37 (± 1.73) years of follow up, 2408 women reported a fracture (excluding high-impact trauma) among 36,217 postmenopausal women from the E3N prospective study. We used Cox regression models to study the interaction between calcium and, respectively, proteins and RNAE, from the 1993 dietary questionnaire, for fracture risk determination, adjusting for potential confounders. Results There was no overall association between fracture risk and total protein or RNAE. However, in the lowest quartile of calcium (< 400 mg/1000 kcal), high protein intake was associated with a significant increased fracture risk (RR = 1.51 for highest versus lowest quartile; 95% CI 1.17–1.94). An increasing fracture risk with increasing animal protein intake was also observed (p trend <0.0001). A similar pattern of interaction for fracture risk was observed between RNAE and calcium. Conclusions In this Western population of post-menopausal women with normal to high protein intake and fairly high calcium intake, there was no overall association between total protein or RNAE and fracture risk. However, there was some evidence that high protein – high acid ash diets were associated with an increased risk of fracture when calcium intake was low (< 400 mg/1000 kcal). PMID:18665794

  8. 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.

  9. Site-specific incorporation of redox active amino acids into proteins

    DOEpatents

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

    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.

  10. 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.

  11. Site-specific incorporation of redox active amino acids into proteins

    DOEpatents

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

    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.

  12. Dilute oxalic acid pretreatment for high total sugar recovery in pretreatment and subsequent enzymatic hydrolysis.

    PubMed

    Qing, Qing; Huang, Meizi; He, Yucai; Wang, Liqun; Zhang, Yue

    2015-12-01

    Oxalic acid was evaluated as an alternative reagent to mineral inorganic acid in pretreatment of corncob to achieve high xylose yield in addition to highly digestible solid residue. A quadratic polynomial model of xylose formation was developed for optimization of pretreatment process by the response surface methodology based on the impact factors of pretreatment temperature, reaction time, acid concentration, and solid-to-liquid ratio. The highest xylose yield was 94.3 % that was obtained under the pretreatment condition of 140 °C for 40 min with 0.5 wt% oxalic acid at a solid loading of 7.5 %. Under these conditions, the xylose yield results of verification experiments were very close to the model prediction, which indicated that the model was applicable. The solid residue generated under this condition also demonstrated a satisfactory enzymatic digestibility and fermentability.

  13. 7 CFR 51.1178 - Maximum anhydrous citric acid permissible for corresponding total soluble solids.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ...) United States Standards for Grades of Florida Oranges and Tangelos Standards for Internal Quality of Common Sweet Oranges (citrus Sinensis (l) Osbeck) § 51.1178 Maximum anhydrous citric acid permissible...

  14. Total phenolics, phenolic acids, isoflavones, and anthocyanins and antioxidant properties of yellow and black soybeans as affected by thermal processing.

    PubMed

    Xu, Baojun; Chang, Sam K C

    2008-08-27

    The effects of boiling and steaming processes on the phenolic components and antioxidant activities of whole yellow (with yellow seed coat and yellow cotyledon) and black (with black seed coat and green cotyledon) soybeans were investigated. As compared to the raw soybeans, all processing methods caused significant (p < 0.05) decreases in total phenolic content (TPC), total flavonoid content (TFC), condensed tannin content (CTC), monomeric anthocyanin content (MAC), DPPH free radical scavenging activity (DPPH), ferric reducing antioxidant power (FRAP), and oxygen radical absorbing capacity (ORAC) in black soybeans. Pressure steaming caused significant (p < 0.05) increases in TPC, CTC, DPPH, FRAP, and ORAC in yellow soybeans. The steaming resulted in a greater retention of TPC, DPPH, FRAP, and ORAC values in both yellow and black soybeans as compared to the boiling treatments. To further investigate the effect of processing on phenolic compounds and elucidate the contribution of these compounds to changes of antioxidant activities, phenolic acids, isoflavones, and anthocyanins were quantitatively determined by HPLC. The pressure steaming treatments caused significant (p < 0.05) increases in gallic acid and 2,3,4-trihydroxybenzoic acid, whereas all treatments caused significant (p < 0.05) decreases in two predominant phenolic acids (chlorogenic acid and trans-cinnamic acid), and total phenolic acids for both yellow and black soybeans. All thermal processing caused significant (p < 0.05) increases in aglucones and beta-glucosides of isoflavones, but caused significant (p < 0.05) decreases in malonylglucosides of isoflavones for both yellow and black soybeans. All thermal processing caused significant (p < 0.05) decreases of cyanidin-3-glucoside and peonidin-3-glucoside in black soybeans. Significant correlations existed between selected phenolic compositions, isoflavone and anthocyanin contents, and antioxidant properties of cooked soybeans.

  15. Decarboxylation of malonyl-(acyl carrier protein) by 3-oxoacyl-(acyl carrier protein) synthases in plant fatty acid biosynthesis.

    PubMed Central

    Winter, E; Brummel, M; Schuch, R; Spener, F

    1997-01-01

    In order to identify regulatory steps in fatty acid biosynthesis, the influence of intermediate 3-oxoacyl-(acyl carrier proteins) (3-oxoacyl-ACPs) and end-product acyl-ACPs of the fatty acid synthase reaction on the condensation reaction was investigated in vitro, using total fatty acid synthase preparations and purified 3-oxoacyl-ACP synthases (KASs; EC 2.3.1.41) from Cuphea lanceolata seeds. KAS I and II in the fatty acid synthase preparations were assayed for the elongation of octanoyl- and hexadecanoyl-ACP respectively, and the accumulation of the corresponding condensation product 3-oxoacyl-ACP was studied by modulating the content of the reducing equivalentS NADH and NADPH. Complete omission of reducing equivalents resulted with either KAS in the abnormal synthesis of acetyl-ACP from malonyl-ACP by a decarboxylation reaction. Supplementation with NADPH or NADH, separately or in combination with recombinant 3-oxoacyl-ACP reductase (EC 1.1.1.100), led to a decrease in the amount of acetyl-ACP and a simultaneous increase in elongation products. This demonstrates that the accumulation of 3-oxoacyl-ACP inhibits the condensation reaction on the one hand, and induces the decarboxylation of malonyl-ACP on the other. By carrying out similar experiments with purified enzymes, this decarboxylation was attributed to the action of KAS. Our data point to a regulatory mechanism for the degradation of malonyl-ACP in plants which is activated by the accumulation of the fatty acid synthase intermediate 3-oxoacyl-ACP. PMID:9020860

  16. Interfacial inhibitors of protein-nucleic acid interactions.

    PubMed

    Pommier, Yves; Marchand, Christophe

    2005-07-01

    This essay develops the paradigm of "Interfacial Inhibitors" (Pommier and Cherfils, TiPS, 2005, 28: 136) for inhibitory drugs beside orthosteric (competitive or non-competitive) and allosteric inhibitors. Interfacial inhibitors bind with high selectivity to a binding site involving two or more macromolecules within macromolecular complexes undergoing conformational changes. Interfacial binding traps (generally reversibly) a transition state of the complex, resulting in kinetic inactivation. The exemplary case of interfacial inhibitor of protein-DNA interface is camptothecin and its clinical derivatives. We will also provide examples generalizing the interfacial inhibitor concept to inhibitors of topoisomerase II (anthracyclines, ellipticines, epipodophyllotoxins), gyrase (quinolones, ciprofloxacin, norfloxacin), RNA polymerases (alpha-amanitin and actinomycin D), and ribosomes (antibiotics such as streptomycin, hygromycin B, tetracycline, kirromycin, fusidic acid, thiostrepton, and possibly cycloheximide). We discuss the implications of the interfacial inhibitor concept for drug discovery.

  17. 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.

  18. Clustering amino acid contents of protein domains: biochemical functions of proteins and implications for origin of biological macromolecules.

    PubMed

    Torshin, I Y

    2001-04-01

    Structural classes of protein domains correlate with their amino acid compositions. Several successful algorithms (that use only amino acid composition) have been elaborated for the prediction of structural class or potential biochemical significance. This work deals with dynamic classification (clustering) of the domains on the basis of their amino acid composition. Amino acid contents of domains from a non-redundant PDB set were clustered in 20-dimensional space of amino acid contents. Despite the variations of an empirical parameter and non-redundancy of the set, only one large cluster (tens-hundreds of proteins) surrounded by hundreds of small clusters (1-5 proteins), was identified. The core of the largest cluster contains at least 64% DNA (nucleotide)-interacting protein domains from various sources. About 90% of the proteins of the core are intracellular proteins. 83% of the DNA/nucleotide interacting domains in the core belong to the mixed alpha-beta folds (a+b, a/b), 14% are all-alpha (mostly helices) and all-beta (mostly beta-strands) proteins. At the same time, when core domains that belong to one organism (E.coli) are considered, over 80% of them prove to be DNA/nucleotide interacting proteins. The core is compact: amino acid contents of domains from the core lie in relatively narrow and specific ranges. The core also contains several Fe-S cluster-binding domains, amino acid contents of the core overlap with ferredoxin and CO-dehydrogenase clusters, the oldest known proteins. As Fe-S clusters are thought to be the first biocatalysts, the results are discussed in relation to contemporary experiments and models dealing with the origin of biological macromolecules. The origin of most primordial proteins is considered here to be a result of co-adsorption of nucleotides and amino acids on specific clays, followed by en-block polymerization of the adsorbed mixtures of amino acids.

  19. Thermodynamics of the interaction of globular proteins with powdered stearic acid in acid pH.

    PubMed

    Mitra, Atanu; Chattoraj, D K; Chakraborty, P

    2006-06-01

    Adsorption isotherms of different globular proteins and gelatin on strearic acid particles have been studied as a function of biopolymer concentration, ionic strength of the medium, and temperature. The effect of neutral salts including CaCl2, Na3PO4, and urea on the adsorption isotherms has been also investigated. It is observed that the extent of adsorption (Gamma2(1)) increases in two steps with the increase of biopolymer concentration (C2) in the bulk. Gamma2(1) increases with an increase of C2 until a steady maximum value Gamma2(m) is reached at a critical concentration C2(m). After initial saturation, Gamma2(1) again increases from Gamma2(m) without reaching any limiting value due to the surface aggregation of the protein. The values of the standard free energy change for adsorption have been calculated on the basis of the Gibbs equation. The standard entropy and enthalpy changes are also calculated.

  20. Effects of one-seed juniper on intake, rumen fermentation, and plasma amino acids in sheep and goats fed supplemental protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We tested the effect of feeding one-seed juniper on total intake, VFA profile, and plasma amino acids (AA) of 12 does and 12 ewes fed sudangrass and a basal diet with no protein supplement (Control; 5% CP) or rumen degradable (SBM; RDP 15% CP) or undegradable (FM; RUP 15% CP) protein supplement. Aft...

  1. A specific protein-enriched enteral formula decreases cortisolemia and improves plasma albumin and amino acid concentrations in elderly patients

    PubMed Central

    2010-01-01

    Background Old age is associated with an involuntary and progressive but physiological loss of muscle mass. The aim of this study was to evaluate the effects of exclusive consumption for 6 months of a protein-enriched enteral diet with a relatively high content of branched-chain amino acids on albuminemia, cortisolemia, plasma amino acids, insulin resistance, and inflammation biomarkers in elderly patients. Methods Thirty-two patients from the Clinical Nutrition Outpatient Unit at our hospital exclusively consumed a protein-enriched enteral diet for 6 months. Data were collected at baseline and at 3 and 6 months on anthropometric and biochemical parameters and on plasma concentrations of amino acids, cortisol, adrenocorticotropic hormone, urea, creatinine, insulin resistance, and inflammation biomarkers. Results The percentage of patients with albumin concentration below normal cut-off values decreased from 18% to 0% by the end of the study. At 6 months, concentrations of total plasma (p = 0.008) and essential amino acids (p = 0.011), especially branched-chain amino acids (p = 0.031), were higher versus baseline values, whereas 3-methylhistidine (p = 0.001), cortisol (p = 0.001) and adrenocorticotropic hormone (p = 0.004) levels were lower. Conclusions Regular intake of specific protein-enriched enteral formula increases plasma essential amino acids, especially branched-chain amino acids, and decreases cortisol and 3-methylhistidine, while plasma urea and creatinine remain unchanged. PMID:20626909

  2. Carvacrol attenuates serum levels of total protein, phospholipase A2 and histamine in asthmatic guinea pig

    PubMed Central

    Boskabady, Mohammad Hossein; Jalali, Sedigheh; Yahyazadeh, Negin; Boskabady, Mostafa

    2016-01-01

    Objective: Pharmacological effects of carvacrol such as its anti-inflammatory activities have been shows. In this study the effects of carvacrol on serum levels of total protein (TP), phospholipase A2 (PLA2) and histamine in sensitized guinea pigs was evaluated. Materials and Methods: Sensitized guinea pigs were given drinking water alone (group S), drinking water containing three concentrations of carvacrol (40, 80 and 160 µg/ml) or dexamethasone. Serum levels of TP, PLA2 and histamine were examined I all sensitized groups as well as a non-sensitized control group (n=6 for each group). Results: In sensitized animals, serum levels of TP, PLA2 and histamine were significantly increased compared to control animals (p<0.05 to p<0.001). Significant reduction in TP, PLA2 and histamine levels were observed in treated groups with the two higher concentrations of carvacrol but dexamethasone treatment only decreased serum level of PLA2 (p<0.05 to p<0.001). Although the effect of the lowest concentration of the extract was less than that of dexamethasone (p<0.05 for TP and p<0.001 for PLA2), the effects of the two higher concentrations on PLA2 were similar to dexamethasone and on TP (p<0.01) and histamine (p<0.001) were higher than those of dexamethasone. Conclusion: These results showed that carvacrol reduced serum levels of TP, PLA2 and histamine in sensitized guinea pigs which may indicate an anti-inflammatory effect of this agent in inflammatory disorders such as asthma. PMID:28078244

  3. Total Leucocyte Count, C-reactive Protein and Neutrophil Count: Diagnostic Aid in Acute Appendicitis

    PubMed Central

    Shafi, Sheikh Muzamil; Afsheen, Misbha; Reshi, Farooq A.

    2009-01-01

    Background/Aim: Acute appendicitis is one of the most common acute intraabdominal affections seen in surgical departments, which can be treated easily if an accurate diagnosis is made in time. Otherwise, delay in diagnosis and treatment can lead to diffuse peritonitis. Materials and Methods: A study was conducted on 110 patients who were operated for acute appendicitis to determine the role and predictive value of the total leucocyte count (TLC), C-reactive protein (CRP) and percentage of neutrophil count in the diagnosis of acute appendicitis. Preoperative TLC, CRP and percentage of neutrophil count were determined and were compared with the results of the histopathology of the removed appendix. Results: Of all the patients studied, 92 had histopathologically positive appendicitis. The TLC was found to be significantly high in 90 patients who proved to have acute appendicitis, whereas CRP was high in only 88 patients and neutrophil percentage was raised in 91; four had a normal CRP level. Thus, TLC had a sensitivity, specificity and positive predictive value of 97.82%, 55.55% and 91.8%, respectively. CRP had a sensitivity, specificity and positive predictive value of 95.6%, 77.77% and 95.6% respectively. Percentage of neutrophil count had a sensitivity, specificity and positive predictive value of 98.9%, 38.88% and 89.21%, respectively. When used in combination, there was a marked improvement in the specificity and the positive predictive value to 88.04% and 98.7%, respectively. Conclusion: The inflammatory markers, i.e., TLC, CRP and neutrophil count can be helpful in the diagnosis when measured together as this increases their specificity and positive predictive value. PMID:19568576

  4. Total syntheses of (-)-alpha-kainic acid and (+)-alpha-allokainic acid via stereoselective C-H insertion and efficient 3,4-stereocontrol.

    PubMed

    Jung, Young Chun; Yoon, Cheol Hwan; Turos, Edward; Yoo, Kyung Soo; Jung, Kyung Woon

    2007-12-21

    Reported herein is a novel approach to the total syntheses of (-)-alpha-kainic acid and (+)-alpha-allokainic acid, where the stereochemistries on C(2), C(3), and C(4) of the pyrrolidine core were introduced efficiently and selectively. A regio- and stereoselective C-H insertion reaction was utilized to prepare the gamma-lactam as an intermediate. A Michael-type cyclization of phenylsulfone with a conjugated acetylenic ketone was developed to prepare the tricyclic ketone as a key intermediate for (-)-alpha-kainic acid. Subsequently, a stereoselective dephenylsulfonylation was carried out successfully to secure the cis relationship at C(3) and C(4) centers. An unprecedented acetylation on the phenylsulfone, followed by a stereoselective dephenylsulfonylation, secured the trans relationship at C(3) and C(4) centers in (+)-alpha-allokainic acid.

  5. Salicylic acid enhances Staphylococcus aureus extracellular adhesin protein expression.

    PubMed

    Alvarez, Lucía P; Barbagelata, María S; Cheung, Ambrose L; Sordelli, Daniel O; Buzzola, Fernanda R

    2011-11-01

    One of the virulence factors required by Staphylococcus aureus at the early stages of infection is Eap, a secreted adhesin that binds many host proteins and is upregulated by the two-component regulatory system saeRS. The S. aureus Newman strain harbors a mutation in saeS that is thought to be responsible for the high level of Eap expression in this strain. This study was designed to ascertain whether salicylic acid (SAL) affects the expression of Eap and the internalization of S. aureus into epithelial cells. The strain Newman treated with SAL exhibited increased levels of eap transcription and protein expression. Furthermore, SAL treatment increased the eap promoter activity. SAL treatment enhanced Eap expression in the Newman and in other S. aureus strains that do not carry the mutation in saeS. Internalization of S. aureus eap and sae mutants into the MAC-T epithelial cells was significantly decreased compared with the wild-type counterparts. In conclusion, we demonstrated that a low concentration of SAL increased S. aureus Eap expression possibly due to enhancement of sae. SAL may create the conditions for S. aureus persistence in the host, not only by decreasing the capsular polysaccharide expression as shown before, but also by enhancing Eap expression.

  6. Crystal growth of proteins, nucleic acids, and viruses in gels.

    PubMed

    Lorber, Bernard; Sauter, Claude; Théobald-Dietrich, Anne; Moreno, Abel; Schellenberger, Pascale; Robert, Marie-Claire; Capelle, Bernard; Sanglier, Sarah; Potier, Noëlle; Giegé, Richard

    2009-11-01

    Medium-sized single crystals with perfect habits and no defect producing intense and well-resolved diffraction patterns are the dream of every protein crystallographer. Crystals of biological macromolecules possessing these characteristics can be prepared within a medium in which mass transport is restricted to diffusion. Chemical gels (like polysiloxane) and physical gels (such as agarose) provide such an environment and are therefore suitable for the crystallisation of biological macromolecules. Instructions for the preparation of each type of gel are given to urge crystal growers to apply diffusive media for enhancing crystallographic quality of their crystals. Examples of quality enhancement achieved with silica and agarose gels are given. Results obtained with other substances forming gel-like media (such as lipidic phases and cellulose derivatives) are presented. Finally, the use of gels in combination with capillary tubes for counter-diffusion experiments is discussed. Methods and techniques implemented with proteins can also be applied to nucleic acids and nucleoprotein assemblies such as viruses.

  7. Small acid soluble proteins for rapid spore identification.

    SciTech Connect

    Branda, Steven S.; Lane, Todd W.; VanderNoot, Victoria A.; Jokerst, Amanda S.

    2006-12-01

    This one year LDRD addressed the problem of rapid characterization of bacterial spores such as those from the genus Bacillus, the group that contains pathogenic spores such as B. anthracis. In this effort we addressed the feasibility of using a proteomics based approach to spore characterization using a subset of conserved spore proteins known as the small acid soluble proteins or SASPs. We proposed developing techniques that built on our previous expertise in microseparations to rapidly characterize or identify spores. An alternative SASP extraction method was developed that was amenable to both the subsequent fluorescent labeling required for laser-induced fluorescence detection and the low ionic strength requirements for isoelectric focusing. For the microseparations, both capillary isoelectric focusing and chip gel electrophoresis were employed. A variety of methods were evaluated to improve the molecular weight resolution for the SASPs, which are in a molecular weight range that is not well resolved by the current methods. Isoelectric focusing was optimized and employed to resolve the SASPs using UV absorbance detection. Proteomic signatures of native wild type Bacillus spores and clones genetically engineered to produce altered SASP patterns were assessed by slab gel electrophoresis, capillary isoelectric focusing with absorbance detection as well as microchip based gel electrophoresis employing sensitive laser-induced fluorescence detection.

  8. Stereocontrolled Total Syntheses of Isodomoic Acids G and H via a Unified Strategy

    PubMed Central

    Denmark, Scott E.; Liu, Jack H.-C.; Muhuhi, Joseck M.

    2010-01-01

    Marine neuroexcitatory compounds isodomoic acids G and H were efficiently synthesized from a common intermediate using a silicon-based cross-coupling reaction. Dividing each target compound into the core fragment and the side-chain fragment enabled the synthesis to be convergent. The trans-2,3-disubstituted pyrrolidine core fragment was accessed through a diastereoselective rhodium-catalyzed carbonylative silylcarbocyclization reaction of a vinylglycine-derived 1,6-enyne. A stereochemically divergent desilylative iodination reaction was developed, converting of the cyclization product to both E- and Z-alkenyl iodides, which would eventually lead to isodomoic acid G and isodomoic acid H, respectively. The late stage alkenyl-alkenyl silicon-based cross-coupling reaction uniting the core alkenyl iodides and the side-chain alkenylsilanol was achieved under mild conditions. Finally, two mild deprotections afforded the target molecules. PMID:21121685

  9. Spaceflight effects on adult rat muscle protein, nucleic acids, and amino acids

    NASA Technical Reports Server (NTRS)

    Steffen, J. M.; Musacchia, X. J.

    1986-01-01

    Exposure to conditions of weightlessness has been associated with decrements in muscle mass and strength. The present studies were undertaken to determine muscle responses at the cellular level. Male Sprague-Dawley rats (360-410 g) were exposed to 7 days of weightlessness during the Spacelab-3 shuttle flight (May 1985). Animals were killed 12 h postflight, and soleus (S), gastrocnemius (G), and extensor digitorum longus (EDL) muscles were excised. Muscle protein, RNA, and DNA were extracted and quantified. Differential muscle atrophy was accompanied by a significant (P less than 0.05) reduction in total protein only in S muscles. There were no significant changes in protein concentration (mg/g) in the muscles examined. In S muscles from flight animals, sarcoplasmic protein accounted for a significantly greater proportion of total protein that in ground controls (37.5 vs. 32.5%). Tissue concentrations (nmol/g) of asparagine-aspartate, glutamine-glutamate, glycine, histidine, and lysine were significantly reduced (from 17 to 63%) in S muscles from flight animals, but only glutamine-glutamate levels were decreased in the G and EDL. Muscle DNA content (microgram) was unchanged in the tissues examined, but S muscle DNA concentration (micrograms/mg) increased 27%. RNA content (micrograms) was significantly (P less than 0.025) reduced in S (-28%) and G(-22%) muscles following spaceflight. These results identify specific alterations in rat skeletal muscle during short term (7-day) exposure to weightlessness and compare favorably with observations previously obtained from ground-based suspension simulations.

  10. Chlorophylls, proteins and fatty acids amounts of arthrospira platensis growing under saline conditions.

    PubMed

    Ayachi, Samah; El Abed, Amor; Dhifi, Wissal; Marzouk, Brahim

    2007-07-15

    Spirulina platensis (Arthrospira platensis) is a Tunisian strain isolated for the first time, in Tunisia, in Oued Essed (Sidi Bou Ali, in Sousse region). Evolution of biomass, proteins, chlorophylls and fatty acids (FA) has been followed during Spirulina growth. Experiments were carried out by varying sodium chloride concentrations in the culture medium in a range from 1 g L(-1) (natural environment) to 60 g L(-1). Results analysis showed an increase in chlorophyll amounts at 15 g L(-1) NaCl in 10 days old cultures but a decrease at high NaCl concentrations. Optimal proteins amounts was observed at 15 g L(-1) NaCl in young cultures (5 and 10 days). FA composition was modified by NaCl and depended on culture age. Cultures exposed to high salinity concentrations showed not only a decrease in growth rate but also a loss in total fatty acids TFA quantities. Samples cultured over 15 days at 30 g L(-1) NaCl rendered optimal quantities of lipids and gamma-linolenic acid.

  11. Fluctuations in phenolic content, ascorbic acid and total carotenoids and antioxidant activity of fruit beverages during storage.

    PubMed

    Castro-López, C; Sánchez-Alejo, E J; Saucedo-Pompa, S; Rojas, R; Aranda-Ruiz, J; Martínez-Avila, G C G

    2016-09-01

    Stability of the total phenolic content, ascorbic acid, total carotenoids and antioxidant activity in eight fruit beverages was analyzed. The influence of storage temperature (4, 8 and 11 °C) during the product shelf-life (20 days) was evaluated. Pomegranate Juice presented the highest values for antioxidant activity by DPPH• assay (552.93 ± 6.00 GAE μg mL(-1)), total carotenoids (3.18 ± 0.11 βCE μg mL(-1)), and total phenolic content (3967.07 ± 2.47 GAE μg mL(-1)); while Splash Blend recorded the highest levels of ascorbic acid (607.39 ± 2.13 AAE μg mL(-1)). The antioxidant capacity was stable at 4 and 8 °C for the first 8 days of storage; while carotenoids and ascorbic acid were slightly degraded through the storage time, possibly due to oxidation and/or reactions with other compounds. The results suggest that the observed variation during testing could be related to storage conditions of the final product.

  12. Effect of total solids content on methane and volatile fatty acid production in anaerobic digestion of food waste.

    PubMed

    Liotta, Flavia; d'Antonio, Giuseppe; Esposito, Giovanni; Fabbricino, Massimiliano; van Hullebusch, Eric D; Lens, Piet N L; Pirozzi, Francesco; Pontoni, Ludovico

    2014-10-01

    This work investigates the role of the moisture content on anaerobic digestion of food waste, as representative of rapidly biodegradable substrates, analysing the role of volatile fatty acid production on process kinetics. A range of total solids from 4.5% to 19.2% is considered in order to compare methane yields and kinetics of reactors operated under wet to dry conditions. The experimental results show a reduction of the specific final methane yield of 4.3% and 40.8% in semi-dry and dry conditions compared with wet conditions. A decreasing trend of the specific initial methane production rate is observed when increasing the total solids concentration. Because of lack of water, volatile fatty acids accumulation occurs during the first step of the process at semi-dry and dry conditions, which is considered to be responsible for the reduction of process kinetic rates. The total volatile fatty acids concentration and speciation are proposed as indicators of process development at different total solids content.

  13. Short communication: Intake, milk production, and milk fatty acid profile of dairy cows fed diets combining fresh forage with a total mixed ration.

    PubMed

    Mendoza, A; Cajarville, C; Repetto, J L

    2016-03-01

    To establish the effects of access time to high-quality temperate fresh forage (FF) on intake and performance of cows fed a total mixed ration (TMR), 9 Holstein cows were assigned to three 3 × 3 Latin squares with 20-d periods with sampling in the last 10 d. The following treatments were evaluated: 0 (T0), 4 (T4), and 8 (T8) h of daily access to FF. Forage (Lolium multiflorum) was cut daily and offered ad libitum beginning at 0800 h in individual stalls, and a TMR was offered ad libitum during the period when cows had no access to FF. Fresh forage dry matter intake (DMI) increased and TMR DMI decreased with the access time to FF, whereas total DMI was 3.0 kg higher for T4 than for T8; no differences were detected with T0. Compared with T0, 8h/d of access to FF decreased milk, protein, and casein yields, and tended to decrease fat and lactose yields, but treatment had no effect on milk composition or feed efficiency. The milk fatty acid profile in T8 had a higher content of rumenic, vaccenic, and linolenic acids compared with T0. We concluded that cows with 4h of access to high-quality FF had similar DMI and milk yields as cows fed only the TMR, but more than 4h of access reduced DMI and performance without changes in feed efficiency, although milk fat had enhanced levels of beneficial fatty acids.

  14. Detection of melamine and cyanuric acid in vegetable protein products used in food production.

    PubMed

    Levinson, Lawrence R; Gilbride, Kimberley A

    2011-05-01

    The multitude of food recalls in 2007 clearly demonstrated that total nitrogen-content (ΣN) determination by means of near-infrared spectroscopy (NIRS) and Kjeldahl-based measurements can be deceived, and should no longer be regarded as a complete quality assurance program for nutritive-protein evaluations. Furthermore, contemporary Canadian-employed analytical tools are precariously limited in their ability to effectively assure a product where there is no a priori knowledge of the environmental toxin(s) involved. In light of these challenges, this study explored a number of analytical techniques used to assess and furthermore assure the quality of vegetable protein products (VPPs). Using liquid chromatography with tandem mass spectrometry (LC/MS/MS) technologies, a combination of VPP-based samples was analyzed for the presence of nitrogen-bearing environmental toxicants. Of the 52 samples tested, involving an assortment of matrices, melamine and cyanuric acid were positively identified (>1 ng/mL) in 22 and 17 samples, respectively. Subsequent high pressure liquid chromatography with ultraviolet/visible (HPLC-UV) amino acid profiling further confirmed the adulteration of those materials contaminated with melamine and melamine-related compounds. Based on the evidence presented herein, LC/MS/MS in combination with HPLC-UV provides for a reliable food safety detection system as applied to VPPs. Moreover, HPLC-UV is indispensable as a stand-alone 1st level of screening to assess the integrity of a VPP or any nutritive protein-based sample.

  15. Assessment on proximate composition, dietary fiber, phytic acid and protein hydrolysis of germinated Ecuatorian brown rice.

    PubMed

    Cáceres, Patricio J; Martínez-Villaluenga, Cristina; Amigo, Lourdes; Frias, Juana

    2014-09-01

    Germinated brown rice (GBR) is considered healthier than brown rice (BR) but its nutritive value has been hardly studied. Since nutritive quality of GBR depends on genetic diversity and germination conditions, six Ecuadorian BR varieties were germinated at 28 and 34 ºC for 48 and 96 h in darkness and proximate composition, dietary fiber fractions, phytic acid content as well as degree of protein hydrolysis and peptide content were studied. Protein, lipids, ash and available carbohydrate ranged 7.3-10.4%, 2.0-4.0%, 0.8-1.5% and 71.6 to 84.0%, respectively, in GBR seedlings. Total dietary fiber increased during germination (6.1-13.6%), with a large proportion of insoluble fraction, while phytic acid was reduced noticeably. In general, protein hydrolysis occurred during germination was more accused at 28 ºC for 48 h. These results suggest that GBR can be consumed directly as nutritive staple food for a large population worldwide contributing to their nutritional requirements.

  16. PROTEIN METABOLISM IN REGENERATING WOUND TISSUE: FUNCTION OF THE SULFUR AMINO ACIDS.

    DTIC Science & Technology

    PROTEINS, *TISSUES(BIOLOGY), METABOLISM, TISSUES(BIOLOGY), REGENERATION(ENGINEERING), WOUNDS AND INJURIES, TISSUES(BIOLOGY), TRACER STUDIES, METHIONINE, COLLAGEN, TYROSINE, BIOSYNTHESIS, AMINO ACIDS .

  17. Total volatile fatty acid concentrations are unreliable estimators of treatment effects on ruminal fermentation in vivo

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Volatile fatty acid concentrations ([VFA], mM) have long been used to assess impact of dietary treatments on ruminal fermentation in vivo. However, discrepancies in statistical results between VFA and VFA pool size (VFAmol), possibly related to ruminal digesta liquid amount (LIQ, kg), suggest issues...

  18. Hydroxyindole Carboxylic Acid-Based Inhibitors for Receptor-Type Protein Tyrosine Protein Phosphatase Beta

    PubMed Central

    Zeng, Li-Fan; Zhang, Ruo-Yu; Bai, Yunpeng; Wu, Li; Gunawan, Andrea M.

    2014-01-01

    Abstract Aims: Protein tyrosine phosphatases (PTPs) play an important role in regulating a wide range of cellular processes. Understanding the role of PTPs within these processes has been hampered by a lack of potent and selective PTP inhibitors. Generating potent and selective probes for PTPs remains a significant challenge because of the highly conserved and positively charged PTP active site that also harbors a redox-sensitive Cys residue. Results: We describe a facile method that uses an appropriate hydroxyindole carboxylic acid to anchor the inhibitor to the PTP active site and relies on the secondary binding elements introduced through an amide-focused library to enhance binding affinity for the target PTP and to impart selectivity against off-target phosphatases. Here, we disclose a novel series of hydroxyindole carboxylic acid-based inhibitors for receptor-type tyrosine protein phosphatase beta (RPTPβ), a potential target that is implicated in blood vessel development. The representative RPTPβ inhibitor 8b-1 (L87B44) has an IC50 of 0.38 μM and at least 14-fold selectivity for RPTPβ over a large panel of PTPs. Moreover, 8b-1 also exhibits excellent cellular activity and augments growth factor signaling in HEK293, MDA-MB-468, and human umbilical vein endothelial cells. Innovation: The bicyclic salicylic acid pharmacophore-based focused library approach may provide a potential solution to overcome the bioavailability issue that has plagued the PTP drug discovery field for many years. Conclusion: A novel method is described for the development of bioavailable PTP inhibitors that utilizes bicyclic salicylic acid to anchor the inhibitors to the active site and peripheral site interactions to enhance binding affinity and selectivity. Antioxid. Redox Signal. 20, 2130–2140. PMID:24180557

  19. [The effect of lime cooking of corn on phytic acid, calcium, total and ionizable iron content].

    PubMed

    Urizar Hernández, A L; Bressani, R

    1997-09-01

    The present study was carried out with the objective to learn about the effect of the nixtamalization process of corn on the content of phytic acid and availability of iron in the lime-cooked corn. For the study, lots of corn with 0, 0.4, 0.8 and 1.20% of lime on the basis of corn weight, in water in the ratio of 3 to 1, and cooking times at each level of lime of 55, 65 and 75 minutes, were processed. Half of the treatments were not soaked after cooking, while the other half were soaked in the cooking solution for a 12-hour period. Statistical analysis of the data and correlations calculated showed that the phytic acid content decreased significantly during the nixtamalization process, affected by the cooking time and the level of lime used, reaching levels of reduction of around 35%. Both the ionizable iron and calcium level increased up to 52-77% and 400-478% respectively. The amount of calcium present in the cooked corn as the result of the lime cooking process, is significantly higher in comparison with the phytic acid content, which may be easily saturated and thus, unavailable to bind iron. An inverse relationship was found between phytic acid and bioavailable iron and its absorption percentage. On the other hand, soaking time did not significantly affected the phytic acid and available iron, although it contributed to a slightly higher Ca accumulation. The amount of ionizable iron was higher at higher levels of lime, which suggested that the nixtamalization process would favor the biological utilization of iron in lime-cooked corn and provide calcium to the diet.

  20. Solvent accessible surface area-based hot-spot detection methods for protein-protein and protein-nucleic acid interfaces.

    PubMed

    Munteanu, Cristian R; Pimenta, António C; Fernandez-Lozano, Carlos; Melo, André; Cordeiro, Maria N D S; Moreira, Irina S

    2015-05-26

    Due to the importance of hot-spots (HS) detection and the efficiency of computational methodologies, several HS detecting approaches have been developed. The current paper presents new models to predict HS for protein-protein and protein-nucleic acid interactions with better statistics compared with the ones currently reported in literature. These models are based on solvent accessible surface area (SASA) and genetic conservation features subjected to simple Bayes networks (protein-protein systems) and a more complex multi-objective genetic algorithm-support vector machine algorithms (protein-nucleic acid systems). The best models for these interactions have been implemented in two free Web tools.

  1. Partial amino acid sequence of human pancreatic stone protein, a novel pancreatic secretory protein.

    PubMed Central

    Montalto, G; Bonicel, J; Multigner, L; Rovery, M; Sarles, H; De Caro, A

    1986-01-01

    Pancreatic stone protein (PSP) is the major organic component of human pancreatic stones. With the use of monoclonal antibody immunoadsorbents, five immunoreactive forms (PSP-S) with close Mr values (14,000-19,000) were isolated from normal pancreatic juice. By CM-Trisacryl M chromatography the lowest-Mr form (PSP-S1) was separated from the others and some of its molecular characteristics were investigated. The Mr of the PSP-S1 polypeptide chain calculated from the amino acid composition was about 16,100. The N-terminal sequences (40 residues) of PSP and PSP-S1 are identical, which suggests that the peptide backbone is the same for both of these polypeptides. The PSP-S1 sequence was determined up to residue 65 and was found to be different from all other known protein sequences. Images Fig. 1. PMID:3541906

  2. Feasibility study for the quantification of total protein content by multiple prompt gamma-ray analysis.

    PubMed

    Toh, Y; Murakami, Y; Furutaka, K; Kimura, A; Koizumi, M; Hara, K; Kin, T; Nakamura, S; Harada, H

    2012-06-01

    Protein is an important nutrient in foods. The classical nitrogen analysis method is the Kjeldahl technique, which is time-consuming and inconvenient. As a convenient method to quantify protein content in biological samples, the feasibility of application of multiple prompt gamma-ray analysis (MPGA) to the quantification was studied. Results for protein content are reported for several reference materials and prove the method to be reliable.

  3. Experimental evolution of a green fluorescent protein composed of 19 unique amino acids without tryptophan.

    PubMed

    Kawahara-Kobayashi, Akio; Hitotsuyanagi, Mitsuhiro; Amikura, Kazuaki; Kiga, Daisuke

    2014-04-01

    At some stage of evolution, genes of organisms may have encoded proteins that were synthesized using fewer than 20 unique amino acids. Similar to evolution of the natural 19-amino-acid proteins GroEL/ES, proteins composed of 19 unique amino acids would have been able to evolve by accumulating beneficial mutations within the 19-amino-acid repertoire encoded in an ancestral genetic code. Because Trp is thought to be the last amino acid included in the canonical 20-amino-acid repertoire, this late stage of protein evolution could be mimicked by experimental evolution of 19-amino-acid proteins without tryptophan (Trp). To further understand the evolution of proteins, we tried to mimic the evolution of a 19-amino-acid protein involving the accumulation of beneficial mutations using directed evolution by random mutagenesis on the whole targeted gene sequence. We created active 19-amino-acid green fluorescent proteins (GFPs) without Trp from a poorly fluorescent 19-amino-acid mutant, S1-W57F, by using directed evolution with two rounds of mutagenesis and selection. The N105I and S205T mutations showed beneficial effects on the S1-W57F mutant. When these two mutations were combined on S1-W57F, we observed an additive effect on the fluorescence intensity. In contrast, these mutations showed no clear improvement individually or in combination on GFPS1, which is the parental GFP mutant composed of 20 amino acids. Our results provide an additional example for the experimental evolution of 19-amino-acid proteins without Trp, and would help understand the mechanisms underlying the evolution of 19-amino-acid proteins. (236 words).

  4. Experimental Evolution of a Green Fluorescent Protein Composed of 19 Unique Amino Acids without Tryptophan

    NASA Astrophysics Data System (ADS)

    Kawahara-Kobayashi, Akio; Hitotsuyanagi, Mitsuhiro; Amikura, Kazuaki; Kiga, Daisuke

    2014-04-01

    At some stage of evolution, genes of organisms may have encoded proteins that were synthesized using fewer than 20 unique amino acids. Similar to evolution of the natural 19-amino-acid proteins GroEL/ES, proteins composed of 19 unique amino acids would have been able to evolve by accumulating beneficial mutations within the 19-amino-acid repertoire encoded in an ancestral genetic code. Because Trp is thought to be the last amino acid included in the canonical 20-amino-acid repertoire, this late stage of protein evolution could be mimicked by experimental evolution of 19-amino-acid proteins without tryptophan (Trp). To further understand the evolution of proteins, we tried to mimic the evolution of a 19-amino-acid protein involving the accumulation of beneficial mutations using directed evolution by random mutagenesis on the whole targeted gene sequence. We created active 19-amino-acid green fluorescent proteins (GFPs) without Trp from a poorly fluorescent 19-amino-acid mutant, S1-W57F, by using directed evolution with two rounds of mutagenesis and selection. The N105I and S205T mutations showed beneficial effects on the S1-W57F mutant. When these two mutations were combined on S1-W57F, we observed an additive effect on the fluorescence intensity. In contrast, these mutations showed no clear improvement individually or in combination on GFPS1, which is the parental GFP mutant composed of 20 amino acids. Our results provide an additional example for the experimental evolution of 19-amino-acid proteins without Trp, and would help understand the mechanisms underlying the evolution of 19-amino-acid proteins. (236 words)

  5. Measurement of total acid number (TAN) and TAN boiling point distribution in petroleum products by electrospray ionization mass spectrometry.

    PubMed

    Qian, Kuangnan; Edwards, Kathleen E; Dechert, Gary J; Jaffe, Stephen B; Green, Larry A; Olmstead, William N

    2008-02-01

    We report a new method for rapid measurement of total acid number (TAN) and TAN boiling point (BP) distribution for petroleum crude and products. The technology is based on negative ion electrospray ionization mass spectrometry (ESI-MS) for selective ionization of petroleum acid and quantification of acid structures and molecular weight distributions. A chip-based nanoelectrospray system enables microscale (<200 mg) and higher throughput (20 samples/h) measurement. Naphthenic acid structures were assigned based on nominal masses of a set of predefined acid structures. Stearic acid is used as an internal standard to calibrate ESI-MS response factors for quantification purposes. With the use of structure-property correlations, boiling point distributions of TAN values can be calculated from the composition. The rapid measurement of TAN BP distributions by ESI is demonstrated for a series of high-TAN crudes and distillation cuts. TAN values determined by the technique agree well with those by the titration method. The distributed properties compare favorably with those measured by distillation and measurement of TAN of corresponding cuts.

  6. Dietary influence on the m. longissimus dorsi fatty acid composition of lambs in relation to protein source.

    PubMed

    Turner, T D; Karlsson, L; Mapiye, C; Rolland, D C; Martinsson, K; Dugan, M E R

    2012-08-01

    Dietary lipid effect, as a consequence of protein supplement, on lamb m. longissimus dorsi fatty acid composition was investigated, with emphasis on biohydrogenation intermediates. Crossbred lambs (White Swedish Landrace × Texel) were fed a barley-based diet without (CON) or with protein supplements including peas (PEA), rapeseed cake (RC) or hempseed cake (HC). The HC diet resulted in the highest muscle 22:6n-3 proportion, with the RC diet being similar (P<0.05). Protein supplement did not affect the c9,t11 conjugated linoleic acid (CLA) proportion, however the HC diet increased some minor CLA isomers, including t10,c12 CLA (P<0.05). The t10-18:1 and total trans-18:1 were lowest for the RC diet (P<0.05), likely relating to rumen conditions and precursor availability. The saturated, monounsaturated and branched-chain fatty acids were largely unaffected by protein supplement. In conclusion, feeding the RC diet lowered the t10-18:1 and total trans-18:1 in meat, and modestly increased 22:6n-3 content. The direction of these changes would be beneficial, making the RC diet the preferred protein supplement; however the magnitude of the changes in the present experiment may not be sufficient to have an impact on human health.

  7. Prebiotic Synthesis of Hydrophobic and Protein Amino Acids

    PubMed Central

    Ring, David; Wolman, Yecheskel; Friedmann, Nadav; Miller, Stanley L.

    1972-01-01

    The formation of amino acids by the action of electric discharges on a mixture of methane, nitrogen, and water with traces of ammonia was studied in detail. The presence of glycine, alanine, α-amino-n-butyric acid, α-aminoisobutyric acid, valine, norvaline, isovaline, leucine, isoleucine, alloisoleucine, norleucine, proline, aspartic acid, glutamic acid, serine, threonine, allothreonine, α-hydroxy-γ-aminobutyric acid, and α,γ-diaminobutyric acid was confirmed by ion-exchange chromatography and gas chromatography-mass spectrometry. All of the primary α-amino acids found in the Murchison Meteorite have been synthesized by this electric discharge experiment. PMID:4501592

  8. Impact of the dietary fatty acid intake on C-reactive protein levels in US adults.

    PubMed

    Mazidi, Mohsen; Gao, Hong-Kai; Vatanparast, Hassan; Kengne, Andre Pascal

    2017-02-01

    Growing evidence suggests that the effects of diet on cardiovascular disease (CVD) occur through mechanisms involving subclinical inflammation. We assessed whether reported dietary fatty acid intake correlates with a serum high-sensitivity C-reactive protein (hs-CRP) concentration in a population-based sample of US men and women.In this cross-sectional analysis, participants were selected from the US National Health and Nutrition Examination Survey (NHANES) and restricted to those with available data on dietary intake, biochemical and anthropometric measurements from 2001 to 2010. All statistical analyses accounted for the survey design and sample weights by using SPSS Complex Samples v22.0 (IBM Corp, Armonk, NY).Of the 17,689 participants analyzed, 8607 (48.3%) were men. The mean age was 45.8 years in the overall sample, 44.9 years in men, and 46.5 years in women (P = 0.047). The age-, race-, and sex-adjusted mean dietary intakes of total polyunsaturated fatty acids (PUFAs), PUFAs 18:2 (octadecadienoic), and PUFAs 18:3 (octadecatrienoic) monotonically decreased across hs-CRP quartiles (P < 0.001), whereas dietary cholesterol increased across hs-CRP quartiles (P < 0.001)This study provides further evidence of an association between fatty acid intake and subclinical inflammation markers. hs-CRP concentrations are likely modulated by dietary fatty acid intake. However, the causality of this association needs to be demonstrated in clinical trials.

  9. Impact of the dietary fatty acid intake on C-reactive protein levels in US adults

    PubMed Central

    Mazidi, Mohsen; Gao, Hong-Kai; Vatanparast, Hassan; Kengne, Andre Pascal

    2017-01-01

    Abstract Growing evidence suggests that the effects of diet on cardiovascular disease (CVD) occur through mechanisms involving subclinical inflammation. We assessed whether reported dietary fatty acid intake correlates with a serum high-sensitivity C-reactive protein (hs-CRP) concentration in a population-based sample of US men and women. In this cross-sectional analysis, participants were selected from the US National Health and Nutrition Examination Survey (NHANES) and restricted to those with available data on dietary intake, biochemical and anthropometric measurements from 2001 to 2010. All statistical analyses accounted for the survey design and sample weights by using SPSS Complex Samples v22.0 (IBM Corp, Armonk, NY). Of the 17,689 participants analyzed, 8607 (48.3%) were men. The mean age was 45.8 years in the overall sample, 44.9 years in men, and 46.5 years in women (P = 0.047). The age-, race-, and sex-adjusted mean dietary intakes of total polyunsaturated fatty acids (PUFAs), PUFAs 18:2 (octadecadienoic), and PUFAs 18:3 (octadecatrienoic) monotonically decreased across hs-CRP quartiles (P < 0.001), whereas dietary cholesterol increased across hs-CRP quartiles (P < 0.001) This study provides further evidence of an association between fatty acid intake and subclinical inflammation markers. hs-CRP concentrations are likely modulated by dietary fatty acid intake. However, the causality of this association needs to be demonstrated in clinical trials. PMID:28207502

  10. 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

  11. Stain-Free total protein staining is a superior loading control to β-actin for Western blots.

    PubMed

    Gilda, Jennifer E; Gomes, Aldrin V

    2013-09-15

    Semi-quantification of proteins using Western blots typically involves normalization against housekeeping genes such as β-actin. More recently, Ponceau S and Coomassie blue staining have both been shown to be suitable alternatives to housekeeping genes as loading controls. Stain-Free total protein staining offers the advantage of no staining or destaining steps. Evaluation of the use of Stain-Free staining as an alternative to β-actin or the protein stain Ponceau S showed that Stain-Free staining was superior to β-actin and as good as or better than Ponceau S staining as a loading control for Western blots.

  12. 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.

  13. Soy protein/soy polysaccharide complex nanogels: folic acid loading, protection, and controlled delivery.

    PubMed

    Ding, Xuzhe; Yao, Ping

    2013-07-09

    In this study, we developed a facile approach to produce nanogels via self-assembly of folic acid, soy protein, and soy polysaccharide. High-pressure homogenization was introduced to break down the original aggregates of soy protein, which benefits the binding of soy protein with soy polysaccharide and folic acid at pH 4.0. After a heat treatment that causes the soy protein denaturation and gelation, folic acid-loaded soy protein/soy polysaccharide complex nanogels were fabricated. The nanogels have a polysaccharide surface that makes the nanogels dispersible in acidic conditions where folic acid is insoluble and soy protein forms precipitates after heating. More importantly, the protein and polysaccharide can inhibit the reactions between dissolved oxygen and folic acid during UV irradiation. After the preparation and storage of the nanogels in the presence of heat, oxygen, and light in acidic conditions, most of the folic acid molecules in the nanogels remain in their natural structure and can be released rapidly at neutral pH, that is, in the intestine. Because most food and beverages are acidic, the nanogels are a suitable delivery system of folic acid in food and beverages.

  14. Identification of Dynamic Changes in Proteins Associated with the Cellular Cytoskeleton after Exposure to Okadaic Acid

    PubMed Central

    Opsahl, Jill A.; Ljostveit, Sonja; Solstad, Therese; Risa, Kristin; Roepstorff, Peter; Fladmark, Kari E.

    2013-01-01

    Exposure of cells to the diarrhetic shellfish poison, okadaic acid, leads to a dramatic reorganization of cytoskeletal architecture and loss of cell-cell contact. When cells are exposed to high concentrations of okadaic acid (100–500 nM), the morphological rearrangement is followed by apoptotic cell death. Okadaic acid inhibits the broad acting Ser/Thr protein phosphatases 1 and 2A, which results in hyperphosphorylation of a large number of proteins. Some of these hyperphosphorylated proteins are most likely key players in the reorganization of the cell morphology induced by okadaic acid. We wanted to identify these phosphoproteins and searched for them in the cellular lipid rafts, which have been found to contain proteins that regulate cytoskeletal dynamics and cell adhesion. By using stable isotope labeling by amino acids in cell culture cells treated with okadaic acid (400 nM) could be combined with control cells before the isolation of lipid rafts. Protein phosphorylation events and translocations induced by okadaic acid were identified by mass spectrometry. Okadaic acid was shown to regulate the phosphorylation status and location of proteins associated with the actin cytoskeleton, microtubules and cell adhesion structures. A large number of these okadaic acid-regulated proteins have previously also been shown to be similarly regulated prior to cell proliferation and migration. Our results suggest that okadaic acid activates general cell signaling pathways that induce breakdown of the cortical actin cytoskeleton and cell detachment. PMID:23708184

  15. BindML/BindML+: Detecting Protein-Protein Interaction Interface Propensity from Amino Acid Substitution Patterns.

    PubMed

    Wei, Qing; La, David; Kihara, Daisuke

    2017-01-01

    Prediction of protein-protein interaction sites in a protein structure provides important information for elucidating the mechanism of protein function and can also be useful in guiding a modeling or design procedures of protein complex structures. Since prediction methods essentially assess the propensity of amino acids that are likely to be part of a protein docking interface, they can help in designing protein-protein interactions. Here, we introduce BindML and BindML+ protein-protein interaction sites prediction methods. BindML predicts protein-protein interaction sites by identifying mutation patterns found in known protein-protein complexes using phylogenetic substitution models. BindML+ is an extension of BindML for distinguishing permanent and transient types of protein-protein interaction sites. We developed an interactive web-server that provides a convenient interface to assist in structural visualization of protein-protein interactions site predictions. The input data for the web-server are a tertiary structure of interest. BindML and BindML+ are available at http://kiharalab.org/bindml/ and http://kiharalab.org/bindml/plus/ .

  16. Blood zinc protoporphyrin, serum total protein, and total cholesterol levels in automobile workshop workers in relation to lead toxicity: Our experience.

    PubMed

    Pachathundikandi, Suneesh Kumar; Varghese, Earaly Thomas

    2006-09-01

    Blood zinc protoporphyrin (ZPP), serum total protein (TP), and total cholesterol (TC) levels in automobile workshop workers in relation to lead toxicity were analysed. In the present study, automobile workshop workers (healthy male workers at an age between 28 and 35 from four major automobile workshops in Kottayam, Kerala State, India) and the control (male healthy adults at an age between 28 and 35 residing at Aymanam, a distant village at Kottayam District, Kerala having reduced or no chance of lead exposure) displayed significant difference in blood lead (BPb) and blood ZZP (BZPP) level. The mean value of BPb in automobile workshop workers was 15.76±0.33 μg/dl, while in the control it was 8.20±0.15 μg/dl. In automobile workshop workers, the mean value of BZPP was 34.2±0.62 μg/dl. The control group exhibited a mean of 11.5±0.22 μg/dl. Automobile workshop workers exhibited significant increase in BZPP was corresponding to the increase in BPb level. The total protein levels estimated in automobile workshop workers showed significant decrease compared to control individuals, but was within the reference range of healthy individuals. The mean value of TP level in automobile workshop workers and control was 6.9±0.13 g/dl and 7.71±0.18 g/dl, respectively. There was no significant difference in blood haemoglobin (BHb) level among the automobile workshop workers and control. The serum TC level in automobile workshop workers showed significant decrease compared to the control individuals, but was with in the reference range of healthy individuals. The mean level of serum TC in automobile workshop workers was 162.00±3.44 mg/dl and the same in control was 172.86±4.32 mg/dl. The present study affirms occupational lead toxicity in automobile workshop workers and its effect on serum protein and cholesterol levels.

  17. Anesthesia with halothane and nitrous oxide alters protein and amino acid metabolism in dogs

    SciTech Connect

    Horber, F.F.; Krayer, S.; Rehder, K.; Haymond, M.W.

    1988-09-01

    General anesthesia in combination with surgery is known to result in negative nitrogen balance. To determine whether general anesthesia without concomitant surgery decreases whole body protein synthesis and/or increases whole body protein breakdown, two groups of dogs were studied: Group 1 (n = 6) in the conscious state and Group 2 (n = 8) during general anesthesia employing halothane (1.5 MAC) in 50% nitrous oxide and oxygen. Changes in protein metabolism were estimated by isotope dilution techniques employing simultaneous infusions of (4,53H)leucine and alpha-(1-14C)-ketoisocaproate (KIC). Total leucine carbon flux was unchanged or slightly increased in the anesthetized animals when compared to the conscious controls, indicating only a slight increase in the rate of proteolysis. However, leucine oxidation was increased (P less than 0.001) by more than 80% in the anesthetized animals when compared with their conscious controls, whereas whole body nonoxidative leucine disappearance, an indicator of whole body protein synthesis, was decreased. The ratio of leucine oxidation to the nonoxidative rate of leucine disappearance, which provides an index of the catabolism of at least one essential amino acid in the postabsorptive state, was more than twofold increased (P less than 0.001) in the anesthetized animals regardless of the tracer employed. These studies suggest that the administration of anesthesia alone, without concomitant surgery, is associated with a decreased rate of whole body protein synthesis and increased leucine oxidation, resulting in increased leucine and protein catabolism, which may be underlying or initiating some of the protein wasting known to occur in patients undergoing surgery.

  18. Expanding the Cyanuric Acid Hydrolase Protein Family to the Fungal Kingdom

    PubMed Central

    Dodge, Anthony G.; Preiner, Chelsea S.

    2013-01-01

    The known enzymes that open the s-triazine ring, the cyanuric acid hydrolases, have been confined almost exclusively to the kingdom Bacteria and are all homologous members of the rare cyanuric acid hydrolase/barbiturase protein family. In the present study, a filamentous fungus, Sarocladium sp. strain CA, was isolated from soil by enrichment culturing using cyanuric acid as the sole source of nitrogen. A reverse-genetic approach identified a fungal cyanuric acid hydrolase gene composed of two exons and one intron. The translated spliced sequence was 39 to 53% identical to previously characterized bacterial cyanuric acid hydrolases. The sequence was used to generate a gene optimized for expression in Escherichia coli and encoding an N-terminally histidine-tagged protein. The protein was purified by nickel affinity and anion-exchange chromatography. The purified protein was shown by 13C nuclear magnetic resonance (13C-NMR) to produce carboxybiuret as the product, which spontaneously decarboxylated to yield biuret and carbon dioxide. The protein was very narrow in substrate specificity, showing activity only with cyanuric acid and N-methyl cyanuric acid. Barbituric acid was an inhibitor of enzyme activity. Sequence analysis identified genes with introns in other fungi from the Ascomycota that, if spliced, are predicted to encode proteins with cyanuric acid hydrolase activity. The Ascomycota cyanuric acid hydrolase homologs are most closely related to cyanuric acid hydrolases from Actinobacteria. PMID:24039269

  19. 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.

  20. Enteral obeticholic acid prevents hepatic cholestasis in total parenteral nutrition-fed neonatal pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Total parenteral nutrition (TPN) is a vital support for neonatal infants with congenital or acquired gastrointestinal (GI) disorders and requiring small bowel resection. An adverse outcome associated with prolonged TPN use is parenteral nutrition associated cholestasis (PNAC). We previously showed t...

  1. Human-specific amino acid changes found in 103 protein-coding genes.

    PubMed

    Kitano, Takashi; Liu, Yu-Hua; Ueda, Shintaroh; Saitou, Naruya

    2004-05-01

    We humans have many characteristics that are different from those of the great apes. These human-specific characters must have arisen through mutations accumulated in the genome of our direct ancestor after the divergence of the last common ancestor with chimpanzee. Gene trees of human and great apes are necessary for extracting these human-specific genetic changes. We conducted a systematic analysis of 103 protein-coding genes for human, chimpanzee, gorilla, and orangutan. Nucleotide sequences for 18 genes were newly determined for this study, and those for the remaining genes were retrieved from the DDBJ/EMBL/GenBank database. The total number of amino acid changes in the human lineage was 147 for 26,199 codons (0.56%). The total number of amino acid changes in the human genome was, thus, estimated to be about 80,000. We applied the acceleration index test and Fisher's synonymous/nonsynonymous exact test for each gene tree to detect any human-specific enhancement of amino acid changes compared with ape branches. Six and two genes were shown to have significantly higher nonsynonymous changes at the human lineage from the acceleration index and exact tests, respectively. We also compared the distribution of the differences of the nonsynonymous substitutions on the human lineage and those on the great ape lineage. Two genes were more conserved in the ape lineage, whereas one gene was more conserved in the human lineage. These results suggest that a small proportion of protein-coding genes started to evolve differently in the human lineage after it diverged from the ape lineage.

  2. [The use of different albumin preparations as calibrators in determining the total protein in blood serum by the biuret method].

    PubMed

    Sigalov, A B; Isaeva, N V; Bezruchkina, S V

    1993-01-01

    The authors have investigated the possibility of using various albumin preparations as calibrators in measurements of human blood serum total protein by the biuret method. Analysis of Precinorm U and Precipath U reference sera has demonstrated that use of various albumin preparations as calibrators may result in significant deviations (as much as 27%) of the resultant values from the due ones.

  3. Laboratory Measurement of Urine Albumin and Urine Total Protein in Screening for Proteinuria in Chronic Kidney Disease

    PubMed Central

    Martin, Helen

    2011-01-01

    Laboratory measurement of urine total protein has been important for the diagnosis and monitoring of renal disease for decades, and since the late 1990s, urine albumin has been measured to determine whether a diabetic patient has incipient nephropathy. Evolving understanding of chronic kidney disease (CKD) and, in particular, the cardiovascular risks that CKD confers, demands more sensitive detection of protein in urine. As well, evidence is now emerging that cardiovascular and all-cause mortality risks are increased at levels within the current ‘normal’ range for urine albumin. Standardisation is essential to permit valid application of universal decision points, and a National Kidney Disease Education Program/International Federation of Clinical Chemistry and Laboratory Medicine (NKDEP/IFCC) Working Party is making progress towards a reference system for urine albumin. In the meantime, available data suggest that Australasian laboratory performance is adequate in terms of precision and accuracy above current decision limits for urine albumin. In contrast, the complexity of proteins in urine makes standardisation of urine total protein measurement impossible. As well, urine total protein measurement is insufficiently sensitive to detect clinically important concentrations of urine albumin. An Australasian Expert Group, the Proteinuria Albuminuria Working Group (PAWG) has proposed that urine albumin/creatinine ratio is measured in a fresh, first morning, spot sample to screen for proteinuria in CKD. Both NKDEP/IFCC and PAWG emphasise the need for standardisation of sample collection and handling. PMID:21611083

  4. Laboratory measurement of urine albumin and urine total protein in screening for proteinuria in chronic kidney disease.

    PubMed

    Martin, Helen

    2011-05-01

    Laboratory measurement of urine total protein has been important for the diagnosis and monitoring of renal disease for decades, and since the late 1990s, urine albumin has been measured to determine whether a diabetic patient has incipient nephropathy. Evolving understanding of chronic kidney disease (CKD) and, in particular, the cardiovascular risks that CKD confers, demands more sensitive detection of protein in urine. As well, evidence is now emerging that cardiovascular and all-cause mortality risks are increased at levels within the current 'normal' range for urine albumin. Standardisation is essential to permit valid application of universal decision points, and a National Kidney Disease Education Program/International Federation of Clinical Chemistry and Laboratory Medicine (NKDEP/IFCC) Working Party is making progress towards a reference system for urine albumin. In the meantime, available data suggest that Australasian laboratory performance is adequate in terms of precision and accuracy above current decision limits for urine albumin. In contrast, the complexity of proteins in urine makes standardisation of urine total protein measurement impossible. As well, urine total protein measurement is insufficiently sensitive to detect clinically important concentrations of urine albumin. An Australasian Expert Group, the Proteinuria Albuminuria Working Group (PAWG) has proposed that urine albumin/creatinine ratio is measured in a fresh, first morning, spot sample to screen for proteinuria in CKD. Both NKDEP/IFCC and PAWG emphasise the need for standardisation of sample collection and handling.

  5. Sorption behavior of polyaramides in relation to isolation of nucleic acids and proteins.

    PubMed

    Liaw, Der-Jang; Yagudaeva, Elena; Prostyakova, Anna; Lazov, Michael; Zybin, Dmitry; Ischenko, Anatoly; Zubov, Vitaly; Chang, Cheng-Hung; Huang, Ying-Chi; Kapustin, Dmitry

    2016-09-01

    The effect of chemical composition and morphology of the surface layers of new polyaramide-containing sorbents on the mechanism of selective sorption of nucleic acids and proteins was investigated as compared to the previously studied sorbents modified with fluoropolymers and polyaniline (high-throughput materials providing one-step isolation of DNA from biological mixtures). A series of silica-based sorbents modified with polyaramides having consistently varying structure and containing the set of "key" structural elements (aromatic units and nitrogen atoms in the backbone, fluorinated groups), and various donor and acceptor moieties was prepared. The chemical composition of the polymer coatings was evaluated by X-ray photoelectron spectroscopy. The surface morphology was studied by scanning probe microscopy. The sorption properties were investigated by passing the mixtures containing DNA, RNA and proteins of different nature through the cartridges containing the obtained sorbents. All the investigated materials weakly retain double-stranded DNA but effectively retain RNA and proteins. The sorption capacity of the sorbents depends on the protein nature. The observed sorption behavior was shown to be determined by the chemical structure and not by the morphology of the polymer coating. It was proposed that similarity of the sorption properties of the series of chemically different polymers could be determined by similar total input of different sorption mechanisms.

  6. Docosahexaenoic acid-mediated protein aggregates may reduce proteasome activity and delay myotube degradation during muscle atrophy in vitro

    PubMed Central

    Shin, Seung Kyun; Kim, Ji Hyeon; Lee, Jung Hoon; Son, Young Hoon; Lee, Min Wook; Kim, Hak Joong; Noh, Sue Ah; Kim, Kwang Pyo; Kim, In-Gyu; Lee, Min Jae

    2017-01-01

    Proteasomes are the primary degradation machinery for oxidatively damaged proteins that compose a class of misfolded protein substrates. Cellular levels of reactive oxygen species increase with age and this cellular propensity is particularly harmful when combined with the age-associated development of various human disorders including cancer, neurodegenerative disease and muscle atrophy. Proteasome activity is reportedly downregulated in these disease conditions. Herein, we report that docosahexaenoic acid (DHA), a major dietary omega-3 polyunsaturated fatty acid, mediates intermolecular protein cross-linkages through oxidation, and the resulting protein aggregates potently reduce proteasomal activity both in vitro and in cultured cells. Cellular models overexpressing aggregation-prone proteins such as tau showed significantly elevated levels of tau aggregates and total ubiquitin conjugates in the presence of DHA, thereby reflecting suppressed proteasome activity. Strong synergetic cytotoxicity was observed when the cells overexpressing tau were simultaneously treated with DHA. Antioxidant N-acetyl cysteine significantly desensitized the cells to DHA-induced oxidative stress. DHA significantly delayed the proteasomal degradation of muscle proteins in a cellular atrophy model. Thus, the results of our study identified DHA as a potent inducer of cellular protein aggregates that inhibit proteasome activity and potentially delay systemic muscle protein degradation in certain pathologic conditions. PMID:28104914

  7. The amino acid sequence of protein CM-3 from Dendroaspis polylepis polylepis (black mamba) venom.

    PubMed

    Joubert, F J

    1985-01-01

    Protein CM-3 from Dendroaspis polylepis polylepis venom was purified by gel filtration and ion exchange chromatography. It comprises 65 amino acids including eight half-cystines. The complete amino acid sequence of protein CM-3 has been elucidated. The sequence (residues 1-50) resembles that of the N-terminal sequence of the subunits of a synergistic type protein and residues 51-65 that of the C-terminal sequence of an angusticeps type protein. Mixtures of protein CM-3 and angusticeps type proteins showed no apparent synergistic effect, in that their toxicity in combination was no greater than the sum of their individual toxicities.

  8. Toward amino acid typing for proteins in FFLUX.

    PubMed

    Fletcher, Timothy L; Popelier, Paul L A

    2017-03-05

    Continuing the development of the FFLUX, a multipolar polarizable force field driven by machine learning, we present a modern approach to atom-typing and building transferable models for predicting atomic properties in proteins. Amino acid atomic charges in a peptide chain respond to the substitution of a neighboring residue and this response can be categorized in a manner similar to atom-typing. Using a machine learning method called kriging, we are able to build predictive models for an atom that is defined, not only by its local environment, but also by its neighboring residues, for a minimal additional computational cost. We found that prediction errors were up to 11 times lower when using a model specific to the correct group of neighboring residues, with a mean prediction of ∼0.0015 au. This finding suggests that atoms in a force field should be defined by more than just their immediate atomic neighbors. When comparing an atom in a single alanine to an analogous atom in a deca-alanine helix, the mean difference in charge is 0.026 au. Meanwhile, the same difference between a trialanine and a deca-alanine helix is only 0.012 au. When compared to deca-alanine models, the transferable models are up to 20 times faster to train, and require significantly less ab initio calculation, providing a practical route to modeling large biological systems. © 2016 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc.

  9. Membrane protein extraction and purification using styrene-maleic acid (SMA) copolymer: effect of variations in polymer structure.

    PubMed

    Morrison, Kerrie A; Akram, Aneel; Mathews, Ashlyn; Khan, Zoeya A; Patel, Jaimin H; Zhou, Chumin; Hardy, David J; Moore-Kelly, Charles; Patel, Roshani; Odiba, Victor; Knowles, Tim J; Javed, Masood-Ul-Hassan; Chmel, Nikola P; Dafforn, Timothy R; Rothnie, Alice J

    2016-12-01

    The use of styrene-maleic acid (SMA) copolymers to extract and purify transmembrane proteins, while retaining their native bilayer environment, overcomes many of the disadvantages associated with conventional detergent-based procedures. This approach has huge potential for the future of membrane protein structural and functional studies. In this investigation, we have systematically tested a range of commercially available SMA polymers, varying in both the ratio of styrene and maleic acid and in total size, for the ability to extract, purify and stabilise transmembrane proteins. Three different membrane proteins (BmrA, LeuT and ZipA), which vary in size and shape, were used. Our results show that several polymers, can be used to extract membrane proteins, comparably to conventional detergents. A styrene:maleic acid ratio of either 2:1 or 3:1, combined with a relatively small average molecular mass (7.5-10 kDa), is optimal for membrane extraction, and this appears to be independent of the protein size, shape or expression system. A subset of polymers were taken forward for purification, functional and stability tests. Following a one-step affinity purification, SMA 2000 was found to be the best choice for yield, purity and function. However, the other polymers offer subtle differences in size and sensitivity to divalent cations that may be useful for a variety of downstream applications.

  10. Relation of plasma calcium to total protein and albumin in African grey (Psittacus erithacus) and Amazon (Amazona spp.) parrots.

    PubMed

    Lumeij, J T

    1990-10-01

    A significant correlation was found between total calcium and albumin concentration in the plasma of 70 African grey parrots (r=0.37; P<0.05). A correlation formula for plasma calcium concentration in the African grey parrot was derived on the basis of the concentration of albumin: Adjusted Ca (mmol/1) = Ca (mmol/1) - 0.015 Albumin (g/1) + 0.4. About 14% of the variability in calcium was attributable to the change in the concentration of plasma albumin concentration (R2=0.137). The correlation between calcium and total protein in African greys and between calcium and albumin and calcium and total protein in Amazons was not significant.

  11. Genetic introduction of a diketone-containing amino acid into proteins.

    PubMed

    Zeng, Huaqiang; Xie, Jianming; Schultz, Peter G

    2006-10-15

    An orthogonal tRNA/aminoacyl-tRNA synthetase pair was evolved that makes possible the site-specific incorporation of an unnatural amino acid bearing a beta-diketone side chain into proteins in Escherichia coli with high translational efficiency and fidelity. Proteins containing this unnatural amino acid can be efficiently and selectively modified with hydroxylamine derivatives of fluorophores and other biophysical probes.

  12. ORAL AND INTRAVENOUSLY ADMINISTERED AMINO ACIDS PRODUCE SIMILAR EFFECTS ON MUSCLE PROTEIN SYNTHESIS IN THE ELDERLY

    PubMed Central

    Rasmussen, B.B.; Wolfe, R.R.; Volpi, E.

    2011-01-01

    BACKGROUND Muscle protein synthesis is stimulated in the elderly when amino acid availability is increased. OBJECTIVE To determine which mode of delivery of amino acids (intravenous vs. oral ingestion) is more effective in stimulating the rate of muscle protein synthesis in elderly subjects. DESIGN Fourteen elderly subjects were assigned to one of two groups. Following insertion of femoral arterial and venous catheters, subjects were infused with a primed, continuous infusion of L-[ring-2H5] phenylalanine. Blood samples and muscle biopsies were obtained to measure muscle protein fractional synthesis rate (FSR) with the precursor-product model, phenylalanine kinetics across the leg with the three-pool model, and whole body phenylalanine kinetics. Protein metabolism parameters were measured in the basal period, and during the administration of oral amino acids (n=8) or a similar amount of intravenous amino acids (n=6). RESULTS Enteral and parenteral amino acid administration increased amino acid arterial concentrations and delivery to the leg to a similar extent in both groups. Muscle protein synthesis as measured by both FSR, and the three-pool model, increased during amino acid administration (P < 0.05 vs. basal) in both groups with no differences between groups. Whole body proteolysis did not change with the oral amino acids whereas it increased slightly during parenteral amino acid administration. CONCLUSIONS Increased amino acid availability stimulates the rate of muscle protein synthesis independent of the route of administration (enteral vs. parenteral). PMID:12459885

  13. Temporal profile of intestinal tissue expression of intestinal fatty acid-binding protein in a rat model of necrotizing enterocolitis

    PubMed Central

    Simões, Ana Leda Bertoncini; Figueira, Rebeca Lopes; Gonçalves, Frances Lilian Lanhellas; Mitidiero, Luís Felipe Tsuyoshi; Silva, Orlando Castro e; Peiró, José Luis; Sbragia, Lourenço

    2016-01-01

    OBJECTIVES: Necrotizing enterocolitis is a severe multifactorial intestinal disorder that primarily affects preterm newborns, causing 20-40% mortality and morbidity. Intestinal fatty acid-binding protein has been reported to be a biomarker for the detection of intestinal injuries. Our aim was to assess intestinal tissue injury and the molecular expression of intestinal fatty acid-binding protein over time in a necrotizing enterocolitis model. METHODS: A total of 144 Newborn rats were divided into two groups: 1) Control, which received breastfeeding (n=72) and 2) Necrotizing Enterocolitis, which received formula feeding and underwent hypoxia and hypothermia (n=72). A total of six time points of ischemia (2 times a day for 3 days; 12 pups for each time point) were examined. Samples were collected for analysis of body weight, morphological and histological characteristics, intestinal weight, intestinal weight/body weight ratio, injury grade, and intestinal fatty acid-binding protein levels. RESULTS: Body and intestinal weights were lower in the Necrotizing Enterocolitis group than in the Control group (p<0.005 and p<0.0005, respectively). The intestinal weight/body weight ratio was higher in the Necrotizing Enterocolitis group than in the Control group (p<0.005) only at the sixth ischemia time point. The Necrotizing Enterocolitis group displayed higher expression of intestinal fatty acid-binding protein (p<0.0005) and showed greater tissue damage than the Control group. CONCLUSION: Intestinal fatty acid-binding protein was an efficient marker of ischemic injury to the intestine and a good correlation was demonstrated between the time of ischemic injury and the grade of intestinal injury. PMID:27464299

  14. A novel amino acid and metabolomics signature in mice overexpressing muscle uncoupling protein 3.

    PubMed

    Aguer, Céline; Piccolo, Brian D; Fiehn, Oliver; Adams, Sean H; Harper, Mary-Ellen

    2017-02-01

    Uncoupling protein 3 (UCP3) is highly selectively expressed in skeletal muscle and is known to lower mitochondrial reactive oxygen species and promote fatty acid oxidation; however, the global impact of UCP3 activity on skeletal muscle and whole-body metabolism have not been extensively studied. We utilized untargeted metabolomics to identify novel metabolites that distinguish mice overexpressing UCP3 in muscle, both at rest and after exercise regimens that challenged muscle metabolism, to potentially unmask subtle phenotypes. Male wild-type (WT) and muscle-specific UCP3-overexpressing transgenic (UCP3 Tg) C57BL/6J mice were compared with or without a 5 wk endurance training protocol at rest or after an acute exercise bout (EB). Skeletal muscle, liver, and plasma samples were analyzed by gas chromatography time-of-flight mass spectrometry. Discriminant metabolites were considered if within the top 99th percentile of variable importance measurements obtained from partial least-squares discriminant analysis models. A total of 80 metabolites accurately discriminated UCP3 Tg mice from WT when modeled within a specific exercise condition (i.e., untrained/rested, endurance trained/rested, untrained/EB, and endurance trained/EB). Results revealed that several amino acids and amino acid derivatives in skeletal muscle and plasma of UCP3 Tg mice (e.g., Asp, Glu, Lys, Tyr, Ser, Met) were significantly reduced after an EB; that metabolites associated with skeletal muscle glutathione/Met/Cys metabolism (2-hydroxybutanoic acid, oxoproline, Gly, and Glu) were altered in UCP3 Tg mice across all training and exercise conditions; and that muscle metabolite indices of dehydrogenase activity were increased in UCP3 Tg mice, suggestive of a shift in tissue NADH/NAD(+) ratio. The results indicate that mitochondrial UCP3 activity affects metabolism well beyond fatty acid oxidation, regulating biochemical pathways associated with amino acid metabolism and redox status. That select

  15. Site specific incorporation of heavy atom-containing unnatural amino acids into proteins for structure determination

    DOEpatents

    Xie, Jianming; Wang, Lei; Wu, Ning; Schultz, Peter G.

    2008-07-15

    Translation systems and other compositions including orthogonal aminoacyl tRNA-synthetases that preferentially charge an orthogonal tRNA with an iodinated or brominated amino acid are provided. Nucleic acids encoding such synthetases are also described, as are methods and kits for producing proteins including heavy atom-containing amino acids, e.g., brominated or iodinated amino acids. Methods of determining the structure of a protein, e.g., a protein into which a heavy atom has been site-specifically incorporated through use of an orthogonal tRNA/aminoacyl tRNA-synthetase pair, are also described.

  16. First total synthesis of (5Z,9Z)-(±)-2-methoxy-5,9-octadecadienoic acid, a marine derived methoxylated analog of taxoleic acid

    PubMed Central

    Carballeira, Néstor M.; O’Neill, Rosann; Silva, Diana

    2008-01-01

    The first total synthesis for the sponge derived (5Z,9Z)-(±)-2-methoxy-5,9-octadecadienoic acid, an analog of taxoleic acid, was accomplished in seven steps and in a 10% overall yield. It was again corroborated that the best strategy to prepare these cis,cis dimethylene interrupted double bonds is the double-alkyne bromide coupling reaction of 1,5-hexadiyne, which provides the advantage of achieving a 100% cis stereochemical purity for both double bonds after hydrogenation under Lindlar conditions. The α-methoxy functionality was best prepared via the Mukaiyama reaction of (4Z,8Z)-heptadecadienal with trimethylsilyl cyanide and triethylamine followed by acid hydrolysis. Selective methylation of the hydroxyl group of (5Z,9Z)-(±)-2-hydroxy-5,9-octadecadienoic acid was achieved with sodium hydride/methyl iodide when tetrahydrofuran was used as solvent. Complete spectral data is presented, for the first time, for this unusual marine α-methoxylated fatty acid. PMID:18761005

  17. Amino Acid and Protein Metabolism in Bermuda Grass During Water Stress 12

    PubMed Central

    Barnett, N. M.; Naylor, A. W.

    1966-01-01

    The ability of Arizona Common and Coastal Bermuda grass [Cynodon dactylon (L.) Pers.] to synthesize amino acids and proteins during water stress was investigated. Amino acids were continually synthesized during the water stress treatments, but protein synthesis was inhibited and protein levels decreased. Water stress induced a 10- to 100-fold accumulation of free proline in shoots and a 2- to 6-fold accumulation of free asparagine, both of which are characteristic responses of water-stressed plants. Valine levels increased, and glutamic acid and alanine levels decreased. 14C labeling experiments showed that free proline turns over more slowly than any other free amino acid during water stress. This proline is readily synthesized and accumulated from glutamic acid. It is suggested that during water stress free proline functions as a storage compound. No significant differences were found in the amino acid and protein metabolism of the 2 varieties of Bermuda grass. PMID:16656387

  18. Determination of sulfur in human hair using high resolution continuum source graphite furnace molecular absorption spectrometry and its correlation with total protein and albumin

    NASA Astrophysics Data System (ADS)

    Ozbek, Nil; Baysal, Asli

    2017-04-01

    Human hair is a valuable contributor for biological monitoring. It is an information storage point to assess the effects of environmental, nutritional or occupational sources on the body. Human proteins, amino acids or other compounds are among the key components to find the sources of different effects or disorders in the human body. Sulfur is a significant one of these compounds, and it has great affinity to some metals and compounds. This property of the sulfur affects the human health positively or negatively. In this manuscript, sulfur was determined in hair samples of autistic and age-match control group children via molecular absorption of CS using a high-resolution continuum source graphite furnace atomic absorption spectrometer. For this purpose, hair samples were appropriately washed and dried at 75 °C. Then samples were dissolved in microwave digestion using HNO3 for sulfur determination. Extraction was performed with HCl hydrolysation by incubation for 24 h at 110 °C for total protein and albumin determination. The validity of the method for the sulfur determination was tested using hair standard reference materials. The results were in the uncertainty limits of the certified values at 95% confidence level. Finally correlation of sulfur levels of autistic children's hair with their total protein and albumin levels were done.

  19. Expression of cationic amino acid transporters, carcass traits, and performance of growing pigs fed low-protein amino acid-supplemented versus high protein diets.

    PubMed

    Morales, A; Grageola, F; García, H; Araiza, A; Zijlstra, R T; Cervantes, M

    2013-10-18

    Free amino acids (AA) appear to be absorbed faster than protein-bound AA (PB-AA). We conducted an experiment to assess the effect of feeding pigs with a partially free (F-AA) or totally PB-AA diet on expression of selected genes and performance of pigs. The expression of cationic AA transporters b(0,+) and CAT-1 in intestinal mucosa, liver, and longissimus (LM) and semitendinosus (SM) muscles, as well as that of myosin in LM and SM, was analyzed. Twelve pigs (31.7 ± 2.7 kg) were used. The F-AA diet was based on wheat, supplemented with 0.59% L-Lys, 0.33% L-Thr, and 0.10% DL-Met. The PB-AA diet was formulated with wheat-soybean meal. Average daily feed intake was 1.53 kg per pig. The expression of b(0,+) and CAT-1 was analyzed in jejunal and ileal mucosa, liver, LM, and SM; myosin expression was also analyzed in both muscles. Pigs fed the PB-AA diet tended to have higher weight gain and feed efficiency (P < 0.10), and had thinner back fat (P = 0.02). The expression of b(0,+) was higher (P < 0.01) in jejunum but lower (P < 0.01) in the liver of pigs fed the F-AA diet; CAT-1 tended to be lower in liver but higher in LM of PB-AA pigs. Myosin expression was not affected. Intestinal AA absorption was faster in pigs fed the F-AA diet, but AA uptake by the liver seemed to be faster in pigs fed the PB-AA. Performance and expression of AA transporters and myosin suggest that the dietary content of free or protein-bound AA does not affect their availability for protein synthesis in pigs.

  20. Apparent Consumption vs. Total Consumption--A Lead-Acid Battery Case Study

    USGS Publications Warehouse

    Wilburn, David R.; Buckingham, David A.

    2006-01-01

    Introduction: This report compares estimates of U.S. apparent consumption of lead with estimates of total U.S. consumption of this mineral commodity from a materials flow perspective. The difference, attributed to the amount of lead contained in imported and exported products, was found to be significant for this sector. The study also assesses the effects of including mineral commodities incorporated in manufactured products on the interpretation of observed trends in minerals consumption and trade. Materials flow is a systems approach to understanding what happens to the materials we use from the time a material is extracted, through its processing and manufacturing, to its ultimate disposition. The U.S. Geological Survey (USGS) provides accurate and detailed mineral production and mineral commodity consumption statistics that are essential for government, nongovernment organizations, and the public to gain a better understanding of how and where materials are used and their effect on the environment and society. Published statistics on mineral apparent consumption are limited to estimates of consumption of raw material forms (ore, concentrate, and [or] refined metal). For this study, apparent consumption is defined as mine production + secondary refined production + imports (concentrates and refined metal) ? exports (concentrates and refined metal) + adjustments for government and industry stock changes. These estimates do not account for the amount of mineral commodities contained in manufactured products that are imported to the United States, nor do they deduct the amount of these mineral commodities contained in manufactured products that are exported from the United States. When imports or exports of manufactured products contribute significantly to the total use of a particular raw material, an estimate of consumption that does not consider the incorporated forms of these mineral commodities within imported or exported manufactured products can be either

  1. Allied Health Chemistry Laboratory: Amino Acids, Insulin, Proteins, and Skin

    ERIC Educational Resources Information Center

    Dever, David F.

    1975-01-01

    Presents a laboratory experiment specifically designed for allied health students. The students construct molecular models of amino acids, extract amino acids from their skin with hot water, and chromatographically analyze the skin extract and hydrolyzed insulin. (MLH)

  2. Partial purification of fatty-acid binding protein by ammonium sulphate fractionation.

    PubMed

    Avanzati, B; Catalá, A

    1983-07-01

    By fractionation of rat liver cytosol with 70% saturation ammonium sulphate, a soluble fraction showing high affinity for oleic acid was obtained. The binding of oleic acid to this fraction was inhibited by flavaspidic acid. The molecular weight of the main protein present in this fraction was 12 000 as determined by SDS-poly-acrylamide-gel electrophoresis. This soluble fraction stimulated the transfer of oleic acid from microsomes to phosphatidylcholine liposomes as demonstrated by a transfer assay in vitro. The behaviour of this fraction is similar to that described for fatty-acid binding protein.

  3. Influence of Bleaching on Flavor of 34% Whey Protein Concentrate and Residual Benzoic Acid Concentration in Dried Whey Proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous studies have shown that bleaching negatively affects the flavor of 70% whey protein concentrate (WPC70), but bleaching effects on lower-protein products have not been established. Benzoyl peroxide (BP), a whey bleaching agent, degrades to benzoic acid (BA) and may elevate BA concentrations...

  4. Partial replacement of saturated fatty acids with almonds or walnuts lowers total plasma cholesterol and low-density-lipoprotein cholesterol.

    PubMed

    Abbey, M; Noakes, M; Belling, G B; Nestel, P J

    1994-05-01

    Sixteen normolipidemic male volunteers aged 41 +/- 9 y (mean +/- SD) consumed a diet providing 36% of energy as fat (92 g fat/d) for 9 wk. A daily supplement of nuts (providing half of the total fat intake) was provided against a common background diet. In the first 3-wk period the background diet was supplemented with raw peanuts (50 g/d), coconut cubes (40 g/d), and a coconut confectionary bar (50 g/d), designed to provide 47 g fat with a ratio of polyunsaturated to monounsaturated to saturated fatty acids (P:M:S) to match the Australian diet (reference diet). During the following 3 wk the background diet was supplemented with monounsaturated fatty acid-rich raw almonds (84 g/d), equivalent to 46 g fat, and during the final 3-wk period the background diet was supplemented with polyunsaturated fatty acid-rich walnuts (68 g/d), equivalent to 46 g fat. Compared with the reference diet there were significant reductions in total and LDL cholesterol, 7% and 10%, respectively, after supplementation with almonds, and 5% and 9%, respectively, after supplementation with walnuts.

  5. 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 .

  6. Maternal micronutrients and omega 3 fatty acids affect placental fatty acid desaturases and transport proteins in Wistar rats.

    PubMed

    Wadhwani, Nisha S; Dangat, Kamini D; Joshi, Asmita A; Joshi, Sadhana R

    2013-03-01

    Adequate supply of LCPUFA from maternal plasma is crucial for fetal normal growth and development. The present study examines the effect of maternal micronutrients (folic acid and vitamin B12) and omega 3 fatty acids on placental mRNA levels of fatty acid desaturases (Δ5 and Δ6) and transport proteins. Pregnant female rats were divided into 6 groups at 2 levels of folic acid both in the presence and absence of vitamin B12. Both the vitamin B12 deficient groups were supplemented with omega 3 fatty acid. Maternal vitamin B12 deficiency reduced placental mRNA and protein levels of Δ5 desaturase, mRNA levels of FATP1 and FATP4 (p<0.05 for all) as compared to control while omega 3 fatty acid supplementation normalized the levels. Our data for the first time indicates that altered maternal micronutrients and omega 3 fatty acids play a key role in regulating fatty acid desaturase and transport protein expression in placenta.

  7. Total tract nutrient digestion and milk fatty acid profile of dairy cows fed diets containing different levels of whole raw soya beans.

    PubMed

    Venturelli, B C; de Freitas Júnior, J E; Takiya, C S; de Araújo, A P C; Santos, M C B; Calomeni, G D; Gardinal, R; Vendramini, T H A; Rennó, F P

    2015-12-01

    Whole oilseeds such as soya beans have been utilized in dairy rations to supply additional fat and protein. However, antinutritional components contained in soya beans, such as trypsin inhibitors and haemagglutinins (lectins) may alter digestibility of nutrients and consequently affect animal performance. The objective of the present experiment was to quantify the effect of different levels of whole raw soya beans in diets of dairy cows on nutrient intake, total tract digestion, nutrient balances and milk yield and composition. Sixteen mid to late-lactation cows (228 ± 20 days in milk; mean ± SD) were used in four replicated 4 × 4 Latin square design experiment with 21-d periods. Cows were assigned to each square according to milk yield and DIM. The animals were randomly allocated to treatments: control (without soya beans addition; CO), WS9, WS18 and WS27, with addition of 9%, 18% and 27% of whole raw soya bean in diet on a dry matter (DM) basis respectively. All diets contained identical forage and concentrate components and consisted of maize silage and concentrate based on ground corn and soya beans at a ratio of 60:40. There were no differences in OM, CP, NDF and NEL intakes (kg/day and MJ/day) among the treatments (p > 0.05). However, DM and NFC intakes were negatively affected (p = 0.04 and p < 0.01, respectively) and ether extract (EE) intake was positively affected (p < 0.01). Total tract digestion increased linearly with whole raw soya beans for EE (p < 0.01) and NDF (p = 0.01). The excretion (kg/day) of digested soya beans grains increased linearly according to addition of whole raw soya beans. However, the nutritive characteristics of excreted grains were not altered. Milk (kg), milk lactose (kg) and protein (kg) yield decreased linearly (p < 0.01, p < 0.01 and p = 0.04, respectively) milk fat content (%) increased linearly (p < 0.01) with whole raw soya beans inclusion. Increasing addition of whole raw soya beans affected milk fatty acid profile

  8. 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

  9. Amino acid supplementation of calf milk replacers containing plasma protein.

    PubMed

    Morrison, S Y; Campbell, J M; Drackley, J K

    2017-03-22

    We determined the effects of calf milk replacers containing 0, 5, or 10% bovine plasma protein (PP), either without or with the supplemental amino acids (AA) Ile and Thr, on growth and health of male Holstein calves (n = 104) for 56 d. Milk replacers were formulated to contain 22% crude protein (CP), 20% fat, and 2.0% Lys. Milk replacers (12.5% solids) were fed at a rate of 1.5% of body weight (BW) on a dry matter basis during wk 1 and 1.75% of BW beginning on d 8. Starter was introduced on d 36 so that effects of PP and AA balance in milk replacers could be isolated. Intake, respiratory scores, and fecal scores were measured daily. Body weight and stature were measured weekly and blood serum samples were obtained during wk 4. Treatments had no effects on intakes of dry matter, CP, or metabolizable energy. During wk 6 and 8, BW was less as PP inclusion increased without AA supplementation compared with the other treatments. In wk 7, calves fed the higher level of PP without AA had lower BW than calves fed either the lower level of PP without supplemented AA or the higher inclusion of PP with supplemented AA. Average daily gain and gain:feed were lowest for calves fed the higher inclusion of PP without supplemented AA; heart girth in wk 7 was smallest for those calves. During the first 21 d, occurrence of scours was greater in calves fed the control milk replacer than in calves fed milk replacers containing the higher inclusion of PP either without or with supplemental AA. Occurrence of scours was also greater for the lower inclusion of PP compared with the higher inclusion of PP when AA were supplemented. Throughout the 56-d experiment, the chance of antibiotic treatment was greater for calves fed the control milk replacer than for all other treatments except the higher inclusion of PP without supplemental AA. Additionally, chance of antibiotic treatment was greater for the higher inclusion of PP without supplemental AA than for other milk replacers with PP. Calves

  10. Secretion of whey acidic protein and cystatin is down regulated at mid-lactation in the red kangaroo (Macropus rufus)

    USGS Publications Warehouse

    Nicholas, K.R.; Fisher, J.A.; Muths, E.; Trott, J.; Janssens, P.A.; Reich, C.; Shaw, D.C.

    2001-01-01

    Milk collected from the red kangaroo (Macropus rufus) between day 100 and 260 of lactation showed major changes in milk composition at around day 200 of lactation, the time at which the pouch young begins to temporarily exit the pouch and eat herbage. The carbohydrate content of milk declined abruptly at this time and although there was only a small increase in total protein content, SDS PAGE analysis of milk revealed asynchrony in the secretory pattern of individual proteins. The levels of ??-lactalbumin, ??-lactoglobulin, serum albumin and transferrin remain unchanged during lactation. In contrast, the protease inhibitor cystatin, and the putative protease inhibitor whey acidic protein (WAP) first appeared in milk at elevated concentrations after approximately 150 days of lactation and then ceased to be secreted at approximately 200 days. In addition, a major whey protein, late lactation protein, was first detected in milk around the time whey acidic protein and cystatin cease to be secreted and was present at least until day 260 of lactation. The co-ordinated, but asynchronous secretion of putative protease inhibitors in milk may have several roles during lactation including tissue remodelling in the mammary gland and protecting specific proteins in milk required for physiological development of the dependent young. ?? 2001 Elsevier Science Inc.

  11. Development of a strategy for the total chemical synthesis of an allergenic protein: the peach LTP Pru p 3.

    PubMed

    Buhler, Sofie; Akkerdaas, Jaap H; A Pertinhez, Thelma; Van Ree, Ronald; Dossena, Arnaldo; Sforza, Stefano; Tedeschi, Tullia

    2017-02-10

    The possibility to obtain allergenic proteins by means of total chemical synthesis would be a big step forward in the development of cures to food allergy and in the study of the mechanism of allergic reactions, because this would allow to achieve control at the molecular level over the structure of the product and to study its relationship with the allergenic activity in fine details. This is instead not possible by using allergens produced by extraction from natural sources or by recombinant DNA techniques. In this work, we aimed to test for the first time the feasibility of the total chemical synthesis of an allergenic protein. Pru p 3, the most studied member of the family of lipid transfer proteins, relevant plant food pan-allergens, was used as model target. Strategies for the convergent assembly of the target protein, starting from five peptide fragments to be bound by means of either native chemical ligation or peptide hydrazide ligation, followed by desulfurization, to achieve ligations at alanine, were developed and tested. All the reaction conditions were set up and optimized. Two large peptides covering the two halves of the protein sequence were synthesized and structurally characterized by means of circular dichroism, and their immunogenicity was proved by means of immunoblot, using antibodies against Pru p 3, and immunoCAP inhibition tests. Finally, the five peptides were bound together to produce the whole protein stretch. The obtained results demonstrate the feasibility of total chemical synthesis as a new way to obtain pure allergens. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

  12. Classification of pseudo pairs between nucleotide bases and amino acids by analysis of nucleotide-protein complexes.

    PubMed

    Kondo, Jiro; Westhof, Eric

    2011-10-01

    Nucleotide bases are recognized by amino acid residues in a variety of DNA/RNA binding and nucleotide binding proteins. In this study, a total of 446 crystal structures of nucleotide-protein complexes are analyzed manually and pseudo pairs together with single and bifurcated hydrogen bonds observed between bases and amino acids are classified and annotated. Only 5 of the 20 usual amino acid residues, Asn, Gln, Asp, Glu and Arg, are able to orient in a coplanar fashion in order to form pseudo pairs with nucleotide bases through two hydrogen bonds. The peptide backbone can also form pseudo pairs with nucleotide bases and presents a strong bias for binding to the adenine base. The Watson-Crick side of the nucleotide bases is the major interaction edge participating in such pseudo pairs. Pseudo pairs between the Watson-Crick edge of guanine and Asp are frequently observed. The Hoogsteen edge of the purine bases is a good discriminatory element in recognition of nucleotide bases by protein side chains through the pseudo pairing: the Hoogsteen edge of adenine is recognized by various amino acids while the Hoogsteen edge of guanine is only recognized by Arg. The sugar edge is rarely recognized by either the side-chain or peptide backbone of amino acid residues.

  13. Seed storage protein deficiency improves sulfur amino acid content in common bean (Phaseolus vulgaris L.): redirection of sulfur from gamma-glutamyl-S-methyl-cysteine.

    PubMed

    Taylor, Meghan; Chapman, Ralph; Beyaert, Ronald; Hernández-Sebastià, Cinta; Marsolais, Frédéric

    2008-07-23

    The contents of sulfur amino acids in seeds of common bean ( Phaseolus vulgaris L.) are suboptimal for nutrition. They accumulate large amounts of a gamma-glutamyl dipeptide of S-methyl-cysteine, a nonprotein amino acid that cannot substitute for methionine or cysteine in the diet. Protein accumulation and amino acid composition were characterized in three genetically related lines integrating a progressive deficiency in major seed storage proteins, phaseolin, phytohemagglutinin, and arcelin. Nitrogen, carbon, and sulfur contents were comparable among the three lines. The contents of S-methyl-cysteine and gamma-glutamyl-S-methyl-cysteine were progressively reduced in the mutants. Sulfur was shifted predominantly to the protein cysteine pool, while total methionine was only slightly elevated. Methionine and cystine contents (mg per g protein) were increased by up to ca. 40%, to levels slightly above FAO guidelines on amino acid requirements for human nutrition. These findings may be useful to improve the nutritional quality of common bean.

  14. Amino acid-selective isotope labeling of proteins for nuclear magnetic resonance study: proteins secreted by Brevibacillus choshinensis.

    PubMed

    Tanio, Michikazu; Tanaka, Rikou; Tanaka, Takeshi; Kohno, Toshiyuki

    2009-03-15

    Here we report the first application of amino acid-type selective (AATS) isotope labeling of a recombinant protein secreted by Brevibacillus choshinensis for a nuclear magnetic resonance (NMR) study. To prepare the 15N-AATS-labeled protein, the transformed B. choshinensis was cultured in 15N-labeled amino acid-containing C.H.L. medium, which is commonly used in the Escherichia coli expression system. The analyses of the 1H-15N heteronuclear single quantum coherence (HSQC) spectra of the secreted proteins with a 15N-labeled amino acid demonstrated that alanine, arginine, asparagine, cysteine, glutamine, histidine, lysine, methionine, and valine are suitable for selective labeling, although acidic and aromatic amino acids are not suitable. The 15N labeling for glycine, isoleucine, leucine, serine, and threonine resulted in scrambling to specific amino acids. These results indicate that the B. choshinensis expression system is an alternative tool for AATS labeling of recombinant proteins, especially secretory proteins, for NMR analyses.

  15. Evolutionary diversification of the avian fatty acid-binding proteins.

    PubMed

    Hughes, Austin L; Piontkivska, Helen

    2011-12-15

    Phylogenetic analysis of avian and other vertebrate fatty acid binding proteins (FABPs) supported the hypothesis that several gene duplications within this family occurred prior to the most recent common ancestor (MRCA) of tetrapods and bony fishes. The chicken genome encodes two liver-expressed FABPs: (1) L-FABP or FABP1; and (2) Lb-FABP. We propose that the latter be designated FABP10, because in our phylogenetic analysis it clustered with zebrafish FABP10. Bioinformatic analysis of across-tissue gene expression patterns in the chicken showed some congruence with phylogenetic relationships. On the basis of expression, chicken FABP genes seemed to form two major groups: (1) a cluster of genes many of which showed predominant expression in the digestive system (FABP1, FABP2, FABP6, FABP10, RBP1, and CRABP1); and (2) a cluster of genes most of which had predominant expression in tissues other than those of the digestive system, including muscle and the central nervous system (FABP3, FABP4, FABP5, FABP7, and PMP2). Since these clusters corresponded to major clusters in the phylogenetic tree as well, it seems a plausible hypothesis that the earliest duplication in the vertebrate FABP family led to the divergence of a gut-specialized gene from a gene expressed mainly in nervous and muscular systems. Data on gene expression in livers of two lines of chickens selected for high growth and low growth showed differences between FABP1 and FABP10 expressions in the liver, supporting the hypothesis of functional divergence between the two chicken liver-expressed FABPs related to food intake.

  16. Amino Acid Composition of Protein-Enriched Dried Pasta:
Is It Suitable for a Low-Carbohydrate Diet?

    PubMed Central

    Vidrih, Rajko

    2015-01-01

    Summary Today, obesity is one of the major health problems, a so-called epidemic of the developed world. Obesity arises through an imbalance between energy intake and energy expenditure, so it is important for products to have a balanced nutritional composition. The aim of this study is to prepare high-protein pasta with high nutritional quality, with emphasis on its amino acid composition, as ordinary durum pasta lacks lysine and threonine. Ordinary durum wheat pasta contains, on average, 77% carbohydrate, and can have even less than 10% protein. It is therefore often excluded from normal energy-restricted diets, and especially from low-carbohydrate diets. In this study pasta that can satisfy the nutritional requirements of a low-carbohydrate diet and is suitable for daily use was developed and evaluated. Protein-enhanced pasta was produced by adding high amounts of plant protein extract (40% dry matter) without (plain high-protein pasta) or with 3% dried spinach powder (high-protein spinach pasta) to durum wheat semolina. According to the sensory analysis data, the addition of 40% of plant protein extract satisfied sensory and nutritional requirements, allowing further development and evaluation for possible marketing. This analysis shows that these high-protein neutral and spinach pasta contain 36.4 and 39.6 g of protein per 100 g of dry mass, 12.07 and 14.70 g of total essential amino acids per 100 g of dry mass, and a high content of branched-chain amino acids, i.e. 5.54 and 6.65 g per 100 g of dry mass, respectively. This therefore represents a true alternative to durum wheat pasta for low-carbohydrate diets. PMID:27904361

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

    PubMed Central

    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. PMID:26208104

  18. 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.

  19. 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.

  20. 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.

  1. Total lipids and fatty acids composition of the coastal and the deep-sea common octopus (Octopus vulgaris) populations: a comparative study.

    PubMed

    Ben-Youssef, Saoussen; Selmi, Salah; Ezzeddine-Najai, Sofia; Sadok, Saloua

    2008-01-01

    The aim of this work was to investigate biochemical differences between Octopus vulgaris caught off costal zone and from the deep-sea of the Golf of Gabès (South coast of Tunisia). In both fishing grounds, octopus total lipids constituted almost 1.5% of wet tissue showing no significant difference (p < 0.05). The percentage distribution of fatty acids was not significantly different, neither between males and females, nor between both areas. Polyunsaturated fatty acids constituted about 50 % of the total fatty acids. Docosahexaenoic (DHA; C22:6 omega 3), eicosapentaenoic (EPA; C20:5 omega 3) and the arachidonic acids (C20:4) were the most important of this group with percentages of 25, 14 and 10% respectively. The saturated fraction constituted almost 30% of the total fatty acids. The most dominant saturated fatty acids were palmitic acid (C16:0) and stearic acid (C18:0), with 18% and 7% respectively. The monounsaturated content was found to contribute only 10% of the total fatty acids. Most of the monounsaturated fat was present as oleic acid (C18:1) and palmitoleic acid (C16:1) with 2.5% and 1.5% respectively. The presence of arachidonic acid in substantial proportions with an omega 3 to omega 6 ratios of 3.9 to 1 is of special interest because of the role of cephalopods in the traditional Mediterranean diet.

  2. Understanding the synergistic effect of arginine and glutamic acid mixtures on protein solubility.

    PubMed

    Shukla, Diwakar; Trout, Bernhardt L

    2011-10-20

    Understanding protein solubility is a key part of physical chemistry. In particular, solution conditions can have a major effect, and the effect of multiple cosolutes is little understood. It has been shown that the simultaneous addition of L-arginine hydrochloride and L-glutamic acid enhances the maximum achievable solubility of several poorly soluble proteins up to 4-8 times (Golovanov et. al, J. Am. Chem. Soc., 2004, 126, 8933-8939) and reduces the intermolecular interactions between proteins. The observed solubility enhancement is negligible for arginine and glutamic acid solutions as compared to the equimolar mixtures. In this study, we have established the molecular mechanism behind this observed synergistic effect of arginine and glutamic acid mixtures using preferential interaction theory and molecular dynamics simulations of Drosophilia Su(dx) protein (ww34). It was found that the protein solubility enhancement is related to the relative increase in the number of arginine and glutamic acid molecules around the protein in the equimolar mixtures due to additional hydrogen bonding interactions between the excipients on the surface of the protein when both excipients are present. The presence of these additional molecules around the protein leads to enhanced crowding, which suppresses the protein association. These results highlight the role of additive-additive interaction in tuning the protein-protein interactions. Furthermore, this study reports a unique behavior of additive solutions, where the presence of one additive in solution affects the concentration of another on the protein surface.

  3. Nucleic acid binding proteins in highly purified Creutzfeldt-Jakob disease preparations.

    PubMed Central

    Sklaviadis, T; Akowitz, A; Manuelidis, E E; Manuelidis, L

    1993-01-01

    The nature of the infectious agent causing human Creutzfeldt-Jakob disease (CJD), a slowly progressive dementia, is controversial. As in scrapie, no agent-specific proteins or nucleic acids have been identified. However, biological features of exponential replication and agent strain variation, as well as physical size and density data, are most consistent with a viral structure--i.e., a nucleic acid-protein complex. It is often assumed that nuclease treatment, which does not reduce infectious titer, leaves no nucleic acids of > 50 bp. However, nucleic acids of 500-6000 bp can be extracted from highly purified infectious complexes with a mass of approximately 1.5 x 10(7) daltons. It was therefore germane to search for nucleic acid binding proteins that might protect an agent genome. We here use Northwestern blotting to show that there are low levels of nonhistone nucleic acid binding proteins in highly purified infectious 120S gradient fractions. Several nucleic acid binding proteins were clearly host encoded, whereas others were apparent only in CJD, but not in parallel preparations from uninfected brain. Small amounts of residual host Gp34 (prion protein) did not bind any 32P-labeled nucleic acid probes. Most of the minor "CJD-specific" proteins had an acidic pI, a characteristic of many viral core proteins. Such proteins deserve further study, as they probably contribute to unique properties of resistance described for these agents. It remains to be seen if any of these proteins are agent encoded. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8516321

  4. Amino acid composition, molecular weight distribution and gel electrophoresis of walnut (Juglans regia L.) proteins and protein fractionations.

    PubMed

    Mao, Xiaoying; Hua, Yufei; Chen, Guogang

    2014-01-27

    As a by-product of oil production, walnut proteins are considered as an additional source of plant protein for human food. To make full use of the protein resource, a comprehensive understanding of composition and characteristics of walnut proteins are required. Walnut proteins have been fractionated and characterized in this study. Amino acid composition, molecular weight distribution and gel electrophoresis of walnut proteins and protein fractionations were analyzed. The proteins were sequentially separated into four fractions according to their solubility. Glutelin was the main component of the protein extract. The content of glutelin, albumin, globulin and prolamin was about 72.06%, 7.54%, 15.67% and 4.73% respectively. Glutelin, albumin and globulin have a balanced content of essential amino acids, except for methionine, with respect to the FAO pattern recommended for adults. SDS-PAGE patterns of albumin, globulin and glutelin showed several polypeptides with molecular weights 14.4 to 66.2 kDa. The pattern of walnut proteins in two-dimension electrophoresis (2-DE) showed that the isoelectric point was mainly in the range of 4.8-6.8. The results of size exclusion chromatogram indicated molecular weight of the major components of walnut proteins were between 3.54 and 81.76 kDa.

  5. 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.

  6. First total synthesis and antileishmanial activity of (Z)-16-methyl-11-heptadecenoic acid, a new marine fatty acid from the sponge Dragmaxia undata

    PubMed Central

    Carballeira, Néstor M.; Montano, Nashbly; Cintrón, Gabriel A.; Márquez, Carmary; Rubio, Celia Fernández; Prada, Christopher Fernández; Balaña-Fouce, Rafael

    2010-01-01

    The first total synthesis for the (Z)-16-methyl-11-heptadecenoic acid, a novel fatty acid from the sponge Dragmaxia undata, was accomplished in seven steps and in a 44% overall yield. The use of (trimethylsilyl)acetylene was key in the synthesis. Based on a previous developed strategy in our laboratory the best synthetic route towards the title compound was first acetylide coupling of (trimethylsilyl)acetylene to the long-chain protected 10-bromo-1-decanol followed by a second acetylide coupling to the short-chain 1-bromo-4-methylpentane, which resulted in higher yields. Complete spectral data is also presented for the first time for this recently discovered fatty acid and the cis double bond stereochemistry of the natural acid was established. The title compound displayed antiprotozoal activity against Leishmania donovani (IC50 = 165.5 ± 23.4 µM) and inhibited the leishmania DNA topoisomerase IB enzyme (LdTopIB) with an IC50 = 62.3 ± 0.7 µM. PMID:21129369

  7. Fast computational methods for predicting protein structure from primary amino acid sequence

    DOEpatents

    Agarwal, Pratul Kumar

    2011-07-19

    The present invention provides a method utilizing primary amino acid sequence of a protein, energy minimization, molecular dynamics and protein vibrational modes to predict three-dimensional structure of a protein. The present invention also determines possible intermediates in the protein folding pathway. The present invention has important applications to the design of novel drugs as well as protein engineering. The present invention predicts the three-dimensional structure of a protein independent of size of the protein, overcoming a significant limitation in the prior art.

  8. Diurnal variations of total carbon, dicarboxylic acids, ketoacids and α-dicarbonyls in aerosols in the northern vicinity of Beijing

    NASA Astrophysics Data System (ADS)

    He, N.; Kawamura, K.; Okuzawa, K.; Kanaya, Y.; Wang, Z. F.

    2013-06-01

    Aerosol samples (TSP, n=58) were collected on day- and night-time basis at Mangshan in the north of Beijing, China in autumn 2007 to better understand the status of air quality and the influence of urban pollutants in the northern vicinity of Beijing. The samples were analyzed for aerosol mass, total carbon (TC), low molecular weight α, ω-dicarboxylic acids (C2-C12), ketoacids (ωC2-ωC9, pyruvic acid), α-dicarbonyls (glyoxal and methylglyoxal), as well as aromatic (phthalic, iso- and tere-phthalic) diacids. Aerosol mass and TC concentrations are higher in daytime than in nighttime. TC/aerosol mass ratios in this study are lower than those reported in megacities in East Asia, but higher than those reported in marine aerosols. Molecular distributions of diacids demonstrated that oxalic (C2) acid was the most abundant species, comprising 38-77% of total diacids, followed by succinic (C4) and malonic (C3) acids. For most compounds, the concentrations were higher in daytime than nighttime, indicating that diacids are produced in daytime by photochemical oxidation of organic precursors emitted from anthropogenic sources in Beijing during the transport to Mangshan area by the northward wind. However, we found that C2 concentrations are higher in nighttime than in daytime. A positive correlation of C2 to glyoxylic acid (ωC2) was obtained at night when relative humidity increased up to 100%, suggesting that aqueous phase production of C2 occurs in nighttime via the oxidation of ωC2. Depletion of C2 by photolysis of Fe-oxalato complexes might be another reason for the lower concentrations of C2 in daytime samples. High phthalic acid/C4 ratios in the aerosol samples suggest that automobile combustion and coal burning products are important sources, which are subjected to photochemical oxidation during the atmospheric transport of urban aerosols from Beijing. In contrast, higher concentrations of methylglyoxal in nighttime than daytime may suggest that isoprene emitted

  9. Acidogenic fermentation characteristics of different types of protein-rich substrates in food waste to produce volatile fatty acids.

    PubMed

    Shen, Dongsheng; Yin, Jun; Yu, Xiaoqin; Wang, Meizhen; Long, Yuyang; Shentu, Jiali; Chen, Ting

    2017-03-01

    In this study, tofu and egg white, representing typical protein-rich substrates in food waste based on vegetable and animal protein, respectively, were investigated for producing volatile fatty acids (VFAs) by acidogenic fermentation. VFA production, composition, conversion pathways and microbial communities in acidogenesis from tofu and egg white with and without hydrothermal (HT) pretreatment were compared. The results showed HT pretreatment could improve the VFA production of tofu but not for egg white. The optimum VFA yields were 0.46g/gVS (tofu with HT) and 0.26g/gVS (egg white without HT), respectively. Tofu could directly produce VFAs through the Stickland reaction, while egg white was converted to lactate and VFAs simultaneously. About 30-40% of total protein remained in all groups after fermentation. Up to 50% of the unconverted soluble protein in the HT groups was protease. More lactate-producing bacteria, mainly Leuconostoc and Lactobacillus, were present during egg white fermentation.

  10. Purification, properties and amino acid sequence of a low-Mr abundant seed protein from pea (Pisum sativum L.).

    PubMed

    Gatehouse, J A; Gilroy, J; Hoque, M S; Croy, R R

    1985-01-01

    The seeds of pea (Pisum sativum L.) contain several proteins in the albumin solubility fraction that are significant components of total cotyledonary protein (5-10%) and are accumulated in developing seeds concurrently with storage-protein synthesis. One of these proteins, of low Mr and designated 'Psa LA', has been purified, characterized and sequenced. Psa LA has an Mr of 11000 and contains polypeptides of Mr 6000, suggesting that the protein molecules are dimeric. The amino acid sequence contains 54 residues, with a high content (10/54) of asparagine/aspartate. It has no inhibitory action towards trypsin or chymotrypsin, and is distinct from the inhibitors of those enzymes found in pea seeds, nor does it inhibit hog pancreatic alpha-amylase. The protein contains no methionine, but significant amounts of cysteine (four residues per polypeptide), suggesting a possible role as a sulphur storage protein. However, its sequence is not homologous with low-Mr (2S) storage proteins from castor bean (Ricinus communis) or rape (Brassica napus). Psa LA therefore represents a new type of low-Mr seed protein.

  11. Protein-surfactant interactions at hydrophobic interfaces studied with total internal reflection fluorescence correlation spectroscopy (TIR-FCS).

    PubMed

    Sonesson, Andreas W; Blom, Hans; Hassler, Kai; Elofsson, Ulla M; Callisen, Thomas H; Widengren, Jerker; Brismar, Hjalmar

    2008-01-15

    The aim of this work was to study the dynamics of proteins near solid surfaces in the presence or absence of competing surfactants by means of total internal reflection fluorescence correlation spectroscopy (TIR-FCS). Two different proteins were studied, bovine serum albumin (BSA) and Thermomyces lanuginosus lipase (TLL). A nonionic/anionic (C12E6/LAS) surfactant composition was used to mimic a detergent formulation and the surfaces used were C18 terminated glass. It was found that with increasing surfactant concentrations the term in the autocorrelation function (ACF) representing surface binding decreased. This suggested that the proteins were competed off the hydrophobic surface by the surfactant. When fitting the measured ACF to a model for surface kinetics, it was seen that with raised C12E6/LAS concentration, the surface interaction rate increased for both proteins. Under these experimental conditions this meant that the time the protein was bound to the surface decreased. At 10 microM C12E6/LAS the surface interaction was not visible for BSA, whereas it was still distinguishable in the ACF for TLL. This indicated that TLL had a higher affinity than BSA for the C18 surface. The study showed that TIR-FCS provides a useful tool to quantify the surfactant effect on proteins adsorption.

  12. Refinement of Generalized Born Implicit Solvation Par