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Sample records for acid sensitive protein

  1. Ascorbic acid glycation of lens proteins produces UVA sensitizers similar to those in human lens

    Soluble calf lens proteins were extensively glycated during a 4 week incubation with ascorbic acid in the presence of oxygen. Amino acids analysis of the dialyzed proteins removed at weekly intervals showed an increasing loss of lysine, arginine and histidine, consistent with the extensive protein cross-linking observed. Irradiation of the dialyzed samples with UVA light (1.0 kJ/cm2 total illumination through a 338 nm cutoff filter) caused an increasing loss of tryptophan, an additional loss of histidine and the production of micromolar concentrations of hydrogen peroxide. No alteration in amino acid content and no photolytic effects were seen in proteins incubated without ascorbic acid in proteins incubated with glucose for 4 weeks. The rate of hydrogen peroxide formation was linear with each glycated sample with a maximum production of 25 nmol/mg protein illuminated. The possibility that the sensitizer activity was due to an ascorbate-induced oxidation of tryptophan was eliminated by the presence of a heavy metal ion chelator during the incubation and by showing equivalent effects with ascorbate-incubated ribonuclease A, which is devoid of tryptophan. The ascorbate-incubated samples displayed increasing absorbance at wavelengths above 300 nm and increasing fluorescence (340/430) as glycation proceeded. The spectra of the 4 week glycated proteins were identical to those obtained with a solubilized water-insoluble fraction from human lens, which is known to have UVA sensitizer activity. (Author)

  2. Direct Channeling of Retinoic Acid between Cellular Retinoic Acid-Binding Protein II and Retinoic Acid Receptor Sensitizes Mammary Carcinoma Cells to Retinoic Acid-Induced Growth Arrest

    Budhu, Anuradha S.; Noy, Noa

    2002-01-01

    Cellular retinoic acid-binding protein II (CRABP-II) is an intracellular lipid-binding protein that associates with retinoic acid with a subnanomolar affinity. We previously showed that CRABP-II enhances the transcriptional activity of the nuclear receptor with which it shares a common ligand, namely, the retinoic acid receptor (RAR), and we suggested that it may act by delivering retinoic acid to this receptor. Here, the mechanisms underlying the effects of CRABP-II on the transcriptional ac...

  3. Molecular characterization and chromosomal assignment of equine cartilage derived retinoic acid sensitive protein (CD-RAP)/melanoma inhibitory activity (MIA)

    Berg, Lise Charlotte; Mata, Xavier; Thomsen, Preben Dybdahl

    2008-01-01

    Cartilage-derived retinoic acid sensitive protein (CD-RAP) also known as melanoma inhibitory activity (MIA) has already been established as a marker for chondrocyte differentiation and a number of cancerous condition sin humans. Studies have also shown that CD-RAP/MIA is a potential marker of joi...

  4. Serum high-sensitivity C-reaction protein and heart fatty acid binding protein level and cardiac accidents in patients with unstable angina pectoris

    朱红秋

    2006-01-01

    Objective To investigate the relationship between serum high-sensitivity C-reaction protein (hs-CRP) and heart fatty acid binding protein (h-FABP) on cardiac accidents in patients with unstable angina pectoris (UAP). Methods Serum levels of hs-CRP, h-FABP, cardiac troponin-Ⅰ(cTn-Ⅰ) and creatine kinase MB isoenzyme (CK-MB) were measured and cardiac accidents within 2 weeks after the test were observed in 74 patients (male

  5. Sensitizing properties of proteins

    Poulsen, Lars K.; Ladics, Gregory S; McClain, Scott;

    2014-01-01

    The scope of allergy risk is diverse considering the myriad ways in which protein allergenicity is affected by physiochemical characteristics of proteins. The complexity created by the matrices of foods and the variability of the human immune system add additional challenges to understanding the...... relationship between sensitization potential and allergy disease. To address these and other issues, an April 2012 international symposium was held in Prague, Czech Republic, to review and discuss the state-of-the-science of sensitizing properties of protein allergens. The symposium, organized by the Protein...... scientists from academia, government, and industry participated in the symposium. Experts provided overviews on known mechanisms by which proteins in food may cause sensitization, discussed experimental models to predict protein sensitizing potential, and explored whether such experimental techniques may be...

  6. Can-CSC-GBE: Developing Cost-sensitive Classifier with Gentleboost Ensemble for breast cancer classification using protein amino acids and imbalanced data.

    Ali, Safdar; Majid, Abdul; Javed, Syed Gibran; Sattar, Mohsin

    2016-06-01

    Early prediction of breast cancer is important for effective treatment and survival. We developed an effective Cost-Sensitive Classifier with GentleBoost Ensemble (Can-CSC-GBE) for the classification of breast cancer using protein amino acid features. In this work, first, discriminant information of the protein sequences related to breast tissue is extracted. Then, the physicochemical properties hydrophobicity and hydrophilicity of amino acids are employed to generate molecule descriptors in different feature spaces. For comparison, we obtained results by combining Cost-Sensitive learning with conventional ensemble of AdaBoostM1 and Bagging. The proposed Can-CSC-GBE system has effectively reduced the misclassification costs and thereby improved the overall classification performance. Our novel approach has highlighted promising results as compared to the state-of-the-art ensemble approaches. PMID:27061661

  7. Influence of berberine combining with atorvastatin on serum high-sensitivity C-reactive protein and adipocyte fatty acid-binding protein in patients with acute ischemic stroke

    Fei-qi ZHU

    2015-01-01

    Full Text Available Objective To observe the influence of berberine combining with atorvastatin on serum high-sensitivity C-reactive protein (hs-CRP and adipocyte fatty acid-binding protein (A-FABP in patients with acute ischemic stroke.  Methods Ischemic stroke patients (N = 55 were randomized into 3 groups: atorvastatin 20 mg/d (N = 28, atorvastatin 40 mg/d (N = 11 and berberine 0.40 g three times a day + atorvastatin 20 mg/d (combined treatment, N = 16. They were treated for 3 months. The expression changes of serum hs-CRP and A-FABP before and after treatment were compared among 3 groups.  Results There were significant decreases between before and 3 months after treatment on the expression of hs-CRP and A-FABP in 3 groups (P = 0.023, 0.000. After treatment, both the expression of hs-CRP and A-FABP significantly decreased, and the decreases were (1.69 ± 2.29 and (281.43 ± 311.05 mg/L in atorvastatin 20 mg/d group, (7.81 ± 12.48 and (321.59 ± 289.35 mg/L in atorvastatin 40 mg/d group, and (2.16 ± 3.34 and (376.55 ± 249.72 mg/L in combined treatment group. However, there was no significant difference among 3 groups (P > 0.05, for all, and there was no correlation between drugs and observation time points (P > 0.05, for all.  Conclusions The effect of berberine combined with atorvastatin on hs-CRP and A-FABP is similar to atorvastation (40 mg/d therapy. DOI: 10.3969/j.issn.1672-6731.2015.01.010

  8. Quantification of proteins using enhanced etching of Ag coated Au nanorods by the Cu2+/bicinchoninic acid pair with improved sensitivity

    Liu, Wenqi; Hou, Shuai; Yan, Jiao; Zhang, Hui; Ji, Yinglu; Wu, Xiaochun

    2015-12-01

    Plasmonic nanosensors show great potential in ultrasensitive detection, especially with the plasmon peak position as the detection modality. Herein, a new sensitive but simple total protein quantification method termed the SPR-BCA assay is demonstrated by combining plasmonic nanosensors with protein oxidation by Cu2+. The easy tuning of localized surface plasmon resonance (LSPR) features of plasmonic nanostructures makes them ideal sensing platforms. We found that the Cu2+/bicinchoninic acid (BCA) pair exhibits accelerated etching of Au@Ag nanorods and results in the LSPR peak shift. A linear relationship between Cu2+ and the LSPR shift is found in a double logarithmic coordinate. Such double logarithm relationship is transferred to the concentration of proteins. Theoretical simulation shows that Au nanorods with large aspect ratios and small core sizes show high detection sensitivity. Via optimized sensor design, we achieved an increased sensitivity (the limit of detection was 3.4 ng ml-1) and a wide working range (0.5 to 1000 μg ml-1) compared with the traditional BCA assay. The universal applicability of our method to various proteins further proves its potential in practical applications.Plasmonic nanosensors show great potential in ultrasensitive detection, especially with the plasmon peak position as the detection modality. Herein, a new sensitive but simple total protein quantification method termed the SPR-BCA assay is demonstrated by combining plasmonic nanosensors with protein oxidation by Cu2+. The easy tuning of localized surface plasmon resonance (LSPR) features of plasmonic nanostructures makes them ideal sensing platforms. We found that the Cu2+/bicinchoninic acid (BCA) pair exhibits accelerated etching of Au@Ag nanorods and results in the LSPR peak shift. A linear relationship between Cu2+ and the LSPR shift is found in a double logarithmic coordinate. Such double logarithm relationship is transferred to the concentration of proteins. Theoretical

  9. Quantification of proteins using enhanced etching of Ag coated Au nanorods by the Cu(2+)/bicinchoninic acid pair with improved sensitivity.

    Liu, Wenqi; Hou, Shuai; Yan, Jiao; Zhang, Hui; Ji, Yinglu; Wu, Xiaochun

    2016-01-14

    Plasmonic nanosensors show great potential in ultrasensitive detection, especially with the plasmon peak position as the detection modality. Herein, a new sensitive but simple total protein quantification method termed the SPR-BCA assay is demonstrated by combining plasmonic nanosensors with protein oxidation by Cu(2+). The easy tuning of localized surface plasmon resonance (LSPR) features of plasmonic nanostructures makes them ideal sensing platforms. We found that the Cu(2+)/bicinchoninic acid (BCA) pair exhibits accelerated etching of Au@Ag nanorods and results in the LSPR peak shift. A linear relationship between Cu(2+) and the LSPR shift is found in a double logarithmic coordinate. Such double logarithm relationship is transferred to the concentration of proteins. Theoretical simulation shows that Au nanorods with large aspect ratios and small core sizes show high detection sensitivity. Via optimized sensor design, we achieved an increased sensitivity (the limit of detection was 3.4 ng ml(-1)) and a wide working range (0.5 to 1000 μg ml(-1)) compared with the traditional BCA assay. The universal applicability of our method to various proteins further proves its potential in practical applications. PMID:26669539

  10. Fatty-acid binding protein 4 gene variants and childhood obesity: potential implications for insulin sensitivity and CRP levels

    Bhattacharjee Rakesh

    2010-02-01

    Full Text Available Abstract Introduction Obesity increases the risk for insulin resistance and metabolic syndrome in both adults and children. FABP4 is a member of the intracellular lipid-binding protein family that is predominantly expressed in adipose tissue, and plays an important role in maintaining glucose and lipid homeostasis. The purpose of this study was to measure FABP4 plasma levels, assess FABP4 allelic variants, and explore potential associations with fasting glucose and insulin levels in young school-age children with and without obesity. Methods A total of 309 consecutive children ages 5-7 years were recruited. Children were divided based on BMI z score into Obese (OB; BMI z score >1.65 and non-obese (NOB. Fasting plasma glucose, lipids, insulin, hsCRP, and FABP4 levels were measured. HOMA was used as correlate of insulin sensitivity. Four SNPs of the human FABP4 gene (rs1051231, rs2303519, rs16909233 and rs1054135, corresponding to several critical regions of the encoding FABP4 gene sequence were genotyped. Results Compared to NOB, circulating FABP4 levels were increased in OB, as were LDL, hsCRP and HOMA. FABP4 levels correlated with BMI, and also contributed to the variance of HOMA and hsCRP, but not serum lipids. The frequency of rs1054135 allelic variant was increased in OB, and was associated with increased FABP4 levels, while the presence of rs16909233 variant allele, although similar in OB and NOB, was associated with increased HOMA values. Conclusions Childhood obesity is associated with higher FABP4 levels that may promote cardiometabolic risk. The presence of selective SNPs in the FABP4 gene may account for increased risk for insulin resistance or systemic inflammation in the context of obesity.

  11. Canine distemper virus neutralization activity is low in human serum and it is sensitive to an amino acid substitution in the hemagglutinin protein.

    Zhang, Xinsheng; Wallace, Olivia L; Domi, Arban; Wright, Kevin J; Driscoll, Jonathan; Anzala, Omu; Sanders, Eduard J; Kamali, Anatoli; Karita, Etienne; Allen, Susan; Fast, Pat; Gilmour, Jill; Price, Matt A; Parks, Christopher L

    2015-08-01

    Serum was analyzed from 146 healthy adult volunteers in eastern Africa to evaluate measles virus (MV) and canine distemper virus (CDV) neutralizing antibody (nAb) prevalence and potency. MV plaque reduction neutralization test (PRNT) results indicated that all sera were positive for MV nAbs. Furthermore, the 50% neutralizing dose (ND50) for the majority of sera corresponded to antibody titers induced by MV vaccination. CDV nAbs titers were low and generally were detected in sera with high MV nAb titers. A mutant CDV was generated that was less sensitive to neutralization by human serum. The mutant virus genome had 10 nucleotide substitutions, which coded for single amino acid substitutions in the fusion (F) and hemagglutinin (H) glycoproteins and two substitutions in the large polymerase (L) protein. The H substitution occurred in a conserved region involved in receptor interactions among morbilliviruses, implying that this region is a target for cross-reactive neutralizing antibodies. PMID:25880113

  12. Impact of dietary betaine and conjugated linoleic acid on insulin sensitivity, protein and fat metabolism of obese pigs.

    Fernández-Fígares, I; Lachica, M; Martín, A; Nieto, R; González-Valero, L; Rodríguez-López, J M; Aguilera, J F

    2012-07-01

    To determine possible mechanisms of action that might explain the nutrient partitioning effect of betaine and conjugated linoleic acid (CLA) in Iberian pigs and to address potential adverse effects, twenty gilts were restrictively fed from 20 to 50 kg BW Control, 0.5% betaine, 1% CLA or 0.5% betaine + 1% CLA diets. Serum hormones and metabolites profile were determined at 30 kg BW and an oral glucose test was performed before slaughter. Pigs were slaughtered at 50 kg BW and livers were obtained for chemical and histological analysis. Decreased serum urea in pigs fed betaine and betaine + CLA diets (11%; P = 0.0001) indicated a more efficient N utilization. The increase in serum triacylglycerol (58% and 28%, respectively; P = 0.0098) indicated that CLA and betaine + CLA could have reduced adipose tissue triacylglycerol synthesis from preformed fatty acids. Serum glucose, low-density lipoprotein (LDL) cholesterol and non-esterified fatty acids were unaffected. CLA and betaine + CLA altered serum lipids profile, although liver of pigs fed CLA diet presented no histopathological changes and triglyceride content was not different from Control pigs. Compared with controls, serum growth hormone decreased (20% to 23%; P = 0.0209) for all treatments. Although serum insulin increased in CLA, and especially in betaine + CLA pigs (28% and 83%; P = 0.0001), indices of insulin resistance were unaffected. In conclusion, CLA, and especially betaine + CLA, induced changes in biochemical parameters and hormones that may partially explain a nutrient partitioning effect in young pigs. Nevertheless, they exhibited weak, although detrimental, effects on blood lipids. Moreover, although livers were chemically and histologically normal, pigs fed CLA diet challenged with a glucose load had higher serum glucose than controls. PMID:23031465

  13. Canine distemper virus neutralization activity is low in human serum and it is sensitive to an amino acid substitution in the hemagglutinin protein

    Serum was analyzed from 146 healthy adult volunteers in eastern Africa to evaluate measles virus (MV) and canine distemper virus (CDV) neutralizing antibody (nAb) prevalence and potency. MV plaque reduction neutralization test (PRNT) results indicated that all sera were positive for MV nAbs. Furthermore, the 50% neutralizing dose (ND50) for the majority of sera corresponded to antibody titers induced by MV vaccination. CDV nAbs titers were low and generally were detected in sera with high MV nAb titers. A mutant CDV was generated that was less sensitive to neutralization by human serum. The mutant virus genome had 10 nucleotide substitutions, which coded for single amino acid substitutions in the fusion (F) and hemagglutinin (H) glycoproteins and two substitutions in the large polymerase (L) protein. The H substitution occurred in a conserved region involved in receptor interactions among morbilliviruses, implying that this region is a target for cross-reactive neutralizing antibodies. - Highlights: • Screened 146 serum samples for measles virus (MV) and canine distemper virus (CDV) neutralizing antibody (nAb). • MV nAb is prevalent in the sera. • CDV neutralizing activity is generally low or absent and when detected it is present in sera with high MV nAb titers. • A neutralization-resistant CDV mutant was isolated using human serum selection. • A mutation was identified in the receptor-binding region of CDV hemagglutinin protein that confers the neutralization resistance

  14. Canine distemper virus neutralization activity is low in human serum and it is sensitive to an amino acid substitution in the hemagglutinin protein

    Zhang, Xinsheng, E-mail: xzhang@iavi.org [AIDS Vaccine Design and Development Laboratory, International AIDS Vaccine Initiative (IAVI), Brooklyn, NY (United States); Molecular and Cellular Biology Program, State University of New York, Brooklyn, NY (United States); Wallace, Olivia L.; Domi, Arban; Wright, Kevin J.; Driscoll, Jonathan [AIDS Vaccine Design and Development Laboratory, International AIDS Vaccine Initiative (IAVI), Brooklyn, NY (United States); Anzala, Omu [Kenya AIDS Vaccine Initiative (KAVI)-Institute of Clinical Research, Nairobi (Kenya); Sanders, Eduard J. [Centre for Geographic Medicine Research, Kenya Medical Research Institute (KEMRI), Kilifi, Kenya & Centre for Clinical Vaccinology and Tropical Medicine, University of Oxford, Headington (United Kingdom); Kamali, Anatoli [MRC/UVRI Uganda Virus Research Unit on AIDS, Masaka and Entebbe (Uganda); Karita, Etienne [Projet San Francisco, Kigali (Rwanda); Allen, Susan [Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA (United States); Fast, Pat [Department of Medical Affairs, International AIDS Vaccine Initiative, NY, NY (United States); Gilmour, Jill [Human Immunology Laboratory, International AIDS Vaccine Initiative, London (United Kingdom); Price, Matt A. [Department of Medical Affairs, International AIDS Vaccine Initiative, NY, NY (United States); Department of Epidemiology and Biostatistics, University of California at San Francisco, San Francisco, CA (United States); Parks, Christopher L. [AIDS Vaccine Design and Development Laboratory, International AIDS Vaccine Initiative (IAVI), Brooklyn, NY (United States); Molecular and Cellular Biology Program, State University of New York, Brooklyn, NY (United States)

    2015-08-15

    Serum was analyzed from 146 healthy adult volunteers in eastern Africa to evaluate measles virus (MV) and canine distemper virus (CDV) neutralizing antibody (nAb) prevalence and potency. MV plaque reduction neutralization test (PRNT) results indicated that all sera were positive for MV nAbs. Furthermore, the 50% neutralizing dose (ND50) for the majority of sera corresponded to antibody titers induced by MV vaccination. CDV nAbs titers were low and generally were detected in sera with high MV nAb titers. A mutant CDV was generated that was less sensitive to neutralization by human serum. The mutant virus genome had 10 nucleotide substitutions, which coded for single amino acid substitutions in the fusion (F) and hemagglutinin (H) glycoproteins and two substitutions in the large polymerase (L) protein. The H substitution occurred in a conserved region involved in receptor interactions among morbilliviruses, implying that this region is a target for cross-reactive neutralizing antibodies. - Highlights: • Screened 146 serum samples for measles virus (MV) and canine distemper virus (CDV) neutralizing antibody (nAb). • MV nAb is prevalent in the sera. • CDV neutralizing activity is generally low or absent and when detected it is present in sera with high MV nAb titers. • A neutralization-resistant CDV mutant was isolated using human serum selection. • A mutation was identified in the receptor-binding region of CDV hemagglutinin protein that confers the neutralization resistance.

  15. EFFECTS OF SIMVASTAIN COMBINED WITH OMEGA-3 FATTY ACIDS ON HIGH SENSITIVE C-REACTIVE PROTEIN,LIPIDEMIA,AND FIBRINOLYSIS IN PATIENTS WITH MIXED DYSLIPIDEMIA

    Heng Hong; Zhi-min Xu; Bao-sen Pang; Liang Cui; Yu Wei; Wen-jing Guo; Yan-ling Mao; Xin-chun Yang

    2004-01-01

    Objective To evaluate the effects of simvastatin combined with omega-3 fatty acids on high sensitive C-reactive protein (HsCRP), lipidemia, and fibrinolysis in coronary heart disease (CHD) and CHD risk equivalent patients with mixed dyslipidemia.Methods A randomized, double-blind placebo controlled and parallel group trial was conducted. Patients with CHD and CHD risk equivalents with mixed dyslipidemia were treated with 10 or 20 mg simvastatin for 6-12 weeks. Following with the treatment of patients whose low-density lipoprotein cholesterol (LDL-ch) reaching goal level (< 100 mg/dL) or close to the goal (< 130 mg/dL), while triglyceride (TG) ≥ 200 mg/dL and < 500 mg/dL, was combined with omega-3fatty acids (3 g/d) or a placebo for 2 months. The effects of the treatment on HsCRP, total cholesterol (TC), LDL-ch, highdensity lipoprotein cholesterol (HDL-ch), TG, lipoprotein (a) [LP (a)], apolipoprotein Al (apoAl), apolipoprotein B (apoB),plasminogen activator inhibitor-1 (PAI-1), and tissue plasminogen activator (tPA) were investigated. Forty patients finished the study with each group consisting of twenty patients.Results (1) There were significant reductions of HsCRP, TG, TC, and TC/HDL-ch, which decreased by 2.16 ± 2.77mg/L (38.5%), 94.0± 65.4 mg/dL (31.1%), 13.3 ± 22.3 mg/dL (6.3%), 0.78 ± 1.60 respectively in the omega-3 fatty acids group (P < 0.01, < 0.001, < 0.05, < 0.05) compared to the baseline. HsCRP and triglyceride reduction were more significant in omega-3 fatty acids group compared to the placebo group (P= 0.021 and 0.011 respectively). (2) In the omega-3 fatty acids group, the values and percentage of TG reduction had a significantly positive relation with HsCRP reduction (r = 0.51and 0.45, P=0.021 and 0.047 respectively).Conclusion In CHD and CHD risk equivalent patients with mixed dyslipidemia, dyslipidemia's therapeutic effect using simvastatin and omega-3 fatty acids may result from not only the combination of lipid adjustment

  16. High sensitive troponin T and heart fatty acid binding protein: Novel biomarker in heart failure with normal ejection fraction?: A cross-sectional study

    Barroso Michael

    2011-07-01

    Full Text Available Abstract Background High sensitive troponin T (hsTnT and heart fatty acid binding protein (hFABP are both markers of myocardial injury and predict adverse outcome in patients with systolic heart failure (SHF. We tested whether hsTnT and hFABP plasma levels are elevated in patients with heart failure with normal ejection fraction (HFnEF. Methods We analyzed hsTnT, hFABP and N-terminal brain natriuretic peptide in 130 patients comprising 49 HFnEF patients, 51 patients with asymptomatic left ventricular diastolic dysfunction (LVDD, and 30 controls with normal diastolic function. Patients were classified to have HFnEF when the diagnostic criteria as recommended by the European Society of Cardiology were met. Results Levels of hs TnT and hFABP were significantly higher in patients with asymptomatic LVDD and HFnEF (both p Conclusion In HFnEF patients, hsTnT and hFABP are elevated independent of coronary artery disease, suggesting that ongoing myocardial damage plays a critical role in the pathophysiology. A combination of biomarkers and echocardiographic parameters might improve diagnostic accuracy and risk stratification of patients with HFnEF.

  17. Sensitivity of Routine Tests for Urine Protein to Hemoglobin

    Jansen, Barbara S.; Lumsden, John H.

    1985-01-01

    Increasing concentrations of canine hemoglobin were added to aliquots of urine and saline to determine the relative sensitivity of several hemoglobin and protein detection methods including commercial reagent strips and sulfosalicylic acid. The hemoglobin detection pads of the reagent strips were 50 times more sensitive than the protein detection pads, indicating the presence of hemoglobin at a concentration of 0.001 g/L whereas the protein pads did not react positively unless the hemoglobin ...

  18. Correlation analysis between the carotid artery ather osclerosis severity of patients with lacunar infarct and the level of serum uric acid, high-sensitivity C-reactive protein

    WANG Shu

    2013-01-01

    Full Text Available Background Nowadays, clinical practice shows that the number of patients with cerebral infarction increases year by year in young and middle-aged adults with unclear causes. This paper aims to investigate the correlation between the level of serum uric acid (UA, high-sensitivity C-reactive protein (hsCRP and the degree of carotid atherosclerosis by detecting the level of UA, hsCRP and carotid intima-media thickness (IMT in young and middle-aged patients with lacunar infarct. Methods By using Holland Philips HT-11 color Doppler ultrasonic diagnostic apparatus with linear type probe and 7.50MHz frequency, IMT of end-diastolic distal and bifurcation of arteria carotis communis and proximal internal carotid artery were measured respectively on 186 patients with definitely diagnosed lacunar infarct. As a result, average values were taken as the IMT values. IMT < 1.00 mm was regarded as negative result and 1.00 mm ≤IMT < 1.20 mm was thickening of carotid artery intima. Carotid artery plaque formation was regarded with echo structure existing in the lumen or abnormal blood flow deficits into the lumen, or local IMT ≥1.20 mm at vertical and horizontal scan. At the same time, the levels of UA and hsCRP were detected, and correlation analysis was made between them and IMT. Results The values of UA, hsCRP and IMT in the case group were higher than that in the control group (P = 0.000. The differences among the case subgroups including IMT thickening group, plaque formation group and IMT normal group were statistically significant (P < 0.01, for all. There was linear correlation between the levels of UA, hsCRP and IMT ( r = 0.923, P = 0.000; r = 0.955, P = 0.008. Conclusion UA and hsCRP involve in the formation of atherosclerosis plaque and play an important role in the first-onset lacunar infarct patients without hypertension and other risk factors.

  19. Phthalocyanides sensitized fragmentation of proteins

    Klementová, S.; Tothová, D.; Revaková, R.; Kasková, M.; Wagnerová, Dana Marie

    2001-01-01

    Roč. 5, č. 1 (2001), s. 13-18. ISSN 0972-0626 R&D Projects: GA ČR GA203/96/1322 Institutional research plan: CEZ:AV0Z4032918 Keywords : phthalocyanides * photosensitied fragmentation of proteins Subject RIV: CA - Inorganic Chemistry

  20. Proximity assays for sensitive quantification of proteins

    Christina Greenwood

    2015-06-01

    Full Text Available Proximity assays are immunohistochemical tools that utilise two or more DNA-tagged aptamers or antibodies binding in close proximity to the same protein or protein complex. Amplification by PCR or isothermal methods and hybridisation of a labelled probe to its DNA target generates a signal that enables sensitive and robust detection of proteins, protein modifications or protein–protein interactions. Assays can be carried out in homogeneous or solid phase formats and in situ assays can visualise single protein molecules or complexes with high spatial accuracy. These properties highlight the potential of proximity assays in research, diagnostic, pharmacological and many other applications that require sensitive, specific and accurate assessments of protein expression.

  1. A single amino acid substitution of the human immunodeficiency virus type 1 capsid protein affects viral sensitivity to TRIM5α

    Shioda Tatsuo

    2010-07-01

    Full Text Available Abstract Background Human immunodeficiency virus type 1 (HIV-1 productively infects only humans and chimpanzees but not Old World monkeys, such as rhesus and cynomolgus (CM monkeys. To establish a monkey model of HIV-1/AIDS, several HIV-1 derivatives have been constructed. We previously reported that efficient replication of HIV-1 in CM cells was achieved after we replaced the loop between α-helices 6 and 7 (L6/7 of the capsid protein (CA with that of SIVmac239 in addition to the loop between α-helices 4 and 5 (L4/5 and vif. This virus (NL-4/5S6/7SvifS was supposed to escape from host restriction factors cyclophilin A, CM TRIM5α, and APOBEC3G. However, the replicative capability of NL-4/5S6/7SvifS in human cells was severely impaired. Results By long-term cultivation of human CEMss cells infected with NL-4/5S6/7SvifS, we succeeded in rescuing the impaired replicative capability of the virus in human cells. Sequence analysis of the CA region of the adapted virus revealed a G-to-E substitution at the 116th position of the CA (G116E. Introduction of this substitution into the molecular DNA clone of NL-4/5S6/7SvifS indeed improved the virus' replicative capability in human cells. Although the G116E substitution occurred during long-term cultivation of human cells infected with NL-4/5S6/7SvifS, the viruses with G116E unexpectedly became resistant to CM, but not human TRIM5α-mediated restriction. The 3-D model showed that position 116 is located in the 6th helix near L4/5 and L6/7 and is apparently exposed to the protein surface. The amino acid substitution at the 116th position caused a change in the structure of the protein surface because of the replacement of G (which has no side chain with E (which has a long negatively charged side chain. Conclusions We succeeded in rescuing the impaired replicative capability of NL-4/5S6/7SvifS and report a mutation that improved the replicative capability of the virus. Unexpectedly, HIV-1 with this

  2. Long-chain omega-3 fatty acids regulate bovine whole-body protein metabolism by promoting muscle insulin signalling to the Akt-mTOR-S6K1 pathway and insulin sensitivity.

    Gingras, Andrée-Anne; White, Phillip James; Chouinard, P Yvan; Julien, Pierre; Davis, Teresa A; Dombrowski, Luce; Couture, Yvon; Dubreuil, Pascal; Myre, Alexandre; Bergeron, Karen; Marette, André; Thivierge, M Carole

    2007-02-15

    The ability of the skeletal musculature to use amino acids to build or renew constitutive proteins is gradually lost with age and this is partly due to a decline in skeletal muscle insulin sensitivity. Since long-chain omega-3 polyunsaturated fatty acids (LCn-3PUFA) from fish oil are known to improve insulin-mediated glucose metabolism in insulin-resistant states, their potential role in regulating insulin-mediated protein metabolism was investigated in this study. Experimental data are based on a switchback design composed of three 5 week experimental periods using six growing steers to compare the effect of a continuous abomasal infusion of LCn-3PUFA-rich menhaden oil with an iso-energetic control oil mixture. Clamp and insulin signalling observations were combined with additional data from a second cohort of six steers. We found that enteral LCn-3PUFA potentiate insulin action by increasing the insulin-stimulated whole-body disposal of amino acids from 152 to 308 micromol kg(-1) h(-1) (P=0.006). The study further showed that in the fed steady-state, chronic adaptation to LCn-3PUFA induces greater activation (P<0.05) of the Akt-mTOR-S6K1 signalling pathway. Simultaneously, whole-body total flux of phenylalanine was reduced from 87 to 67 micromol kg(-1) h(-1) (P=0.04) and oxidative metabolism was decreased (P=0.05). We conclude that chronic feeding of menhaden oil provides a novel nutritional mean to enhance insulin-sensitive aspects of protein metabolism. PMID:17158167

  3. Long-chain omega-3 fatty acids regulate bovine whole-body protein metabolism by promoting muscle insulin signalling to the Akt–mTOR–S6K1 pathway and insulin sensitivity

    Gingras, Andrée-Anne; White, Phillip James; Chouinard, P Yvan; Julien, Pierre; Davis, Teresa A; Dombrowski, Luce; Couture, Yvon; Dubreuil, Pascal; Myre, Alexandre; Bergeron, Karen; Marette, André; Thivierge, M Carole

    2007-01-01

    The ability of the skeletal musculature to use amino acids to build or renew constitutive proteins is gradually lost with age and this is partly due to a decline in skeletal muscle insulin sensitivity. Since long-chain omega-3 polyunsaturated fatty acids (LCn–3PUFA) from fish oil are known to improve insulin-mediated glucose metabolism in insulin-resistant states, their potential role in regulating insulin-mediated protein metabolism was investigated in this study. Experimental data are based on a switchback design composed of three 5 week experimental periods using six growing steers to compare the effect of a continuous abomasal infusion of LCn–3PUFA-rich menhaden oil with an iso-energetic control oil mixture. Clamp and insulin signalling observations were combined with additional data from a second cohort of six steers. We found that enteral LCn–3PUFA potentiate insulin action by increasing the insulin-stimulated whole-body disposal of amino acids from 152 to 308 μmol kg−1 h−1 (P = 0.006). The study further showed that in the fed steady-state, chronic adaptation to LCn–3PUFA induces greater activation (P < 0.05) of the Akt–mTOR–S6K1 signalling pathway. Simultaneously, whole-body total flux of phenylalanine was reduced from 87 to 67 μmol kg−1 h−1 (P = 0.04) and oxidative metabolism was decreased (P = 0.05). We conclude that chronic feeding of menhaden oil provides a novel nutritional mean to enhance insulin-sensitive aspects of protein metabolism. PMID:17158167

  4. High-Sensitivity C-Reactive Protein Complements Plasma Epstein-Barr Virus Deoxyribonucleic Acid Prognostication in Nasopharyngeal Carcinoma: A Large-Scale Retrospective and Prospective Cohort Study

    Tang, Lin-Quan [Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University, Guangzhou (China); Department of Nasopharyngeal Carcinoma, Sun Yat-sen University, Guangzhou (China); Li, Chao-Feng [Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University, Guangzhou (China); Department of Information Technology, Sun Yat-sen University, Guangzhou (China); Chen, Qiu-Yan; Zhang, Lu [Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University, Guangzhou (China); Department of Nasopharyngeal Carcinoma, Sun Yat-sen University, Guangzhou (China); Lai, Xiao-Ping; He, Yun; Xu, Yun-Xiu-Xiu; Hu, Dong-Peng; Wen, Shi-Hua; Peng, Yu-Tuan [ZhongShan School of Medicine, Sun Yat-sen University, Guangzhou (China); Chen, Wen-Hui [Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University, Guangzhou (China); Liu, Huai; Guo, Shan-Shan; Liu, Li-Ting [Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University, Guangzhou (China); Department of Nasopharyngeal Carcinoma, Sun Yat-sen University, Guangzhou (China); Li, Jing [Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University, Guangzhou (China); Zhang, Jing-Ping [Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University, Guangzhou (China); Department of Clinical Laboratory, Sun Yat-sen University, Guangzhou (China); and others

    2015-02-01

    Purpose: To evaluate the effects of combining the assessment of circulating high-sensitivity C-reactive protein (hs-CRP) with that of Epstein-Barr virus DNA (EBV DNA) in the pretherapy prognostication of nasopharyngeal carcinoma (NPC). Patients and Methods: Three independent cohorts of NPC patients (training set of n=3113, internal validation set of n=1556, and prospective validation set of n=1668) were studied. Determinants of disease-free survival, distant metastasis–free survival, and overall survival were assessed by multivariate analysis. Hazard ratios and survival probabilities of the patient groups, segregated by clinical stage (T1-2N0-1M0, T3-4N0-1M0, T1-2N2-3M0, and T3-4N2-3M0) and EBV DNA load (low or high) alone, and also according to hs-CRP level (low or high), were compared. Results: Elevated hs-CRP and EBV DNA levels were significantly correlated with poor disease-free survival, distant metastasis–free survival, and overall survival in both the training and validation sets. Associations were similar and remained significant after excluding patients with cardiovascular disease, diabetes, and chronic hepatitis B. Patients with advanced-stage disease were segregated by high EBV DNA levels and high hs-CRP level into a poorest-risk group, and participants with either high EBV DNA but low hs-CRP level or high hs-CRP but low EBV DNA values had poorer survival compared with the bottom values for both biomarkers. These findings demonstrate a significant improvement in the prognostic ability of conventional advanced NPC staging. Conclusion: Baseline plasma EBV DNA and serum hs-CRP levels were significantly correlated with survival in NPC patients. The combined interpretation of EBV DNA with hs-CRP levels led to refinement of the risks for the patient subsets, with improved risk discrimination in patients with advanced-stage disease.

  5. High-Sensitivity C-Reactive Protein Complements Plasma Epstein-Barr Virus Deoxyribonucleic Acid Prognostication in Nasopharyngeal Carcinoma: A Large-Scale Retrospective and Prospective Cohort Study

    Purpose: To evaluate the effects of combining the assessment of circulating high-sensitivity C-reactive protein (hs-CRP) with that of Epstein-Barr virus DNA (EBV DNA) in the pretherapy prognostication of nasopharyngeal carcinoma (NPC). Patients and Methods: Three independent cohorts of NPC patients (training set of n=3113, internal validation set of n=1556, and prospective validation set of n=1668) were studied. Determinants of disease-free survival, distant metastasis–free survival, and overall survival were assessed by multivariate analysis. Hazard ratios and survival probabilities of the patient groups, segregated by clinical stage (T1-2N0-1M0, T3-4N0-1M0, T1-2N2-3M0, and T3-4N2-3M0) and EBV DNA load (low or high) alone, and also according to hs-CRP level (low or high), were compared. Results: Elevated hs-CRP and EBV DNA levels were significantly correlated with poor disease-free survival, distant metastasis–free survival, and overall survival in both the training and validation sets. Associations were similar and remained significant after excluding patients with cardiovascular disease, diabetes, and chronic hepatitis B. Patients with advanced-stage disease were segregated by high EBV DNA levels and high hs-CRP level into a poorest-risk group, and participants with either high EBV DNA but low hs-CRP level or high hs-CRP but low EBV DNA values had poorer survival compared with the bottom values for both biomarkers. These findings demonstrate a significant improvement in the prognostic ability of conventional advanced NPC staging. Conclusion: Baseline plasma EBV DNA and serum hs-CRP levels were significantly correlated with survival in NPC patients. The combined interpretation of EBV DNA with hs-CRP levels led to refinement of the risks for the patient subsets, with improved risk discrimination in patients with advanced-stage disease

  6. Human Protein and Amino Acid Requirements.

    Hoffer, L John

    2016-05-01

    Human protein and amino acid nutrition encompasses a wide, complex, frequently misunderstood, and often contentious area of clinical research and practice. This tutorial explains the basic biochemical and physiologic principles that underlie our current understanding of protein and amino acid nutrition. The following topics are discussed: (1) the identity, measurement, and essentiality of nutritional proteins; (2) the definition and determination of minimum requirements; (3) nutrition adaptation; (4) obligatory nitrogen excretion and the minimum protein requirement; (5) minimum versus optimum protein intakes; (6) metabolic responses to surfeit and deficient protein intakes; (7) body composition and protein requirements; (8) labile protein; (9) N balance; (10) the principles of protein and amino acid turnover, including an analysis of the controversial indicator amino acid oxidation technique; (11) general guidelines for evaluating protein turnover articles; (12) amino acid turnover versus clearance; (13) the protein content of hydrated amino acid solutions; (14) protein requirements in special situations, including protein-catabolic critical illness; (15) amino acid supplements and additives, including monosodium glutamate and glutamine; and (16) a perspective on the future of protein and amino acid nutrition research. In addition to providing practical information, this tutorial aims to demonstrate the importance of rigorous physiologic reasoning, stimulate intellectual curiosity, and encourage fresh ideas in this dynamic area of human nutrition. In general, references are provided only for topics that are not well covered in modern textbooks. PMID:26796095

  7. Animal models for protein respiratory sensitizers.

    Ward, Marsha D W; Selgrade, Maryjane K

    2007-01-01

    Protein induced respiratory hypersensitivity, particularly atopic disease in general, and allergic asthma in particular, has increased dramatically over the last several decades in the US and other industrialized nations as a result of ill-defined changes in living conditions in modern western society. In addition, work-related asthma has become the most frequently diagnosed occupational respiratory illness. Animal models have demonstrated great utility in developing an understanding of the etiology and mechanisms of many diseases. A few models been developed as predictive models to identify a protein as an allergen or to characterize its potency. Here we describe animal models that have been used to investigate and identify protein respiratory sensitizers. In addition to prototypical experimental design, methods for exposure route, sample collection, and endpoint assessment are described. Some of the most relevant endpoints in assessing the potential for a given protein to induce atopic or allergic asthma respiratory hypersensitivity are the development of cytotropic antibodies (IgE, IgG1), eosinophil influx into the lung, and airway hyperresponsiveness to the sensitizing protein and/or to non-antigenic stimuli (Mch). The utility of technologies such as PCR and multiplexing assay systems is also described. These models and methods have been used to elucidate the potential for protein sources to induce allergy, identify environmental conditions (pollutants) to impact allergy responsiveness, and establish safe exposure limits. As an example, data are presented from an experiment designed to compare the allergenicity of a fungal biopesticide Metarhizium anisopliae (MACA) crude extract with the one of its components, conidia (CON) extract. PMID:17161304

  8. Interaction of rat hormone-sensitive lipase with adipocyte lipid-binding protein

    Shen, Wen-Jun; Sridhar, Kunju; Bernlohr, David A.; Fredric B Kraemer

    1999-01-01

    Hormone-sensitive lipase (HSL) is a cytosolic neutral lipase that functions as the rate-limiting enzyme for the mobilization of free fatty acids in adipose tissue. By using the yeast two-hybrid system to examine the potential interaction of HSL with other cellular proteins, evidence is provided to demonstrate a direct interaction of HSL with adipocyte lipid-binding protein (ALBP), a member of the family of intracellular lipid-binding proteins that binds fatty acids, retinoids, and other hydro...

  9. EFFECT OF ACIDITY ON ACID-SENSITIVE UV CURING SYSTEM

    Qi-dao Chen; Bing Wu; Xiao-yin Hong

    1999-01-01

    By using diphenyliodonium salts with different counterions as photo acid generators (PAGs), the effect of acidity on ring-opening polymerization of epoxy monomers and polycondensation of polyol with hexamethoxymethyl melamine (HMMM) was studied. The result shows that the rate of ring-opening polymerization is evidently dependent on the acidity of the acid and strong photo-generated acid is required.However, there is a leveling effect in the polycondensation system; if the photo-generated acid is stronger than protonated HMMM, the acidity does not obviously affect the polycondensation rate.

  10. Cytokines: muscle protein and amino acid metabolism

    van Hall, Gerrit

    2012-01-01

    raises TNF-α and IL-6 to moderate levels, has only identified IL-6 as a potent cytokine, decreasing systemic amino acid levels and muscle protein metabolism. The marked decrease in circulatory and muscle amino acid concentrations was observed with a concomitant reduction in both the rates of muscle...... IL-6 on the regulation of muscle protein metabolism but indirectly via IL-6 reducing amino acid availability. SUMMARY: Recent studies suggest that the best described cytokines TNF-α and IL-6 are unlikely to be the major direct mediators of muscle protein loss in inflammatory diseases. However, these...

  11. Protein and ligand adaptation in a retinoic acid binding protein.

    Pattanayek, R.; Newcomer, M E

    1999-01-01

    A retinoic acid binding protein isolated from the lumen of the rat epididymis (ERABP) is a member of the lipocalin superfamily. ERABP binds both the all-trans and 9-cis isomers of retinoic acid, as well as the synthetic retinoid (E)-4-[2-(5,6,7,8)-tetrahydro-5,5,8,8-tetramethyl-2 napthalenyl-1 propenyl]-benzoic acid (TTNPB), a structural analog of all-trans retinoic acid. The structure of ERABP with a mixture of all-trans and 9-cis retinoic acid has previously been reported. To elucidate any ...

  12. Liver Fatty Acid Binding Protein and Obesity

    Atshaves, B.P.; Martin, G G; Hostetler, H.A.; McIntosh, A.L.; Kier, A B; Schroeder, F.

    2010-01-01

    While low levels of unesterified long chain fatty acids (LCFAs) are normal metabolic intermediates of dietary and endogenous fat, LCFAs are also potent regulators of key receptors/enzymes, and at high levels become toxic detergents within the cell. Elevated levels of LCFAs are associated with diabetes, obesity, and metabolic syndrome. Consequently, mammals evolved fatty acid binding proteins (FABPs) that bind/sequester these potentially toxic free fatty acids in the cytosol and present them f...

  13. Factors Affecting Sensitivity of Variable Charge Soils to Acid Rain

    WANGJING-HUA

    1995-01-01

    The sensitivity of a large number of variable charge soils to acid rain was evaluated through examining pH-H2SO4 input curves.Two derivative parameters,the consumption of hydrogen ions by the soil and the acidtolerant limit as defined as the quantity of sulfuric acid required to bring the soil to pH 3.5 in a 0.001mol L-1 Ca(NO3)2 solution,were used.The sensitivity of variable charge soils was higher than that of constant charge soils,due to the predominance of kaolinite in clay mineralogical composition.Among these soils the sensitivity was generally of the order lateritic red soil>red soil> latosol.For a given type of soil within the same region the sensitivity was affected by parent material,due to differences in clay minerals and texture.The sensitivity of surface soil may be lower or higher than that of subsiol,depending on whether organic matter or texture plays the dominant role in determining the buffering capacity.Paddy soils consumed more acid within lower range of acid input when compared with upland soils,due to the presence of more exchangeable bases,but consumed less acid within higher acid input range,caused by the decrease in clay content.

  14. Trends in protein and nucleic acid electroanalysis

    Paleček, Emil

    Seville, 2008. s. 1. [The 59th Annual Meeting of the International Society of Electrochemistry. 07.09.2008-12.09.2008, Seville] Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : protein electroanalysis * nucleic acid electroanalysis Subject RIV: AQ - Safety, Health Protection, Human - Machine

  15. AMINO ACIDS AUGMENT MUSCLE PROTEIN SYNTHESIS IN NEONATAL PIGS DURING ENDOTOXEMIA BY MODULATING TRANSLATION INITIATION

    In adults, sepsis reduces protein synthesis in skeletal muscle by restraining translation. The effect of sepsis on amino acid-stimulated muscle protein synthesis has not been determined in neonates, a population who is highly anabolic and whose muscle protein synthesis rates are uniquely sensitive ...

  16. Momilactone sensitive proteins in Arabidopsis thaliana.

    Kato-Noguchi, Hisashi; Kitajima, Shinya

    2015-05-01

    The labdane-related diterpenoid, momilactone B has potent growth inhibitory activity and was demonstrated to play a particularly critical role in the allelopathy of rice (Oryza sativa L.). However, there is limited information available about the mode of action of momilactone B on the growth inhibition. The present research describes the effects of momilactone B on protein expression in the early development of Arabidopsis thaliana seedling, which was determined by two-dimensional electrophoresis and MALDI-TOFMS. Momilactone B inhibited the accumulation of subtilisin-like serine protease, amyrin synthase LUP2, β-glucosidase and malate synthase at 1 h after the momilactone application. Those proteins are involved in the metabolic turnover and the production of intermediates needed for cell structures resulting in plant growth and development. Momilactone B also inhibited the breakdown of cruciferin 2, which is essential for seed germination and seedling growth to construct cell structures. Momilactone B induced the accumulation of translationally controlled tumor protein, glutathione S-transferase and 1-cysteine peroxiredoxin 1. These proteins are involved in stress responses and increased stress tolerance. In addition, glutathione S-transferase has the activity of herbicide detoxification and 1-cysteine peroxiredoxin 1 has inhibitory activity for seed germination under unfavorable conditions. The present research suggests that momilactone B may inhibit the seedling growth by the inhibition of the metabolic turnover and the production of intermediates for cell structures. In addition, momilactone induced proteins associated with plant defense responses. PMID:26058145

  17. Analysis of protein-nucleic acid interactions by photochemical cross-linking and mass spectrometry

    Steen, Hanno; Jensen, Ole Nørregaard

    2002-01-01

    . Mass spectrometry (MS) has emerged as a sensitive and efficient analytical technique for determination of such cross-linking sites in proteins. The present review of the field describes a number of MS-based approaches for the characterization of cross-linked protein-nucleic acid complexes and for......Photochemical cross-linking is a commonly used method for studying the molecular details of protein-nucleic acid interactions. Photochemical cross-linking aids in defining nucleic acid binding sites of proteins via subsequent identification of cross-linked protein domains and amino acid residues...... sequencing of peptide-nucleic acid heteroconjugates. The combination of photochemical cross-linking and MS provides a fast screening method to gain insights into the overall structure and formation of protein-oligonucleotide complexes. Because the analytical methods are continuously refined and protein...

  18. Trans fatty acids, insulin sensitivity and type 2 diabetes

    Risérus, Ulf

    2006-01-01

    Trans fatty acids (TFA) could affect cell membrane functions, and may therefore influence peripheral insulin sensitivity and the risk of developing type 2 diabetes. It is important to understand whether low amounts of TFA consumed during long periods may promote insulin resistance and have clinically relevant effects on diabetes risk. Data from controlled intervention studies examining the effects of TFA on insulin sensitivity and type 2 diabetes are reviewed. The results show no consistent e...

  19. Protein and Amino Acid Requirements during Pregnancy.

    Elango, Rajavel; Ball, Ronald O

    2016-07-01

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

  20. Circulating docosahexaenoic acid levels are associated with fetal insulin sensitivity.

    Jin-Ping Zhao

    Full Text Available BACKGROUND: Arachidonic acid (AA; C20∶4 n-6 and docosahexaenoic acid (DHA; C22∶6 n-3 are important long-chain polyunsaturated fatty acids (LC-PUFA in maintaining pancreatic beta-cell structure and function. Newborns of gestational diabetic mothers are more susceptible to the development of type 2 diabetes in adulthood. It is not known whether low circulating AA or DHA is involved in perinatally "programming" this susceptibility. This study aimed to assess whether circulating concentrations of AA, DHA and other fatty acids are associated with fetal insulin sensitivity or beta-cell function, and whether low circulating concentrations of AA or DHA are involved in compromised fetal insulin sensitivity in gestational diabetic pregnancies. METHODS AND PRINCIPAL FINDINGS: In a prospective singleton pregnancy cohort, maternal (32-35 weeks gestation and cord plasma fatty acids were assessed in relation to surrogate indicators of fetal insulin sensitivity (cord plasma glucose-to-insulin ratio, proinsulin concentration and beta-cell function (proinsulin-to-insulin ratio in 108 mother-newborn pairs. Cord plasma DHA levels (in percentage of total fatty acids were lower comparing newborns of gestational diabetic (n = 24 vs. non-diabetic pregnancies (2.9% vs. 3.5%, P = 0.01. Adjusting for gestational age at blood sampling, lower cord plasma DHA levels were associated with lower fetal insulin sensitivity (lower glucose-to-insulin ratio, r = 0.20, P = 0.036; higher proinsulin concentration, r = -0.37, P <0.0001. The associations remained after adjustment for maternal and newborn characteristics. Cord plasma saturated fatty acids C18∶0 and C20∶0 were negatively correlated with fetal insulin sensitivity, but their levels were not different between gestational diabetic and non-diabetic pregnancies. Cord plasma AA levels were not correlated with fetal insulin sensitivity. CONCLUSION: Low circulating DHA levels are associated with

  1. Increased sensitivity to gamma irradiation in bacteria lacking protein HU.

    Boubrik, F; Rouviere-Yaniv, J.

    1995-01-01

    The heterodimeric HU protein, isolated from Escherichia coli, is associated with the bacterial nucleoid and shares some properties with both histones and HMG proteins. It is the prototype of small bacterial DNA binding proteins with a pleiotropic role in the cell. HU participates in several biological processes like cell division, initiation of DNA replication, transposition, and other biochemical functions. We show here that bacteria lacking HU are extremely sensitive to gamma irradiation. E...

  2. Alleviation of temperature-sensitive secretion defect of Pseudomonas fluorescens ATP-binding cassette (ABC) transporter, TliDEF, by a change of single amino acid in the ABC protein, TliD.

    Eom, Gyeong Tae; Oh, Joon Young; Park, Ji Hyun; Lim, Hye Jin; Lee, So Jeong; Kim, Eun Young; Choi, Ji-Eun; Jegal, Jonggeon; Song, Bong Keun; Yu, Ju-Hyun; Song, Jae Kwang

    2016-09-01

    An ABC transporter, TliDEF, from Pseudomonas fluorescens SIK W1, mediates the secretion of its cognate lipase, TliA, in a temperature-dependent secretion manner; the TliDEF-mediated secretion of TliA was impossible at the temperatures over 33°C. To isolate a mutant TliDEF capable of secreting TliA at 35°C, the mutagenesis of ABC protein (TliD) was performed. The mutated tliD library where a random point mutation was introduced by error-prone PCR was coexpressed with the wild-type tliE, tliF and tliA in Escherichia coli. Among approximately 10,000 colonies of the tliD library, we selected one colony that formed transparent halo on LB-tributyrin plates at 35°C. At the growth temperature of 35°C, the selected mutant TliD showed 1.75 U/ml of the extracellular lipase activity, while the wild-type TliDEF did not show any detectable lipase activity in the culture supernatant of E. coli. Moreover, the mutant TliD also showed higher level of TliA secretion than the wild-type TliDEF at other culture temperatures, 20°C, 25°C and 30°C. The mutant TliD had a single amino acid change (Ser287Pro) in the predicted transmembrane region in the membrane domain of TliD, implying that the corresponding region of TliD was important for causing the temperature-dependent secretion of TliDEF. These results suggested that the property of ABC transporter could be changed by the change of amino acid in the ABC protein. PMID:27033673

  3. Depletion of abundant plasma proteins by poly(N-isopropylacrylamide-acrylic acid) hydrogel particles

    Such-Sanmartín, Gerard; Ventura-Espejo, Estela; Jensen, Ole N

    2014-01-01

    the application of pH-sensitive poly(N-isopropylacrylamide-acrylic acid) hydrogel particles for removal of abundant plasma proteins, prior to proteome analysis by MS. Protein depletion occurs by two separate mechanisms: (1) hydrogel particles incubated with low concentrations of plasma capture...

  4. Amino acid protein composition of grain of common wheat mutants

    The quantitative ratio of some amino acids changes in the grain of soft wheat morphological mutants with a high protein content. The soft wheat mutants developed, valuable for breeding, surpass the initial varieties in total protein content and the content of some amino acids in grain. The relative content of some amino acids in protein varies, and the stable sum of irreplaceable amino acids in it is retained

  5. Inhibition of protein kinase C intracerebroventricularly attenuates sensitization

    Mrowczynski, Oliver Daniel

    2012-01-01

    Drug relapse, mediated by drug-associated memories, is a major problem associated with addiction. Protein kinase C (PKC) is a family of protein kinase enzymes that has been implicated in learning and memory with regards to addiction. This study used a PKC inhibitor, chelerythrine (10nmol), to investigate the effects of blocking PKC throughout the brain on addiction related memories. Cocaine (15mg/kg) induced locomotor sensitization, used to model the transition from casual to compulsive use, ...

  6. Photo-CIDNP studies of amino acids and proteins

    The ultimate aim of the research described in this thesis is the development of methods with which ope may study the structure and function of proteins on a molecular level. This is done with the help of a combination of NMR (Nuclear Magnetic Resonance) and flash photolysis, in which light initiated reactions between a chromophore and an amino acid induce abnormal NMR intensities. Chapters 1, 2 and 3: In the first chapter, a brief introduction of CIDNP and its application to proteins is given, followed by a short description of each chapter. The second chapter is an introductory review, covering the basics of the NMR experiment in the first part, and the theory behind the CIDNP phenomenon in the second. Chapter three describes the experimental apparatus and methods. Chapter 4: Photosensitization The light initiated chemical reaction between photosensitizer and amino acid residue is studied in detail for the case of FMN (flavinmononucleotide) and the amino acids tyrosine, tryptophan and histidine. An introduction is given of further sensitizers which have been found to generate CIDNP on amino acids, and which are used in later chapters. Chapter 5: CIDNP of Amino Acids and Proteins The typical CIDNP spectra of the amino acids tyrosine, tryptophan and histidine are introduced and elucidated in the first half of this chapter. Photo-CIDNP on the proteins ribonuclease A and Hen Egg White Lysozyme with the photosensitizers FMN, thionin and eosin Y are described in the second half. Chapter 6: CIDNP in Micellar Solutions The presence of detergent, below and above the critical micelle concentration, is shown to affect CIDNP intensities, due to electrostatic interactions between charged dye and detergent molecules. In the last part of this chapter, photo-CIDNP experiments with the membrane protein gramicidin A, incorporated in detergent and lipid micelles, are described. Chapter 7: CIDNP Study of the Tryptophan Radical CIDNP spectra are characteristic of the transient radical

  7. Photo-CIDNP studies of amino acids and proteins

    Lopez, J.J

    2001-07-01

    The ultimate aim of the research described in this thesis is the development of methods with which ope may study the structure and function of proteins on a molecular level. This is done with the help of a combination of NMR (Nuclear Magnetic Resonance) and flash photolysis, in which light initiated reactions between a chromophore and an amino acid induce abnormal NMR intensities. Chapters 1, 2 and 3: In the first chapter, a brief introduction of CIDNP and its application to proteins is given, followed by a short description of each chapter. The second chapter is an introductory review, covering the basics of the NMR experiment in the first part, and the theory behind the CIDNP phenomenon in the second. Chapter three describes the experimental apparatus and methods. Chapter 4: Photosensitization The light initiated chemical reaction between photosensitizer and amino acid residue is studied in detail for the case of FMN (flavinmononucleotide) and the amino acids tyrosine, tryptophan and histidine. An introduction is given of further sensitizers which have been found to generate CIDNP on amino acids, and which are used in later chapters. Chapter 5: CIDNP of Amino Acids and Proteins The typical CIDNP spectra of the amino acids tyrosine, tryptophan and histidine are introduced and elucidated in the first half of this chapter. Photo-CIDNP on the proteins ribonuclease A and Hen Egg White Lysozyme with the photosensitizers FMN, thionin and eosin Y are described in the second half. Chapter 6: CIDNP in Micellar Solutions The presence of detergent, below and above the critical micelle concentration, is shown to affect CIDNP intensities, due to electrostatic interactions between charged dye and detergent molecules. In the last part of this chapter, photo-CIDNP experiments with the membrane protein gramicidin A, incorporated in detergent and lipid micelles, are described. Chapter 7: CIDNP Study of the Tryptophan Radical CIDNP spectra are characteristic of the transient radical

  8. Sensitive ligand-based protein quantification using immuno-PCR

    Hansen, Marcus Celik; Nederby, Line; Henriksen, Mads Okkels-Birk; Hansen, Maria; Nyvold, Charlotte Guldborg

    2014-01-01

    Quantitative PCR (qPCR) of reverse-transcribed mRNA has revolutionized gene expression analyses. qPCR analysis is based on the prevalent assumption that mRNA transcript numbers provide an adequate measure of specific biomarker expression. However, taking the complexity of protein turnover into...... account, there is a need to correlate qPCR-derived transcriptional patterns with protein translational patterns so as to not leave behind important pathobiological details. One emerging approach in protein analysis is PCR-coupled protein quantification, often denoted as immuno-PCR (iPCR), which targets...... soluble proteins. Here we review recent trends and applications in iPCR assays that may bridge the gap between classical enzyme-linked immunosorbent assays and mass spectrometry methodologies in terms of sensitivity and multiplexing....

  9. What makes ribosome-mediated transcriptional attenuation sensitive to amino Acid limitation?

    2005-06-01

    Full Text Available Ribosome-mediated transcriptional attenuation mechanisms are commonly used to control amino acid biosynthetic operons in bacteria. The mRNA leader of such an operon contains an open reading frame with "regulatory" codons, cognate to the amino acid that is synthesized by the enzymes encoded by the operon. When the amino acid is in short supply, translation of the regulatory codons is slow, which allows transcription to continue into the structural genes of the operon. When amino acid supply is in excess, translation of regulatory codons is rapid, which leads to termination of transcription. We use a discrete master equation approach to formulate a probabilistic model for the positioning of the RNA polymerase and the ribosome in the attenuator leader sequence. The model describes how the current rate of amino acid supply compared to the demand in protein synthesis (signal determines the expression of the amino acid biosynthetic operon (response. The focus of our analysis is on the sensitivity of operon expression to a change in the amino acid supply. We show that attenuation of transcription can be hyper-sensitive for two main reasons. The first is that its response depends on the outcome of a race between two multi-step mechanisms with synchronized starts: transcription of the leader of the operon, and translation of its regulatory codons. The relative change in the probability that transcription is aborted (attenuated can therefore be much larger than the relative change in the time it takes for the ribosome to read a regulatory codon. The second is that the general usage frequencies of codons of the type used in attenuation control are small. A small percentage decrease in the rate of supply of the controlled amino acid can therefore lead to a much larger percentage decrease in the rate of reading a regulatory codon. We show that high sensitivity further requires a particular choice of regulatory codon among several synonymous codons for the

  10. What makes ribosome-mediated transcriptional attenuation sensitive to amino acid limitation?

    Johan Elf

    2005-06-01

    Full Text Available Ribosome-mediated transcriptional attenuation mechanisms are commonly used to control amino acid biosynthetic operons in bacteria. The mRNA leader of such an operon contains an open reading frame with "regulatory" codons, cognate to the amino acid that is synthesized by the enzymes encoded by the operon. When the amino acid is in short supply, translation of the regulatory codons is slow, which allows transcription to continue into the structural genes of the operon. When amino acid supply is in excess, translation of regulatory codons is rapid, which leads to termination of transcription. We use a discrete master equation approach to formulate a probabilistic model for the positioning of the RNA polymerase and the ribosome in the attenuator leader sequence. The model describes how the current rate of amino acid supply compared to the demand in protein synthesis (signal determines the expression of the amino acid biosynthetic operon (response. The focus of our analysis is on the sensitivity of operon expression to a change in the amino acid supply. We show that attenuation of transcription can be hyper-sensitive for two main reasons. The first is that its response depends on the outcome of a race between two multi-step mechanisms with synchronized starts: transcription of the leader of the operon, and translation of its regulatory codons. The relative change in the probability that transcription is aborted (attenuated can therefore be much larger than the relative change in the time it takes for the ribosome to read a regulatory codon. The second is that the general usage frequencies of codons of the type used in attenuation control are small. A small percentage decrease in the rate of supply of the controlled amino acid can therefore lead to a much larger percentage decrease in the rate of reading a regulatory codon. We show that high sensitivity further requires a particular choice of regulatory codon among several synonymous codons for the

  11. What makes ribosome-mediated transcriptional attenuation sensitive to amino acid limitation?

    Elf, Johan; Ehrenberg, Måns

    2005-06-01

    Ribosome-mediated transcriptional attenuation mechanisms are commonly used to control amino acid biosynthetic operons in bacteria. The mRNA leader of such an operon contains an open reading frame with "regulatory" codons, cognate to the amino acid that is synthesized by the enzymes encoded by the operon. When the amino acid is in short supply, translation of the regulatory codons is slow, which allows transcription to continue into the structural genes of the operon. When amino acid supply is in excess, translation of regulatory codons is rapid, which leads to termination of transcription. We use a discrete master equation approach to formulate a probabilistic model for the positioning of the RNA polymerase and the ribosome in the attenuator leader sequence. The model describes how the current rate of amino acid supply compared to the demand in protein synthesis (signal) determines the expression of the amino acid biosynthetic operon (response). The focus of our analysis is on the sensitivity of operon expression to a change in the amino acid supply. We show that attenuation of transcription can be hyper-sensitive for two main reasons. The first is that its response depends on the outcome of a race between two multi-step mechanisms with synchronized starts: transcription of the leader of the operon, and translation of its regulatory codons. The relative change in the probability that transcription is aborted (attenuated) can therefore be much larger than the relative change in the time it takes for the ribosome to read a regulatory codon. The second is that the general usage frequencies of codons of the type used in attenuation control are small. A small percentage decrease in the rate of supply of the controlled amino acid can therefore lead to a much larger percentage decrease in the rate of reading a regulatory codon. We show that high sensitivity further requires a particular choice of regulatory codon among several synonymous codons for the same amino acid. We

  12. Effects of arachidonic acid on ATP-sensitive K+ current in murine colonic smooth muscle cells.

    Jun, Jae Yeoul; Yeum, Cheol Ho; Park, Yoo Whan; Jang, In Youb; Kong, In Deok; Sim, Jae Hoon; So, Insuk; Kim, Ki Whan; You, Ho Jin

    2002-09-01

    The effects of arachidonic acid (AA) and the mechanism through which it modulates ATP-sensitive K+ (K(ATP)) currents were examined in single smooth muscle cells of murine proximal colon. In the current-clamping mode, AA and glibenclamide induced depolarization of membrane potential. Using 0.1 mM ATP and 140 mM K+ solution in the pipette and 90 mM K+ in the bath solution at a -80 mV of holding potential, pinacidil activated the glibenclamide-sensitive inward current. The potential of these currents was reversed to near the equilibrium potential of K+ by 60 mM K+ in the bath solution. AA inhibited K(ATP) currents in a dose-dependent manner. This inhibition was not changed when 1 mM GDPbetaS was present in the pipette. Chelerythrine, protein kinase C inhibitor, did not block the AA effects. Superoxide dismutase and metabolic inhibitors (indomethacin and nordihydroguaiacretic acid) of AA did not affect the AA-induced inhibition. Eicosatetraynoic acid, a nonmetabolizable analogue of AA, inhibited the K(ATP) currents. These results suggest that AA-induced inhibition of K(ATP) currents is not mediated by G-protein or protein kinase C activation. The inhibitory action is likely to be a possible mechanism of AA-induced membrane depolarization. PMID:12396031

  13. Insulin sensitivity is independent of lipid binding protein trafficking at the plasma membrane in human skeletal muscle

    Jordy, Andreas Børsting; Serup, Annette Karen; Karstoft, Kristian;

    2014-01-01

    The aim of the present study was to investigate lipid-induced regulation of lipid binding proteins in human skeletal muscle and the impact hereof on insulin sensitivity. Eleven healthy male subjects underwent a 3-day hyper-caloric and high-fat diet regime. Muscle biopsies were taken before and...... increased fatty acid availability. This suggests a time dependency in the up-regulation of FAT/CD36 and FABPpm protein during high availability of plasma fatty acids. Furthermore, we did not detect FATP1 and FATP4 protein in giant sarcolemmal vesicles obtained from human skeletal muscle. In conclusion, this...... study shows that a short-term lipid-load increases mRNA content of key lipid handling proteins in human muscle. However, decreased insulin sensitivity after high-fat diet is not accompanied with relocation of FAT/CD36 or FABPpm protein to the sarcolemma. Finally, FATP1 and FATP4 protein could was...

  14. The clinical significance of fatty acid binding proteins

    Barbara Choromańska; Piotr Myśliwiec; Jacek Dadan; Hady Razak Hady; Adrian Chabowski

    2011-01-01

    Excessive levels of free fatty acids are toxic to cells. The human body has evolved a defense mechanism in the form of small cytoplasmic proteins called fatty acid binding proteins (FABPs) that bind long-chain fatty acids (LCFA), and then refer them to appropriate intracellular disposal sites (oxidation in mitochondria and peroxisomes or storage in the endoplasmic reticulum). So far, nine types of these proteins have been described, and their name refers to the place in which they were first ...

  15. Microspectrophotometric quantitation of nucleic acid and protein in irradiated epidermis

    Nucleic acid and proteins of newborn rat tail epidermis subjected to local X-irradiation were microspectrophotometrically studied. Feulgen, gallocyanine chrom-alum and naphthol yellow S methods were performed for demonstration of DNA, total nucleic acid and proteins respectively. The amount of proteins and total nucleic acid increases concomitantly with reactional acanthosis. However, the proteins and nucleic acid decrease as from day 3 post-irradiation. A tentative interpretation of the results would point to a giantization of the epidermic cells not only caused by aqueous imbition but also by an actual increase of the cellular protoplasm. (orig.)

  16. Microspectrophotometric quantitation of nucleic acid and protein in irradiated epidermis.

    Conti, C J; Giménez, I B; Cabrini, R L

    1976-03-01

    Nucleic acid and proteins of newborn rat tail subjected to local X-irradiation were microspectrophotometrically studied. Feulgen, gallocyanine chrom-alum and naphthol yellow S methods were performed for demonstration of DNA, total nucleic acid and proteins respectively. The amount of proteins and total nucleic acid increases concomitantly with reactional acanthosis. However, the proteins and nucleic acid decrease as from day 3 post-irradiation. A tentative interpretation of the results would point to a giantization of the epidermic cells not only caused by aqueous imbition but also by an actual increase of the cellular protoplasm. PMID:1258094

  17. Roles of Intrinsic Disorder in Protein-Nucleic Acid Interactions

    Dyson, H. Jane

    2011-01-01

    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 t...

  18. Prevalence of soy protein hypersensitivity in cow's milk protein-sensitive children in Korea.

    Ahn, Kang-Mo; Han, Young-Shin; Nam, Seung-Yeon; Park, Hwa-young; Shin, Mee-Yong; Lee, Sang-Il

    2003-01-01

    This study was aimed to evaluate the prevalence of soy protein hypersensitivity in cow's milk protein-sensitive children in Korea. A total of 1,363 patients with atopic dermatitis, urticaria, enterocolitis syndrome, bronchial asthma or allergic rhinitis were recruited. First, we estimated the prevalence of sensitization to soy in children sensitized to cow's milk. Specific IgE levels > 0.7 kU/L by CAP assay were considered positive. Next, the prevalence of soy allergy in cow's milk allergy (C...

  19. Identify Secretory Protein of Malaria Parasite with Modified Quadratic Discriminant Algorithm and Amino Acid Composition.

    Feng, Yong-E

    2016-06-01

    Malaria parasite secretes various proteins in infected red blood cell for its growth and survival. Thus identification of these secretory proteins is important for developing vaccine or drug against malaria. In this study, the modified method of quadratic discriminant analysis is presented for predicting the secretory proteins. Firstly, 20 amino acids are divided into five types according to the physical and chemical characteristics of amino acids. Then, we used five types of amino acids compositions as inputs of the modified quadratic discriminant algorithm. Finally, the best prediction performance is obtained by using 20 amino acid compositions, the sensitivity of 96 %, the specificity of 92 % with 0.88 of Mathew's correlation coefficient in fivefold cross-validation test. The results are also compared with those of existing prediction methods. The compared results shown our method are prominent in the prediction of secretory proteins. PMID:26286010

  20. Shedding light on proteins, nucleic acids, cells, humans and fish

    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.

  1. Development of a Quantitative BRET Affinity Assay for Nucleic Acid-Protein Interactions.

    Vickers, Timothy A; Crooke, Stanley T

    2016-01-01

    Protein-nucleic acid interactions play a crucial role in the regulation of diverse biological processes. Elucidating the roles that protein-nucleic acid complexes play in the regulation of transcription, translation, DNA replication, repair and recombination, and RNA processing continues to be a crucial aspect of understanding of cell biology and the mechanisms of disease. In addition, proteins have been demonstrated to interact with antisense oligonucleotide therapeutics in a sequence and chemistry dependent manner, influencing ASO potency and distribution in cells and in vivo. While many assays have been developed to measure protein-nucleic acid interactions, many suffer from lack of throughput and sensitivity, or challenges with protein purification and scalability. In this report we present a new BRET assay for the analysis of DNA-protein interactions which makes use of an extremely bright luciferase as a tag for the binding protein, along with a long-wavelength fluorophore conjugated to the nucleic acid. The resulting assay is high throughput, sensitive, does not require protein purification, and even allows for quantitative characterization of these interactions within the biologically relevant context of whole cells. PMID:27571227

  2. Intumescent features of nucleic acids and proteins

    Highlights: • The combustion resistance of DNA and caseins to different heat fluxes was studied. • Upon heating, DNA and caseins exhibited an intumescent behaviour. • The char derived from DNA was more stable and coherent than that from caseins. - Abstract: Are nucleic acids and proteins intumescent molecules? In order to get an answer, in the present manuscript, powders of deoxyribose nucleic acids (DNA) and caseins have been exposed to different heat fluxes under a cone calorimeter source and to the direct application of a propane flame. Under these conditions, DNA and caseins exhibited a typical intumescent behaviour, generating a coherent expanded cellular carbonaceous residue (char), extremely resistant to heat exposure. The resulting volumetric expansion as well as the resistance of the formed char turned out to be dependent on (i) the chemical structure of the chosen biomacromolecule, (ii) the evolution of ammonia and (iii) the adopted heat flux in cone calorimetry tests (namely, 25, 35, 50 and 75 kW/m2). The presence of ribose units within the DNA backbone determined the formation of highly expanded and coherent residues as compared to those obtained from caseins. Indeed, under a heat flux of 35 kW/m2, when a carbon source (i.e. common cane sugar) was added to caseins, the resulting char was similar to that formed by DNA. Furthermore, the char expansion was ascribed to the evolution of ammonia released by these biomacromolecules upon heating, as detected by thermogravimetry coupled to infrared spectroscopy, and confirmed by scanning electron microscopy experiments performed on the bubbles present in the residues of flammability tests

  3. Intumescent features of nucleic acids and proteins

    Alongi, Jenny, E-mail: jenny.alongi@polito.it; Cuttica, Fabio; Blasio, Alessandro Di; Carosio, Federico; Malucelli, Giulio

    2014-09-10

    Highlights: • The combustion resistance of DNA and caseins to different heat fluxes was studied. • Upon heating, DNA and caseins exhibited an intumescent behaviour. • The char derived from DNA was more stable and coherent than that from caseins. - Abstract: Are nucleic acids and proteins intumescent molecules? In order to get an answer, in the present manuscript, powders of deoxyribose nucleic acids (DNA) and caseins have been exposed to different heat fluxes under a cone calorimeter source and to the direct application of a propane flame. Under these conditions, DNA and caseins exhibited a typical intumescent behaviour, generating a coherent expanded cellular carbonaceous residue (char), extremely resistant to heat exposure. The resulting volumetric expansion as well as the resistance of the formed char turned out to be dependent on (i) the chemical structure of the chosen biomacromolecule, (ii) the evolution of ammonia and (iii) the adopted heat flux in cone calorimetry tests (namely, 25, 35, 50 and 75 kW/m{sup 2}). The presence of ribose units within the DNA backbone determined the formation of highly expanded and coherent residues as compared to those obtained from caseins. Indeed, under a heat flux of 35 kW/m{sup 2}, when a carbon source (i.e. common cane sugar) was added to caseins, the resulting char was similar to that formed by DNA. Furthermore, the char expansion was ascribed to the evolution of ammonia released by these biomacromolecules upon heating, as detected by thermogravimetry coupled to infrared spectroscopy, and confirmed by scanning electron microscopy experiments performed on the bubbles present in the residues of flammability tests.

  4. Structure and behaviour of proteins, nucleic acids and viruses from vibrational Raman optical activity

    Barron, L.D.; Blanch, E.W.; McColl, I.H.; Syme, C.D.; Hecht, L.; Nielsen, Kurt

    stacking arrangement and the mutual orientation of the sugar and base rings around the C-N glycosidic link. The ROA spectra of intact viruses provide information on the folds of the coat proteins and the nucleic acid structure. The large number of structure-sensitive bands in protein ROA spectra is...... aqueous solution. Protein ROA spectra provide information on the secondary and tertiary structure of the polypeptide backbone, hydration, side chain conformation and structural elements present in denatured states. Nucleic acid ROA spectra provide information on the sugar ring conformation, the base...

  5. Prediction of protein motions from amino acid sequence and its application to protein-protein interaction

    Wako Hiroshi

    2010-07-01

    Full Text Available Abstract Background Structural flexibility is an important characteristic of proteins because it is often associated with their function. The movement of a polypeptide segment in a protein can be broken down into two types of motions: internal and external ones. The former is deformation of the segment itself, but the latter involves only rotational and translational motions as a rigid body. Normal Model Analysis (NMA can derive these two motions, but its application remains limited because it necessitates the gathering of complete structural information. Results In this work, we present a novel method for predicting two kinds of protein motions in ordered structures. The prediction uses only information from the amino acid sequence. We prepared a dataset of the internal and external motions of segments in many proteins by application of NMA. Subsequently, we analyzed the relation between thermal motion assessed from X-ray crystallographic B-factor and internal/external motions calculated by NMA. Results show that attributes of amino acids related to the internal motion have different features from those related to the B-factors, although those related to the external motion are correlated strongly with the B-factors. Next, we developed a method to predict internal and external motions from amino acid sequences based on the Random Forest algorithm. The proposed method uses information associated with adjacent amino acid residues and secondary structures predicted from the amino acid sequence. The proposed method exhibited moderate correlation between predicted internal and external motions with those calculated by NMA. It has the highest prediction accuracy compared to a naïve model and three published predictors. Conclusions Finally, we applied the proposed method predicting the internal motion to a set of 20 proteins that undergo large conformational change upon protein-protein interaction. Results show significant overlaps between the

  6. Temperature-Sensitive Mutants of Escherichia coli Requiring Saturated and Unsaturated Fatty Acids for Growth: Isolation and Properties

    Harder, Mark E.; Beacham, Ifor R.; Cronan, John E.; Beacham, Kathryn; Honegger, Joy L.; Silbert, David F.

    1972-01-01

    A procedure is described for selection of temperature-sensitive mutants affecting fatty-acid synthesis based upon radiation suicide of wild-type organisms by tritiated acetate selectively incorporated into fatty acids. At 37°, two of the mutants extensively incorporate fatty-acid supplements provided in the medium, and grow for extended periods only when a trans-unsaturated or a combination of saturated and cis-unsaturated fatty acids is available. In vivo fatty-acid synthesis, measured by [14C]acetate incorporation, is temperature-sensitive in these strains relative to protein synthesis and other non-lipid macromolecular syntheses using acetate. The biochemical nature of these mutations has not been identified. PMID:4564200

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

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

    2012-01-01

    Liver fatty acid binding protein (L-FABP), a cytosolic protein most abundant in liver, is associated with intracellular transport of fatty acids, nuclear signaling, and regulation of intracellular lipolysis. Among the members of the intracellular lipid binding protein family, L-FABP is of particular interest as it can i), bind two fatty acid molecules simultaneously and ii), accommodate a variety of bulkier physiological ligands such as bilirubin and fatty acyl CoA. To better understand the p...

  8. Contact sensitizers induce skin inflammation via ROS production and hyaluronic acid degradation.

    Philipp R Esser

    Full Text Available BACKGROUND: Allergic contact dermatitis (ACD represents a severe health problem with increasing worldwide prevalence. It is a T cell-mediated skin disease induced by protein-reactive organic and inorganic chemicals. A key feature of contact allergens is their ability to trigger an innate immune response that leads to skin inflammation. Previous evidence from the mouse contact hypersensitivity (CHS model suggests a role for endogenous activators of innate immune signaling. Here, we analyzed the role of contact sensitizer induced ROS production and concomitant changes in hyaluronic acid metabolism on CHS responses. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed in vitro and in vivo ROS production using fluorescent ROS detection reagents. HA fragmentation was determined by gel electrophoresis. The influence of blocking ROS production and HA degradation by antioxidants, hyaluronidase-inhibitor or p38 MAPK inhibitor was analyzed in the murine CHS model. Here, we demonstrate that organic contact sensitizers induce production of reactive oxygen species (ROS and a concomitant breakdown of the extracellular matrix (ECM component hyaluronic acid (HA to pro-inflammatory low molecular weight fragments in the skin. Importantly, inhibition of either ROS-mediated or enzymatic HA breakdown prevents sensitization as well as elicitation of CHS. CONCLUSIONS/SIGNIFICANCE: These data identify an indirect mechanism of contact sensitizer induced innate inflammatory signaling involving the breakdown of the ECM and generation of endogenous danger signals. Our findings suggest a beneficial role for anti-oxidants and hyaluronidase inhibitors in prevention and treatment of ACD.

  9. Redox-Mediated Suberoylanilide Hydroxamic Acid Sensitivity in Breast Cancer

    Chiaradonna, Ferdinando; Barozzi, Iros; Miccolo, Claudia; Bucci, Gabriele; Palorini, Roberta; Fornasari, Lorenzo; Botrugno, Oronza A.; Pruneri, Giancarlo; Masullo, Michele; Passafaro, Alfonso; Galimberti, Viviana E.; Fantin, Valeria R.; Richon, Victoria M.; Pece, Salvatore; Viale, Giuseppe; Di Fiore, Pier Paolo; Draetta, Giulio; Pelicci, Pier Giuseppe

    2015-01-01

    Abstract Aims: Vorinostat (suberoylanilide hydroxamic acid; SAHA) is a histone deacetylase inhibitor (HDACi) approved in the clinics for the treatment of T-cell lymphoma and with the potential to be effective also in breast cancer. We investigated the responsiveness to SAHA in human breast primary tumors and cancer cell lines. Results: We observed a differential response to drug treatment in both human breast primary tumors and cancer cell lines. Gene expression analysis of the breast cancer cell lines revealed that genes involved in cell adhesion and redox pathways, especially glutathione metabolism, were differentially expressed in the cell lines resistant to SAHA compared with the sensitive ones, indicating their possible association with drug resistance mechanisms. Notably, such an association was also observed in breast primary tumors. Indeed, addition of buthionine sulfoximine (BSO), a compound capable of depleting cellular glutathione, significantly enhanced the cytotoxicity of SAHA in both breast cancer cell lines and primary breast tumors. Innovation: We identify and validate transcriptional differences in genes involved in redox pathways, which include potential predictive markers of sensitivity to SAHA. Conclusion: In breast cancer, it could be relevant to evaluate the expression of antioxidant genes that may favor tumor resistance as a factor to consider for potential clinical application and treatment with epigenetic drugs (HDACis). Antioxid. Redox Signal. 23, 15–29. PMID:25897982

  10. Amino acid sequences of proteins from Leptospira serovar pomona

    Alves Selmo F

    2000-01-01

    Full Text Available 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.

  11. Protein structure-sensitive electrocatalysis at dithiothreitol-modified electrodes

    Ostatná, Veronika; Černocká, Hana; Paleček, Emil

    2010-01-01

    Roč. 132, č. 27 (2010), s. 9408-9413. ISSN 0002-7863 R&D Projects: GA AV ČR(CZ) KJB100040901; GA ČR(CZ) GP202/07/P497; GA MŠk(CZ) LC06035 Grant ostatní: GA AV ČR(CZ) KAN400310651 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : constant current chronopotentiometry * mercury and solid amalgam electrodes * structure-sensitive protein analysis Subject RIV: BO - Biophysics Impact factor: 9.019, year: 2010

  12. Structure and behaviour of proteins, nucleic acids and viruses from vibrational Raman optical activity

    Barron, L.D.; Blanch, E.W.; McColl, I.H.; Syme, C.D.; Hecht, L.; Nielsen, Kurt

    aqueous solution. Protein ROA spectra provide information on the secondary and tertiary structure of the polypeptide backbone, hydration, side chain conformation and structural elements present in denatured states. Nucleic acid ROA spectra provide information on the sugar ring conformation, the base...... stacking arrangement and the mutual orientation of the sugar and base rings around the C-N glycosidic link. The ROA spectra of intact viruses provide information on the folds of the coat proteins and the nucleic acid structure. The large number of structure-sensitive bands in protein ROA spectra is...... especially favourable for fold determination using pattern recognition techniques. This article gives a brief account of the ROA technique and presents the ROA spectra of a selection of proteins, nucleic acids and viruses that illustrate the applications of ROA spectroscopy in biomolecular research....

  13. Protein microarray: sensitive and effective immunodetection for drug residues

    Zer Cindy

    2010-02-01

    Full Text Available Abstract Background Veterinary drugs such as clenbuterol (CL and sulfamethazine (SM2 are low molecular weight ( Results The artificial antigens were spotted on microarray slides. Standard concentrations of the compounds were added to compete with the spotted antigens for binding to the antisera to determine the IC50. Our microarray assay showed the IC50 were 39.6 ng/ml for CL and 48.8 ng/ml for SM2, while the traditional competitive indirect-ELISA (ci-ELISA showed the IC50 were 190.7 ng/ml for CL and 156.7 ng/ml for SM2. We further validated the two methods with CL fortified chicken muscle tissues, and the protein microarray assay showed 90% recovery while the ci-ELISA had 76% recovery rate. When tested with CL-fed chicken muscle tissues, the protein microarray assay had higher sensitivity (0.9 ng/g than the ci-ELISA (0.1 ng/g for detection of CL residues. Conclusions The protein microarrays showed 4.5 and 3.5 times lower IC50 than the ci-ELISA detection for CL and SM2, respectively, suggesting that immunodetection of small molecules with protein microarray is a better approach than the traditional ELISA technique.

  14. Interaction of milk whey protein with common phenolic acids

    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.

  15. Informational Way to Protein Alphabet: Entropic Classification of Amino Acids

    Gorban, A N; Popova, T

    2007-01-01

    What are proteins made from, as the working parts of the living cells protein machines? To answer this question, we need a technology to disassemble proteins onto elementary func-tional details and to prepare lumped description of such details. This lumped description might have a multiple material realization (in amino acids). Our hypothesis is that informational approach to this problem is possible. We propose a way of hierarchical classification that makes the primary structure of protein maximally non-random. The first steps of the suggested research program are realized: the method and the analysis of optimal informational protein binary alphabet. The general method is used to answer several specific questions, for example: (i) Is there a syntactic difference between Globular and Membrane proteins? (ii) Are proteins random sequences of amino acids (a long discussion)? For these questions, the answers are as follows: (i) There exists significant syntactic difference between Globular and Membrane proteins,...

  16. Small acid soluble proteins for rapid spore identification.

    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.

  17. Activation of pheromone-sensitive neurons is mediated by conformational activation of pheromone-binding protein.

    Laughlin, John D; Ha, Tal Soo; Jones, David N M; Smith, Dean P

    2008-06-27

    Detection of volatile odorants by olfactory neurons is thought to result from direct activation of seven-transmembrane odorant receptors by odor molecules. Here, we show that detection of the Drosophila pheromone, 11-cis vaccenyl acetate (cVA), is instead mediated by pheromone-induced conformational shifts in the extracellular pheromone-binding protein, LUSH. We show that LUSH undergoes a pheromone-specific conformational change that triggers the firing of pheromone-sensitive neurons. Amino acid substitutions in LUSH that are predicted to reduce or enhance the conformational shift alter sensitivity to cVA as predicted in vivo. One substitution, LUSH(D118A), produces a dominant-active LUSH protein that stimulates T1 neurons through the neuronal receptor components Or67d and SNMP in the complete absence of pheromone. Structural analysis of LUSH(D118A) reveals that it closely resembles cVA-bound LUSH. Therefore, the pheromone-binding protein is an inactive, extracellular ligand converted by pheromone molecules into an activator of pheromone-sensitive neurons and reveals a distinct paradigm for detection of odorants. PMID:18585358

  18. Representation of protein-sequence information by amino acid subalphabets

    Andersen, C.A.F.; Brunak, Søren

    2004-01-01

    -sequence information, using machine learning strategies, where the primary goal is the discovery of novel powerful representations for use in AI techniques. In the case of proteins and the 20 different amino acids they typically contain, it is also a secondary goal to discover how the current selection of amino acids......-which now are common in proteins-might have emerged from simpler selections, or alphabets, in use earlier during the evolution of living organisms....

  19. FLU, an amino acid substitution model for influenza proteins

    Gascuel Olivier; Le Quang; Dang Cuong; Le Vinh

    2010-01-01

    Abstract Background The amino acid substitution model is the core component of many protein analysis systems such as sequence similarity search, sequence alignment, and phylogenetic inference. Although several general amino acid substitution models have been estimated from large and diverse protein databases, they remain inappropriate for analyzing specific species, e.g., viruses. Emerging epidemics of influenza viruses raise the need for comprehensive studies of these dangerous viruses. We p...

  20. Interactions between acidic proteins and crystals: stereochemical requirements in biomineralization.

    Addadi, L; Weiner, S

    1985-01-01

    Acidic matrix macromolecules are intimately involved in biological crystal growth. In vitro experiments, in which crystals of calcium dicarboxylate salts were grown in the presence of aspartic acid-rich proteins, revealed a stereochemical property common to all the interacting faces. Calcite crystals are nucleated on stereochemically analogous faces when proteins are adsorbed onto a rigid substrate. The importance of this property in biomineralization is discussed.

  1. Natural ligand binding and transfer from liver fatty acid binding protein (LFABP) to membranes.

    De Gerónimo, Eduardo; Hagan, Robert M; Wilton, David C; Córsico, Betina

    2010-09-01

    Liver fatty acid-binding protein (LFABP) is distinctive among fatty acid-binding proteins because it binds more than one molecule of long-chain fatty acid and a variety of diverse ligands. Also, the transfer of fluorescent fatty acid analogues to model membranes under physiological ionic strength follows a different mechanism compared to most of the members of this family of intracellular lipid binding proteins. Tryptophan insertion mutants sensitive to ligand binding have allowed us to directly measure the binding affinity, ligand partitioning and transfer to model membranes of natural ligands. Binding of fatty acids shows a cooperative mechanism, while acyl-CoAs binding presents a hyperbolic behavior. Saturated fatty acids seem to have a stronger partition to protein vs. membranes, compared to unsaturated fatty acids. Natural ligand transfer rates are more than 200-fold higher compared to fluorescently-labeled analogues. Interestingly, oleoyl-CoA presents a markedly different transfer behavior compared to the rest of the ligands tested, probably indicating the possibility of specific targeting of ligands to different metabolic fates. PMID:20541621

  2. Protein evolution via amino acid and codon elimination

    Goltermann, Lise; Larsen, Marie Sofie Yoo; Banerjee, Rajat;

    2010-01-01

    a common buried residue, Phe, from the green fluorescent protein (GFP), while retaining activity. A GFP variant containing 11 Phe residues was used as starting scaffold to generate 10 separate variants in which each Phe was replaced individually (in one construct two adjacent Phe residues were changed......BACKGROUND: Global residue-specific amino acid mutagenesis can provide important biological insight and generate proteins with altered properties, but at the risk of protein misfolding. Further, targeted libraries are usually restricted to a handful of amino acids because there is an exponential...... correlation between the number of residues randomized and the size of the resulting ensemble. Using GFP as the model protein, we present a strategy, termed protein evolution via amino acid and codon elimination, through which simplified, native-like polypeptides encoded by a reduced genetic code were obtained...

  3. Contact sensitizers modulate the arachidonic acid metabolism of PMA-differentiated U-937 monocytic cells activated by LPS.

    Del Bufalo, Aurélia; Bernad, José; Dardenne, Christophe; Verda, Denis; Meunier, Jean Roch; Rousset, Françoise; Martinozzi-Teissier, Silvia; Pipy, Bernard

    2011-10-01

    For the effective induction of a hapten-specific T cell immune response toward contact sensitizers, in addition to covalent-modification of skin proteins, the redox and inflammatory statuses of activated dendritic cells are crucial. The aim of this study was to better understand how sensitizers modulate an inflammatory response through cytokines production and COX metabolism cascade. To address this purpose, we used the human monocytic-like U-937 cell line differentiated by phorbol myristate acetate (PMA) and investigated the effect of 6 contact sensitizers (DNCB, PPD, hydroquinone, propyl gallate, cinnamaldehyde and eugenol) and 3 non sensitizers (lactic acid, glycerol and tween 20) on the production of pro-inflammatory cytokines (IL-1β and TNF-α) and on the arachidonic acid metabolic profile after bacterial lipopolysaccharide (LPS) stimulation. Our results showed that among the tested molecules, all sensitizers specifically prevent the production of PMA/LPS-induced COX-2 metabolites (PGE(2,) TxB(2) and PGD(2)), eugenol and cinnamaldehyde inhibiting also the production of IL-1β and TNF-α. We further demonstrated that there is no unique PGE(2) inhibition mechanism: while the release of arachidonic acid (AA) from membrane phospholipids does not appear do be a target of modulation, COX-2 expression and/or COX-2 enzymatic activity are the major steps of prostaglandin synthesis that are inhibited by sensitizers. Altogether these results add a new insight into the multiple biochemical effects described for sensitizers. PMID:21807015

  4. Phthalic Acid Chemical Probes Synthesized for Protein-Protein Interaction Analysis

    Chin-Jen Wu

    2013-06-01

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

  5. Modifications of proteins by polyunsaturated fatty acid peroxidation products

    Refsgaard, Hanne; Tsai, Lin; Stadtman, Earl

    2000-01-01

    -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......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(lll)/O-2] depends on the degree of unsaturation of the fatty acid. The fatty acid...... 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 (alpha,beta-unsaturated aldehydes) with lysine...

  6. REGULATION OF CARDIAC AND SKELETAL MUSCLE PROTEIN SYNTHESIS BY INDIVIDUAL BRANCHED-CHAIN AMINO ACIDS IN NEONATAL PIGS

    Skeletal muscle grows at a very rapid rate in the neonatal pig, due in part to an enhanced sensitivity of protein synthesis to the postprandial rise in amino acids. An increase in leucine alone stimulates protein synthesis in skeletal muscle of the neonatal pig; however, the effect of isoleucine and...

  7. Competitive Binding to Cuprous Ions of Protein and BCA in the Bicinchoninic Acid Protein Assay

    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 increa...

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

    Jillian L Blatti; Joris Beld; Behnke, Craig A; Michael Mendez; 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...

  9. Buffer Interference with Protein Dynamics: A Case Study on Human Liver Fatty Acid Binding Protein

    Long, Dong; Yang, Daiwen

    2009-01-01

    Selection of suitable buffer types is often a crucial step for generating appropriate protein samples for NMR and x-ray crystallographic studies. Although the possible interaction between MES buffer (2-(N-morpholino)ethanesulfonic acid) and proteins has been discussed previously, the interaction is usually thought to have no significant effects on the structures of proteins. In this study, we demonstrate the direct, albeit weak, interaction between MES and human liver fatty acid binding prote...

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

    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 i...

  11. Purification of an acidic recombinant protein from transgenic tobacco

    Holler, Christopher J.

    2007-01-01

    Tobacco has been studied as a host for producing recombinant therapeutic proteins on a large-scale, commercial basis. However, the proteins expressed in tobacco usually need to be purified to high yield and purity from large amounts of biomass in order for their production to be commercially viable. The methods needed to purify proteins from tobacco are very challenging and not well studied. The objective of this research was to develop a process for the purification of the acidic model pr...

  12. The recombinase protein is a torque sensitive molecular switch

    Atwell, Scott; Migliozzi, Daniel; Viovy, Jean-Louis; Cappello, Giovanni

    2016-01-01

    How a nano-searcher finds its nano-target is a general problem in non-equilibrium statistical physics. It becomes vital when the searcher is a damaged DNA fragment trying to find its counterpart on the intact homologous chromosome. If the two copies are paired, that intact homologous sequence serves as a template to reconstitute the damaged DNA sequence, enabling the cell to survive without genetic mutations. To succeed, the search must stop only when the perfect homology is found. The biological process that ensures such a genomic integrity is called Homologous Recombination and is promoted by the Recombinase proteins. In this article, we use torque-sensitive magnetic tweezers to measure the free-energy landscape of the human Recombinase hRad51 protein assembled a DNA fragment. Based on our measurements we model the hRad51/DNA complex as an out-of-equilibrium two-state system and provide a thermodynamical description of Homologous Recombination. With this dynamical two-state model, we suggest a mechanism by ...

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

    Jillian L Blatti

    Full Text Available 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.

  14. Can electrophoretic types of listeria monocytogenes induce different sensitivity to lactic acid bacteria bacteriocins?

    Dimitrijević Mirjana

    2005-01-01

    Full Text Available The aim of this study was to find if electrophoretic types (ETs of Listeria monocytogenes, typed by multilocus enzyme electrophoresis (MEE, can induce different sensitivity to lactic acid bacteria bacteriocins. Bacteriocins are extracellular peptides or protein molecules, produced by lactic acid bacteria, which not only have bactericidal or bacteriostatic effects, on usually closely related bacterial strains, but also they may have destructive effects on some not so closely related Gram positive bacteria, for example Listeria monocytogenes. Listeria monocytogenes is commonly found in the intestines of humans and animals, in milk, soil, leafy vegetables and in food processing environments. These bacteria have been isolated in a variety of foods, including raw and cooked poultry, meat, seafood, salads and sandwiches. Many techniques for typing of Listeria monocytogenes in foodstuffs, have been developed for the purpose of identification of the origin of infection for epidemiological and epizootological studies. Among the 98 examined isolates of Listeria monocytogenes (50 clinical/human and 48 from food of animal origin 32 electrophoretic types have been detected. Bacteriocins, which we have used in the study, originated from the following lacic acid bacteria: Lactobacillus sake 148, Lactococcus UW, Lactobacillus sake 706, Pediococcus 347 and Lactobacillus sake 265. In this study, on the basis of a dendogram, our results indicate that a reliable relationship between genetic distance of Listeria monocytogenes electrophoretic types and their sensitivity to lactic acid bacteria bacteriocins cannot been found. MEE may, however, be of future benefit in establishing links between isolates from human disease cases and thus be useful in establishing the epidemiology of not only sporadic cases, but of outbreaks of listeriosis, as well.

  15. Evaluation of regional acid sensitivity predictions using field data: issues of scale and heterogeneity

    B. Reynolds

    2001-01-01

    Full Text Available An acid waters sensitivity map has been widely used as a background resource for assessing potential impacts of forest management on stream water acidity within Wales. The validity of this approach has been explored by comparing predicted acid sensitivity with the observed acid neutralisation capacity (ANC in stream runoff using data sets from two regional surveys of a major area of acidification concern in Wales. For catchments in excess of 20 ha, the acid sensitivity predictions are generally upheld, although biologically important extremes of acidity are not predicted. For smaller catchments of 2 to 5 ha area, the large observed variations in ANC are poorly predicted from the acid sensitivity map; there is no statistically significant relationship between predicted sensitivity and measured baseflow and stormflow ANC values for the catchments. This reflects the high degree of heterogeneity within catchments and the complexity of soil and groundwater interactions. The maps offer a starting point for assessing sensitivity. However, they provide no more than a very crude indication at the scale of interest to forestry managers. It is concluded that a more appropriate route for assessing sensitivity is via field measurement and the direct use of water quality and biological information. Keywords: streams, forestry, acidification, acid sensitivity mapping, ANC, Wales

  16. Macromolecular mimicry of nucleic acid and protein

    Nautrup Pedersen, Gitte; Nyborg, Jens; Clark, Brian F

    1999-01-01

    of the concept of macromolecular mimicry. Macromolecular mimicry has further been proposed among initiation and release factors, thereby adding a new element to the description of protein synthesis in bacteria. Such mimicry has also been observed in other biological processes such as autoimmunity, DNA repair...

  17. [Amino acid composition of rice grain proteins].

    Peruanskiĭ, Iu V; Savich, I M

    1976-01-01

    The composition of the major reserve proteins of rice grain--globulins, prolamines and glutelins--was examined in four rice varieties (Dubovsky 129, Kuban 3, Alakul, Ushtobinsky). Globulins proved to be most heterogeneous whereas glutelins appeared to be least heterogeneous. In regards to the ratio of components globulins showed high variability and glutelins displayed high stability. PMID:1005365

  18. Contact sensitizers modulate the arachidonic acid metabolism of PMA-differentiated U-937 monocytic cells activated by LPS

    For the effective induction of a hapten-specific T cell immune response toward contact sensitizers, in addition to covalent-modification of skin proteins, the redox and inflammatory statuses of activated dendritic cells are crucial. The aim of this study was to better understand how sensitizers modulate an inflammatory response through cytokines production and COX metabolism cascade. To address this purpose, we used the human monocytic-like U-937 cell line differentiated by phorbol myristate acetate (PMA) and investigated the effect of 6 contact sensitizers (DNCB, PPD, hydroquinone, propyl gallate, cinnamaldehyde and eugenol) and 3 non sensitizers (lactic acid, glycerol and tween 20) on the production of pro-inflammatory cytokines (IL-1β and TNF-α) and on the arachidonic acid metabolic profile after bacterial lipopolysaccharide (LPS) stimulation. Our results showed that among the tested molecules, all sensitizers specifically prevent the production of PMA/LPS-induced COX-2 metabolites (PGE2, TxB2 and PGD2), eugenol and cinnamaldehyde inhibiting also the production of IL-1β and TNF-α. We further demonstrated that there is no unique PGE2 inhibition mechanism: while the release of arachidonic acid (AA) from membrane phospholipids does not appear do be a target of modulation, COX-2 expression and/or COX-2 enzymatic activity are the major steps of prostaglandin synthesis that are inhibited by sensitizers. Altogether these results add a new insight into the multiple biochemical effects described for sensitizers. - Highlights: → We investigated how contact sensitizers modulate an inflammatory response. → We used macrophage-differentiated cell line, U-937 treated with PMA/LPS. → Sensitizers specifically inhibit the production of COX metabolites (PGE2, TxB2). → Several mechanisms of inhibition: COX-2 expression/enzymatic activity, isomerases. → New insight in the biochemical properties of sensitizers.

  19. Non-protein amino acids in peptide design

    S Aravinda; N Shamala; Rituparna S Roy; P Balaram

    2003-10-01

    An overview of the use of non-protein amino acids in the design of conformationally well-defined peptides, based on work from the author’s laboratory, is discussed. The crystal structures of several designed oligopeptides illustrate the use -aminoisobutyric acid (Aib) in the construction of helices, D-amino acids in the design of helix termination segments and DPro-Xxx segments for nucleating of -hairpin structures. - and -amino acid residues have been used to expand the range of designed polypeptide structures.

  20. ENZYME DIGEST AND ACID HYDROLYZED INDEX OF PROTEIN QUALITY EVALUATION

    H.Mohammadiha P. Mostafavi

    1984-08-01

    Full Text Available A pancreatopeptidase (Elastase digest index was devised for a rapid and accurate estimation of protein quality. This index was calculated on the basis of all the amino acids released by an in-vitro Elastase digestion, acid hydrolyses of same sample and the residue of enzyme hydrolyzed. The amino acids were determined by Thin-Layer Chromatography. Samples used were cooked white kidneybeans, cooked and over-heated soybean powder, and skimmed milk powder. Good correlation was observed between elastase index value and their biological values reported in the literature from feeding trials. The pattern of aminoacids released by acid and by enzyme hydrolysis was about the same.

  1. Sensitization of Salmonella typhi towards gamma-radiation by ascorbic acid

    The sensitivity of a S. typhi culture to γ-radiation has been examined. The D10 value, when the culture was irradiated in phosphate buffer, was found to be 70 krad. The addition of ascorbic acid (at a concentration of 0.57 mM) during irradiation, sensitized this organism quite markedly towards γ-radiation. This was evident from the observation that the D10 value was reduced to 20 krad when cells were irradiated in the ascorbic acid. Ascorbic acid was converted to its oxidized form during the radiation process. Irradiation under nitrogen instead of air inhibited the sensitization afforded by ascorbic acid. The population of cells surviving after irradiation in the presence of ascorbic acid showed a higher activity of catalase than its buffer-irradiated counterpart. The implication of the role of hydrogen peroxide (one of the possible intermediates formed during the oxidation of ascorbic acid) in sensitization is discussed. (author)

  2. 慢性心力衰竭患者心型脂肪酸结合蛋白与超敏C-反应蛋白的变化及其相关性%The relationship of heart-type fatty acid binding protein and high-sensitivity C-reactive protein in patients with chronic heart failure

    李洁琪; 李晓翔; 吴立荣; 方颖; 李屏

    2009-01-01

    Objective To examine clinical significance and relativity of heart-type fatty acid binding protein (H-FABP) and high-sensitivity C-reactive protein (hs-CRP) in patients with chronic heart failure. Methods Ser-um concentrations of H-FABP and hs-CRP were measured in 60 patients with chronic heart failure and 30 control subjects. Left ventricular ejection fraction (LVEF) was examined by Doppler echocardio graphic in all subjects. Re-sults Serum concentrations of H-FABP and hs-CRP were higher in patients with chronic heart failure than in con-trol subjects[(6.11±1.49)μg/L vs (4.24±1.40)μg/L,and (12.77±3.65)mg/L vs(4.85±1.35) mg/L,t=5.746 and 7.543,P<0.01] but LVEF was lower in patients with chronic heart failure than in control subjects [(42.13±6.55) % vs (61.50±3.89) %,t=-14.902,P<0.01]. In CHF subgroups,H-FABP and hs-CRP lev-el increased with advancing NYHA class (F=26.288 and 351.784,P<0.01) but LVEF decreased (F=252.834,P<0.01). The serum H-FABP concentrations had a positive correlation with serum hs-CRP concentrations (r=0.801,P<0.01),and a negative correlation with LVEF (r=-0.718,P<0.01) ;serum hs-CRP concentrations had a negative correlation with LVEF(r=-0.881,P<0.01). Conclusion Serum H-FABP and hs-CRP levels are in-creased with the worsening of CHF. H-FABP and hs-CRP level are pnsitiviely related. The quantitative determination of serum concentrations of H-FABP and hs-CRP is valuable for risk stratification in patients with chronic heart fail-ure.%目的 观察慢性心力衰竭(CHF)患者血清心型脂肪酸结合蛋白(H-FABP)和超敏C-反应蛋白(hs-CRP)的浓度变化,并探讨其相关性及临床意义.方法 选择不同心功能级别的CHF患者60例及同期健康体检者30例,测定其血清H-FABP及hs-CRP的浓度,同时用彩色多普勒超声测定左心室射血分数(LVEF).结果 CHF组H-FABP[(6.11±1.49)μg/L]及ks-CRP[(12.77±3.65)mg/L]的浓度均较对照组[分别为(4.24±1.40)μg/L和(4.85±1.35)mg/L]升高(t值分别为5

  3. Effects of three liquid diets on nutrition-sensitive plasma proteins of tube-fed elderly men.

    Feller, A G; Caindec, N; Rudman, I W; Rudman, D

    1990-06-01

    The effects on three nutrition-sensitive plasma proteins of isocaloric feedings with three enteral formulas were compared in 10 tube-fed male nursing home residents. The enteral products were Isocal (based on whole protein), Peptamen (based on a mixture of oligopeptides), and Vivonex T.E.N. (based on free amino acids). The nutrition-sensitive plasma proteins were albumin, transferrin, and retinol-binding protein. After observation during four weeks of feeding with Isocal, each subject was then monitored during four weeks of Peptamen and four weeks of Vivonex T.E.N. The latter two products were alternated in a crossover design. The shift of Isocal to Peptamen did not significantly (P greater than .05) influence the serum level of albumin, transferrin, or retinol-binding protein. In contrast, the shift of Isocal to Vivonex T.E.N. or of Peptamen to Vivonex caused a significant (P less than .05) decline in all three plasma proteins, the kinetics of their reductions corresponding to their known half-lives. The behavior of the three nutrition-sensitive plasma proteins suggests that in elderly nursing home men without gastrointestinal disease the nutritional value of the protein component of the three formulas follows the order Isocal = Peptamen greater than Vivonex T.E.N. However, this conclusion will require confirmation by nitrogen balance studies. PMID:2113546

  4. FLU, an amino acid substitution model for influenza proteins

    Gascuel Olivier

    2010-04-01

    Full Text Available Abstract Background The amino acid substitution model is the core component of many protein analysis systems such as sequence similarity search, sequence alignment, and phylogenetic inference. Although several general amino acid substitution models have been estimated from large and diverse protein databases, they remain inappropriate for analyzing specific species, e.g., viruses. Emerging epidemics of influenza viruses raise the need for comprehensive studies of these dangerous viruses. We propose an influenza-specific amino acid substitution model to enhance the understanding of the evolution of influenza viruses. Results A maximum likelihood approach was applied to estimate an amino acid substitution model (FLU from ~113, 000 influenza protein sequences, consisting of ~20 million residues. FLU outperforms 14 widely used models in constructing maximum likelihood phylogenetic trees for the majority of influenza protein alignments. On average, FLU gains ~42 log likelihood points with an alignment of 300 sites. Moreover, topologies of trees constructed using FLU and other models are frequently different. FLU does indeed have an impact on likelihood improvement as well as tree topologies. It was implemented in PhyML and can be downloaded from ftp://ftp.sanger.ac.uk/pub/1000genomes/lsq/FLU or included in PhyML 3.0 server at http://www.atgc-montpellier.fr/phyml/. Conclusions FLU should be useful for any influenza protein analysis system which requires an accurate description of amino acid substitutions.

  5. Mapping structures of proteins and nucleic acids

    Methods and algorithms for analysis of surfaces of globular and fibrillar proteins, DNA, and RNA have been developed. These methods of constructing cards of fragments of these objects, in the cylindrical projection original developed herein, essentially expand possibilities for studying distribution of charges and a relief of a surface of biological structures. This approach essentially supplements qualitative characteristics of methods of visualization of biopolymer structures

  6. Swelling of Bacterial Cellulose-Acrylic Acid Hydrogels: Sensitivity Towards External Stimuli

    This study evaluated various environmental factors affecting the swelling degree of bacterial cellulose-acrylic acid hydrogels. Aqueous bacterial cellulose-acrylic acid (4:1) mixtures were prepared and subjected to electron beam irradiation at 30 and 50 kGy. Swelling rate under influenced of pH, temperature and ionic strength was investigated from 1 to 24 hours. Swelling degree of hydrogels was dependent on irradiation dose: those synthesized at 50 kGy exhibited significant higher swelling degree (p<0.0001) in methanol (619 %) compared to water (510 %) at room temperature after 24 hours. External ionic strength affected swelling, for example elevation in sodium chloride concentration decreased swelling degree. Hydrogels were also sensitive to pH: swelling increased with increasing pH and was optimal at pH 7. Swelling also increased with increasing temperature from 25 to 50 degree Celsius. In conclusion, the ability of electron irradiated bacterial cellulose-acrylic acid hydrogels to respond to various external environment make it a material to be developed as an active delivery system for drugs, proteins and hormones. (author)

  7. Ser649 and Ser650 are the major determinants of protein kinase A-mediated activation of human hormone-sensitive lipase against lipid substrates

    Krintel, Christian; Osmark, Peter; Larsen, Martin Rask; Resjö, Svante; Logan, Derek T; Holm, Cecilia

    2008-01-01

    Hormone-sensitive lipase (HSL) is a key enzyme in the mobilization of fatty acids from stored triacylglycerols. Its activity is regulated by reversible protein phosphorylation. In rat HSL Ser563, Ser659 and Ser660 have been shown to be phosphorylated by protein kinase A (PKA) in vitro as well as in...

  8. Urinary liver-type fatty acid-binding protein predicts adverse outcomes in acute kidney injury

    Ferguson, Michael A.; Vaidya, Vishal S.; Waikar, Sushrut S.; Collings, Fitz B.; Sunderland, Kelsey E.; Gioules, Costas J.; Bonventre, Joseph V.

    2009-01-01

    Acute kidney injury (AKI) is a common condition with significant associated morbidity and mortality. The insensitivity and non-specificity of traditional markers of renal dysfunction prevent timely diagnosis, estimation of the severity of renal injury, and the administration of possible therapeutic agents. Here, we determine the prognostic ability of urinary liver-type fatty acid-binding protein (L-FABP), and further characterize its sensitivity and specificity as a biomarker of AKI. Initial ...

  9. Value of eight-amino-acid matches in predicting the allergenicity status of proteins: an empirical bioinformatic investigation

    ThirumalaiswamySekhar Arvind

    2009-10-01

    Full Text Available Abstract The use of biotechnological techniques to introduce novel proteins into food crops (transgenic or GM crops has motivated investigation into the properties of proteins that favor their potential to elicit allergic reactions. As part of the allergenicity assessment, bioinformatic approaches are used to compare the amino-acid sequence of candidate proteins with sequences in a database of known allergens to predict potential cross reactivity between novel food proteins and proteins to which people have become sensitized. Two criteria commonly used for these queries are searches over 80-amino-acid stretches for >35% identity, and searches for 8-amino-acid contiguous matches. We investigated the added value provided by the 8-amino-acid criterion over that provided by the >35%-identity-over-80-amino-acid criterion, by identifying allergens pairs that only met the former criterion, but not the latter criterion. We found that the allergen-sequence pairs only sharing 8-amino-acid identity, but not >35% identity over 80 amino acids, were unlikely to be cross reactive allergens. Thus, the common search for 8-amino-acid identity between novel proteins and known allergens appears to be of little additional value in assessing the potential allergenicity of novel proteins.

  10. Physical activity and high-sensitivity C-reactive protein.

    Plaisance, Eric P; Grandjean, Peter W

    2006-01-01

    Cardiovascular disease (CVD) remains one of the leading causes of death and disability in developed countries around the world despite the documented success of lifestyle and pharmacological interventions. This illustrates the multifactorial nature of atherosclerosis and the use of novel inflammatory markers as an adjunct to risk factor reduction strategies. As evidence continues to accumulate that inflammation is involved in all stages of the development and progression of atherosclerosis, markers of inflammation such as high-sensitivity C-reactive protein (CRP) may provide additional information regarding the biological status of the atherosclerotic lesion. Recent investigations suggest that physical activity reduces CRP levels. Higher levels of physical activity and cardiorespiratory fitness are consistently associated with 6-35% lower CRP levels. Longitudinal training studies that have demonstrated reductions in CRP concentrations range from 16% to 41%, an effect that may be independent of baseline levels of CRP, body composition or weight loss. The average change in CRP associated with physical activity appears to be at least as good, if not better, than currently prescribed pharmacological interventions in similar populations. The primary purpose of this review will be to present evidence from both cross-sectional and longitudinal investigations that physical activity lowers CRP levels in a dose-response manner. Finally, this review will examine factors such as body composition, sex, blood sample timing, diet and smoking, which may influence the CRP response to physical activity. PMID:16646631

  11. Fatty acid induced remodeling within the human liver fatty acid-binding protein.

    Sharma, Ashwani; Sharma, Amit

    2011-09-01

    We crystallized human liver fatty acid-binding protein (LFABP) in apo, holo, and intermediate states of palmitic acid engagement. Structural snapshots of fatty acid recognition, entry, and docking within LFABP support a heads-in mechanism for ligand entry. Apo-LFABP undergoes structural remodeling, where the first palmitate ingress creates the atomic environment for placement of the second palmitate. These new mechanistic insights will facilitate development of pharmacological agents against LFABP. PMID:21757748

  12. Fatty Acid Induced Remodeling within the Human Liver Fatty Acid-binding Protein*

    Sharma, Ashwani; Sharma, Amit

    2011-01-01

    We crystallized human liver fatty acid-binding protein (LFABP) in apo, holo, and intermediate states of palmitic acid engagement. Structural snapshots of fatty acid recognition, entry, and docking within LFABP support a heads-in mechanism for ligand entry. Apo-LFABP undergoes structural remodeling, where the first palmitate ingress creates the atomic environment for placement of the second palmitate. These new mechanistic insights will facilitate development of pharmacological agents against ...

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

    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 deg. C, while each substitution of Asp with Glu increased the Tm of ETRX by about 1.5 deg. 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

  14. Controlled overproduction of proteins by lactic acid bacteria

    Kuipers, Oscar P; Ruyter, Pascalle G.G.A. de; Kleerebezem, Michiel; de Vos, Willem M

    1997-01-01

    Lactic acid bacteria are widely used in industrial food fermentations, contributing to flavour, texture and preservation of the fermented products. Here we describe recent advances in the development of controlled gene expression systems, which allow the regulated overproduction of any desirable protein by lactic acid bacteria. Some systems benefit from the fact that the expression vectors, marker genes and inducing factors can be used directly in food applications since they are all derived ...

  15. Structural studies of nucleic acids and proteins involved in nucleic acid recognition

    Russo Krauss, Irene

    2010-01-01

    This PhD thesis focuses on the structural analysis of the protein-nucleic acid recognition. In particular the research work has been focalized on two different kinds of proteins and their nucleotide ligands. The first part concerns the structural characterization of complexes between human α-thrombin, a protein of physiological and pathological relevance, and two oligonucleotide aptamers (the so called thrombin binding aptamer and a modified version of it), which adopt a G-quadruplex fold. Th...

  16. Mass spectral characterization of a protein-nucleic acid photocrosslink.

    Golden, M. C.; Resing, K. A.; Collins, B. D.; Willis, M. C.; Koch, T H

    1999-01-01

    A photocrosslink between basic fibroblast growth factor (bFGF155) and a high affinity ssDNA oligonucleotide was characterized by positive ion electrospray ionization mass spectrometry (ESIMS). The DNA was a 61-mer oligonucleotide photoaptamer bearing seven bromodeoxyuridines, identified by in vitro selection. Specific photocrosslinking of the protein to the oligonucleotide was achieved by 308 nm XeCl excimer laser excitation. The cross-linked protein nucleic acid complex was proteolyzed with ...

  17. Oxidation of amino acids and proteins by peroxynitrite

    Recent studies suggested that proteins exposed to free radicals and other strong oxidants generated by living organisms may be the source of damage to tissues even at sites distant from the original point of generation of the reactive species. In examining the ability of biologically significant oxidizing agents to generate protein peroxides, the authors have studied protein peroxidation by peroxynitrite (ONOO-), known to be a potential source of tissue damage. Treatment of bovine serum albumin, Iysozyme, apotransferrin, insulin or human serum albumin with peroxynitrous acid (POXNA) led to formation of hydroperoxide groups on the proteins, detected by their reaction with iodide. Under optimum conditions, up to one peroxide group formed on each molecule of protein. Hydroxyl radical scavengers, antioxidants, or metal chelators, were unable to affect the quantities of peroxides generated by POXNA. These findings suggest that the oxidation was not mediated by the hydroxyl free radicals. The iodide assay cannot be applied to the measurement of peroxides in presence of nitrite, which is a contaminant of most solutions of POXNA. Nitrite can be easily removed from proteins by molecular filtration, but this method cannot be applied to amino acids. Therefore an amino acid peroxide test based on chemiluminescence was used, which shows promise for general peroxide detection

  18. A comparative study of ribosomal proteins: linkage between amino acid distribution and ribosomal assembly

    r-proteins in assembly does not appear to depend on these electrostatics interactions. Additionally, because thermophiles and mesophiles exhibit significantly different amino acid compositions in their sequences but not in the identities of contact sites, we conclude that this electrostatic component of interaction is insensitive to temperature and is not the determining factor differentiating the temperature sensitivity of ribosome assembly

  19. Acid-induced changes of brain protein buffering

    Kraig, Richard P.; Wagner, Robert J.

    1987-01-01

    Excessive cellular acidosis is thought to enhance destruction of brain from ischemia. Protein denaturation may contribute to such injury although the behavior of brain proteins to acidosis is poorly defined. As a first approach to detect acid-induced changes in brain proteins and to characterize buffer content, homogenates were acidified for 20 min (as low as pH 3.1), returned to baseline pH (6.9), and then titrated. Titration curves show a significant (P < 0.0001) and permanent increase in b...

  20. Swelling characteristics of hydroxyethylmethacrylate/ methacrylic acid pH -sensitive hydrogel as a drug delivery system

    M. Falamarzian- J. Varshosaz

    1996-08-01

    Full Text Available Hydroxyethyl methacrylate /methacrylic acid (HEMA/MAA copolymer cross-linked with ethylenglycol dimethacrylate was prepared by a bulk.free radical polymerization method. The results indicate that this polymer is a pH -sensitive hydrogel which is collapsed in the acidic medium but completely swollen in the alkaline and neutral pH . it was determined that a proportion of 40% of MAA, the ionizing monomer of this hydrogel, was the best concentration among the different percentages used which showed a non-Fickian water transport mechanism. Increasing MAA content from 20 to 70% was accompanied with a change in water transport mechanism from Fickian to non-Fickian. However, increasing the percentage of MAA from 40 to 70 didn't improve the swelling capacity of this polymer. Pore size determination by a solute exclusion technique, showed the greatest distribution in the hydrogel with 40% MAA compared to other percentages of this monomer used. About 75% of the pores were less than 16.5 A in diameter in this polymer which is important specially in loading the hydrogel with macromoiecular drugs like proteines.

  1. Heterologous protein display on the cell surface of lactic acid bacteria mediated by the s-layer protein

    Han Lanlan

    2011-10-01

    Full Text Available Abstract Background Previous studies have revealed that the C-terminal region of the S-layer protein from Lactobacillus is responsible for the cell wall anchoring, which provide an approach for targeting heterologous proteins to the cell wall of lactic acid bacteria (LAB. In this study, we developed a new surface display system in lactic acid bacteria with the C-terminal region of S-layer protein SlpB of Lactobacillus crispatus K2-4-3 isolated from chicken intestine. Results Multiple sequence alignment revealed that the C-terminal region (LcsB of Lb. crispatus K2-4-3 SlpB had a high similarity with the cell wall binding domains SA and CbsA of Lactobacillus acidophilus and Lb. crispatus. To evaluate the potential application as an anchoring protein, the green fluorescent protein (GFP or beta-galactosidase (Gal was fused to the N-terminus of the LcsB region, and the fused proteins were successfully produced in Escherichia coli, respectively. After mixing them with the non-genetically modified lactic acid bacteria cells, the fused GFP-LcsB and Gal-LcsB were functionally associated with the cell surface of various lactic acid bacteria tested. In addition, the binding capacity could be improved by SDS pretreatment. Moreover, both of the fused proteins could simultaneously bind to the surface of a single cell. Furthermore, when the fused DNA fragment of gfp:lcsB was inserted into the Lactococcus lactis expression vector pSec:Leiss:Nuc, the GFP could not be secreted into the medium under the control of the nisA promoter. Western blot, in-gel fluorescence assay, immunofluorescence microscopy and SDS sensitivity analysis confirmed that the GFP was successfully expressed onto the cell surface of L. lactis with the aid of the LcsB anchor. Conclusion The LcsB region can be used as a functional scaffold to target the heterologous proteins to the cell surfaces of lactic acid bacteria in vitro and in vivo, and has also the potential for biotechnological

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

    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. PMID:21762678

  3. Monomeric Tartrate Resistant Acid Phosphatase Induces Insulin Sensitive Obesity

    Lång, Pernilla; van Harmelen, Vanessa; Rydén, Mikael; Kaaman, Maria; Parini, Paolo; Carneheim, Claes; Cassady, A. Ian; Hume, David A.; Andersson, Göran; Arner, Peter

    2008-01-01

    Background Obesity is associated with macrophage infiltration of adipose tissue, which may link adipose inflammation to insulin resistance. However, the impact of inflammatory cells in the pathophysiology of obesity remains unclear. Tartrate resistant acid phosphatase (TRAP) is an enzyme expressed by subsets of macrophages and osteoclasts that exists either as an enzymatically inactive monomer or as an active, proteolytically processed dimer. Principal Findings Using mice over expressing TRAP...

  4. Protein and amino acid metabolism in skeletal muscle

    Wu, Guoyao.

    1989-01-01

    Isolated chick extensor digitorum communis (EDC) muscles and, in some experiments, rat skeletal muscles were used to study a number of aspects of protein and amino acid metabolism. (1) Chick EDC muscles synthesize and release large amounts of alanine and glutamine, which indirectly obtain their amino groups from branched-chain amino acids (BCAA). (2) Acetoacetate or DL-{beta}-hydroxybutyrate (4 mM) decrease (P < 0.01) alanine synthesis and BCAA transamination in EDC muscles from 24-h fasted chicks by decreasing (P < 0.01) intracellular concentrations of pyruvate due to inhibition of glycolysis. (3) Glutamine is extensively degraded in skeletal muscles from both chicks and rats, thus challenging the traditional view that glutamine oxidation is negligible in skeletal muscle. The cytosolic glutamine aminotransferases L and K in the rat and the mitochondrial phosphate-activated glutaminase in the chick play important roles in the conversion of glutamine to {alpha}-ketoglutarate for further oxidation. (4) Although methionine has been reported to be extensively transaminated in rat skeletal muscle preparations in the absence of other amino acids, transamination of methionine is absent or negligible in chick and rat skeletal muscles in the presence of physiological concentrations of amino acids. (5) Glutamine at 1.0-15 mM increases (P < 0.01) protein synthesis ({sup 3}H-phenylalanine incorporation), and at 10.0-15.0 mM decreases (P < 0.05) protein degradation ({sup 3}H-phenylalanine release from prelabelled protein in vivo) in EDC muscles from fed chicks as compared to muscles incubated in the absence of glutamine. (6) Acetoacetate or DL-{beta}-hydroxybutyrate (4 mM) has a small but significant inhibitory effect (P < 0.05) on the rate of protein synthesis, but has no effect (P > 0.05) on the rate of protein degradation in EDC muscles from fed chicks.

  5. pH-sensitive liposomes: acid-induced liposome fusion.

    Connor, J.; Yatvin, M B; Huang, L.

    1984-01-01

    Sonicated unilamellar liposomes containing phosphatidylethanolamine and palmitoylhomocysteine fuse rapidly when the medium pH is lowered from 7 to 5. Liposome fusion was demonstrated by (i) mixing of the liposomal lipids as shown by resonance energy transfer, (ii) gel filtration, and (iii) electron microscopy. The pH-sensitive fusion of liposomes was observed only when palmitoylhomocysteine (greater than or equal to 20 mol%) was present in the liposomes. The presence of phosphatidyl-ethanolam...

  6. Random amino acid mutations and protein misfolding lead to Shannon limit in sequence-structure communication.

    Andreas Martin Lisewski

    Full Text Available The transmission of genomic information from coding sequence to protein structure during protein synthesis is subject to stochastic errors. To analyze transmission limits in the presence of spurious errors, Shannon's noisy channel theorem is applied to a communication channel between amino acid sequences and their structures established from a large-scale statistical analysis of protein atomic coordinates. While Shannon's theorem confirms that in close to native conformations information is transmitted with limited error probability, additional random errors in sequence (amino acid substitutions and in structure (structural defects trigger a decrease in communication capacity toward a Shannon limit at 0.010 bits per amino acid symbol at which communication breaks down. In several controls, simulated error rates above a critical threshold and models of unfolded structures always produce capacities below this limiting value. Thus an essential biological system can be realistically modeled as a digital communication channel that is (a sensitive to random errors and (b restricted by a Shannon error limit. This forms a novel basis for predictions consistent with observed rates of defective ribosomal products during protein synthesis, and with the estimated excess of mutual information in protein contact potentials.

  7. Development of a glucose-sensitive drug delivery device: Microencapsulated liposomes and poly(2-ethylacrylic acid)

    Kanokpanont, Sorada

    The current study is the development a self-regulated, glucose responsive drug delivery system, using dioleoylphosphatidylcholine (DOPC) liposomes, a pH sensitive polymer, poly (2-ethylacrylic acid)(PEAA), and the feed back reaction of glucose with glucose oxidase enzyme (GO). The thesis investigates the use of PEAR and liposomes to work inside a microcapsule in response to the glucose level of the environment, by following the release of fluorescence probes, 8-aminonapthalene-1,3,6-trisulfonic acid, disodium salt/p-xylene-bis-pyridimuim bromide (ANTS/DPX) and a model protein, myoglobin. The continuing studies of PEAR and liposome interaction indicated an evidence of the previous hypothesis of two-mode release at different pHs. Differential scanning calorimetric studies of DOPC and PEAA complexes revealed the possibility of polymer adsorption to the liposomes in the pH range 5.5--7.0 and insertion in the liposome bilayer at pH optimal concentration in the capsules. The pH reduction inside the capsule due to GO reaction showed positive results for the use of GO in a non-buffered system. The procedure of liquid-core alginate capsules was modified to facilitate the pH-responsive release of ANTS/DPX and myoglobin. The capsules responded to high blood glucose concentration by releasing myoglobin within 30 minutes. Although more studies are required to improve the response of the system to the normal blood glucose and to control the total protein release from negative controls, the results of in vitro release experiments confirmed the hypothesis that an idealized feedback control insulin delivery system is feasible.

  8. Clinical applications of immunofixation: a more sensitive technique for the detection of Bence Jones protein.

    Whicher, J T; Hawkins, L.; Higginson, J.

    1980-01-01

    Immunofixation in agarose gel has been compared with agarose electrophoresis for the detection of Bence Jones protein in urine. The technique has a sensitivity between five and 10 times greater than electrophoresis and allows the identification of multiple Bence Jones proteins and Bence Jones proteins with fast mobility in the presence of other urinary proteins. In four out of 12 patients studied, Bence Jones protein was undetectable by electrophoresis of 300 times concentrated urine but was ...

  9. Sequence and structural features of binding site residues in protein-protein complexes: comparison with protein-nucleic acid complexes

    Selvaraj S

    2011-10-01

    Full Text Available Abstract Background Protein-protein interactions are important for several cellular processes. Understanding the mechanism of protein-protein recognition and predicting the binding sites in protein-protein complexes are long standing goals in molecular and computational biology. Methods We have developed an energy based approach for identifying the binding site residues in protein–protein complexes. The binding site residues have been analyzed with sequence and structure based parameters such as binding propensity, neighboring residues in the vicinity of binding sites, conservation score and conformational switching. Results We observed that the binding propensities of amino acid residues are specific for protein-protein complexes. Further, typical dipeptides and tripeptides showed high preference for binding, which is unique to protein-protein complexes. Most of the binding site residues are highly conserved among homologous sequences. Our analysis showed that 7% of residues changed their conformations upon protein-protein complex formation and it is 9.2% and 6.6% in the binding and non-binding sites, respectively. Specifically, the residues Glu, Lys, Leu and Ser changed their conformation from coil to helix/strand and from helix to coil/strand. Leu, Ser, Thr and Val prefer to change their conformation from strand to coil/helix. Conclusions The results obtained in this study will be helpful for understanding and predicting the binding sites in protein-protein complexes.

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

    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. PMID:27080133

  11. Retinoic acid increases the sensitivity of the rat embryo fibroblast transformation assay.

    Halazonetis, T D; Daugherty, C; Leder, P

    1988-01-01

    The rat embryo fibroblast focus assay is used to evaluate the transforming potential of several oncogenes. The sensitivity of this assay increased fivefold when retinoic acid was added to tissue culture media. Retinoic acid probably acts by selectively inhibiting the proliferation of nontransformed cells.

  12. Cross-sensitization patterns in guinea pigs between cinnamaldehyde, cinnamyl alcohol and cinnamic acid

    Weibel, H; Hansen, J; Andersen, Klaus Ejner

    1989-01-01

    Guinea pig maximization tests (GPMT) were performed with cinnamon substances. There was a certain degree of cross-reactivity between cinnamaldehyde, cinnamyl alcohol and cinnamic acid as animals sensitized to cinnamaldehyde reacted to the challenge with the three substances. Animals sensitized to...

  13. Liver mitochondria contain an ATP-dependent, vanadate-sensitive pathway for the degradation of proteins.

    Desautels, M; Goldberg, A L

    1982-01-01

    A large fraction (30-50%) of the various proteins synthesized within isolated rat liver mitochondria were degraded to amino acids within 60 min after synthesis. Incomplete mitochondrial polypeptides resulting from the incorporation of puromycin were degraded even more extensively (80% per hr). Protein breakdown was measured by the appearance of acid-soluble radioactivity and by the disappearance of labeled polypeptides detected on NaDodSO4/polyacrylamide gel electrophoresis. The amino acids g...

  14. Cholesterol-lowering effect of rice bran protein containing bile acid-binding proteins.

    Wang, Jilite; Shimada, Masaya; Kato, Yukina; Kusada, Mio; Nagaoka, Satoshi

    2015-01-01

    Dietary plant protein is well known to reduce serum cholesterol levels. Rice bran is a by-product of rice milling and is a good source of protein. The present study examined whether feeding rats a high-cholesterol diet containing 10% rice bran protein (RBP) for 10 d affected cholesterol metabolism. Rats fed dietary RBP had lower serum total cholesterol levels and increased excretion of fecal steroids, such as cholesterol and bile acids, than those fed dietary casein. In vitro assays showed that RBP strongly bound to taurocholate, and inhibited the micellar solubility of cholesterol, compared with casein. Moreover, the bile acid-binding proteins of the RBP were eluted by a chromatographic column conjugated with cholic acid, and one of them was identified as hypothetical protein OsJ_13801 (NCBI accession No. EAZ29742) using MALDI-TOF mass spectrometry analysis. These results suggest that the hypocholesterolemic action of the RBP may be caused by the bile acid-binding proteins. PMID:25374002

  15. IgE-mediated soy protein sensitization in children with cow`s milk allergy

    Agustina Santi; Mohammad Juffrie; Sumadiono

    2012-01-01

    Background Soy-based formula as an alternative to cow’s milk formula is preferable to extensively hydrolyzed protein formula because of the lower cost and more acceptable taste. However, cow’s milk allergy patients can subsequently develop a sensitivity to soy protein. Objective To compare soy protein sensitization in children with and without an allergy to cow’s milk. Methods This study was conducted in Yogyakarta from September 2007 until March 2008. Subjects were children aged belo...

  16. Long-term trends in water chemistry of acid-sensitive Swedish lakes show slow recovery from historic acidification

    Martyn N. Futter; Valinia, Salar; Löfgren, Stefan; Köhler, Stephan J.; Fölster, Jens

    2014-01-01

    Long-term (1987–2012) water quality monitoring in 36 acid-sensitive Swedish lakes shows slow recovery from historic acidification. Overall, strong acid anion concentrations declined, primarily as a result of declines in sulfate. Chloride is now the dominant anion in many acid-sensitive lakes. Base cation concentrations have declined less rapidly than strong acid anion concentrations, leading to an increase in charge balance acid neutralizing capacity. In many lakes, modeled organic acidity is...

  17. Noninvasive measurement of fecal calprotectin and serum amyloid A combined with intestinal fatty acid-binding protein in necrotizing enterocolitis.

    Reisinger, K.W.; Zee, D.C. van der; Brouwers, H.A.A.; Kramer, B.W.; Heurn, L.W.E. van; Buurman, W.A.; Derikx, J.P.

    2012-01-01

    BACKGROUND: Diagnosis of necrotizing enterocolitis (NEC), prevalent in premature infants, remains challenging. Enterocyte damage in NEC can be assessed by intestinal fatty acid-binding protein (I-FABP), with a sensitivity of 93% and a specificity of 90%. Numerous markers of inflammation are known, s

  18. Noninvasive measurement of fecal calprotectin and serum amyloid A combined with intestinal fatty acid-binding protein in necrotizing enterocolitis

    Reisinger, Kostan W.; Van der Zee, David C.; Brouwers, Hens A. A.; Kramer, Boris W.; van Heurn, L. W. Ernest; Buurman, Wim A.; Derikx, Joep P. M.

    2012-01-01

    Background: Diagnosis of necrotizing enterocolitis (NEC), prevalent in premature infants, remains challenging. Enterocyte damage in NEC can be assessed by intestinal fatty acid-binding protein (I-FABP), with a sensitivity of 93% and a specificity of 90%. Numerous markers of inflammation are known, s

  19. A novel regulatory mechanism for whey acidic protein gene expression.

    Chen, L.H.; Bissell, M J

    1989-01-01

    When primary mouse mammary epithelial cells (PMME) are cultured on a basement membrane type matrix, they undergo extensive morphogenesis leading to the formation of 3-dimensional alveoli-like spherical structures surrounding a closed lumen. We show for the first time that cells cultured on basement membrane-type matrix express high levels of whey acidic protein (WAP) mRNA and secrete the protein into the lumen. The expression of WAP appears to be dependent upon the formation of the alveoli-li...

  20. A fatty-acid-binding protein from wheat kernels

    Castagnaro, Atilio; García Olmedo, Francisco

    1994-01-01

    A protein of about 7 kDa (W-FABP) has been isolated from mature wheat kernels by H2O extraction and gel filtration of the extract, followed by two steps of high-performance liquid chromatography. The N-terminal amino acid sequence has been determined up to the 28th residue and found to be identical (except for positions 4 and 5) to that deduced from a barley cDNA (EMBL X15257), which had been improperly classified as a non-specific lipid transfer protein (LTP2). Similarly with LTPs, W-FABP do...

  1. Induction of DNA damage by oxidised amino acids and proteins

    Luxford, Catherine; Dean, Roger T; Davies, Michael Jonathan

    2002-01-01

    Exposure of amino acids, peptides and proteins to radicals in the presence of O2 generates hydroperoxides in a dose-dependent manner. These hydroperoxides are stable in the absence of exogenous catalysts (e.g. heat, light, redox-active transition metal ions), but decompose rapidly in the presence...... of these agents to give a variety of radicals including alkoxyl (RO*), peroxyl (ROO*) and carbon-centred (R) species. These radicals are shown to react with DNA to give DNA-protein cross-links and single strand breaks....

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

    Chieko Iwao

    Full Text Available 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.

  3. Topological features of proteins from amino acid residue networks

    Alves, N A; Alves, Nelson Augusto; Martinez, Alexandre Souto

    2006-01-01

    Topological properties of native folds are obtained from statistical analysis of 160 low homology proteins covering the four structural classes. This is done analysing one, two and three-vertex joint distribution of quantities related to the corresponding network of amino acid residues. Emphasis on the amino acid residue hydrophobicity leads to the definition of their center of mass as vertices in this contact network model with interactions represented by edges. The network analysis helps us to interpret experimental results such as hydrophobic scales and fraction of buried accessible surface area in terms of the network connectivity. To explore the vertex type dependent correlations, we build a network of hydrophobic and polar vertices. This procedure presents the wiring diagram of the topological structure of globular proteins leading to the following attachment probabilities between hydrophobic-hydrophobic 0.424(5), hydrophobic-polar 0.419(2) and polar-polar 0.157(3) residues.

  4. The clinical significance of fatty acid binding proteins

    Barbara Choromańska

    2011-11-01

    Full Text Available Excessive levels of free fatty acids are toxic to cells. The human body has evolved a defense mechanism in the form of small cytoplasmic proteins called fatty acid binding proteins (FABPs that bind long-chain fatty acids (LCFA, and then refer them to appropriate intracellular disposal sites (oxidation in mitochondria and peroxisomes or storage in the endoplasmic reticulum. So far, nine types of these proteins have been described, and their name refers to the place in which they were first identified or where they can be found in the greatest concentration. The most important FABPs were isolated from the liver (L-FABP, heart (H-FABP, intestine (I-FABP, brain (B-FABP, epidermis (E-FABP and adipocytes (A-FABP. Determination of H-FABP is used in the diagnosis of myocardial infarction, and L-FABP in kidney lesions of different etiologies. It is postulated that FABPs play an important role in the pathogenesis of metabolic diseases. Elevated levels of A-FABP have been found in the pericardial fat tissue and were associated with cardiac dysfunction in obese people. A rise in A-FABP has been observed in patients with type II diabetes. I-FABP is known as a marker of cell damage in the small intestine. Increased concentration of B-FABP has been associated with human brain tumors such as glioblastoma and astrocytoma, as well as with neurodegenerative diseases (Alzheimer’s, Parkinson’s and other disorders of cognitive function. The aim of this work was to present current data on the clinical significance of fatty acid binding proteins.

  5. Quantitation of glial fibrillary acidic protein in human brain tumours

    Rasmussen, S; Bock, E; Warecka, K;

    1980-01-01

    The glial fibrillary acidic protein (GFA) content of 58 human brain tumours was determined by quantitative immunoelectrophoresis, using monospecific antibody against GFA. Astrocytomas, glioblastomas, oligodendrogliomas, spongioblastomas, ependymomas and medulloblastomas contained relatively high...... amounts of GFA, up to 85 times the concentration in parietal grey substance of normal human brain. GFA was not found in neurinomas, meningiomas, adenomas of the hypophysis, or in a single case of metastasis of adenocarcinoma. Non-glial tumours of craniopharyngioma and haemangioblastoma were infiltrated by...

  6. The Protein and Nucleic Acid (PAN) Facility at Stanford University

    Eckart, M.; Kosovilka, N.; Sanchez, A; Tran, Y; Walker, P; Winant, R.; Zuo, E.; Patel, S.

    2010-01-01

    The Protein and Nucleic Acid (PAN) Facility (http://pan.stanford.edu) at Stanford University's Beckman Center is a multifaceted biotechnology fee-for-service laboratory providing services to the Stanford scientific community, other non-profit and biopharmaceutical organizations. The Facility's mission is to be adaptable and responsive to the changing needs of biomedical research by providing basic science investigators continued access to key tools and applications in an efficient and cost ef...

  7. Interactions of human mannose-binding protein with lipoteichoic acids.

    Polotsky, V Y; Fischer, W; Ezekowitz, R A; Joiner, K A

    1996-01-01

    We explored the interaction of human recombinant mannose-binding protein and lipoteichoic acids (LTAs) by enzyme-linked immunosorbent assay. The best ligand was Micrococcus luteus lipomannan, followed by Enterococcus spp. LTA containing mono-, di-, and oligoglucosyl substituents. LTAs lacking terminal sugars (those of Streptococcus pyogenes and Staphylococcus aureus) or containing galactosyl substituents (those of Listeria spp. and Lactococcus spp.) were poor ligands. These results are consis...

  8. A rapid and sensitive high-throughput screening method to identify compounds targeting protein–nucleic acids interactions

    Alonso, Nicole; Guillen, Roboan; Chambers, Jeremy W.; Leng, Fenfei

    2015-01-01

    DNA-binding and RNA-binding proteins are usually considered ‘undruggable’ partly due to the lack of an efficient method to identify inhibitors from existing small molecule repositories. Here we report a rapid and sensitive high-throughput screening approach to identify compounds targeting protein–nucleic acids interactions based on protein–DNA or protein–RNA interaction enzyme-linked immunosorbent assays (PDI-ELISA or PRI-ELISA). We validated the PDI-ELISA method using the mammalian high-mobi...

  9. Characterisation of a cell wall-anchored protein of Staphylococcus saprophyticus associated with linoleic acid resistance

    King Nathan P

    2012-01-01

    Full Text Available Abstract Background The Gram-positive bacterium Staphylococcus saprophyticus is the second most frequent causative agent of community-acquired urinary tract infections (UTI, accounting for up to 20% of cases. A common feature of staphylococci is colonisation of the human skin. This involves survival against innate immune defenses including antibacterial unsaturated free fatty acids such as linoleic acid which act by disrupting bacterial cell membranes. Indeed, S. saprophyticus UTI is usually preceded by perineal skin colonisation. Results In this study we identified a previously undescribed 73.5 kDa cell wall-anchored protein of S. saprophyticus, encoded on plasmid pSSAP2 of strain MS1146, which we termed S. saprophyticus surface protein F (SssF. The sssF gene is highly prevalent in S. saprophyticus clinical isolates and we demonstrate that the SssF protein is expressed at the cell surface. However, unlike all other characterised cell wall-anchored proteins of S. saprophyticus, we were unable to demonstrate a role for SssF in adhesion. SssF shares moderate sequence identity to a surface protein of Staphylococcus aureus (SasF recently shown to be an important mediator of linoleic acid resistance. Using a heterologous complementation approach in a S. aureus sasF null genetic background, we demonstrate that SssF is associated with resistance to linoleic acid. We also show that S. saprophyticus strains lacking sssF are more sensitive to linoleic acid than those that possess it. Every staphylococcal genome sequenced to date encodes SssF and SasF homologues. Proteins in this family share similar predicted secondary structures consisting almost exclusively of α-helices in a probable coiled-coil formation. Conclusions Our data indicate that SssF is a newly described and highly prevalent surface-localised protein of S. saprophyticus that contributes to resistance against the antibacterial effects of linoleic acid. SssF is a member of a protein family

  10. Prediction of flexible/rigid regions from protein sequences using k-spaced amino acid pairs

    Ruan Jishou

    2007-04-01

    characterized by accuracies below 70%. Finally, the Naïve Bayes method is shown to provide the highest sensitivity for the prediction of flexible regions, while FlexRP and SVM give the highest sensitivity for rigid regions. Conclusion A new sequence representation that uses k-spaced amino acid pairs is shown to be the most efficient in the prediction of the flexible/rigid regions of protein sequences. The proposed FlexRP method provides the highest prediction accuracy of about 80%. The experimental tests show that the FlexRP and SVM methods achieved high overall accuracy and the highest sensitivity for rigid regions, while the best quality of the predictions for flexible regions is achieved by the Naïve Bayes method.

  11. Hyperdimensional analysis of amino acid pair distributions in proteins.

    Svend B Henriksen

    Full Text Available 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.

  12. Sensitive and direct electrochemical detection of double-stranded DNA utilizing alkaline phosphatase-labelled zinc finger proteins.

    Noh, Soodong; Ha, Dat Thinh; Yang, Haesik; Kim, Moon-Soo

    2015-06-21

    Direct detection of double-stranded DNA (dsDNA) using zinc finger proteins (ZFPs) is of great importance in biomedical applications such as identifying pathogens and circulating DNAs. However, its sensitivity is still not sufficiently high because limited signalling labels can be conjugated or fused. Herein, we report sensitive and direct detection of dsDNA using (i) alkaline phosphatase (ALP) as a fast catalytic label conjugated to ZFPs along with (ii) electrochemical measurement of an ALP product (l-ascorbic acid) at the indium-tin oxide electrode with a high signal-to-background ratio. ALP is simply conjugated to a ZFP through lysine residues in a ZFP purification tag, a maltose binding protein (MBP). Sandwich-type electrochemical detection of dsDNA allows a detection limit of ca. 100 fM without using DNA amplification. PMID:25969923

  13. Mouse whey acidic protein is a novel member of the family of 'four-disulfide core' proteins.

    Hennighausen, L G; Sippel, A E

    1982-01-01

    Unlike in other mammalian species, the major whey protein in mouse is not alpha-lactalbumin, but a cysteine rich, acidic protein with a molecular weight of 14.0 kDa. We have deduced the amino acid sequence of this mouse acidic of whey protein from the nucleotide sequence of cloned cDNA. The positions of the half cysteines suggest that mouse whey acidic protein (WAP) is a two domain protein, very similar in structure to the plant lectin wheat germ agglutinin and the hypothalamic carrier protei...

  14. Uric acid: a modulator of prostate cells and activin sensitivity.

    Sangkop, Febbie; Singh, Geeta; Rodrigues, Ely; Gold, Elspeth; Bahn, Andrew

    2016-03-01

    Elevated serum uric acid (SUA) or urate is associated with inflammation and gout. Recent evidence has linked urate to cancers, but little is known about urate effects in prostate cancer. Activins are inflammatory cytokines and negative growth regulators in the prostate. A hallmark of prostate cancer progression is activin insensitivity; however, mechanisms underlying this are unclear. We propose that elevated SUA is associated with prostate cancer counteracting the growth inhibitory effects of activins. The expression of activins A and B, urate transporter GLUT9 and tissue urate levels were examined in human prostate disease. Intracellular and secreted urate and GLUT9 expression were assessed in human prostate cancer cell lines. Furthermore, the effects of urate and probenecid, a known urate transport inhibitor, were determined in combination with activin A. Activin A expression was increased in low-grade prostate cancer, whereas activin B expression was reduced in high-grade prostate cancer. Intracellular urate levels decreased in all prostate pathologies, while GLUT9 expression decreased in benign prostatic hyperplasia, prostatitis and high-grade prostate cancer. Activin responsive LNCaP cells had higher intracellular and lower secreted urate levels than activin-insensitive PC3 cells. GLUT9 expression in prostate cancer cells was progressively lower than in prostate epithelial cells. Elevated extracellular urate was growth promoting in vitro, which was abolished by the gout medication probenecid, and it antagonized the growth inhibitory effects of activins. This study shows for the first time that a change in plasma or intracellular urate levels, possibly involving GLUT9 and a urate efflux transporter, has an impact on prostate cancer cell growth, and that lowering SUA levels in prostate cancer is likely to be therapeutically beneficial. PMID:26910779

  15. A highly sensitive method for detection of molybdenum-containing proteins

    A highly sensitive method for detection of molybdenum-containing proteins in gels after electrophoresis has been developed. The method involves in vitro labeling of the proteins with the radioactive isotope 185W. The method used to detect molybdenum-accumulating proteins in lupine seeds, xanthine dehydrogenase and another molybdenum-containing protein in wheat, barley, and pea seedlings, and nitrate reductase and xanthine dehydrogenase in bacteroides from lupine nodules. Nitrogenase could not be detected by the method. 16 refs., 5 figs

  16. Interaction of bovine serum albumin protein with self assembled monolayer of mercaptoundecanoic acid

    Poonia, Monika; Agarwal, Hitesh; Manjuladevi, V.; Gupta, R. K.

    2016-05-01

    Detection of proteins and other biomolecules in liquid phase is the essence for the design of a biosensor. The sensitivity of a sensor can be enhanced by the appropriate functionalization of the sensing area so as to establish the molecular specific interaction. In the present work, we have studied the interaction of bovine serum albumin (BSA) protein with a chemically functionalized surface using a quartz crystal microbalance (QCM). The gold-coated quartz crystals (AT-cut/5 MHz) were functionalized by forming self-assembled monolayer (SAM) of 11-Mercaptoundecanoic acid (MUA). The adsorption characteristics of BSA onto SAM of MUA on quartz crystal are reported. BSA showed the highest affinity for SAM of MUA as compared to pure gold surface. The SAM of MUA provides carboxylated surface which enhances not only the adsorption of the BSA protein but also a very stable BSA-MUA complex in the liquid phase.

  17. Influence of irradiation on protein and amino acids in laboratory rodent diet

    The effect of irradiation treatment on the protein quality and constituent amino acids of laboratory rodent diets is reviewed and compared with other methods of sterilization - autoclaving and ethylene oxide fumigation. Gamma irradiation has been shown to have minimal influence on total protein, protein quality and total and available amino acid levels. Autoclaving reduces amino acid availability and consequently protein quality. Limited evidence shows reduction of certain available amino acids following ethylene oxide fumigation. (author)

  18. Detection of C',Cα correlations in proteins using a new time- and sensitivity-optimal experiment

    Sensitivity- and time-optimal experiment, called COCAINE (CO-CA In- and aNtiphase spectra with sensitivity Enhancement), is proposed to correlate chemical shifts of 13C' and 13Cα spins in proteins. A comparison of the sensitivity and duration of the experiment with the corresponding theoretical unitary bounds shows that the COCAINE experiment achieves maximum possible transfer efficiency in the shortest possible time, and in this sense the sequence is optimal. Compared to the standard HSQC, the COCAINE experiment delivers a 2.7-fold gain in sensitivity. This newly proposed experiment can be used for assignment of backbone resonances in large deuterated proteins effectively bridging 13C' and 13Cα resonances in adjacent amino acids. Due to the spin-state selection employed, the COCAINE experiment can also be used for efficient measurements of one-bond couplings (e.g. scalar and residual dipolar couplings) in any two-spin system (e.g. the N/H in the backbone of protein)

  19. High-sensitivity detection of proteins using gel electrophoresis and atomic force microscopy

    We have developed a method to detect specific proteins with a high sensitivity using a gel electrophoresis method and force measurement of atomic force microscopy (AFM). Biotinylated proteins were separated by electrophoresis and fixed with cross-linking chemicals on the gel, followed by direct force measurement between the biotinylated proteins on the gel and a streptavidin-modified tip of an AFM cantilever. We were able to achieve a high enough sensitivity to detect the picogram order of the biotinylated proteins by evaluating the frequency of the interaction force larger than 100 pN in the force profile, which corresponds to the rupture force of interaction between streptavidin and biotin.

  20. Novel humic acid-bonded magnetite nanoparticles for protein immobilization

    The present paper is the first report that introduces (i) a useful methodology for chemical immobilization of humic acid (HA) to aminopropyltriethoxysilane-functionalized magnetite iron oxide nanoparticles (APS-MNPs) and (ii) human serum albumin (HSA) binding to the obtained material (HA-APS-MNPs). The newly prepared magnetite nanoparticle was characterized by using Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and elemental analysis. Results indicated that surface modification of the bare magnetite nanoparticles (MNPs) with aminopropyltriethoxysilane (APS) and HA was successfully performed. The protein binding studies that were evaluated in batch mode exhibited that HA-APS-MNPs could be efficiently used as a substrate for the binding of HSA from aqueous solutions. Usually, recovery values higher than 90% were found to be feasible by HA-APS-MNPs, while that value was around 2% and 70% in the cases of MNPs and APS-MNPs, respectively. Hence, the capacity of MNPs was found to be significantly improved by immobilization of HA. Furthermore, thermal degradation of HA-APS-MNPs and HSA bonded HA-APS-MNPs was evaluated in terms of the Horowitz–Metzger equation in order to determine kinetic parameters for thermal decomposition. Activation energies calculated for HA-APS-MNPs (20.74 kJ mol−1) and HSA bonded HA-APS-MNPs (33.42 kJ mol−1) implied chemical immobilization of HA to APS-MNPs, and tight interactions between HA and HA-APS-MNPs. - Highlights: • A new magnetite nanoparticle based humic acid was prepared for the first time. • Protein binding studies of magnetite nanoparticle based humic acid were performed. • Kinetic parameters of protein and/or humic acid bonded nanoparticles were evaluated

  1. Cytosolic fatty acid-binding proteins: subjects and tools in metabolic research

    Fatty acid-binding proteins (FABPs) are major targets for specific binding of fatty acids in vivo. They constitute a widely expressed family of genetically related, small cytosolic proteins which very likely mediate intracellular transport of free long chain fatty acids. Genetic inhibition of FABP expression in vivo should therefore provide a useful tool to investigate and engineer fatty acid metabolism. (orig.)

  2. Sensitive and Selective Determination of Orotic Acid in Biological Specimens Using a Novel Fluorogenic Reaction.

    Yin, Sheng; Dragusha, Shpend; Ejupi, Valon; Shibata, Takayuki; Kabashima, Tsutomu; Kai, Masaaki

    2015-07-01

    Orotic acid is an intermediate in the synthesis pathway of uridine-5'-monophosphate, and increases in body fluids of patients suffering from hereditary disorders such as orotic aciduria and hyperammonemia. In this study, we developed a spectrofluorometric method with or without high-performance liquid chromatography for the selective and sensitive quantification of orotic acid in human biological specimens, using 4-trifluoromethylbenzamidoxime (4-TFMBAO) as a fluorogenic reagent. This reagent provided intensive fluorescence for only orotic acid amongst 62 compounds including structurally related bio-substances such as nucleic acid bases, nucleosides, nucleotides, amino acids, vitamins, bilirubin, uric acid, urea, creatine, creatinine and sugars. Under optimized reaction conditions, orotic acid was reacted with 4-TFMBAO, K3[Fe(CN)6] and K2CO3 in an aqueous solution. The fluorescence produced from the orotic acid derivative was measured at an excitation of 340 nm and an emission of 460 nm. A concentration of 1.2 μM orotic acid per 1.0 mM creatinine in normal urine and 0.64 nmol orotic acid per 5.0 × 10(5) HeLa cells were determined by this method. The present method permitted the facile quantification of orotic acid in healthy human urine and cultured HeLa cells by spectrofluorometry and/or high-performance liquid chromatography. PMID:26026930

  3. Systemic sensitization with the protein allergen ovalbumin augments local sensitization in atopic dermatitis

    Yoo J

    2014-02-01

    Full Text Available Jane Yoo,1 Anne M Manicone,2 John K McGuire,3 Ying Wang,2 William C Parks2 1Center for Lung Biology, Department of Medicine, Division of Dermatology, 2Division of Pulmonary and Critical Care Medicine, 3Department of Pediatrics, Division Critical Care Medicine, University of Washington, Seattle, WA, USA Abstract: Mouse models of atopic dermatitis based on epicutaneous sensitization have shed light on the role of epicutaneous allergen entry in the development of respiratory and gastrointestinal allergy. However, the contribution of non-cutaneous modes of sensitization to skin diseases has not been evaluated. We assessed if systemic ovalbumin administration, in conjunction with local sensitization, could prime for a robust inflammatory response. Furthermore, we attempted to elucidate important aspects of disease pathogenesis previously unaddressed in mouse models. Mice that underwent intraperitoneal ovalbumin sensitization prior to epicutaneous challenge demonstrated an acute (Th2-polarized atopic dermatitis-like phenotype upon local challenge. The inflammatory response was strikingly more robust than in mice that underwent epicutaneous sensitization alone. The lesional infiltrate contained a dendritic cell population that corresponded phenotypically with inflammatory dendritic epidermal cells of significance in human disease. Finally, in accordance with observations in human atopic dermatitis, there was an increase in cluster of differentiation (CD 103 (αE subunit-expressing CD4+ T lymphocytes. However, the absence of CD103 on approximately 50% of infiltrating cells argues against a primary role for the αEβ7 integrin in tissue homing. In conclusion, we present a mouse model of atopic dermatitis that reveals novel insights into the pathogenesis of this complex disease. Keywords: atopic dermatitis, mouse model, ovalbumin, sensitization, Th2, dendritic cells

  4. Sensory-motor responses to mechanical stimulation of the esophagus after sensitization with acid

    Asbj(ф)rn Mlohr Drewes; Hariprasad Reddy; Camilla Staahl; Jan Pedersen; Peter Funch-Jensen; Lars Arendt-Nielsen; Hans Gregersen

    2005-01-01

    AIM: Sensitization most likely plays an important role in chronic pain disorders, and such sensitization can be mimicked by experimental acid perfusion of the esophagus.The current study systematically investigated the sensory and motor responses of the esophagus to controlled mechanical stimuli before and after sensitization.METHODS: Thirty healthy subjects were included.Distension of the distal esophagus with a balloon was performed before and after perfusion with 0.1 mol/L hydrochloric acid for 30 min. An impedance planimetry system was used to measure cross-sectional area,volume, pressure, and tension during the distensions. A new model allowed evaluation of the phasic contractions by the tension during contractions as a function of the initial muscle length before the contraction (comparable to the Frank-Starling law for the heart). Length-tension diagrams were used to evaluate the muscle tone before and after relaxation of the smooth muscle with butylscopolamine.RESULTS: The sensitization resulted in allodynia and hyperalgesia to the distension volumes, and the degree of sensitization was related to the infused volume of acid. Furthermore, a nearly 50% increase in the evoked referred pain was seen after sensitization. The mechanical analysis demonstrated hyper-reactivity of the esophagus following acid perfusion, with an increased number and force of the phasic contractions, but the muscle tone did not change.CONCLUSION: Acid perfusion of the esophagus sensitizes the sensory pathways and facilitates secondary contractions.The new model can be used to study abnormal sensorymotor mechanisms in visceral organs.

  5. Comparative sensitivity of 125I-protein A and enzyme-conjugated antibodies for detection of immunoblotted proteins

    Immunoblotting is a powerful technique for the detection of small amounts of immunologically interesting proteins in unpurified preparations. Iodinated protein A (PA) has been widely used as a second antibody for detection of proteins; however, it does not bind equally well to immunoglobulins from different species nor does it bind to all subclasses of immunoglobulin G (IgG). We compared the sensitivity of [125I]PA with those of both horseradish peroxidase-conjugated second antibodies (HRP) and glucose oxidase-anti-glucose oxidase (GAG) soluble complexes for visualizing bovine serum albumin, human IgG, or human C3 which was either dot blotted or electroblotted to nitrocellulose. [125I]PA was uniformly 10- to 100-fold less sensitive than either HRP or GAG. GAG was more sensitive than HRP except for C3 (electroblotting) and bovine serum albumin and IgG (dot blotting), in which they were equivalent. In general, dot blotting was 10- to 1000-fold more sensitive than electroblotting. Although relative sensitivities varied depending on the proteins analyzed and the antisera used, GAG appeared to be superior to [125I]PA and HRP for detection of immunoblotted proteins

  6. Composite motifs integrating multiple protein structures increase sensitivity for function prediction.

    Chen, Brian Y; Bryant, Drew H; Cruess, Amanda E; Bylund, Joseph H; Fofanov, Viacheslav Y; Kristensen, David M; Kimmel, Marek; Lichtarge, Olivier; Kavraki, Lydia E

    2007-01-01

    The study of disease often hinges on the biological function of proteins, but determining protein function is a difficult experimental process. To minimize duplicated effort, algorithms for function prediction seek characteristics indicative of possible protein function. One approach is to identify substructural matches of geometric and chemical similarity between motifs representing known active sites and target protein structures with unknown function. In earlier work, statistically significant matches of certain effective motifs have identified functionally related active sites. Effective motifs must be carefully designed to maintain similarity to functionally related sites (sensitivity) and avoid incidental similarities to functionally unrelated protein geometry (specificity). Existing motif design techniques use the geometry of a single protein structure. Poor selection of this structure can limit motif effectiveness if the selected functional site lacks similarity to functionally related sites. To address this problem, this paper presents composite motifs, which combine structures of functionally related active sites to potentially increase sensitivity. Our experimentation compares the effectiveness of composite motifs with simple motifs designed from single protein structures. On six distinct families of functionally related proteins, leave-one-out testing showed that composite motifs had sensitivity comparable to the most sensitive of all simple motifs and specificity comparable to the average simple motif. On our data set, we observed that composite motifs simultaneously capture variations in active site conformation, diminish the problem of selecting motif structures, and enable the fusion of protein structures from diverse data sources. PMID:17951837

  7. Identification of cDNA clones encoding HMG 2, a major protein of the mexican axolotl hydrocortisone-sensitive thymocytes.

    de Guerra, A; Guillet, F; Charlemagne, J; Fellah, J S

    1995-01-01

    We have identified and analyzed cDNA clones encoding a major 26 kDa protein of the HMG1-2 family which is abundant in the cytoplasm and nucleus of axolotl hydrocortisone-sensitive thymocytes. The axolotl HMG2 protein is very similar to proteins belonging to the HMG1-2 family, from teleost fish to mammals. All the molecular features of the HMG1-2 proteins are conserved, including the high proportion of basic and aromatic residues, and the characteristic acidic C-terminus tail. The 3'-untranslated region of the HMG2 axolotl cDNA is also similar to the avian and mammalian HMG2 3'-UT sequences, suggesting that some selective events have acted at the DNA level to conserve this region, which could be important in the differential expression of the HMG1 and HMG2 genes. The axolotl HMG2 protein contains the two well conserved HMG boxes which are thought to be the DNA-binding domains of the molecule. Axolotl thymocytes and spleen cells contain almost identical amounts of HMG2 mRNAs but HMG2 polypeptide is undetectable in spleen cells using anti-26 kDa antibodies. The reason for the accumulation of HMG1-2 molecules in vertebrate hydrocortisone-sensitive thymocytes is discussed, as well as their possible role in apoptosis. PMID:8654668

  8. Swelling characteristics of hydroxyethylmethacrylate/ methacrylic acid pH -sensitive hydrogel as a drug delivery system

    M. Falamarzian- J. Varshosaz

    1996-01-01

    Hydroxyethyl methacrylate /methacrylic acid (HEMA/MAA) copolymer cross-linked with ethylenglycol dimethacrylate was prepared by a bulk.free radical polymerization method. The results indicate that this polymer is a pH -sensitive hydrogel which is collapsed in the acidic medium but completely swollen in the alkaline and neutral pH . it was determined that a proportion of 40% of MAA, the ionizing monomer of this hydrogel, was the best concentration among the different percentages used which sho...

  9. Conjugated Linoleic Acid in Humans: Regulation of Adiposity and Insulin Sensitivity1,2

    Brown, J. Mark; McIntosh, Michael K.

    2003-01-01

    Conjugated linoleic acid (CLA) isomers, a group of positional and geometric isomers of linoleic acid [18:2(n-6)], have been studied extensively due to their ability to modulate cancer, atherosclerosis, obesity, immune function and diabetes in a variety of experimental models. The purpose of this review was to examine CLA’s isomer-specific regulation of adiposity and insulin sensitivity in humans and in cultures of human adipocytes. It has been clearly demonstrated that specific CLA isomers or...

  10. α-Lipoic Acid Reduces Hypertension and Increases Baroreflex Sensitivity in Renovascular Hypertensive Rats

    Queiroz, Thyago; Guimarães, Drielle; Mendes-Junior, Leônidas; Braga, Valdir

    2012-01-01

    Renovascular hypertension has robust effects on control of blood pressure, including an impairment in baroreflex mechanisms, which involves oxidative stress. Although α-lipoic acid (LA) has been described as a potent antioxidant, its effect on renovascular hypertension and baroreflex sensitivity (BRS) has not been investigated. In the present study we analyzed the effects caused by chronic treatment with LA on blood pressure, heart rate and baroreflex sensitivity (sympathetic and parasympathe...

  11. Analysis of Salicylic Acid Induced Proteins in Rice

    1999-01-01

    An analysis using SDS-PAGE of acidic and basic protein fractions extracted from rice seedling treated with salicylic acid (SA) yielded several new proteins, some of which are similar in relative molecular mass to PR-1a,c, PR-2, 2e and PR-3d, 3e of tobacco.Direct assays for peroxidases and β-1,3-glucanases demonstrated that the activities of the two enzymes in the rice seedlings increased rapidly with time after SA treatment, reaching a maximum 6 days after treatment.Disease resistance tests showed that SA treated rice seedlings stunted the development of blight lesions and displayed higher resistance to rice blight pathogen (Xanthomonas oryzea pv.oryzea).The data suggest that the treatment with SA, even for plants with high endogenous SA levels such as rice, may induce the appearance of new proteins and the formation of disease resistance.The results contribute to the analysis of the SA role in rice systemic acquired resistance.

  12. Human Skeletal Muscle Protein Metabolism Responses to Amino Acid Nutrition.

    Mitchell, W Kyle; Wilkinson, Daniel J; Phillips, Bethan E; Lund, Jonathan N; Smith, Kenneth; Atherton, Philip J

    2016-07-01

    Healthy individuals maintain remarkably constant skeletal muscle mass across much of adult life, suggesting the existence of robust homeostatic mechanisms. Muscle exists in dynamic equilibrium whereby the influx of amino acids (AAs) and the resulting increases in muscle protein synthesis (MPS) associated with the intake of dietary proteins cancel out the efflux of AAs from muscle protein breakdown that occurs between meals. Dysregulated proteostasis is evident with aging, especially beyond the sixth decade of life. Women and men aged 75 y lose muscle mass at a rate of ∼0.7% and 1%/y, respectively (sarcopenia), and lose strength 2- to 5-fold faster (dynapenia) as muscle "quality" decreases. Factors contributing to the disruption of an otherwise robust proteostatic system represent targets for potential therapies that promote healthy aging. Understanding age-related impairments in anabolic responses to AAs and identifying strategies to mitigate these factors constitute major areas of interest. Numerous studies have aimed to identify 1) the influence of distinct protein sources on absorption kinetics and muscle anabolism, 2) the latency and time course of MPS responses to protein/AAs, 3) the impacts of protein/AA intake on muscle microvascular recruitment, and 4) the role of certain AAs (e.g., leucine) as signaling molecules, which are able to trigger anabolic pathways in tissues. This review aims to discuss these 4 issues listed, to provide historical and modern perspectives of AAs as modulators of human skeletal muscle protein metabolism, to describe how advances in stable isotope/mass spectrometric approaches and instrumentation have underpinned these advances, and to highlight relevant differences between young adults and older individuals. Whenever possible, observations are based on human studies, with additional consideration of relevant nonhuman studies. PMID:27422520

  13. Amino acid profiles and digestible indispensable amino acid scores of proteins from the prioritized key foods in Bangladesh.

    Shaheen, Nazma; Islam, Saiful; Munmun, Sarah; Mohiduzzaman, Md; Longvah, Thingnganing

    2016-12-15

    Concentrations of standard amino acids were determined in the composite samples (representing 30 agro-ecological zones of Bangladesh) of six prioritized key dietary protein sources: Oryza sativa (rice), Triticum aestivum (wheat flour), Lens culinaris (lentils), Pangusius pangusius (pangas), Labeo rohita (rohu) and Oreochromis mossambicus (tilapia). Digestible indispensable amino acid scores (DIAAS) was calculated using published data on amino acids' digestibility to evaluate the protein quality of these foods. Indispensable amino acid (IAA) contents (mg IAA/g protein), found to be highest in pangas (430) and lowest in wheat (336), of all these analyzed foods exceeded the FAO recommended daily allowance (277mg IAA/g protein) and contributed on average 40% to total amino acid contents. Untruncated DIAAS values ranged from 51% (lysine) in wheat to 106% (histidine) in pangas and distinguished pangas, rohu, and tilapia containing 'excellent quality' protein (DIAAS>100%) with potential to complement lower quality protein of cereals, fruits, and vegetables. PMID:27451158

  14. Chemical characteristics and acid sensitivity of boreal headwater lakes in northwest Saskatchewan

    Jean S. BIRKS

    2010-08-01

    Full Text Available Boreal ecosystems in northwest Saskatchewan may be threatened by acidification as this area is downwind of atmospheric emissions sources from regional oil sands mining operations. To evaluate the status of lakes in this region, a survey of 259 headwater lakes was conducted during 2007–2008 within ~300 km of Fort McMurray, Alberta. Acid sensitivity by ecoregion increased from Mid-Boreal Upland to Churchill River Upland to Athabasca Plain, with 60% of lakes classified as sensitive (50–200 μeq L–1 acid neutralizing capacity (ANC, and 8% as very sensitive (<50 μeq L–1 ANC to acid deposition. Organic anions dominated the acidity balance in most lakes, but non-marine sulphate varied positively with lake elevation and % upland cover (r2 = 0.24. Base cation concentrations (Ca, Mg, K, Na were correlated with % deciduous forest in the catchment area (r2 = 0.33, while dissolved organic carbon (DOC was related most strongly to % bog and lake flushing variables (r2 = 0.53. Variation in runoff coefficients derived by isotope mass balance corresponded with catchment area attributes that proxy controls on evaporation, infiltration and storage, and showed some ecoregional differences. The findings have implications for assignment of runoff values required to calculate critical loads of acidity. Although acidification appears not to be significantly advanced, many dilute oligotrophic lakes with pH 6.0 to pH 6.5 are vulnerable to acid deposition.

  15. Cocaine-induced behavioral sensitization decreases the expression of endocannabinoid signaling-related proteins in the mouse hippocampus.

    Blanco, Eduardo; Galeano, Pablo; Palomino, Ana; Pavón, Francisco J; Rivera, Patricia; Serrano, Antonia; Alen, Francisco; Rubio, Leticia; Vargas, Antonio; Castilla-Ortega, Estela; Decara, Juan; Bilbao, Ainhoa; de Fonseca, Fernando Rodríguez; Suárez, Juan

    2016-03-01

    In the reward mesocorticolimbic circuits, the glutamatergic and endocannabinoid systems are implicated in neurobiological mechanisms underlying cocaine addiction. However, the involvement of both systems in the hippocampus, a critical region to process relational information relevant for encoding drug-associated memories, in cocaine-related behaviors remains unknown. In the present work, we studied whether the hippocampal gene/protein expression of relevant glutamate signaling components, including glutamate-synthesizing enzymes and metabotropic and ionotropic receptors, and the hippocampal gene/protein expression of cannabinoid type 1 (CB1) receptor and endocannabinoid metabolic enzymes were altered following acute and/or repeated cocaine administration resulting in conditioned locomotion and locomotor sensitization. Results showed that acute cocaine administration induced an overall down-regulation of glutamate-related gene expression and, specifically, a low phosphorylation level of GluA1. In contrast, locomotor sensitization to cocaine produced an up-regulation of several glutamate receptor-related genes and, specifically, an increased protein expression of the GluN1 receptor subunit. Regarding the endocannabinoid system, acute and repeated cocaine administration were associated with an increased gene/protein expression of CB1 receptors and a decreased gene/protein expression of the endocannabinoid-synthesis enzymes N-acyl phosphatidylethanolamine D (NAPE-PLD) and diacylglycerol lipase alpha (DAGLα). These changes resulted in an overall decrease in endocannabinoid synthesis/degradation ratios, especially NAPE-PLD/fatty acid amide hydrolase and DAGLα/monoacylglycerol lipase, suggesting a reduced endocannabinoid production associated with a compensatory up-regulation of CB1 receptor. Overall, these findings suggest that repeated cocaine administration resulting in locomotor sensitization induces a down-regulation of the endocannabinoid signaling that could

  16. Protein phosphatase 2A associates with Rb2/p130 and mediates retinoic acid-induced growth suppression of ovarian carcinoma cells

    Vuocolo, Scott; Purev, Enkhtsetseg; Zhang, Dongmei;

    2003-01-01

    Levels of Rb2/p130 protein are increased 5-10-fold following all-trans-retinoic acid (ATRA) treatment of the retinoid-sensitive ovarian adenocarcinoma cell line CAOV3, but not the retinoid-resistant adenocarcinoma cell line SKOV3. We found that this increase in Rb2/p130 protein levels in ATRA...

  17. A split horseradish peroxidase for the detection of intercellular protein-protein interactions and sensitive visualization of synapses.

    Martell, Jeffrey D; Yamagata, Masahito; Deerinck, Thomas J; Phan, Sébastien; Kwa, Carolyn G; Ellisman, Mark H; Sanes, Joshua R; Ting, Alice Y

    2016-07-01

    Intercellular protein-protein interactions (PPIs) enable communication between cells in diverse biological processes, including cell proliferation, immune responses, infection, and synaptic transmission, but they are challenging to visualize because existing techniques have insufficient sensitivity and/or specificity. Here we report a split horseradish peroxidase (sHRP) as a sensitive and specific tool for the detection of intercellular PPIs. The two sHRP fragments, engineered through screening of 17 cut sites in HRP followed by directed evolution, reconstitute into an active form when driven together by an intercellular PPI, producing bright fluorescence or contrast for electron microscopy. Fusing the sHRP fragments to the proteins neurexin (NRX) and neuroligin (NLG), which bind each other across the synaptic cleft, enabled sensitive visualization of synapses between specific sets of neurons, including two classes of synapses in the mouse visual system. sHRP should be widely applicable to studying mechanisms of communication between a variety of cell types. PMID:27240195

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

    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. PMID:24648165

  19. Assay of urinary protein-bound sialic acid can differentiate steroidsensitive nephrotic syndrome from steroid-resistant cases

    Niranjan Gopal

    2016-01-01

    Full Text Available The protein selectivity index as measured from the ratio of urinary immunoglobulin to albumin failed to differentiate between steroid-sensitive (SS and steroid-resistant (SR cases of nephrotic syndrome (NS. Sialic acid contributes negative charges to many plasma proteins. The negative charge is a determinant of protein excretion rate. The prognostic significance of assay of urinary excretion of protein-bound sialic acid in NS has not been evaluated. Hence, the present study was designed to evaluate whether measurement of urinary protein bound sialic acid (UPBSA can be used as a marker to differentiate SS from SR cases of NS. The urine samples of 70 (47 SS and 23 SR pediatric NS children were assayed for UPBSA by Aminoff′s method. The levels were compared and the receiver-operator curve was drawn to determine the optimum cutoff point to differentiate among the groups before starting the therapy. The excretion of UPBSA in SR cases of NS was significantly higher than that of SS cases (P<0.05. The optimum cutoff limit for UPBSA was 2.71 μg/mg of proteins with 75% sensitivity and 75.5% specificity for differentiating SS cases from SR cases (area under the plasma- concentration time curve = 0.814, P = 0.009. We conclude that UPBSA can differentiate SR cases from SS cases of NS in pediatric patients and may help in predicting the response to steroid therapy.

  20. Fast TiO2Sensitization Using the Semisquaric Acid as Anchoring Group

    D. Pugliese

    2013-01-01

    Full Text Available Metal-free dye molecules for dye-sensitized solar cells application can avoid some of the typical drawbacks of common metal-based sensitizers, that are high production costs, relatively low molar extinction coefficient in the visible region, limited availability of precursors, and waste disposal issues. Recently we have proposed an innovative organic dye based on a simple hemi-squaraine molecule (CT1. In the present work, the effect of the sensitization time of the TiO2 photoelectrode in the dye solution is studied with the aim of optimizing the performance of CT1-based DSCs. Moreover, the addition of the chenodeoxycholic acid (CDCA as coadsorbent in the dye solution at different concentrations is investigated. Both CT1-sensitized mesoporous TiO2 photoanodes and complete solar cells have been fully characterized in their electrical and absorption properties. We have found that the best photoconversion performances are obtained with 1 hour of impregnation time and a 1 mM CDCA concentration. The very fast kinetics in dye adsorption, with optimal sensitization steps almost 15 times faster than conventional Ru-based sensitizers, confirms the theoretical predictions and indicates a strong interaction of the semisquaric acid group with the anatase surface. This result suggests that this small molecule can be a promising sensitizer even in a continuous industrial process.

  1. The variable detergent sensitivity of proteases that are utilized for recombinant protein affinity tag removal

    Vergis, James M.; Michael C. Wiener

    2011-01-01

    Recombinant proteins typically include one or more affinity tags to facilitate purification and/or detection. Expression constructs with affinity tags often include an engineered protease site for tag removal. Like other enzymes, the activities of proteases can be affected by buffer conditions. The buffers used for integral membrane proteins contain detergents, which are required to maintain protein solubility. We examined the detergent sensitivity of six commonly-used proteases (Enterokinase...

  2. Dye-Sensitized and Localized Surface Plasmon Resonance Enhanced Visible-Light Photoelectrochemical Biosensors for Highly Sensitive Analysis of Protein Kinase Activity.

    Yan, Zhiyong; Wang, Zonghua; Miao, Zhuang; Liu, Yang

    2016-01-01

    A novel visible-light photoelectrochemical (PEC) biosensor based on localized surface plasmon resonance (LSPR) enhancement and dye sensitization was fabricated for highly sensitive analysis of protein kinase activity with ultralow background. In this strategy, DNA conjugated gold nanoparticles (DNA@AuNPs) were assembled on the phosphorylated kemptide modified TiO2/ITO electrode through the chelation between Zr(4+) ions and phosphate groups, then followed by the intercalation of [Ru(bpy)3](2+) into DNA grooves. The adsorbed [Ru(bpy)3](2+) can harvest visible light to produce excited electrons that inject into the TiO2 conduction band to form photocurrent under visible light irradiation. In addition, the photocurrent efficiency was further improved by the LSPR of AuNPs under the irradiation of visible light. Moreover, because of the excellent conductivity and large surface area of AuNPs that facilitate electron-transfer and accommodate large number of [Ru(bpy)3](2+), the photocurrent was significantly amplified, affording an extremely sensitive PEC analysis of kinase activity with ultralow background signals. The detection limit of as-proposed PEC biosensor was 0.005 U mL(-1) (S/N = 3). The biosensor also showed excellent performances for quantitative kinase inhibitor screening and PKA activities detection in MCF-7 cell lysates under forskolin and ellagic acid stimulation. The developed dye-sensitization and LSPR enhancement visible-light PEC biosensor shows great potential in protein kinases-related clinical diagnosis and drug discovery. PMID:26648204

  3. Compositional changes of proteins and amino acids in germinating coffee seeds

    Milton Massao Shimizu; Paulo Mazzafera

    2000-01-01

    Endosperm is the main reserve tissue in coffee seeds. Coffee (Coffea arabica L.) seeds were germinated for six weeks and qualitative and quantitative changes in amino acids and proteins were investigated. The total content of free amino acids were reduced during germination, however, protein content remained constant. SDS-PAGE profiles showed that legumin-like proteins became less stained in the last weeks. Asparagine, glutamic acid, aspartic acid, alanine and lysine were the major free amino...

  4. Photocleavable DNA barcode-antibody conjugates allow sensitive and multiplexed protein analysis in single cell

    Agasti, Sarit S.; Liong, Monty; Peterson, Vanessa M.; Lee, Hakho; Weissleder, Ralph

    2012-01-01

    DNA barcoding is an attractive technology as it allows sensitive and multiplexed target analysis. However, DNA barcoding of cellular proteins remains challenging, primarily because barcode amplification and readout techniques are often incompatible with the cellular microenvironment. Here, we describe the development and validation of a photocleavable DNA barcode-antibody conjugate method for rapid, quantitative and multiplexed detection of proteins in single live cells. Following target binding, this method allows DNA barcodes to be photoreleased in solution, enabling easy isolation, amplification and readout. As a proof of principle, we demonstrate sensitive and multiplexed detection of protein biomarkers in a variety of cancer cells. PMID:23092113

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

    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. PMID:25096519

  6. Strains of Lactococcus lactis with a partial pyrimidine requirement show sensitivity toward aspartic acid

    Wadskov-Hansen, Steen Lyders Lerche; Martinussen, Jan

    2009-01-01

    that the partial pyrimidine requirement can be explained by a low specific activity of the pyrimidine biosynthetic enzymes. In conclusion, L. lactis LM0230 during the process of plasmid- and prophage-curing has acquired a partial pyrimidine requirement resulting in sensitivity toward aspartic acid....

  7. PUFAs acutely affect triacylglycerol-derived skeletal muscle fatty acid uptake and increase postprandial insulin sensitivity

    Jans, A.; Konings, E.; Goossens, G.H.; Bouwman, F.G.; Moors, C.C.; Boekschoten, M.V.; Afman, L.A.; Muller, M.R.; Mariman, E.C.; Blaak, E.E.

    2012-01-01

    Background: Dietary fat quality may influence skeletal muscle lipid processing and fat accumulation, thereby modulating insulin sensitivity. Objective: The objective was to examine the acute effects of meals with various fatty acid (FA) compositions on skeletal muscle FA processing and postprandial

  8. Comparison of C-reactive protein and high-sensitivity C-reactive protein levels in patients on hemodialysis

    Imed Helal; Lilia Zerelli; Madiha Krid; Fethi ElYounsi; Hedi Ben Maiz; Bechir Zouari; Jaouida Adelmoula; Adel Kheder

    2012-01-01

    Chronic inflammation is highly prevalent in patients on hemodialysis (HD), as evidenced by increased levels of C-reactive protein (CRP). We compared CRP to high-sensitivity C-reactive protein (hs-CRP) to determine whether it has any clinical implications and prognostic significance in terms of mortality. CRP was measured using a standard immunoturbidometric assay on the COBAS; INTEGRA system and hs-CRP was measured using the Dade Behring on the Konelab Nephelometer in 50 patients on HD. CRP (...

  9. Folic acid sensitive birth defects in association with intrauterine exposure to folic acid antagonists

    Meijer, W.M.; Walle, H.E.K.de; Kerstjens-Frederikse, W.S; de Jong-van den Berg, Lolkje Theodora Wilhelmina

    2005-01-01

    Since the protective effect of folic acid (FA) on birth defects is well known, it is reasonable to assume intrauterine exposure to FA antagonists increases the risk on these defects. We have therefore performed case-control analyses to investigate the risk of intrauterine exposure to FA antagonists,

  10. Major latex protein-like protein 43 (MLP43) functions as a positive regulator during abscisic acid responses and confers drought tolerance in Arabidopsis thaliana

    WANG, YANPING; Yang, Li; Chen, Xi; Ye, Tiantian; Zhong, Bao; Liu, Ruijie; Wu, Yan; Chan, Zhulong

    2015-01-01

    Drought stress is one of the disadvantageous environmental conditions for plant growth and reproduction. Given the importance of abscisic acid (ABA) to plant growth and abiotic stress responses, identification of novel components involved in ABA signalling transduction is critical. In this study, we screened numerous Arabidopsis thaliana mutants by seed germination assay and identified a mutant mlp43 (major latex protein-like 43) with decreased ABA sensitivity in seed germination. The mlp43 m...

  11. Study of Bcl-2 siRNA Enhancement of Sensitivity of HL-60 Cells to All Trans Retinoic Acid

    Haiyan Hu; Yuan Zhang; Dongmei He

    2008-01-01

    OBJECTIVE To study whether siRNA targeting against the Bcl-2gene can enhance sensitivity of HL-60 cells to all trans retinoic acid (ATRA).METHODS siRNA, which is a leading sequence selected by previous experiments, was transferred into HL-60 cells. At 6 h after transfection, the cells were cultured with ATRA. The cell growth of the HL-60 cells was measured by the MTT assay at 24,48, 72 h. The level of the Bcl-2 protein and ROS (reactive oxygen species) as well as membrane potential of the mitochondria were determined by flowcytometry.RESULTS siRNA significantly increased the inhibitory effect of ATRA on growth of the HL-60 cells. The combination of siRNA with ATRA resulted in a decrease in the Bcl-2 protein level and an increase in the ROS level as well as significantly lowering the mitochondrial membrane potential of the HL-60 cells (P < 0.05).CONCLUSION Effective siRNA targeting of Bcl-2 increases the sensitivity of HL-60 leukemic cells to ATRA by inhibiting the expression of the Bcl-2 protein.

  12. Enterocyte Fatty Acid Binding Proteins (FABPs): Different Functions of Liver- and Intestinal- FABPs in the Intestine

    Gajda, Angela M.; Storch, Judith

    2014-01-01

    Fatty acid binding proteins (FABP) are highly abundant cytosolic proteins that are expressed in most mammalian tissues. In the intestinal enterocyte, both Liver- (LFABP; FABP1) and Intestinal-fatty acid binding proteins (IFABP; FABP2) are expressed. These proteins display high affinity binding for long chain fatty acids (FA) and other hydrophobic ligands, thus they are believed to be involved with uptake and trafficking of lipids in the intestine. In vitro studies have identified differences ...

  13. Sensitive red protein calcium indicators for imaging neural activity.

    Dana, Hod; Mohar, Boaz; Sun, Yi; Narayan, Sujatha; Gordus, Andrew; Hasseman, Jeremy P; Tsegaye, Getahun; Holt, Graham T; Hu, Amy; Walpita, Deepika; Patel, Ronak; Macklin, John J; Bargmann, Cornelia I; Ahrens, Misha B; Schreiter, Eric R; Jayaraman, Vivek; Looger, Loren L; Svoboda, Karel; Kim, Douglas S

    2016-01-01

    Genetically encoded calcium indicators (GECIs) allow measurement of activity in large populations of neurons and in small neuronal compartments, over times of milliseconds to months. Although GFP-based GECIs are widely used for in vivo neurophysiology, GECIs with red-shifted excitation and emission spectra have advantages for in vivo imaging because of reduced scattering and absorption in tissue, and a consequent reduction in phototoxicity. However, current red GECIs are inferior to the state-of-the-art GFP-based GCaMP6 indicators for detecting and quantifying neural activity. Here we present improved red GECIs based on mRuby (jRCaMP1a, b) and mApple (jRGECO1a), with sensitivity comparable to GCaMP6. We characterized the performance of the new red GECIs in cultured neurons and in mouse, Drosophila, zebrafish and C. elegans in vivo. Red GECIs facilitate deep-tissue imaging, dual-color imaging together with GFP-based reporters, and the use of optogenetics in combination with calcium imaging. PMID:27011354

  14. Effect of basic additives on sensitivity and diffusion of acid in chemical amplification resists

    Asakawa, Koji; Ushirogouchi, Tohru; Nakase, Makoto

    1995-06-01

    The effect of amine additives in chemical amplification resists is discussed. Phenolic amines such as 4-aminophenol and 2-(4-aminophenyl)-2-(4-hydroxyphenyl) propane were investigated as model compounds from the viewpoint of sensitivity, diffusion and resolution. Equal molar amounts of acid and amine deactivated at the very beginning of post-exposure bake, and could not participate in decomposing the inhibitor as a catalyst. Only the acid which survived from the deactivation diffuses in the resist, decomposing the inhibitors from the middle to late stage of PEB. The basic additives reduce the diffusion range of the acid, especially for long-range diffusion, resulting in higher contrast at the interfaces between the exposed and unexposed areas. In addition, the amine concentration required is found to be less than the concentration which causes the resist sensitivity to start decreasing.

  15. Amino-modified tetraphenylethene derivatives as nucleic acid stain: relationship between the structure and sensitivity.

    Xu, Li; Zhu, Zece; Wei, Danqing; Zhou, Xiang; Qin, Jingui; Yang, Chuluo

    2014-10-22

    A series of new amino-functionalized tetraphenylethene (TPE) derivatives were designed and synthesized to study the effect of molecular structures on the detection of nucleic acid. Contrastive studies revealed that the number of binding groups, the length of hydrophobic linking arm and the configuration of TPE molecule all play important roles on the sensitivity of the probes in nucleic acid detection. Z-TPE3 with two binding amino groups, long linking arms, and cis configuration was found to be the most sensitive dye in both solution and gel matrix. Z-TPE3 is able to stain dsDNA with the lowest amount of 1 ng and exclusively stain 40 ng of short oligonucleotide with only 10 nt. This work is of important significance for the further design of TPE probes as biosensors with higher sensitivity. PMID:25279446

  16. Identification and characterization of a double-stranded RNA- reovirus temperature-sensitive mutant defective in minor core protein mu2.

    Coombs, K M

    1996-01-01

    A newly identified temperature-sensitive mutant whose defect was mapped to the reovirus M1 gene (minor core protein mu2) was studied to better understand the functions of this virion protein. Sequence determination of the Ml gene of this mutant (tsH11.2) revealed a predicted methionine-to-threonine alteration at amino acid 399 and a change from proline to histidine at amino acid 414. The mutant made normal amounts of single-stranded RNA, both in in vitro transcriptase assays and in infected c...

  17. The Investigation of Virginiamycin-Added Fungal Fermentation on the Size and Immunoreactivity of Heat-Sensitive Soy Protein

    Liyan Chen

    2015-01-01

    Full Text Available The usage of soy protein for young monogastric animals is restricted due to potential allergens and high molecular weight. The investigation of fungi fermentation effect on soy protein has been interrupted by substrate sterilization. Virginiamycin at 0.05% was added together with Aspergillus oryzae for solid state fermentation (SSF in unsterilized soy meal (SM. When compared to A. oryzae SSF alone, virginiamycin did not cause the interference of fungal fermentation but elucidated the protein degradation. SDS-PAGE results showed that both α and α′ subunits of β-conglycinin were degraded significantly. In addition, western blot results showed that the immunoreactive signals of soy protein were considerably reduced in virginiamycin-added fermentation with unsterilized SM. Furthermore, fungal fermentation increased total protein and essential amino acid contents, suggesting the value enhancement of SM products. Taken together, this study demonstrated for the first time that virginiamycin could help investigate fermentation effect on heat-sensitive soy protein. Fermented SM has several potential applications in feed industry.

  18. Membrane fractionation of herring marinade for separation and recovery of fats, proteins, amino acids, salt, acetic acid and water

    Fjerbæk Søtoft, Lene; Lizarazu, Juncal Martin; Razi Parjikolaei, Behnaz;

    2015-01-01

    containing sugars, amino acids and smaller peptides and a NF permeate containing salt and acetic acid ready for reuse. 42% of the spent marinade is recovered to substitute fresh water and chemicals. The Waste water amount is reduced 62.5%. Proteins are concentrated 30 times, while amino acids and smaller...

  19. HIGH SENSITIVE C-REACTIVE PROTEIN IN CEREBROVASCULAR ISCHEMIA

    Padmalatha

    2016-02-01

    Full Text Available BACKGROUND Cerebrovascular ischemia is recognized as a major health problem, which causes significant morbidity and mortality. The main pathophysiology of ischemic stroke is atherosclerosis of cerebral vessels. Hs-CRP is a sensitive marker of inflammation tissue injury in the arterial wall, which contributes to atherosclerosis. In this study, we aim to investigate the association of hs-CRP in patients with ischemic stroke and to correlate hs-CRP levels with possible risk factors of ischemic stroke and to assess the prognostic value of hs-CRP in ischemic stroke. METHODS In the present case control study after meeting inclusion and exclusion criteria, 50 patients with acute ischemic stroke admitted in the medical ward, King George Hospital, during the period between April 2014 and October 2014 and 40 asymptomatic age and sex matched control subjects were included. RESULTS The mean hs-CRP value in cases is 3.78+5.28mg/dl and in controls is 0.425+0.305mg/dl. Mean hs-CRP value is higher (3.78mg/dl in cases when compared to controls (0.425mg/dl, which is statistically significant. P admitted with severe degree of weakness (0-1/5 power with mean hs-CRP value of 4.28+4.07 without significant improvement in the power at the time of discharge; 8(16%> with mean hs-CRP value of 10.43+7.74 were expired. CONCLUSION Acute ischemic patients had higher mean hs-CRP values when compared to healthy asymptomatic control subjects P0.05. Higher mean hs-CRP values were associated with poor outcome after acute ischemic stroke. P<0.001.

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

    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?...

  1. Bile salt recognition by human liver fatty acid binding protein.

    Favretto, Filippo; Santambrogio, Carlo; D'Onofrio, Mariapina; Molinari, Henriette; Grandori, Rita; Assfalg, Michael

    2015-04-01

    Fatty acid binding proteins (FABPs) act as intracellular carriers of lipid molecules, and play a role in global metabolism regulation. Liver FABP (L-FABP) is prominent among FABPs for its wide ligand repertoire, which includes long-chain fatty acids as well as bile acids (BAs). In this work, we performed a detailed molecular- and atomic-level analysis of the interactions established by human L-FABP with nine BAs to understand the binding specificity for this important class of cholesterol-derived metabolites. Protein-ligand complex formation was monitored using heteronuclear NMR, steady-state fluorescence spectroscopy, and mass spectrometry. BAs were found to interact with L-FABP with dissociation constants in the narrow range of 0.6-7 μm; however, the diverse substitution patterns of the sterol nucleus and the presence of side-chain conjugation resulted in complexes endowed with various degrees of conformational heterogeneity. Trihydroxylated BAs formed monomeric complexes in which single ligand molecules occupied similar internal binding sites, based on chemical-shift perturbation data. Analysis of NMR line shapes upon progressive addition of taurocholate indicated that the binding mechanism departed from a simple binary association equilibrium, and instead involved intermediates along the binding path. The co-linear chemical shift behavior observed for L-FABP complexes with cholate derivatives added insight into conformational dynamics in the presence of ligands. The observed spectroscopic features of L-FABP/BA complexes, discussed in relation to ligand chemistry, suggest possible molecular determinants of recognition, with implications regarding intracellular BA transport. Our findings suggest that human L-FABP is a poorly selective, universal BA binder. PMID:25639618

  2. Dysferlin Binds SNAREs (Soluble N-Ethylmaleimide-sensitive Factor (NSF) Attachment Protein Receptors) and Stimulates Membrane Fusion in a Calcium-sensitive Manner.

    Codding, Sara J; Marty, Naomi; Abdullah, Nazish; Johnson, Colin P

    2016-07-01

    Resealing of tears in the sarcolemma of myofibers is a necessary step in the repair of muscle tissue. Recent work suggests a critical role for dysferlin in the membrane repair process and that mutations in dysferlin are responsible for limb girdle muscular dystrophy 2B and Miyoshi myopathy. Beyond membrane repair, dysferlin has been linked to SNARE-mediated exocytotic events including cytokine release and acid sphingomyelinase secretion. However, it is unclear whether dysferlin regulates SNARE-mediated membrane fusion. In this study we demonstrate a direct interaction between dysferlin and the SNARE proteins syntaxin 4 and SNAP-23. In addition, analysis of FRET and in vitro reconstituted lipid mixing assays indicate that dysferlin accelerates syntaxin 4/SNAP-23 heterodimer formation and SNARE-mediated lipid mixing in a calcium-sensitive manner. These results support a function for dysferlin as a calcium-sensing SNARE effector for membrane fusion events. PMID:27226605

  3. TPA-inducible proteins may account for sensitivity to promotion of transformation

    The preneoplastic JB6 mouse epidermal cell system includes cell lines sensitive (P+) or resistant (P-) to tumor promoter induced neoplastic transformation. The authors investigated whether a difference in TPA-inducible proteins may explain this differential sensitivity. The synthesis of a 39 Kd cytoplasmic protein (Major Excreted Protein) was TPA-inducible, but to a similar extent in both P+ and P- cells. TPA stimulated phosphorylation but not synthesis of the previously described stress protein pp80 in both P+ and P- cells from 1 to 5 hr after treatment. Pulse labelling of P+ and P- cells with 35S-methionine revealed a TPA dependent P+ specific transient increase in the synthesis of 58Kd protein. Induction was observed at 1 hr, and returned to basal levels by 4 hr and 20 hr, in nuclear and cytoplasmic fractions, respectively. This protein is not phosphorylated in response to TPA treatment. P+ cells differ from P- cells in one or more genes that specify sensitivity to promotion of transformation, designated pro genes. Antibodies to three peptides representing the pro-1 open reading frame were used in immunoprecipitation and Western blotting to isolate the pro-1 gene product. A 43 Kd protein was immunologically responsive to the pro-1 peptide antibodies, and showed an increased signal 40 min after TPA treatment. Since the predicted molecular weight of a pro-1 gene product is only 7 Kd, the possibility of a modification of the protein by poly(ADP-ribosylation) or glycosylation is being investigated

  4. Amino acid distance matrices and classifications for different protein secondary structure

    Zhang, L; Guan, S; Zheng, W M; Zhang, Li-mei; Liu, Xin; Guan, Shan; Zheng, Wei-Mou

    2003-01-01

    The property of an amino acid is different according to the variation of protein secondary structure. Each central amino acid corresponds to a position specific amino acid distribution around it. Based on the probability distribution for the central amino acid, we get amino acid distance matrices and classifications for helix, sheet, coil and turn. It is observed that evident discrepancy exists in amino acid distance for different protein secondary structure. Some obvious difference between the distance matrices and blocks substitution matrix(BLOSUM) is found which can tell the difference of amino acid property between in certain protein secondary structure and the whole protein database. The classification of amino acid alphabets for specific protein secondary structure provide a clue for observing these difference.

  5. Predicting DNA-binding sites of proteins from amino acid sequence

    Wu Feihong

    2006-05-01

    Full Text Available Abstract Background Understanding the molecular details of protein-DNA interactions is critical for deciphering the mechanisms of gene regulation. We present a machine learning approach for the identification of amino acid residues involved in protein-DNA interactions. Results We start with a Naïve Bayes classifier trained to predict whether a given amino acid residue is a DNA-binding residue based on its identity and the identities of its sequence neighbors. The input to the classifier consists of the identities of the target residue and 4 sequence neighbors on each side of the target residue. The classifier is trained and evaluated (using leave-one-out cross-validation on a non-redundant set of 171 proteins. Our results indicate the feasibility of identifying interface residues based on local sequence information. The classifier achieves 71% overall accuracy with a correlation coefficient of 0.24, 35% specificity and 53% sensitivity in identifying interface residues as evaluated by leave-one-out cross-validation. We show that the performance of the classifier is improved by using sequence entropy of the target residue (the entropy of the corresponding column in multiple alignment obtained by aligning the target sequence with its sequence homologs as additional input. The classifier achieves 78% overall accuracy with a correlation coefficient of 0.28, 44% specificity and 41% sensitivity in identifying interface residues. Examination of the predictions in the context of 3-dimensional structures of proteins demonstrates the effectiveness of this method in identifying DNA-binding sites from sequence information. In 33% (56 out of 171 of the proteins, the classifier identifies the interaction sites by correctly recognizing at least half of the interface residues. In 87% (149 out of 171 of the proteins, the classifier correctly identifies at least 20% of the interface residues. This suggests the possibility of using such classifiers to identify

  6. Protein phosphatase 2A regulates central sensitization in the spinal cord of rats following intradermal injection of capsaicin

    Fang Li

    2006-03-01

    Full Text Available Abstract Background Intradermal injection of capsaicin into the hind paw of rats induces spinal cord central sensititzation, a process in which the responsiveness of central nociceptive neurons is amplified. In central sensitization, many signal transduction pathways composed of several cascades of intracellular enzymes are involved. As the phosphorylation state of neuronal proteins is strictly controlled and balanced by the opposing activities of protein kinases and phosphatases, the involvement of phosphatases in these events needs to be investigated. This study is designed to determine the influence of serine/threonine protein phosphatase type 2A (PP2A on the central nociceptive amplification process, which is induced by intradermal injection of capsaicin in rats. Results In experiment 1, the expression of PP2A protein in rat spinal cord at different time points following capsaicin or vehicle injection was examined using the Western blot method. In experiment 2, an inhibitor of PP2A (okadaic acid, 20 nM or fostriecin, 30 nM was injected into the subarachnoid space of the spinal cord, and the spontaneous exploratory activity of the rats before and after capsaicin injection was recorded with an automated photobeam activity system. The results showed that PP2A protein expression in the spinal cord was significantly upregulated following intradermal injection of capsaicin in rats. Capsaicin injection caused a significant decrease in exploratory activity of the rats. Thirty minutes after the injection, this decrease in activity had partly recovered. Infusion of a phosphatase inhibitor into the spinal cord intrathecal space enhanced the central sensitization induced by capsaicin by making the decrease in movement last longer. Conclusion These findings indicate that PP2A plays an important role in the cellular mechanisms of spinal cord central sensitization induced by intradermal injection of capsaicin in rats, which may have implications in

  7. IgE-mediated soy protein sensitization in children with cow`s milk allergy

    Agustina Santi

    2012-02-01

    Full Text Available Background Soy-based formula as an alternative to cow’s milk formula is preferable to extensively hydrolyzed protein formula because of the lower cost and more acceptable taste. However, cow’s milk allergy patients can subsequently develop a sensitivity to soy protein. Objective To compare soy protein sensitization in children with and without an allergy to cow’s milk. Methods This study was conducted in Yogyakarta from September 2007 until March 2008. Subjects were children aged below 4 years with an atopic history. Subjects were divided into 2 groups: those with a positive skin prick test to cow’s milk and those with a negative skin prick test to cow’s milk (control group. Both groups were given soy formula and tested at 6 weeks for sensitization to soy. Results There were 45 children in each group. Age, sex, and atopic history were similar in both groups. We found no soy protein sensitization (negative skin prick results in all subjects from both groups. Conclusion Risk of immunoglobulin E-mediated sensitization to soy protein was not proven in children with cow’s milk allergy. [Paediatr Indones. 2012;52:67-71].

  8. Thermophysical properties of starch and whey protein composite prepared in presence of organic acid and esters

    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...

  9. Amino acid composition and thermal stability of protein structures: the free energy geography of the Protein Data Bank

    Deiana, Antonio; Shimizu, Kana; Giansanti, Andrea

    2010-01-01

    We study the combined influence of amino acid composition and chain length on the thermal stability of protein structures. A new parameterization of the internal free energy is considered, as the sum of hydrophobic effect, hydrogen-bond and de-hydration energy terms. We divided a non-redundant selection of protein structures from the Protein Data Bank into three groups: i) rich in order-promoting residues (OPR proteins); ii) rich in disorder-promoting residues (DPR proteins); iii) belonging t...

  10. Assessment of lake sensitivity to acidic deposition in national parks of the Rocky Mountains

    Nanus, L.; Williams, M.W.; Campbell, D.H.; Tonnessen, K.A.; Blett, T.; Clow, D.W.

    2009-01-01

    The sensitivity of high-elevation lakes to acidic deposition was evaluated in five national parks of the Rocky Mountains based on statistical relations between lake acid-neutralizing capacity concentrations and basin characteristics. Acid-neutralizing capacity (ANC) of 151 lakes sampled during synoptic surveys and basin-characteristic information derived from geographic information system (GIS) data sets were used to calibrate the statistical models. The explanatory basin variables that were considered included topographic parameters, bedrock type, and vegetation type. A logistic regression model was developed, and modeling results were cross-validated through lake sampling during fall 2004 at 58 lakes. The model was applied to lake basins greater than 1 ha in area in Glacier National Park (n = 244 lakes), Grand Teton National Park (n = 106 lakes), Great Sand Dunes National Park and Preserve (n = 11 lakes), Rocky Mountain National Park (n = 114 lakes), and Yellowstone National Park (n = 294 lakes). Lakes that had a high probability of having an ANC concentration 3000 m, with 80% of the catchment bedrock having low buffering capacity. The modeling results indicate that the most sensitive lakes are located in Rocky Mountain National Park and Grand Teton National Park. This technique for evaluating the lake sensitivity to acidic deposition is useful for designing long-term monitoring plans and is potentially transferable to other remote mountain areas of the United States and the world.

  11. Phosphorylation of acidic ribosomal proteins from rabbit reticulocytes by a ribosome-associated casein kinase

    Issinger, O G

    1977-01-01

    Two acidic proteins from 80-S ribosomes were isolated and purified to homogeneity. The purified acidic proteins could be phosphorylated by casein kinase using [gamma-32P]ATP and [gamma-32P]GTP as a phosphoryl donor. The proteins became phosphorylated in situ, too. Sodium dodecyl sulfate...

  12. S100A10 protein expression is associated with oxaliplatin sensitivity in human colorectal cancer cells

    Suzuki Sayo

    2011-12-01

    Full Text Available Abstract Background Individual responses to oxaliplatin (L-OHP-based chemotherapy remain unpredictable. The objective of our study was to find candidate protein markers for tumor sensitivity to L-OHP from intracellular proteins of human colorectal cancer (CRC cell lines. We performed expression difference mapping (EDM analysis of whole cell lysates from 11 human CRC cell lines with different sensitivities to L-OHP by using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS, and identified a candidate protein by liquid chromatography/mass spectrometry ion trap time-of-flight (LCMS-IT-TOF. Results Of the qualified mass peaks obtained by EDM analysis, 41 proteins were differentially expressed in 11 human colorectal cancer cell lines. Among these proteins, the peak intensity of 11.1 kDa protein was strongly correlated with the L-OHP sensitivity (50% inhibitory concentrations (P R2 = 0.80. We identified this protein as Protein S100-A10 (S100A10 by MS/MS ion search using LCMS-IT-TOF. We verified its differential expression and the correlation between S100A10 protein expression levels in drug-untreated CRC cells and their L-OHP sensitivities by Western blot analyses. In addition, S100A10 protein expression levels were not correlated with sensitivity to 5-fluorouracil, suggesting that S100A10 is more specific to L-OHP than to 5-fluorouracil in CRC cells. S100A10 was detected in cell culture supernatant, suggesting secretion out of cells. Conclusions By proteomic approaches including SELDI technology, we have demonstrated that intracellular S100A10 protein expression levels in drug-untreated CRC cells differ according to cell lines and are significantly correlated with sensitivity of CRC cells to L-OHP exposure. Our findings provide a new clue to searching predictive markers of the response to L-OHP, suggesting that S100A10 is expected to be one of the candidate protein markers.

  13. Overexpression of the human HAP1 protein sensitizes cells to the lethal effect of bioreductive drugs.

    Prieto-Alamo, M J; Laval, F

    1999-03-01

    Abasic sites (AP sites) are generated in DNA either directly by DNA-damaging agents or by DNA glycosylases acting during base excision repair. These sites are repaired in human cells by the HAP1 protein, which, besides its AP-endonuclease activity, also possesses a redox function. To investigate the ability of HAP1 protein to modulate cell resistance to DNA-damaging agents, CHO cells were transfected with HAP1 cDNA, resulting in stable expression of the protein in the cell nuclei. The sensitivity of the transfected cells to the toxic effect of various agents, e.g. methylmethane sulfonate, bleomycin and H2O2, was not modified. However, the transfected cells became more sensitive to killing by mitomycin C, porfiromycin, daunorubicin and aziridinyl benzoquinone, drugs that are activated by reduction. To test whether the redox function of HAP1 protein was involved in this increased cytotoxicity, we have constructed a mutated HAP1 protein endowed with normal AP-endonuclease activity but deleted for redox function. When this mutated protein was expressed in the cells, elevated AP-endonuclease activity was measured, but sensitization to the lethal effects of compounds requiring bioreduction was no longer observed. These results suggest that HAP1 protein, besides its involvement in DNA repair, is able to activate bioreduction of alkylating drugs used in cancer chemotherapy. PMID:10190555

  14. Fatty acid specificity of hormone-sensitive lipase. Implication in the selective hydrolysis of triacylglycerols.

    Raclot, T; Holm, C; Langin, D

    2001-12-01

    The selective mobilization of fatty acids from white fat cells depends on their molecular structure, in particular the degree of unsaturation. The present study was designed to examine if the release of fatty acids by hormone-sensitive lipase (HSL) in vitro i) is influenced by the amount of unsaturation, ii) depends on the temperature, and iii) could explain the selective pattern of fatty acid mobilization and notably the preferential mobilization of certain highly unsaturated fatty acids. Recombinant rat and human HSL were incubated with a lipid emulsion. The hydrolysis of 35 individual fatty acids, ranging in chain length from 12 to 24 carbon atoms and in unsaturation from 0 to 6 double bonds was measured. Fatty acid composition of in vitro released NEFA was compared with that of fat cell triacylglycerols (TAG), the ratio % NEFA/% TAG being defined as the relative hydrolysis. The relative hydrolysis of individual fatty acids differed widely, ranging from 0.44 (24:1n-9) to 1.49 (18:1n-7) with rat HSL, and from 0.38 (24:1n-9) to 1.67 (18:1n-7) with human HSL. No major difference was observed between rat and human HSL. The relative release was dependent on the number of double bonds according to chain length. The amount of fatty acid released by recombinant rat HSL was decreased but remained robust at 4 degrees C compared with 37 degrees C, and the relative hydrolysis of some individual fatty acids was affected. The relative hydrolysis of fatty acids moderately, weakly, and highly mobilized by adipose tissue in vivo was similar and close to unity in vitro. We conclude that i) the release of fatty acids by HSL is only slightly affected by their degree of unsaturation, ii) the ability of HSL to efficiently and selectively release fatty acids at low temperature could reflect a cold adaptability for poikilotherms or hibernators when endogenous lipids are needed, and iii) the selectivity of fatty acid hydrolysis by HSL does not fully account for the selective pattern of

  15. Smart protein biogate as a mediator to regulate competitive host-guest interaction for sensitive ratiometric electrochemical assay of prion

    Yu, Peng; Zhang, Xiaohua; Zhou, Jiawan; Xiong, Erhu; Li, Xiaoyu; Chen, Jinhua

    2015-11-01

    A novel competitive host-guest strategy regulated by protein biogate was developed for sensitive and selective analysis of prion protein. The methylene blue (MB)-tagged prion aptamer (MB-Apt) was introduced to the multiwalled carbon nanotubes-β-cyclodextrins (MWCNTs-β-CD) composites-modified glassy carbon (GC) electrode through the host-guest interaction between β-CD and MB. In the absence of prion, MB-Apt could be displaced by ferrocenecarboxylic acid (FCA) due to its stronger binding affinity to β-CD, resulting in a large oxidation peak of FCA. However, in the presence of prion, the specific prion-aptamer interaction drove the formation of protein biogate to seal the cavity of β-CD, which hindered the guest displacement of MB by FCA and resulted in the oxidation peak current of MB (IMB) increased and that of FCA (IFCA) decreased. The developed aptasensor showed good response towards the target (prion protein) with a low detection limit of 160 fM. By changing the specific aptamers, this strategy could be easily extended to detect other proteins, showing promising potential for extensive applications in bioanalysis.

  16. Determination of complex formation constants by phase sensitive alternating current polarography: Cadmium-polymethacrylic acid and cadmium-polygalacturonic acid.

    Garrigosa, Anna Maria; Gusmão, Rui; Ariño, Cristina; Díaz-Cruz, José Manuel; Esteban, Miquel

    2007-10-15

    The use of phase sensitive alternating current polarography (ACP) for the evaluation of complex formation constants of systems where electrodic adsorption is present has been proposed. The applicability of the technique implies the previous selection of the phase angle where contribution of capacitive current is minimized. This is made using Multivariate Curve Resolution by Alternating Least Squares (MCR-ALS) in the analysis of ACP measurements at different phase angles. The method is checked by the study of the complexation of Cd by polymethacrylic (PMA) and polygalacturonic (PGA) acids, and the optimal phase angles have been ca. -10 degrees for Cd-PMA and ca. -15 degrees for Cd-PGA systems. The goodness of phase sensitive ACP has been demonstrated comparing the determined complex formation constants with those obtained by reverse pulse polarography, a technique that minimizes the electrode adsorption effects on the measured currents. PMID:19073101

  17. Single cell protein production by penicillium expansum incorporating of acid hydrolysate of rice husk in medium

    The aim of the research work is to bioconversion of rice husk to single cell protein by penicillium expansum. The rice husk was degraded chemically using sulphuric acid and perchloric acid with various concentrations (0.15, 0.30, 0.45, and 0.60 N) to fermentable sugars and these were used as substrate for the production of single cell protein by penicillium expansum. It was observed that the amount of single cell protein is higher in case of perchloric acid hydrolysate in comparison to sulphuric acid hydrolysate, while the protein content of single cell protein is higher in sulphuric acid hydrolysate. The single cell protein of penicillium expansum contains nearly all essential amino acids while it free from aflatoxin. (author)

  18. ATP analog-sensitive Pat1 protein kinase for synchronous fission yeast meiosis at physiological temperature

    Cipak, Lubos; Hyppa, Randy; Smith, Gerald; Gregan, Juraj

    2012-01-01

    To study meiosis, synchronous cultures are often indispensable, especially for physical analyses of DNA and proteins. A temperature-sensitive allele of the Pat1 protein kinase (pat1-114) has been widely used to induce synchronous meiosis in the fission yeast Schizosaccharomyces pombe, but pat1-114-induced meiosis differs from wild-type meiosis, and some of these abnormalities might be due to higher temperature needed to inactivate the Pat1 kinase. Here, we report an ATP analog-sensitive allel...

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

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

    2014-01-01

    Enhanced bio-hydrogen production from protein wastewater by altering protein structure and amino acids acidification type via pH control was investigated. The hydrogen production reached 205.2 mL/g-protein when protein wastewater was pretreated at pH 12 and then fermented at pH 10. The mechanism studies showed that pH 12 pretreatment significantly enhanced protein bio-hydrolysis during the subsequent fermentation stage as it caused the unfolding of protein, damaged the protein hydrogen bondin...

  20. Folic acid deficiency increases chromosomal instability, chromosome 21 aneuploidy and sensitivity to radiation-induced micronuclei

    Folic acid deficiency can lead to uracil incorporation into DNA, hypomethylation of DNA, inefficient DNA repair and increase chromosome malsegregation and breakage. Because ionising radiation increases demand for efficient DNA repair and also causes chromosome breaks we hypothesised that folic acid deficiency may increase sensitivity to radiation-induced chromosome breakage. We tested this hypothesis by using the cytokinesis-block micronucleus assay in 10 day WIL2-NS cell cultures at four different folic acid concentrations (0.2, 2, 20, and 200 nM) that span the 'normal' physiological range in humans. The study showed a significant dose-dependent increase in frequency of binucleated cells with micronuclei and/or nucleoplasmic bridges with decreasing folic acid concentration (P < 0.0001, P = 0.028, respectively). These biomarkers of chromosomal instability were also increased in cells irradiated (1.5 Gy γ-rays) on day 9 relative to un-irradiated controls (P < 0.05). Folic acid deficiency and γ-irradiation were shown to have a significant interactive effect on frequency of cells containing micronuclei (two-way ANOVA, interaction P 0.0039) such that the frequency of radiation-induced micronucleated cells (i.e. after subtracting base-line frequency of un-irradiated controls) increased with decreasing folic acid concentration (P-trend < 0.0001). Aneuploidy of chromosome 21, apoptosis and necrosis were increased by folic acid deficiency but not by ionising radiation. The results of this study show that folate status has an important impact on chromosomal stability and is an important modifying factor of cellular sensitivity to radiation-induced genome damage

  1. Activation of G proteins by GIV-GEF is a pivot point for insulin resistance and sensitivity.

    Ma, Gary S; Lopez-Sanchez, Inmaculada; Aznar, Nicolas; Kalogriopoulos, Nicholas; Pedram, Shabnam; Midde, Krishna; Ciaraldi, Theodore P; Henry, Robert R; Ghosh, Pradipta

    2015-11-15

    Insulin resistance (IR) is a metabolic disorder characterized by impaired insulin signaling and cellular glucose uptake. The current paradigm for insulin signaling centers upon the insulin receptor (InsR) and its substrate IRS1; the latter is believed to be the sole conduit for postreceptor signaling. Here we challenge that paradigm and show that GIV/Girdin, a guanidine exchange factor (GEF) for the trimeric G protein Gαi, is another major hierarchical conduit for the metabolic insulin response. By virtue of its ability to directly bind InsR, IRS1, and phosphoinositide 3-kinase, GIV serves as a key hub in the immediate postreceptor level, which coordinately enhances the metabolic insulin response and glucose uptake in myotubes via its GEF function. Site-directed mutagenesis or phosphoinhibition of GIV-GEF by the fatty acid/protein kinase C-theta pathway triggers IR. Insulin sensitizers reverse phosphoinhibition of GIV and reinstate insulin sensitivity. We also provide evidence for such reversible regulation of GIV-GEF in skeletal muscles from patients with IR. Thus GIV is an essential upstream component that couples InsR to G-protein signaling to enhance the metabolic insulin response, and impairment of such coupling triggers IR. We also provide evidence that GIV-GEF serves as therapeutic target for exogenous manipulation of physiological insulin response and reversal of IR in skeletal muscles. PMID:26378251

  2. Interaction of basic amino acids, polypeptides and proteins with heparin

    A study has been made of the relative binding affinities for heparin of L-lysine, L-arginine, poly-L-lysine, poly-L-arginine, protamine, thrombin and antithrombin III. The destruction by different concentrations of organic cations of the heparin-methylene blue complex was determined by visible absorption spectroscopy. The strength of binding of the basic amino acids and polypeptides increased with increasing molecular weight, and decreased at low pH. Pulse radiolysis experiments were used to show the effects of increasing concentrations of L-lysine, poly-L-lysine and protamine sulphate on the reaction of e-sub(aq) with the heparin-methylene blue complex. The results indicate that the binding of basic amino acids and polypeptides to heparin is ionic, and that the enhanced rate of association of antithrombin III to thrombin in the presence of heparin is due to partial or complete charge neutralization of the basic groups in the proteins. (U.K.)

  3. Amino acid analysis of sub-picomolar amounts of proteins by precolumn fluorescence derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate.

    Masuda, Akiko; Dohmae, Naoshi

    2011-12-01

    Amino acid analysis (AAA) method is the most accurate methodology for absolute quantification of proteins. The conventional postcolumn method employing ninhydrin labeling of amino acids, which is adopted in automatic amino acid analyzer, is limited by low sensitivity. Therefore, a highly sensitive AAA method is required to confirm the data obtained from mass spectrometry or N-terminal sequence analysis. To increase the sensitivity of AAA, an analytical method based on precolumn derivatization with fluorescent 6-aminoquinolyl-carbamyl (AQC) reagent and separation of the AQC-amino acid derivatives by ion-pair chromatography using a reversed-phase column is reported herein. The sensitive analysis of low abundance proteins requires strict prevention of environmental contamination. In this review, we provide a protocol for high sensitivity amino acid analysis and show that the amino acid composition of bovine serum albumin below 100 ng, i.e., 1.5 pmol, determined using the presented method, matched with the theoretical composition in with low standard deviations. These results suggest that the current AAA method is potentially applicable for highly sensitive analysis as a complement to mass spectrometry-based proteomics. PMID:22281536

  4. Determination of damage-free crystal structure of an X-ray sensitive protein using XFEL

    We report a method of femtosecond crystallography for determining radiation damage-free crystal structures of large proteins at atomic resolution. The name of the method is 'serial femtosecond rotation crystallography' (SF-ROX). Here, we demonstrate experimental details of SF-ROX and 1.9-Å radiation damage-free structure analysis of bovine cytochrome c oxidase, a large (420-kDa), highly radiation-sensitive membrane protein. (author)

  5. Crystal growth of proteins, nucleic acids, and viruses in gels.

    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. PMID:20005247

  6. Biomimetic PEG hydrogels crosslinked with minimal plasmin-sensitive tri-amino acid peptides

    Jo, Suk Jo; Rizzi, Simone C.; Ehrbar, Martin; Weber, Franz E.; Hubbell, Jeffrey A.; Lutolf, Matthias P.

    2010-01-01

    Semi-synthetic, proteolytically degradable polymer hydrogels have proven effective as scaffolds to augment bone and skin regeneration in animals. However, high costs due to expensive peptide building blocks pose a significant hurdle towards broad clinical usage of these materials. Here we demonstrate that tri-amino acid peptides bearing lysine (or arginine), flanked by two cysteine residues for crosslinking, are adequate as minimal plasmin-sensitive peptides in poly(ethylene glycol)-based hyd...

  7. Impact of anticipated climate change on recovery from acidification of an acid-sensitive forested catchment

    Hruška, Jakub; Lamačová, Anna; Oulehle, Filip; Krám, Pavel; Farda, Aleš; Chuman, Tomáš

    Volume 1. 1. Brno: Global Change Research Centre, Academy of Sciences of the Czech Republic, v. v. i, 2015 - (Urban, O.; Klem, K.), s. 150-162 ISBN 978-80-87902-14-1 R&D Projects: GA MŠk(CZ) LO1415 Institutional support: RVO:67179843 Keywords : climate change * acid-sensitive forested * stream- water chemistry * forest management Subject RIV: EH - Ecology, Behaviour

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

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

    2016-01-01

    Background The acyl carrier protein (ACP) is an essential and ubiquitous component of microbial synthesis of fatty acids, the natural precursor to biofuels. Natural fatty acids usually contain long chains of 16 or more carbon atoms. Shorter carbon chains, with increased fuel volatility, are desired for internal combustion engines. Engineering the length specificity of key proteins in fatty acid metabolism, such as ACP, may enable microbial synthesis of these shorter chain fatty acids. Results...

  9. Quantitative evaluation of proteins with bicinchoninic acid (BCA): resonance Raman and surface-enhanced resonance Raman scattering-based methods.

    Chen, Lei; Yu, Zhi; Lee, Youngju; Wang, Xu; Zhao, Bing; Jung, Young Mee

    2012-12-21

    A rapid and highly sensitive bicinchoninic acid (BCA) reagent-based protein quantitation tool was developed using competitive resonance Raman (RR) and surface-enhanced resonance Raman scattering (SERRS) methods. A chelation reaction between BCA and Cu(+), which is reduced by protein in an alkaline environment, is exploited to create a BCA-Cu(+) complex that has strong RR and SERRS activities. Using these methods, protein concentrations in solutions can be quantitatively measured at concentrations as low as 50 μg mL(-1) and 10 pg mL(-1). There are many advantages of using RR and SERRS-based assays. These assays exhibit a much wider linear concentration range and provide an additional one (RR method) to four (SERRS method) orders of magnitude increase in detection limits relative to UV-based methods. Protein-to-protein variation is determined using a reference to a standard curve at concentrations of BSA that exhibits excellent recoveries. These novel methods are extremely accurate in detecting total protein concentrations in solution. This improvement in protein detection sensitivity could yield advances in the biological sciences and medical diagnostic field and extend the applications of reagent-based protein assay techniques. PMID:23099478

  10. Intravenous supplementation of acetate, glucose or essential amino acids to an energy and protein deficient diet in lactating dairy goats

    Safayi, S.; Nielsen, M. O.

    2013-01-01

    In the present experiment we aimed to study, if milk synthesis is more sensitive toward deficiency in supply of amino acids in early (EL) versus late lactation (LL), and if energy yielding substrates in the form of acetate (but not glucose) can contribute to sustain milk (protein) synthesis, when...... amino acid supply is suboptimal. Goats were fed a basal diet deficient in energy (90% of requirements) and protein (80% of requirements), and were randomly allocated to 4 treatments in a balanced 4 x 4 Latin square design. The treatments consisted of 4-d continuous intravenous infusions of isoosmotic...... for differential protein energy recommendations for ruminants across the lactation period. (C) 2012 Elsevier B.V. All rights reserved....

  11. UVA photolysis using the protein-bound sensitizers present in human lens

    This research was undertaken to demonstrate that the protein-bound chromophores in aged human lens can act as sensitizers for protein damage by UVA light. The water-insoluble (WI) proteins from pooled human and bovine lenses were solubilized by sonication in water and illuminated with UV light similar in output to that transmitted by the cornea. Analysis of the irradiated proteins showed a linear decrease in sulfhydryl groups with a 30% loss after 2 h. No loss was seen when native α-crystallin was irradiated under the same conditions. A 25% loss of histidine residues was also observed with the human lens WI fraction, and sodium dodecyl sulfate polyacrylamide gels indicated considerable protein cross-linking. Similar photodamage was seen with a WI fraction from old bovine lenses. While the data show the presence of UVA sensitizers, some histidine destruction and protein cross-linking were also obtained with α-crystallin and with lysozyme which argue that part of the histidine loss in the human WISS was likely due to tryptophan acting as a sensitizer. (Author)

  12. UVA photolysis using the protein-bound sensitizers present in human lens

    Ortwerth, B.J.; Olesen, P.R. (Missouri Univ., Columbia, MO (United States). Mason Inst. of Ophthalmology)

    1994-07-01

    This research was undertaken to demonstrate that the protein-bound chromophores in aged human lens can act as sensitizers for protein damage by UVA light. The water-insoluble (WI) proteins from pooled human and bovine lenses were solubilized by sonication in water and illuminated with UV light similar in output to that transmitted by the cornea. Analysis of the irradiated proteins showed a linear decrease in sulfhydryl groups with a 30% loss after 2 h. No loss was seen when native [alpha]-crystallin was irradiated under the same conditions. A 25% loss of histidine residues was also observed with the human lens WI fraction, and sodium dodecyl sulfate polyacrylamide gels indicated considerable protein cross-linking. Similar photodamage was seen with a WI fraction from old bovine lenses. While the data show the presence of UVA sensitizers, some histidine destruction and protein cross-linking were also obtained with [alpha]-crystallin and with lysozyme which argue that part of the histidine loss in the human WISS was likely due to tryptophan acting as a sensitizer. (Author).

  13. Nutritional analyses for proteins and amino acids in beans (Phaseolus sp.

    Wathelet B.

    1999-01-01

    Full Text Available The chemical index is a good estimator of seed protein quality of Phaseolus beans. In order to estimate this value, a protein hydrolysis and amino acid quantification are realised. The problems inherent to these techniques are presented.

  14. Novel redox-sensing modules : Accessory protein- and nucleic acid-mediated signaling

    Siedenburg, Gabriele; Groves, Matthew R; Ortiz de Orué Lucana, Darío

    2012-01-01

    SIGNIFICANCE: Organisms have evolved both enzymatic and nonenzymatic pathways to prevent oxidative damage to essential macromolecules, including proteins and nucleic acids. Pathways modulated by different protein-based sensory and regulatory modules ensure a rapid and appropriate response. RECENT AD

  15. Experimental Evolution of a Green Fluorescent Protein Composed of 19 Unique Amino Acids without Tryptophan

    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)

  16. Impact of oral vancomycin on gut microbiota, bile acid metabolism, and insulin sensitivity

    Vrieze, Anne; Out, Carolien; Fuentes, Susana;

    2014-01-01

    BACKGROUND & AIMS: Obesity has been associated with changes in the composition and function of the intestinal microbiota. Modulation of the microbiota by antibiotics also alters bile acid and glucose metabolism in mice. Hence, we hypothesized that short term administration of oral antibiotics in...... humans would affect fecal microbiota composition and subsequently bile acid and glucose metabolism. METHODS: In this single blinded randomized controlled trial, 20 male obese subjects with metabolic syndrome were randomized to 7 days of amoxicillin 500 mg t.i.d. or 7 days of vancomycin 500 mg t.i.d. At...... baseline and after 1 week of therapy, fecal microbiota composition (Human Intestinal Tract Chip phylogenetic microarray), fecal and plasma bile acid concentrations as well as insulin sensitivity (hyperinsulinemic euglycemic clamp using [6,6-(2)H2]-glucose tracer) were measured. RESULTS: Vancomycin reduced...

  17. Nucleic acid and protein extraction from electropermeabilized E. coli cells on a microfluidic chip

    Matos, T.; Senkbeil, Silja; Mendonça, A; Queiroz, J. A.; Kutter, Jörg Peter; Bulow, L.

    2013-01-01

    Due to the extensive use of nucleic acid and protein analysis of bacterial samples, there is a need for simple and rapid extraction protocols for both plasmid DNA and RNA molecules as well as reporter proteins like the green fluorescent protein (GFP). In this report, an electropermeability technique has been developed which is based on exposing E. coli cells to low voltages to allow extraction of nucleic acids and proteins. The flow-through electropermeability chip used consists of a microflu...

  18. Investigation on protein content and amino acid composition in the kernels of some sunflower lines

    Nenova N.; Drumeva M.

    2012-01-01

    This study took into account the protein content in the kernel of ten lines derived from interspecific hybrids Helianthus annuus (line 2607) × Helianthus resinosus and Helianthus annuus (line 2607) × Helianthus salicifolius. The amino acid composition of storage protein was also studied. The protein in the new lines exceeded the protein in the parental forms with up to 10.6%. The essential amino acids lysine, valine, threonine and phenylalanine had higher a...

  19. A nonionic surfactant-decorated liquid crystal sensor for sensitive and selective detection of proteins.

    Wang, Yi; Hu, Qiongzheng; Tian, Tongtong; Gao, Yan'an; Yu, Li

    2016-09-21

    Proteins are responsible for most biochemical events in human body. It is essential to develop sensitive and selective methods for the detection of proteins. In this study, liquid crystal (LC)-based sensor for highly selective and sensitive detection of lysozyme, concanavalin A (Con A), and bovine serum albumin (BSA) was constructed by utilizing the LC interface decorated with a nonionic surfactant, dodecyl β-d-glucopyranoside. A change of the LC optical images from bright to dark appearance was observed after transferring dodecyl β-d-glucopyranoside onto the aqueous/LC interface due to the formation of stable self-assembled surfactant monolayer, regardless of pH and ion concentrations studied in a wide range. The optical images turned back from dark to bright appearance after addition of lysozyme, Con A and BSA, respectively. Noteworthy is that these proteins can be further distinguished by adding enzyme inhibitors and controlling incubation temperature of the protein solutions based on three different interaction mechanisms between proteins and dodecyl β-d-glucopyranoside, viz. enzymatic hydrolysis, specific saccharide binding, and physical absorption. The LC-based sensor decorated with dodecyl β-d-glucopyranoside shows high sensitivity for protein detection. The limit of detection (LOD) for lysozyme, Con A and BSA reaches around 0.1 μg/mL, 0.01 μg/mL and 0.001 μg/mL, respectively. These results might provide new insights into increasing selectivity and sensitivity of LC-based sensors for the detection of proteins. PMID:27590553

  20. Pyruvate carboxylase as a sensitive protein biomarker for exogenous steroid chemicals

    Assessing protein responses to endocrine disrupting chemicals is critical for understanding the mechanisms of chemical action and for the assessment of hazards. In this study, the response of the liver proteome of male rare minnows (Gobiocypris rarus) treated with 17β-estradiol (E2) and females treated with 17α-methyltestosterone (MT) were analyzed. A total of 23 and 24 proteins were identified with differential expression in response to E2 and MT, respectively. Pyruvate carboxylase (PC) was the only common differentially expressed protein in both males and females after E2- and MT-treatments. The mRNA as well as the protein levels of PC were significantly down-regulated compared with that of the controls (p < 0.05). Our results suggest that endocrine disruptors interfere with genes and proteins of the TCA cycle and PC may be a sensitive biomarker of exposure to exogenous steroid chemicals in the liver of fish. - Highlights: • The hepatic proteomes of rare minnow (Gobiocypris rarus) exposed to E2 and MT were analyzed. • Differentially expressed proteins (23 and 24 respectively) were identified following E2 and MT exposure. • Four differentially expressed proteins associated with chemical stimulus were characterized. • PC was identified as a responsive biomarker for both estrogens and androgens. - Our results suggest PC may be a sensitive biomarker of exposure to exogenous steroid chemicals in the liver of fish

  1. Prognostic value of high sensitive C-reactive protein in subjects with silent myocardial ischemia

    Mouridsen, Mette; Intzilakis, Theodoros; Binici, Zeynep;

    2012-01-01

    OBJECTIVES: The aim of this study was to evaluate the prognostic value of high sensitive C-reactive protein (CRP) in subjects with silent myocardial ischemia (SMI). DESIGN: In total, 678 healthy men and women aged 55 to 75 years with no history of cardiovascular disease or stroke were included. H...

  2. Identification of Dynamic Changes in Proteins Associated with the Cellular Cytoskeleton after Exposure to Okadaic Acid

    Peter Roepstorff

    2013-05-01

    Full Text Available 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.

  3. (E)-Propyl α-Cyano-4-Hydroxyl Cinnamylate: A High Sensitive and Salt Tolerant Matrix for Intact Protein Profiling by MALDI Mass Spectrometry

    Wang, Sheng; Xiao, Zhaohui; Xiao, Chunsheng; Wang, Huixin; Wang, Bing; Li, Ying; Chen, Xuesi; Guo, Xinhua

    2016-04-01

    Low-abundance samples and salt interference are always of great challenges for the practical protein profiling by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Herein, a series of carboxyl-esterified derivatives of α-cyano-4-hydroxycinnamic acid (CHCA) were synthesized and evaluated as matrices for MALDI-MS analysis of protein. Among them, (E)-propyl α-cyano-4-hydroxyl cinnamylate (CHCA-C3) was found to exhibit excellent assay performance for intact proteins by improving the detection sensitivity 10 folds compared with the traditional matrices [i.e., super2,5-dihydroxybenzoic acid (superDHB), sinapic acid (SA), and CHCA]. In addition, CHCA-C3 was shown to have high tolerance to salts, the ion signal of myoglobin was readily detected even in the presence of urea (8 M), NH4HCO3 (2 M), and KH2PO4 (500 mM), meanwhile sample washability was robust. These achievements were mainly attributed to improved ablation ability and increased hydrophobicity or affinity of CHCA-C3 to proteins in comparison with hydrophilic matrixes, leading to more efficient ionization of analyte. Furthermore, direct analysis of proteins from crude egg white demonstrated that CHCA-C3 was a highly efficient matrix for the analysis of low-abundance proteins in complex biological samples. These outstanding performances indicate the tremendous potential use of CHCA-C3 in protein profiling by MALDI-MS.

  4. Scale-free behaviour of amino acid pair interactions in folded proteins

    Petersen, Steffen B.; Neves-Petersen, Maria Teresa; Mortensen, Rasmus J.;

    2012-01-01

    The protein structure is a cumulative result of interactions between amino acid residues interacting with each other through space and/or chemical bonds. Despite the large number of high resolution protein structures, the ‘‘protein structure code’’ has not been fully identified. Our manuscript...... presents a novel approach to protein structure analysis in order to identify rules for spatial packing of amino acid pairs in proteins. We have investigated 8706 high resolution non-redundant protein chains and quantified amino acid pair interactions in terms of solvent accessibility, spatial and sequence...... are in buried a-helices or b-strands, in a spatial distance of 3.8–4.3A° and in a sequence distance .4 residues. We speculate that the scale free organization of the amino acid pair interactions in the 8D protein structure combined with the clear dominance of pairs of Ala, Ile, Leu and Val is...

  5. Improving the sensitivity of protein microarray by evanescent-field-induced fluorescence

    WANG Li-qiang; LU Zu-kang

    2005-01-01

    To improve the sensitivity of protein microarray, a prism surface replaces the surface of the common microscope slide.The protein targets arrayed on the surface are hybridized and labelled by fluorescent probes. Evanescent excitation occurs when the convergent laser reaches the surface, and a photomultiplier tube detects the emitted fluorescent signal. A two-dimensional actuator scans the whole surface to achieve planar laser excitation and fluorescence collection. The penetration depth of the evanescent field into the protein targets is only some hundred nanometers and can be controlled by different incident angle of the laser beam, so the undesired background signals are reduced dramatically and the detection sensitivity is improved by a factor of 50 to 100 comparing to confocal excitation. This approach can detect low abundance analytes without signal amplification.

  6. APPLICATION OF NONIONIC TEMPERATURE SENSITIVE HYDROGEL FOR CONCENTRATION OF PROTEIN AQUEOUS SOLUTION

    SUN Yishi; QIU Zhiyong; HONG Yaoliang

    1992-01-01

    Six different N-alkyl substituted acrylamide nonionic hydrogels were prepared and their swelling characteristics were measured. Poly N-isopropyl acrylamide (PNIPA) and poly N-n-propylacrylamide (PNNPA) temperature sensitive hydrogels were chosen as the nonionic temperature sensitive hydrogels for concentration of very dilute aqueous protein solution. The separation properties of PNIPA and PNNPA hydrogels with different network dimensions were studied and the modification of the hydrogels was surveyed in order to decrease their surface adsorption of protein molecules. The experimental results of the concentration of BSA (Bovin serum albumin) dilute aqueous solution by hydroxylpropyl methacrylate (HPMA) copolymerized PNIPA hydrogel were given. The value and the limitation of concentration of dilute aqueous protein solution by this method was evaluated.

  7. [Effect of proteolysis inhibitors on the incorporation of labelled amino acids into proteins].

    Konikova, A S; Korotkina, R N

    1975-01-01

    Role of peptide bond breaks in the incorporation of amino acids into proteins in a "protein--amino acid" system is investigated. For this purpose the incorporation of labelled amino acids into trypsin under the inhibition of its autolysis by a specific inhibitor from soybean and epsilon-amino-caproic acid is studied. The trypsin inhibitor from soybean is found to suppress considerably the incorporation of 14C-glycine, 14C-lysine and 14C-methionine into crystal trypsin and not to affect the incorporation of labelled amino acids into chomotrypsin, papain and carboxypeptidase. Epsilon-Aminocaproic acid inhibited 14C-glycine incorporation into crystal trypsin by 40% and did not change its incorporation level into serum albumin. The dependency of amino acid incorporation level into trypsin on the activity of autolysis in the "protein--amino acid" system is demonstrated. PMID:1212456

  8. Geological and hydrochemical sensitivity of the eastern United States to acid precipitation

    Hendrey, G.R.; Galloway, J.N.; Norton, S.A.; Schofield, C.L.; Shaffer, P.W.; Burns, D.A.

    1980-03-01

    A new analysis of bedrock geology maps of the eastern US constitutes a simple model for predicting areas which might be impacted by acid precipitation and it allows much greater resolution for detecting sensitivity than has previously been available for the region. Map accuracy has been verified by examining current alkalinities and pH's of waters in several test states, including Maine, New Hampshire, New York, Virginia and North Carolina. In regions predicted to be highly sensitive, alkalinities in upstream sites were generally low. Many areas of the eastern US are pinpointed in which some of the surface waters, especially upstream reaches, may be sensitive to acidification. Pre-1970 data were compared to post-1975 data, revealing marked declines in both alkalinity and pH of sensitive waters of two states tested, North Carolina, where pH and alkalinity have decreased in 80% of 38 streams and New Hampshire, where pH in 90% of 49 streams and lakes has decreased since 1949. These sites are predicted to be sensitive by the geological map on the basis of their earlier alkalinity values. The map is to be improved by the addition of a soils component.

  9. Regulation of extracellular copper-binding proteins in copper-resistant and copper-sensitive mutants of Vibrio alginolyticus.

    Harwood, V J; Gordon, A S

    1994-01-01

    Extracellular proteins of wild-type Vibrio alginolyticus were compared with those of copper-resistant and copper-sensitive mutants. One copper-resistant mutant (Cu40B3) constitutively produced an extracellular protein with the same apparent molecular mass (21 kDa) and chromatographic behavior as copper-binding protein (CuBP), a copper-induced supernatant protein which has been implicated in copper detoxification in wild-type V. alginolyticus. Copper-sensitive V. alginolyticus mutants displaye...

  10. Branched-chain Amino Acid Biosensing Using Fluorescent Modified Engineered Leucine/Isoleucine/Valine Binding Protein

    Koji Sode

    2007-06-01

    Full Text Available A novel fluorescence sensing system for branched-chain amino acids (BCAAswas developed based on engineered leucine/isoleucine/valine-binding proteins (LIVBPsconjugated with environmentally sensitive fluorescence probes. LIVBP was cloned fromEscherichia coli and Gln149Cys, Gly227Cys, and Gln254Cys mutants were generated bygenetic engineering. The mutant LIVBPs were then modified with environmentallysensitive fluorophores. Based on the fluorescence intensity change observed upon thebinding of the ligands, the MIANS-conjugated Gln149Cys mutant (Gln149Cys-M showedthe highest and most sensitive response. The BCAAs Leu, Ile, and Val can each bemonitored at the sub-micromolar level using Gln149Cys-M. Measurements were alsocarried out on a mixture of BCAFAs and revealed that Gln149Cys-M-based measurementis not significantly affected by the change in the molar ratio of Leu, Ile and Val in thesample. Its high sensitivity and group-specific molecular recognition ability make the newsensing system ideally suited for the measurement of BCAAs and the determination of theFischer ratio, an indicator of hepatic disease involving metabolic dysfunction.

  11. Amino acid and protein turnover in human skeletal muscle

    Vesali, Rokhsareh Farrah

    2005-01-01

    Critically ill patients are characterised by a severe net protein catabolism. The rate of muscle protein loss is in the magnitude of 10% per week. A consequence of muscle wasting is increased weakness, which is associated with high rates of mortality and morbidity. Protein wasting is a result of either a decrease of protein synthesis or an increase of protein degradation or a combination of both. To understand the underlying mechanisms determinations of both protein synthesi...

  12. Experimentally Testing the Hypothesis of a Limited Amino Acid Repertoire in Primitive Proteins

    Akanuma, S.; Nakajima, Y.; Yokobori, S.; Yamagishi, A.

    2013-11-01

    It has been argued that a fewer amino acids were used in primitive proteins and later the repertoire increased up to 20. To test this hypothesis experimentally, we restricted the amino acid usage of a reconstructed, ancestral protein to reduced sets.

  13. Insulin-Sensitizing Effects of Omega-3 Fatty Acids: Lost in Translation?

    Lalia, Antigoni Z; Lanza, Ian R

    2016-01-01

    Omega-3 polyunsaturated fatty acids (n-3 PUFA) of marine origin, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), have been long studied for their therapeutic potential in the context of type 2 diabetes, insulin resistance, and glucose homeostasis. Glaring discordance between observations in animal and human studies precludes, to date, any practical application of n-3 PUFA as nutritional therapeutics against insulin resistance in humans. Our objective in this review is to summarize current knowledge and provide an up-to-date commentary on the therapeutic value of EPA and DHA supplementation for improving insulin sensitivity in humans. We also sought to discuss potential mechanisms of n-3 PUFA action in target tissues, in specific skeletal muscle, based on our recent work, as well as in liver and adipose tissue. We conducted a literature search to include all preclinical and clinical studies performed within the last two years and to comment on representative studies published earlier. Recent studies support a growing consensus that there are beneficial effects of n-3 PUFA on insulin sensitivity in rodents. Observational studies in humans are encouraging, however, the vast majority of human intervention studies fail to demonstrate the benefit of n-3 PUFA in type 2 diabetes or insulin-resistant non-diabetic people. Nevertheless, there are still several unanswered questions regarding the potential impact of n-3 PUFA on metabolic function in humans. PMID:27258299

  14. Seasonal profiles of leaf ascorbic acid content and redox state in ozone-sensitive wildflowers

    Cutleaf coneflower (Rudbeckia laciniata L.), crown-beard (Verbesina occidentalis Walt.), and tall milkweed (Asclepias exaltata L.) are wildflower species native to Great Smoky Mountains National Park (U.S.A.). Natural populations of each species were analyzed for leaf ascorbic acid (AA) and dehydroascorbic acid (DHA) to assess the role of ascorbate in protecting the plants from ozone stress. Tall milkweed contained greater quantities of AA (7-10 μmol g-1 fresh weight) than crown-beard (2-4 μmol g-1 fresh weight) or cutleaf coneflower (0.5-2 μmol g-1 fresh weight). DHA was elevated in crown-beard and cutleaf coneflower relative to tall milkweed suggesting a diminished capacity for converting DHA into AA. Tall milkweed accumulated AA in the leaf apoplast (30-100 nmol g-1 fresh weight) with individuals expressing ozone foliar injury symptoms late in the season having less apoplast AA. In contrast, AA was not present in the leaf apoplast of either crown-beard or cutleaf coneflower. Unidentified antioxidant compounds were present in the leaf apoplast of all three species. Overall, distinct differences in antioxidant metabolism were found in the wildflower species that corresponded with differences in ozone sensitivity. - Wildflower species exhibit differences in ascorbic acid content and redox status that affect ozone sensitivity

  15. Long-term development of acid deposition (1880–2030 in sensitive freshwater regions in Europe

    W. Schöpp

    2003-01-01

    Full Text Available Time series of the deposition of acidifying substances are a pre-requisite for the study of the acidification and recovery of ecosystems such as surface waters. This paper reports the derivation and calculation of deposition trends of the potentially acidifying compounds SO2, NOx and NH3 in sensitive freshwater regions in Europe studied in the EU-funded RECOVER: 2010 project. The time interval covered is 151 years: from 1880, which can be considered as the pre-industrial era in most countries, to 2030, taking into account the consequences of current emission reduction agreements in Europe. The historic and predicted emissions for European countries are used to calculate the deposition development in the study areas, using meteorologically averaged atmospheric source-receptor transfer coefficients derived from the EMEP Lagrangian acid deposition model. These time series were used as driving forces for the application of the dynamic acidification model MAGIC to study the acidification and recovery of sensitive freshwater ecosystems in Europe. Keywords: acid deposition, historic depositions, sensitive lake regions, Europe

  16. Genome-wide screening for genes associated with valproic acid sensitivity in fission yeast.

    Lili Zhang

    Full Text Available We have been studying the action mechanisms of valproic acid (VPA in fission yeast Schizosaccharomyces pombe by developing a genetic screen for mutants that show hypersensitivity to VPA. In the present study, we performed a genome-wide screen of 3004 haploid deletion strains and confirmed 148 deletion strains to be VPA sensitive. Of the 148 strains, 93 strains also showed sensitivity to another aliphatic acids HDAC inhibitor, sodium butyrate (SB, and 55 strains showed sensitivity to VPA but not to SB. Interestingly, we found that both VPA and SB treatment induced a marked increase in the transcription activity of Atf1 in wild-type cells. However, in clr6-1, a mutant allele the clr6(+ gene encoding class I HDAC, neither VPA- nor SB induced the activation of Atf1 transcription activity. We also found that VPA, but not SB, caused an increase in cytoplasmic Ca(2+ level. We further found that the cytoplasmic Ca(2+ increase was caused by Ca(2+ influx from extracellular medium via Cch1-Yam8 channel complex. Altogether, our present study indicates that VPA and SB play similar but distinct roles in multiple physiological processes in fission yeast.

  17. Enhanced insulin sensitivity associated with provision of mono and polyunsaturated fatty acids in skeletal muscle cells involves counter modulation of PP2A.

    Francesca Nardi

    Full Text Available AIMS/HYPOTHESIS: Reduced skeletal muscle insulin sensitivity is a feature associated with sustained exposure to excess saturated fatty acids (SFA, whereas mono and polyunsaturated fatty acids (MUFA and PUFA not only improve insulin sensitivity but blunt SFA-induced insulin resistance. The mechanisms by which MUFAs and PUFAs institute these favourable changes remain unclear, but may involve stimulating insulin signalling by counter-modulation/repression of protein phosphatase 2A (PP2A. This study investigated the effects of oleic acid (OA; a MUFA, linoleic acid (LOA; a PUFA and palmitate (PA; a SFA in cultured myotubes and determined whether changes in insulin signalling can be attributed to PP2A regulation. PRINCIPAL FINDINGS: We treated cultured skeletal myotubes with unsaturated and saturated fatty acids and evaluated insulin signalling, phosphorylation and methylation status of the catalytic subunit of PP2A. Unlike PA, sustained incubation of rat or human myotubes with OA or LOA significantly enhanced Akt- and ERK1/2-directed insulin signalling. This was not due to heightened upstream IRS1 or PI3K signalling nor to changes in expression of proteins involved in proximal insulin signalling, but was associated with reduced dephosphorylation/inactivation of Akt and ERK1/2. Consistent with this, PA reduced PP2Ac demethylation and tyrosine307phosphorylation - events associated with PP2A activation. In contrast, OA and LOA strongly opposed these PA-induced changes in PP2Ac thus exerting a repressive effect on PP2A. CONCLUSIONS/INTERPRETATION: Beneficial gains in insulin sensitivity and the ability of unsaturated fatty acids to oppose palmitate-induced insulin resistance in muscle cells may partly be accounted for by counter-modulation of PP2A.

  18. Site specific incorporation of heavy atom-containing unnatural amino acids into proteins for structure determination

    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.

  19. Cyclin B synthesis and rapamycin-sensitive regulation of protein synthesis during starfish oocyte meiotic divisions.

    Lapasset, Laure; Pradet-Balade, Bérengère; Vergé, Valérie; Lozano, Jean-Claude; Oulhen, Nathalie; Cormier, Patrick; Peaucellier, Gérard

    2008-11-01

    Translation of cyclin mRNAs represents an important event for proper meiotic maturation and post-fertilization mitoses in many species. Translational control of cyclin B mRNA has been described to be achieved through two separate but related mechanisms: translational repression and polyadenylation. In this paper, we evaluated the contribution of global translational regulation by the cap-dependent translation repressor 4E-BP (eukaryotic initiation factor 4E-binding protein) on the cyclin B protein synthesis during meiotic maturation of the starfish oocytes. We used the immunosupressant drug rapamycin, a strong inhibitor of cap-dependent translation, to check for the involvement of this protein synthesis during this physiological process. Rapamycin was found to prevent dissociation of 4E-BP from the initiation factor eIF4E and to suppress correlatively a burst of global protein synthesis occurring at the G2/M transition. The drug had no effect on first meiotic division but defects in meiotic spindle formation prevented second polar body emission, demonstrating that a rapamycin-sensitive pathway is involved in this mechanism. While rapamycin affected the global protein synthesis, the drug altered neither the specific translation of cyclin B mRNA nor the expression of the Mos protein. The expression of these two proteins was correlated with the phosphorylation and the dissociation of the cytoplasmic polyadenylation element-binding protein from eIF4E. PMID:18361417

  20. Interaction of perfluoroalkyl acids with human liver fatty acid-binding protein.

    Sheng, Nan; Li, Juan; Liu, Hui; Zhang, Aiqian; Dai, Jiayin

    2016-01-01

    Perfluoroalkyl acids (PFAAs) are highly persistent and bioaccumulative, resulting in their broad distribution in humans and the environment. The liver is an important target for PFAAs, but the mechanisms behind PFAAs interaction with hepatocyte proteins remain poorly understood. We characterized the binding of PFAAs to human liver fatty acid-binding protein (hL-FABP) and identified critical structural features in their interaction. The binding interaction of PFAAs with hL-FABP was determined by fluorescence displacement and isothermal titration calorimetry (ITC) assay. Molecular simulation was conducted to define interactions at the binding sites. ITC measurement revealed that PFOA/PFNA displayed a moderate affinity for hL-FABP at a 1:1 molar ratio, a weak binding affinity for PFHxS and no binding for PFHxA. Moreover, the interaction was mainly mediated by electrostatic attraction and hydrogen bonding. Substitution of Asn111 with Asp caused loss of binding affinity to PFAA, indicating its crucial role for the initial PFAA binding to the outer binding site. Substitution of Arg122 with Gly caused only one molecule of PFAA to bind to hL-FABP. Molecular simulation showed that substitution of Arg122 increased the volume of the outer binding pocket, making it impossible to form intensive hydrophobic stacking and hydrogen bonds with PFOA, and highlighting its crucial role in the binding process. The binding affinity of PFAAs increased significantly with their carbon number. Arg122 and Asn111 played a pivotal role in these interactions. Our findings may help understand the distribution pattern, bioaccumulation, elimination, and toxicity of PFAAs in humans. PMID:25370009

  1. Lipid recognition propensities of amino acids in membrane proteins from atomic resolution data

    Morita Mizuki

    2011-12-01

    Full Text Available Abstract Background Protein-lipid interactions play essential roles in the conformational stability and biological functions of membrane proteins. However, few of the previous computational studies have taken into account the atomic details of protein-lipid interactions explicitly. Results To gain an insight into the molecular mechanisms of the recognition of lipid molecules by membrane proteins, we investigated amino acid propensities in membrane proteins for interacting with the head and tail groups of lipid molecules. We observed a common pattern of lipid tail-amino acid interactions in two different data sources, crystal structures and molecular dynamics simulations. These interactions are largely explained by general lipophilicity, whereas the preferences for lipid head groups vary among individual proteins. We also found that membrane and water-soluble proteins utilize essentially an identical set of amino acids for interacting with lipid head and tail groups. Conclusions We showed that the lipophilicity of amino acid residues determines the amino acid preferences for lipid tail groups in both membrane and water-soluble proteins, suggesting that tightly-bound lipid molecules and lipids in the annular shell interact with membrane proteins in a similar manner. In contrast, interactions between lipid head groups and amino acids showed a more variable pattern, apparently constrained by each protein's specific molecular function.

  2. Lipid recognition propensities of amino acids in membrane proteins from atomic resolution data

    Protein-lipid interactions play essential roles in the conformational stability and biological functions of membrane proteins. However, few of the previous computational studies have taken into account the atomic details of protein-lipid interactions explicitly. To gain an insight into the molecular mechanisms of the recognition of lipid molecules by membrane proteins, we investigated amino acid propensities in membrane proteins for interacting with the head and tail groups of lipid molecules. We observed a common pattern of lipid tail-amino acid interactions in two different data sources, crystal structures and molecular dynamics simulations. These interactions are largely explained by general lipophilicity, whereas the preferences for lipid head groups vary among individual proteins. We also found that membrane and water-soluble proteins utilize essentially an identical set of amino acids for interacting with lipid head and tail groups. We showed that the lipophilicity of amino acid residues determines the amino acid preferences for lipid tail groups in both membrane and water-soluble proteins, suggesting that tightly-bound lipid molecules and lipids in the annular shell interact with membrane proteins in a similar manner. In contrast, interactions between lipid head groups and amino acids showed a more variable pattern, apparently constrained by each protein's specific molecular function

  3. Carbon nanofiber-based luminol-biotin probe for sensitive chemiluminescence detection of protein.

    Baj, Stefan; Krawczyk, Tomasz; Pradel, Natalia; Azam, Md Golam; Shibata, Takayuki; Dragusha, Shpend; Skutil, Krzysztof; Pawlyta, Miroslawa; Kai, Masaaki

    2014-01-01

    A carbon nanofiber-based luminol-biotin probe was synthesized for the sensitive chemiluminescence (CL) detection of a target protein by grafting luminol and biotin onto an oxidized carbon nanofiber. This carbon nanofiber was prepared by chemical vapor-deposition with methane in the presence of the Ni-Cu-MgO catalyst, which was followed by oxidization with HNO3-H2SO4 to produce a carboxyl group on the surface of the nanofiber. The material was grafted with luminol and biotin by means of a standard carbodiimide activation of COOH groups to produce corresponding amides. The substance was water-soluble and thus could be utilized as a sensitive CL probe for a protein assay. The probe showed highly specific affinity towards the biotin-labeled antibody via a streptavidin-biotin interaction. The detection limit for this model assay was approximately 0.2 pmol of the biotinized IgG spotted on a polyvinylidene fluoride (PVDF) membrane. Nonspecific binding to other proteins was not observed. Therefore, the synthesized carbon nanofiber-based CL probe may be useful for a sensitive and specific analysis of the target protein. PMID:25382040

  4. Proteomic identification of early salicylate- and flg22-responsive redox-sensitive proteins in Arabidopsis

    Liu, Pei

    2015-02-27

    Accumulation of reactive oxygen species (ROS) is one of the early defense responses against pathogen infection in plants. The mechanism about the initial and direct regulation of the defense signaling pathway by ROS remains elusive. Perturbation of cellular redox homeostasis by ROS is believed to alter functions of redox-sensitive proteins through their oxidative modifications. Here we report an OxiTRAQ-based proteomic study in identifying proteins whose cysteines underwent oxidative modifications in Arabidopsis cells during the early response to salicylate or flg22, two defense pathway elicitors that are known to disturb cellular redox homeostasis. Among the salicylate- and/or flg22-responsive redox-sensitive proteins are those involved in transcriptional regulation, chromatin remodeling, RNA processing, post-translational modifications, and nucleocytoplasmic shuttling. The identification of the salicylate-/flg22-responsive redox-sensitive proteins provides a foundation from which further study can be conducted toward understanding biological significance of their oxidative modifications during the plant defense response.

  5. Sorption of zinc by novel pH-sensitive hydrogels based on chitosan, itaconic acid and methacrylic acid

    Milosavljevic, Nedeljko B.; Ristic, Mirjana D.; Peric-Grujic, Aleksandra A.; Filipovic, Jovanka M. [Faculty of Technology and Metallurgy, University of Belgrade, Karnegijeva 4, 11120 Belgrade (Serbia); Strbac, Svetlana B. [ICTM-Institute of Electrochemistry, University of Belgrade, P.O.B. 815, 11001 Belgrade (Serbia); Rakocevic, Zlatko Lj. [INS Vinca, Laboratory for Atomic Physics, University of Belgrade, P.O.B. 522, Mike Alasa 12-14, 11001 Belgrade (Serbia); Kalagasidis Krusic, Melina T., E-mail: meli@tmf.bg.ac.rs [Faculty of Technology and Metallurgy, University of Belgrade, Karnegijeva 4, 11120 Belgrade (Serbia)

    2011-08-30

    Highlights: {yields} A removal of Zn{sup 2+} ions by pH-sensitive Ch/IA/MAA hydrogel from aqueous solutions was studied. {yields} SEM/EDX analysis and AFM surface topography indicate that sorption takes place on the surface of the hydrogel and in the bulk. {yields} FTIR spectra of the Ch/IA/MAA hydrogel, free and Zn-loaded, indicate that -NH{sub 2}, -OH and -COOH groups are involved in the sorption process. {yields} The negative values of free energy and enthalpy indicated that the adsorption is spontaneous and exothermic one. {yields} The adsorption capacities did not show any significant decrease after the third reuse cycle. - Abstract: Novel pH-sensitive hydrogels based on chitosan, itaconic acid and methacrylic acid were applied as adsorbents for the removal of Zn{sup 2+} ions from aqueous solution. In batch tests, the influence of solution pH, contact time, initial metal ion concentration and temperature was examined. The sorption was found pH dependent, pH 5.5 being the optimum value. The adsorption process was well described by the pseudo-second order kinetic. The hydrogels were characterized by spectral (Fourier transform infrared-FTIR) and structural (SEM/EDX and atomic force microscopy-AFM) analyses. The surface topography changes were observed by atomic force microscopy, while the changes in surface composition were detected using phase imaging AFM. The negative values of free energy and enthalpy indicated that the adsorption is spontaneous and exothermic one. The best fitting isotherms were Langmuir and Redlich-Peterson and it was found that both linear and nonlinear methods were appropriate for obtaining the isotherm parameters. However, the increase of temperature leads to higher adsorption capacity, since swelling degree increased with temperature.

  6. Sorption of zinc by novel pH-sensitive hydrogels based on chitosan, itaconic acid and methacrylic acid

    Highlights: → A removal of Zn2+ ions by pH-sensitive Ch/IA/MAA hydrogel from aqueous solutions was studied. → SEM/EDX analysis and AFM surface topography indicate that sorption takes place on the surface of the hydrogel and in the bulk. → FTIR spectra of the Ch/IA/MAA hydrogel, free and Zn-loaded, indicate that -NH2, -OH and -COOH groups are involved in the sorption process. → The negative values of free energy and enthalpy indicated that the adsorption is spontaneous and exothermic one. → The adsorption capacities did not show any significant decrease after the third reuse cycle. - Abstract: Novel pH-sensitive hydrogels based on chitosan, itaconic acid and methacrylic acid were applied as adsorbents for the removal of Zn2+ ions from aqueous solution. In batch tests, the influence of solution pH, contact time, initial metal ion concentration and temperature was examined. The sorption was found pH dependent, pH 5.5 being the optimum value. The adsorption process was well described by the pseudo-second order kinetic. The hydrogels were characterized by spectral (Fourier transform infrared-FTIR) and structural (SEM/EDX and atomic force microscopy-AFM) analyses. The surface topography changes were observed by atomic force microscopy, while the changes in surface composition were detected using phase imaging AFM. The negative values of free energy and enthalpy indicated that the adsorption is spontaneous and exothermic one. The best fitting isotherms were Langmuir and Redlich-Peterson and it was found that both linear and nonlinear methods were appropriate for obtaining the isotherm parameters. However, the increase of temperature leads to higher adsorption capacity, since swelling degree increased with temperature.

  7. Efficient DNP NMR of membrane proteins: sample preparation protocols, sensitivity, and radical location.

    Liao, Shu Y; Lee, Myungwoon; Wang, Tuo; Sergeyev, Ivan V; Hong, Mei

    2016-03-01

    Although dynamic nuclear polarization (DNP) has dramatically enhanced solid-state NMR spectral sensitivities of many synthetic materials and some biological macromolecules, recent studies of membrane-protein DNP using exogenously doped paramagnetic radicals as polarizing agents have reported varied and sometimes surprisingly limited enhancement factors. This motivated us to carry out a systematic evaluation of sample preparation protocols for optimizing the sensitivity of DNP NMR spectra of membrane-bound peptides and proteins at cryogenic temperatures of ~110 K. We show that mixing the radical with the membrane by direct titration instead of centrifugation gives a significant boost to DNP enhancement. We quantify the relative sensitivity enhancement between AMUPol and TOTAPOL, two commonly used radicals, and between deuterated and protonated lipid membranes. AMUPol shows ~fourfold higher sensitivity enhancement than TOTAPOL, while deuterated lipid membrane does not give net higher sensitivity for the membrane peptides than protonated membrane. Overall, a ~100 fold enhancement between the microwave-on and microwave-off spectra can be achieved on lipid-rich membranes containing conformationally disordered peptides, and absolute sensitivity gains of 105-160 can be obtained between low-temperature DNP spectra and high-temperature non-DNP spectra. We also measured the paramagnetic relaxation enhancement of lipid signals by TOTAPOL and AMUPol, to determine the depths of these two radicals in the lipid bilayer. Our data indicate a bimodal distribution of both radicals, a surface-bound fraction and a membrane-bound fraction where the nitroxides lie at ~10 Å from the membrane surface. TOTAPOL appears to have a higher membrane-embedded fraction than AMUPol. These results should be useful for membrane-protein solid-state NMR studies under DNP conditions and provide insights into how biradicals interact with phospholipid membranes. PMID:26873390

  8. Cellular fatty acid composition, protein profile and antimicrobial activity of Bacillus sp., isolated from fish gut

    Pushparaj Sujith; Baskaran Rohini; Singaram Jayalakshmi

    2014-01-01

    Objective: To purify and partially characterize the antimicrobial compounds from bacteriaBacillus sp., isolated from fish gut. Methods: Protein and fatty acids were isolated from the bacteria and checked for the presence of antibacterial activity. Protein has been purified to apparent homogeneity from the supernatants of culture by means of ammonium sulphate precipitation followed by dialysis. Fourier transform infrared spectroscopy analyses were performed for proteins to identify the functional groups.Results:sulfate polyacrylamide gel electrophoresis. Fatty acids were extracted and subjected to gas chromatographic analysis.Conclusions:Protein showed an apparent molecular mass 56, 47 and 39 kDa on sodium dodecyl acids and proteins which holds promise for the development of new drugs. The antimicrobial activity of the bacteria might be due to the presence of fatty acids and proteins which holds promise for the development of new drugs.

  9. Spinal afferent neurons projecting to the rat lung and pleura express acid sensitive channels

    Kummer Wolfgang

    2006-07-01

    Full Text Available Abstract Background The acid sensitive ion channels TRPV1 (transient receptor potential vanilloid receptor-1 and ASIC3 (acid sensing ion channel-3 respond to tissue acidification in the range that occurs during painful conditions such as inflammation and ischemia. Here, we investigated to which extent they are expressed by rat dorsal root ganglion neurons projecting to lung and pleura, respectively. Methods The tracer DiI was either injected into the left lung or applied to the costal pleura. Retrogradely labelled dorsal root ganglion neurons were subjected to triple-labelling immunohistochemistry using antisera against TRPV1, ASIC3 and neurofilament 68 (marker for myelinated neurons, and their soma diameter was measured. Results Whereas 22% of pulmonary spinal afferents contained neither channel-immunoreactivity, at least one is expressed by 97% of pleural afferents. TRPV1+/ASIC3- neurons with probably slow conduction velocity (small soma, neurofilament 68-negative were significantly more frequent among pleural (35% than pulmonary afferents (20%. TRPV1+/ASIC3+ neurons amounted to 14 and 10% respectively. TRPV1-/ASIC3+ neurons made up between 44% (lung and 48% (pleura of neurons, and half of them presumably conducted in the A-fibre range (larger soma, neurofilament 68-positive. Conclusion Rat pleural and pulmonary spinal afferents express at least two different acid-sensitive channels that make them suitable to monitor tissue acidification. Patterns of co-expression and structural markers define neuronal subgroups that can be inferred to subserve different functions and may initiate specific reflex responses. The higher prevalence of TRPV1+/ASIC3- neurons among pleural afferents probably reflects the high sensitivity of the parietal pleura to painful stimuli.

  10. Terbium fluorescence as a sensitive, inexpensive probe for UV-induced damage in nucleic acids

    Graphical abstract: -- Highlights: •Simple, inexpensive, mix-and-read assay for positive detection of DNA damage. •Recognition of undamaged DNA via hybridization to a hairpin probe. •Terbium(III) fluorescence reports the amount of damage by binding to ssDNA. •Tb/hairpin is a highly selective and sensitive fluorescent probe for DNA damage. -- Abstract: Much effort has been focused on developing methods for detecting damaged nucleic acids. However, almost all of the proposed methods consist of multi-step procedures, are limited, require expensive instruments, or suffer from a high level of interferences. In this paper, we present a novel simple, inexpensive, mix-and-read assay that is generally applicable to nucleic acid damage and uses the enhanced luminescence due to energy transfer from nucleic acids to terbium(III) (Tb3+). Single-stranded oligonucleotides greatly enhance the Tb3+ emission, but duplex DNA does not. With the use of a DNA hairpin probe complementary to the oligonucleotide of interest, the Tb3+/hairpin probe is applied to detect ultraviolet (UV)-induced DNA damage. The hairpin probe hybridizes only with the undamaged DNA. However, the damaged DNA remains single-stranded and enhances the intrinsic fluorescence of Tb3+, producing a detectable signal directly proportional to the amount of DNA damage. This allows the Tb3+/hairpin probe to be used for sensitive quantification of UV-induced DNA damage. The Tb3+/hairpin probe showed superior selectivity to DNA damage compared to conventional molecular beacons probes (MBs) and its sensitivity is more than 2.5 times higher than MBs with a limit of detection of 4.36 ± 1.2 nM. In addition, this probe is easier to synthesize and more than eight times cheaper than MBs, which makes its use recommended for high-throughput, quantitative analysis of DNA damage

  11. Molecular mechanism of recombinant liver fatty acid binding protein's antioxidant activity

    Yan, Jing; Gong, Yuewen; She, Yi-Min; Wang, Guqi; Roberts, Michael S; Burczynski, Frank J.

    2009-01-01

    Hepatocytes expressing liver fatty acid binding protein (L-FABP) are known to be more resistant to oxidative stress than those devoid of this protein. The mechanism for the observed antioxidant activity is not known. We examined the antioxidant mechanism of a recombinant rat L-FABP in the presence of a hydrophilic (AAPH) or lipophilic (AMVN) free radical generator. Recombinant L-FABP amino acid sequence and its amino acid oxidative products following oxidation were identified by MALDI quadrup...

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

    Brown, Laura D.; Rozance, Paul J.; Thorn, Stephanie R.; FRIEDMAN, Jacob E.; Hay, William W.

    2012-01-01

    Placental insufficiency decreases fetal amino acid uptake from the placenta, plasma insulin concentrations, and protein accretion, thus compromising normal fetal growth trajectory. We tested whether acute supplementation of amino acids or insulin into the fetus with intrauterine growth restriction (IUGR) would increase net fetal protein accretion rates. Late-gestation IUGR and control (CON) fetal sheep received acute, 3-h infusions of amino acids (with euinsulinemia), insulin (with euglycemia...

  13. Fast and Sensitive Method for Determination of Domoic Acid in Mussel Tissue

    Elena Barbaro

    2016-01-01

    Full Text Available Domoic acid (DA, a neurotoxic amino acid produced by diatoms, is the main cause of amnesic shellfish poisoning (ASP. In this work, we propose a very simple and fast analytical method to determine DA in mussel tissue. The method consists of two consecutive extractions and requires no purification steps, due to a reduction of the extraction of the interfering species and the application of very sensitive and selective HILIC-MS/MS method. The procedural method was validated through the estimation of trueness, extract yield, precision, detection, and quantification limits of analytical method. The sample preparation was also evaluated through qualitative and quantitative evaluations of the matrix effect. These evaluations were conducted both on the DA-free matrix spiked with known DA concentration and on the reference certified material (RCM. We developed a very selective LC-MS/MS method with a very low value of method detection limit (9 ng g−1 without cleanup steps.

  14. A simple and highly sensitive assay of perfluorooctanoic acid based on resonance light scattering technique

    Zhang, Fang; Zheng, Yonghong; Liang, Jiaman; Long, Sha; Chen, Xianping; Tan, Kejun

    2016-04-01

    A simple, highly sensitive resonance light scattering (RLS) method for the detection of perfluorooctanoic acid (PFOA) has been developed based on the interaction with crystal violet (CV). It was found that PFOA can form complexes with CV in acid medium resulting in remarkable enhancement of the RLS intensity of the system. And the enhanced RLS intensities are in proportion to the concentration of PFOA in the range of 0.1-25.0 μmol/L (R2 = 0.9998), with a detection limit of 11.0 nmol/L (S/N = 3). In this work, the optimum reaction conditions and the interferences of foreign substances were investigated. The reaction mechanism between CV and PFOA was also studied by the absorption spectrum and scanning electron microscope (SEM). This method is successfully applied to the determination of PFOA in tap water and Jialing river water samples with RSD ≤ 4.04%.

  15. Fast and Sensitive Method for Determination of Domoic Acid in Mussel Tissue.

    Barbaro, Elena; Zangrando, Roberta; Barbante, Carlo; Gambaro, Andrea

    2016-01-01

    Domoic acid (DA), a neurotoxic amino acid produced by diatoms, is the main cause of amnesic shellfish poisoning (ASP). In this work, we propose a very simple and fast analytical method to determine DA in mussel tissue. The method consists of two consecutive extractions and requires no purification steps, due to a reduction of the extraction of the interfering species and the application of very sensitive and selective HILIC-MS/MS method. The procedural method was validated through the estimation of trueness, extract yield, precision, detection, and quantification limits of analytical method. The sample preparation was also evaluated through qualitative and quantitative evaluations of the matrix effect. These evaluations were conducted both on the DA-free matrix spiked with known DA concentration and on the reference certified material (RCM). We developed a very selective LC-MS/MS method with a very low value of method detection limit (9 ng g(-1)) without cleanup steps. PMID:26904720

  16. Influence of Bleaching on Flavor of 34% Whey Protein Concentrate and Residual Benzoic Acid Concentration in Dried Whey Proteins

    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...

  17. Hippocampal kindling alters the concentration of glial fibrillary acidic protein and other marker proteins in rat brain

    Hansen, A; Jørgensen, Ole Steen; Bolwig, T G;

    1990-01-01

    The effect of hippocampal kindling on neuronal and glial marker proteins was studied in the rat by immunochemical methods. In hippocampus, pyriform cortex and amygdala there was an increase in glial fibrillary acidic protein (GFAP), indicating reactive gliosis, and an increase in the glycolytic e...

  18. Morphology-dependent Electrochemical Enhancements of Porous Carbon as Sensitive Determination Platform for Ascorbic Acid, Dopamine and Uric Acid

    Cheng, Qin; Ji, Liudi; Wu, Kangbing; Zhang, Weikang

    2016-02-01

    Using starch as the carbon precursor and different-sized ZnO naoparticles as the hard template, a series of porous carbon materials for electrochemical sensing were prepared. Experiments of scanning electron microscopy, transmission electron microscopy and Nitrogen adsorption-desorption isotherms reveal that the particle size of ZnO has big impacts on the porous morphology and surface area of the resulting carbon materials. Through ultrasonic dispersion of porous carbon and subsequent solvent evaporation, different sensing interfaces were constructed on the surface of glassy carbon electrode (GCE). The electrochemical behaviors of ascorbic acid (AA), dopamine (DA) and uric acid (UA) were studied. On the surface of porous carbon materials, the accumulation efficiency and electron transfer ability of AA, DA and UA are improved, and consequently their oxidation signals enhance greatly. Moreover, the interface enhancement effects of porous carbon are also controlled by the particle size of hard template. The constructed porous carbon interface displays strong signal amplification ability and holds great promise in constructing a sensitive platform for the simultaneous determination of AA, DA and UA.

  19. Susceptibility of antibiotic-resistant and antibiotic-sensitive foodborne pathogens to acid anionic sanitizers.

    Lopes, J A

    1998-10-01

    Acid anionic sanitizers for treatment of fruits and vegetables were prepared using ingredients generally recognized as safe by the U.S. Food and Drug Administration or anionic surfactants and organic acid food additives. They met the regulatory definition as sanitizers by showing bactericidal efficacy of 99.999% in 30 s against Staphylococcus aureus ATCC 6538 and Escherichia coli ATCC 11229. These sanitizers showed a broad spectrum of microbicidal activity against both gram-positive and gram-negative bacteria. Antibiotic-sensitive and resistant strains of Listeria monocytogenes and Salmonella typhimurium were equally susceptible to these sanitizers. The acid anionic sanitizers showed microbicidal efficacy equal to that of hypochlorite against Aeromonas hydrophila, E. coli O157:H7, L. monocytogenes, Pseudomonas aeruginosa, S. typhimurium, and S. aureus. Unlike most other sanitizers, these agents do not covalently react with organic components of food; unlike cationic agents, they do not leave residues. The acid anionic sanitizers are prepared using stable, biodegradable, and nontoxic ingredients. Rapid microbicidal activity and the ease of storage, transportation, and use make these sanitizers an attractive alternative to hypochlorite for sanitizing fruits and vegetables. PMID:9798163

  20. Purification of an N-ethylmaleimide-sensitive protein catalyzing vesicular transport.

    Block, M.R.; Glick, B S; Wilcox, C A; Wieland, F. T.; Rothman, J E

    1988-01-01

    N-Ethylmaleimide (NEM) inhibits protein transport between successive compartments of the Golgi stack in a cell-free system. After inactivation of the Golgi membranes by NEM, transport can be rescued by adding back an appropriately prepared cytosol fraction. This complementation assay has allowed us to purify the NEM-sensitive factor, which we term NSF. The NEM-sensitive factor is a tetramer of 76-kDa subunits, and appears to act catalytically, one tetramer leading to the metabolism of numerou...

  1. Detection of IgG sensitization of red cells with 125I staphylococcal protein A

    Most cases of immune hemolytic anemia are associated with a positive direct antiglobulin test. However, in some cases, the antiglobulin test is not sensitive enough to detect low levels of red-cell bound antibodies. This report describes a method using radiolabelled purified staphylococcal protein A which is capable of detecting IgG sensitization of red cells beyond the threshold of serologic techniques. It is less cumbersome than previously described methods and does not require antibody purification procedures. Its effectiveness was demonstrated for the detection of red-cell alloantibodies and in evaluation of patients with acquired hemolytic anemias associated with a negative direct antiglobulin test

  2. An oleic acid-capped CdSe quantum-dot sensitized solar cell

    In this letter, we report an oleic acid (OA)-capped CdSe quantum-dot sensitized solar cell (QDSSC) with an improved performance. The TiO2/OA-CdSe photoanode in a two-electrode device exhibited a photon-to-current conversion efficiency of 17.5% at 400 nm. At AM1.5G irradiation with 100 mW/cm2 light intensity, the QDSSCs based on OA-capped CdSe showed a power conversion efficiency of about 1%. The function of OA was to increase QD loading, extend the absorption range and possibly suppress the surface recombination.

  3. Acid Hydrolysis of Wheat Gluten Induces Formation of New Epitopes but Does Not Enhance Sensitizing Capacity by the Oral Route: A Study in “Gluten Free” Brown Norway Rats

    Kroghsbo, Stine; Andersen, Nanna Birch; Rasmussen, Tina Frid; Jacobsen, Susanne; Madsen, Charlotte Bernhard

    2014-01-01

    BackgroundAcid hydrolyzed wheat proteins (HWPs) are used in the food and cosmetic industry as emulsifiers. Cases of severe food allergic reactions caused by HWPs have been reported. Recent data suggest that these reactions are caused by HWPs produced by acid hydrolysis.ObjectivesTo examine the sensitizing capacity of gluten proteins per se when altered by acid or enzymatic hydrolysis relative to unmodified gluten in rats naïve to gluten.MethodsHigh IgE-responder Brown Norway (BN) rats bred on...

  4. Radiation damage of proteins in the solid state: changes of amino acid composition in catalase

    Catalase has been irradiated with 100 keV electrons under conditions simulating the hazards of electron microscopic imaging. Amino acid analysis reveals a definite pattern of amino acid destruction which is well correlated with the particular chemical structures of amino acid side chains. This pattern appears to be distinctly different from the sensitivity pattern for monoamino acid systems which is ascribed to intramolecular energy transfer and selective attack of liberated radicals

  5. Phase Sensitive X-Ray Diffraction Imaging Study of Protein Crystals

    Hu, Z. W.

    2003-01-01

    The study of defects and growth of protein crystals is of importance in providing a fundamental understanding of this important category of systems and the rationale for crystallization of better ordered crystals for structural determination and drug design. Yet, as a result of the extremely weak scattering power of x-rays in protein and other biological macromolecular crystals, the extinction lengths for those crystals are extremely large and, roughly speaking, of the order of millimeters on average compared to the scale of micrometers for most small molecular crystals. This has significant implication for x-ray diffraction and imaging study of protein crystals, and presents an interesting challenge to currently available x-ray analytical techniques. We proposed that coherence-based phase sensitive x-ray diffraction imaging could provide a way to augment defect contrast in x-ray diffraction images of weakly diffracting biological macromolecular crystals. I shall examine the principles and ideas behind this approach and compare it to other available x-ray topography and diffraction methods. I shall then present some recent experimental results in two model protein systems-cubic apofemtin and tetragonal lysozyme crystals to demonstrate the capability of the coherence-based imaging method in mapping point defects, dislocations, and the degree of perfection of biological macromolecular crystals with extreme sensitivity. While further work is under way, it is intended to show that the observed new features have yielded important information on protein crystal perfection and nucleation and growth mechanism otherwise unobtainable.

  6. Amino Acid Molecular Units: Building Primary and Secondary Protein Structures

    Aparecido R. Silva

    2008-05-01

    Full Text Available In order to guarantee the learning quality and suitable knowledge  use  about structural biology, it is fundamental to  exist, since the beginning of  students’ formation, the possibility of clear visualization of biomolecule structures. Nevertheless, the didactic books can only bring  schematic  drawings; even more elaborated figures and graphic computation  do not permit the necessary interaction.  The representation of three-dimensional molecular structures with ludic models, built with representative units, have supplied to the students and teachers a successfully experience to  visualize such structures and correlate them to the real molecules.  The design and applicability of the representative units were discussed with researchers and teachers before mould implementation.  In this stage  it  will be presented the  developed  kit  containing the  representative  plastic parts of the main amino acids.  The kit can demonstrate the interaction among the amino acids  functional groups  (represented by colors, shapes,  sizes and  the peptidic bonds between them  facilitating the assembly and visuali zation of the primary and secondary protein structure.  The models were designed for  Ca,  amino,  carboxyl groups  and  hydrogen. The  lateral chains have  well defined models that represent their geometrical shape.  The completed kit set  will be presented in this meeting (patent requested.  In the last phase of the project will be realized  an effective evaluation  of the kit  as a facilitative didactic tool of the teaching/learning process in the Structural Molecular Biology area.

  7. High-sensitive factor I and C-reactive protein based biomarkers for coronary artery disease

    Zhao, Qing; Du, Jian-Shi; Han, Dong-Mei; Ma, Ying

    2014-01-01

    An analysis of high-sensitive factor I and C-reactive proteins as biomarkers for coronary artery disease has been performed from 19 anticipated cohort studies that included 21,567 participants having no information about coronary artery disease. Besides, the clinical implications of statin therapy initiated due to assessment of factor I and C-reactive proteins have also been modeled during studies. The measure of risk discrimination (C-index) was increased (by 0.0101) as per the prognostic mo...

  8. Highly sensitive evanescent wave combination tapered fiber optic fluorosensor for protein detection

    Nardone, Vincent; Kapoor, Rakesh

    2008-02-01

    In this paper we are reporting the development of a highly sensitive evanescent wave combination tapered fiber optic fluorosensor. We have demonstrated detection of 5 pM Bovine Serum Albumin (BSA) protein using these fiber optic sensors. The sensor can be easily adopted for detection of other proteins. Six identical probes were prepared and affinity pure Goat anti-BSA antibodies were immobilized on the probe surface. We could detect signal from all the probes kept in 5 pM to 1 nM BSA solution while no signal was detected from the probes kept in 20 nM labeled ESA solution.

  9. Analysis of the protein-protein interactions between the human acidic ribosomal P-proteins: evaluation by the two hybrid system

    Tchórzewski, M; Boldyreff, B; Issinger, O;

    2000-01-01

    function of these proteins, we are the first to have precisely analyzed mutual interactions among human P-proteins, employing the two hybrid system. The human acidic ribosomal P-proteins, (P1 or P2,) were fused to the GAL4 binding domain (BD) as well as the activation domain (AD), and analyzed in yeast...... forms the 60 S ribosomal stalk: P0-(P1/P2)(2). Additionally, mutual interactions among human and yeast P-proteins were analyzed. Heterodimer formation could be observed between human P2 and yeast P1 proteins.......The surface acidic ribosomal proteins (P-proteins), together with ribosomal core protein P0 form a multimeric lateral protuberance on the 60 S ribosomal subunit. This structure, also called stalk, is important for efficient translational activity of the ribosome. In order to shed more light on the...

  10. Optimizing scoring function of protein-nucleic acid interactions with both affinity and specificity.

    Zhiqiang Yan

    Full Text Available Protein-nucleic acid (protein-DNA and protein-RNA recognition is fundamental to the regulation of gene expression. Determination of the structures of the protein-nucleic acid recognition and insight into their interactions at molecular level are vital to understanding the regulation function. Recently, quantitative computational approach has been becoming an alternative of experimental technique for predicting the structures and interactions of biomolecular recognition. However, the progress of protein-nucleic acid structure prediction, especially protein-RNA, is far behind that of the protein-ligand and protein-protein structure predictions due to the lack of reliable and accurate scoring function for quantifying the protein-nucleic acid interactions. In this work, we developed an accurate scoring function (named as SPA-PN, SPecificity and Affinity of the Protein-Nucleic acid interactions for protein-nucleic acid interactions by incorporating both the specificity and affinity into the optimization strategy. Specificity and affinity are two requirements of highly efficient and specific biomolecular recognition. Previous quantitative descriptions of the biomolecular interactions considered the affinity, but often ignored the specificity owing to the challenge of specificity quantification. We applied our concept of intrinsic specificity to connect the conventional specificity, which circumvents the challenge of specificity quantification. In addition to the affinity optimization, we incorporated the quantified intrinsic specificity into the optimization strategy of SPA-PN. The testing results and comparisons with other scoring functions validated that SPA-PN performs well on both the prediction of binding affinity and identification of native conformation. In terms of its performance, SPA-PN can be widely used to predict the protein-nucleic acid structures and quantify their interactions.

  11. High Protein Intake Improves Insulin Sensitivity but Exacerbates Bone Resorption in Immobility (WISE Study)

    Heer, Martina; Smith, Scott M.; Frings-Meuthen, Petra; Zwart, Sara R.; Baecker, Natalie

    2012-01-01

    Inactivity, like bed rest (BR), causes insulin resistance (IR) and bone loss even in healthy subjects. High protein intake seems to mitigate this IR but might exacerbate bone loss. We hypothesized that high protein intake (animal:vegetable protein ratio: 60:40), isocaloric, compared to the control group plus high potassium intake would prevent IR without affecting bone turnover. After a 20-day ambulatory adaptation to controlled confinement and diet, 16 women participated in a 60-day, 6 deg head-down-tilt BR and were assigned randomly to one of the two groups. Control subjects (CON, n=8) received 1g/kg body mass/d dietary protein. Nutrition subjects (NUT, n=8) received 1.45g/kg body mass/d dietary protein plus 7.2g branched chain amino acids per day during BR. All subjects received 1670 kcal/d. Bed rest decreased glucose disposal by 35% (pprotein intake prevented insulin resistance, but exacerbated bed rest induced increase in bone resorption markers C-telopeptide (> 30%) and Ntelopeptide (>20%) (both: pprotein intake. We conclude from these results that high protein intake might positively affect glucose tolerance, but might also foster bone loss. Further long-duration studies are mandatory before high protein intake for diabetic patients, who have an increased fracture risk, might be recommended.

  12. G-protein activation, identification and immunolocalization in pheromone-sensitive sensilla trichodea of moths.

    Laue, M; Maida, R; Redkozubov, A

    1997-04-01

    Electrophysiological in situ recordings from pheromone-sensitive sensilla trichodea of Bombyx mori males with a recording pipette which contained G-protein-activating fluoride, showed receptor cell activity similar to that evoked by pheromone stimulation. This suggests that G-proteins might be physiologically active in olfactory sensilla of insects in situ. Biochemical experiments using specific antibodies revealed the presence of G-protein, belonging to the Gq family, in antennal preparations. Similar G-protein was identified in sensory hair preparations of Antheraea pernyi which contained only cuticle, sensillum lymph and dendritic material. Moreover, the absence of this G-protein in pure sensillum lymph preparations indicates its association with the receptive dendrites. This particular association could be shown by immunolabelling studies at the ultrastructural level. Strong specific labelling of membranes of receptor-cell dendrites was found in all types of olfactory sensilla present on the antenna of the silkmoths. Additional specific labelling of apical membranes of auxiliary cells, epidermal cells and membranes forming the axon/glia interface demonstrated that this G-protein is not restricted to the sensory dendrites and that other signal-transduction pathways could be present at these membranes. In summary, the experiments imply a participation of G-protein of the Gq family in signal transduction of olfactory receptor cells in moths. PMID:9042782

  13. Down-Regulation of AKT Signalling by Ursolic Acid Induces Intrinsic Apoptosis and Sensitization to Doxorubicin in Soft Tissue Sarcoma

    Villar, Victor Hugo; Vögler, Oliver; Barceló, Francisca; Martín-Broto, Javier; Martínez-Serra, Jordi; Ruiz-Gutiérrez, Valentina; Alemany, Regina

    2016-01-01

    Several important biological activities have been attributed to the pentacyclic triterpene ursolic acid (UA), being its antitumoral effect extensively studied in human adenocarcinomas. In this work, we focused on the efficacy and molecular mechanisms involved in the antitumoral effects of UA, as single agent or combined with doxorubicin (DXR), in human soft tissue sarcoma cells. UA (5–50 μM) strongly inhibited (up to 80%) the viability of STS cells at 24 h and its proliferation in soft agar, with higher concentrations increasing apoptotic death up to 30%. UA treatment (6–9 h) strongly blocked the survival AKT/GSK3β/β-catenin signalling pathway, which led to a concomitant reduction of the anti-apoptotic proteins c-Myc and p21, altogether resulting in the activation of intrinsic apoptosis. Interestingly, UA at low concentrations (10–15 μM) enhanced the antitumoral effects of DXR by up to 2-fold, while in parallel inhibiting DXR-induced AKT activation and p21 expression, two proteins implicated in antitumoral drug resistance and cell survival. In conclusion, UA is able to induce intrinsic apoptosis in human STS cells and also to sensitize these cells to DXR by blocking the AKT signalling pathway. Therefore, UA may have beneficial effects, if used as nutraceutical adjuvant during standard chemotherapy treatment of STS. PMID:27219337

  14. Down-Regulation of AKT Signalling by Ursolic Acid Induces Intrinsic Apoptosis and Sensitization to Doxorubicin in Soft Tissue Sarcoma.

    Victor Hugo Villar

    Full Text Available Several important biological activities have been attributed to the pentacyclic triterpene ursolic acid (UA, being its antitumoral effect extensively studied in human adenocarcinomas. In this work, we focused on the efficacy and molecular mechanisms involved in the antitumoral effects of UA, as single agent or combined with doxorubicin (DXR, in human soft tissue sarcoma cells. UA (5-50 μM strongly inhibited (up to 80% the viability of STS cells at 24 h and its proliferation in soft agar, with higher concentrations increasing apoptotic death up to 30%. UA treatment (6-9 h strongly blocked the survival AKT/GSK3β/β-catenin signalling pathway, which led to a concomitant reduction of the anti-apoptotic proteins c-Myc and p21, altogether resulting in the activation of intrinsic apoptosis. Interestingly, UA at low concentrations (10-15 μM enhanced the antitumoral effects of DXR by up to 2-fold, while in parallel inhibiting DXR-induced AKT activation and p21 expression, two proteins implicated in antitumoral drug resistance and cell survival. In conclusion, UA is able to induce intrinsic apoptosis in human STS cells and also to sensitize these cells to DXR by blocking the AKT signalling pathway. Therefore, UA may have beneficial effects, if used as nutraceutical adjuvant during standard chemotherapy treatment of STS.

  15. Highly sensitive detection of S-nitrosylated proteins by capillary gel electrophoresis with laser induced fluorescence.

    Wang, Siyang; Circu, Magdalena L; Zhou, Hu; Figeys, Daniel; Aw, Tak Y; Feng, June

    2011-09-23

    S-nitrosylated proteins are biomarkers of oxidative damage in aging and Alzheimer's disease (AD). Here, we report a new method for detecting and quantifying nitrosylated proteins by capillary gel electrophoresis with laser induced fluorescence detection (CGE-LIF). Dylight 488 maleimide was used to specifically label thiol group (SH) after switching the S-nitrosothiol (S-NO) to SH in cysteine using the "fluorescence switch" assay. In vitro nitrosylation model-BSA subjected to S-nitrosoglutathione (GSNO) optimized the labeling reactions and characterized the response of the LIF detector. The method proves to be highly sensitive, detecting 1.3 picomolar (pM) concentration of nitrosothiols in nanograms of proteins, which is the lowest limit of detection of nitrosothiols reported to date. We further demonstrated the direct application of this method in monitoring protein nitrosylation damage in MQ mediated human colon adenocarcinoma cells. The nitrosothiol amounts in MQ treated and untreated cells are 14.8±0.2 and 10.4±0.5 pmol/mg of proteins, respectively. We also depicted nitrosylated protein electrophoretic profiles of brain cerebrum of 5-month-old AD transgenic (Tg) mice model. In Tg mice brain, 15.5±0.4 pmol of nitrosothiols/mg of proteins was quantified while wild type contained 11.7±0.3 pmol/mg proteins. The methodology is validated to quantify low levels of S-nitrosylated protein in complex protein mixtures from both physiological and pathological conditions. PMID:21820121

  16. Localization and quantification of carbon-centered radicals on any amino acid of a protein

    A general strategy to localize and quantify carbon-centered radicals within proteins is described. The methodology was first exemplified on amino acids and then on a peptide. This method is applicable to any protein system regardless of size, and the site of hydrogen abstraction by OH radical on all residues within proteins is easily and accurately detected. (authors)

  17. The effect of amino acid repertoire on the protein structure universe

    Vymětal, Jiří; Tretyachenko, V.; Hlouchová, Klára; Vondrášek, Jiří

    Praha: Ústav organické chemie a biochemie AV ČR, v. v. i, 2014. s. 76. ISBN 978-80-86241-51-7. [Prague Protein Spring Meeting 2014: Proteins and their interactions /3./. 09.05.2014-11.05.2014, Praha] Institutional support: RVO:61388963 Keywords : amino acids * protein structures Subject RIV: CE - Biochemistry

  18. High Sensitivity Bacillus thuringiensis Cry1Ac Protein Detections Using Fluorescein Diacetate Nanoparticles.

    Liu, Cui; Zhou, Zhen; Zou, Linling; Cao, Yuan-Cheng; Liu, Jun'An; Lin, Yongjun

    2016-03-01

    A highly sensitive transgenic protein analysis method was proposed here based on fluorescein diacetate (FDA). First, FDA was prepared by the ball mill to harvest the nano-sized organic particles. Further examines showed that the FDA size can be controlled by the speed of centrifugation which can obtain FDA in well-distributed size. Cy3 antibody immobilization tests showed that the proteins can attach onto the FDA particles while keep bioactivities. FDA and Cry1Ac antibody immunoassay tests showed that when the FDA particle was in 150 nm, the linear range was 0.01 ng/L-30 μg/mL. And it has the lower detection limitation of 0.01 ng/L, which is 100 times more sensitive than the ELISA methods. These results indicate that the FDA related immunoassays are the promising approach in the transgenic analysis. PMID:26642804

  19. Improved method for simultaneous isolation of proteins and nucleic acids.

    Chey, Soroth; Claus, Claudia; Liebert, Uwe Gerd

    2011-04-01

    Guanidinium thiocyanate-phenol-chloroform extraction (GTPC extraction) is widely used in molecular biology for isolating DNA, RNA, and proteins. Protein isolation by commercially available GTPC solutions is time consuming and the resulting pellets are only incompletely soluble. In this study ethanol-bromochloropropane-water was used for precipitation of proteins from the phenol-ethanol phase after GTPC extraction of RNA and DNA. The precipitated proteins can be readily dissolved in 4% SDS for subsequent analysis. This technique allows a fast (30min) and efficient (with a protein recovery of up to 95%) extraction of proteins for the study of transcriptional and posttranscriptional events from the same sample. PMID:21094121

  20. Expression of liver fatty acid binding protein in hepatocellular carcinoma.

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

    2016-04-01

    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

  1. Algal Photobiology: A Rich Source of Unusual Light Sensitive Proteins for Synthetic Biology and Optogenetics.

    Kianianmomeni, Arash; Hallmann, Armin

    2016-01-01

    The light absorption system in eukaryotic (micro)algae includes highly sensitive photoreceptors, which change their conformation in response to different light qualities on a subsecond time scale and induce physiological and behavioral responses. Some of the light sensitive modules are already in use to engineer and design photoswitchable tools for control of cellular and physiological activities in living organisms with various degrees of complexity. Thus, identification of new light sensitive modules will not only extend the source material for the generation of optogenetic tools but also foster the development of new light-based strategies in cell signaling research. Apart from searching for new proteins with suitable light-sensitive modules, smaller variants of existing light-sensitive modules would be helpful to simplify the construction of hybrid genes and facilitate the generation of mutated and chimerized modules. Advances in genome and transcriptome sequencing as well as functional analysis of photoreceptors and their interaction partners will help to discover new light sensitive modules. PMID:26965114

  2. Microwave-assisted Weak Acid Hydrolysis of Proteins

    Miyeong Seo

    2012-06-01

    Full Text Available Myoglobin was hydrolyzed by microwave-assisted weak acid hydrolysis with 2% formic acid at 37 oC, 50 oC, and100 oC for 1 h. The most effective hydrolysis was observed at 100 oC. Hydrolysis products were investigated using matrixassistedlaser desorption/ionization time-of-flight mass spectrometry. Most cleavages predominantly occurred at the C-termini ofaspartyl residues. For comparison, weak acid hydrolysis was also performed in boiling water for 20, 40, 60, and 120 min. A 60-min weak acid hydrolysis in boiling water yielded similar results as a 60-min microwave-assisted weak acid hydrolysis at100 oC. These results strongly suggest that microwave irradiation has no notable enhancement effect on acid hydrolysis of proteinsand that temperature is the major factor that determines the effectiveness of weak acid hydrolysis.

  3. Influence of amino acids, dietary protein, and physical activity on muscle mass development in humans

    Dideriksen, Kasper; Reitelseder, Søren; Holm, Lars

    2013-01-01

    of muscle protein synthesis (response). In addition to the protein amount, the protein digestibility and, hence, the availability of its constituent amino acids is decisive for the response. In this regard, recent studies have provided in-depth knowledge about the time-course of the muscle protein...... synthetic response dependent on the characteristics of the protein ingested. The effect of protein intake on muscle protein accretion can further be stimulated by prior exercise training. In the ageing population, physical training may counteract the development of "anabolic resistance" and restore the...

  4. No relationship between lung function and high-sensitive C-reactive protein in adolescence

    Nybo, Mads; Hansen, Henrik Steen; Siersted, Hans Christian; Rasmussen, Finn

    2010-01-01

      Several studies on adults have indicated that lower spirometric lung function may be associated with increased systemic inflammation, but no studies have investigated if this association is already present in adolescence. Objective:  We explored the temporal relationship between changes in lung...... function and concentrations of plasma C-reactive protein (CRP) in a population-based cohort study at ages 14 and 20 years using a high-sensitivity CRP assay....

  5. The Capsule Sensitizes Streptococcus pneumoniae to α-Defensins Human Neutrophil Proteins 1 to 3▿

    Beiter, Katharina; Wartha, Florian; Hurwitz, Robert; Normark, Staffan; Zychlinsky, Arturo; Henriques-Normark, Birgitta

    2008-01-01

    Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. Its polysaccharide capsule causes resistance to phagocytosis and interferes with the innate immune system's ability to clear infections at an early stage. Nevertheless, we found that encapsulated pneumococci are sensitive to killing by a human neutrophil granule extract. We fractionated the extract by high-performance liquid chromatography and identified α-defensins by mass spectrometry as the proteins responsible...

  6. High temperature, drought and their interaction induced protein alterations in sensitive and tolerant wheat varieties

    Vikender Kaur, Reena Mahla And R.K.Behl

    2014-01-01

    Two contrasting wheat (Triticum aestivum L.) cultivars WH730 (high temperature tolerant) and UP2565 (high temperature sensitive) were tested for differential response to combined and individually applied high temperature (HT) and drought (D) stress at seedling stage for peptide profile. Initial profile of the stress induced peptides was outlined via SDS electrophoresis of leaf extracts. Electrophoretic pattern of proteins revealed expression of new bands as well as disappearance of certain ot...

  7. Retinoic acid disrupts the Golgi apparatus and increases the cytosolic routing of specific protein toxins

    1994-01-01

    All-trans retinoic acid can specifically increase receptor mediated intoxication of ricin A chain immunotoxins more than 10,000 times, whereas fluid phase endocytosis of ricin A chain alone or ricin A chain immunotoxins was not influenced by retinoic acid. The immunotoxin activation by retinoic acid does not require RNA or protein synthesis and is not a consequence of increased receptor binding of the immunotoxin. Vitamin D3 and thyroid hormone T3, that activate retinoic acid receptor (RAR) c...

  8. Cinnamic Acid and Its Derivatives Inhibit Fructose-Mediated Protein Glycation

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

    2012-01-01

    Cinnamic acid and its derivatives have shown a variety of pharmacologic properties. However, little is known about the antiglycation properties of cinnamic acid and its derivatives. The present study sought to characterize the protein glycation inhibitory activity of cinnamic acid and its derivatives in a bovine serum albumin (BSA)/fructose system. The results demonstrated that cinnamic acid and its derivatives significantly inhibited the formation of advanced glycation end products (AGEs) by...

  9. In Vitro Binding Capacity of Bile Acids by Defatted Corn Protein Hydrolysate

    Pierre Claver Irakoze

    2011-02-01

    Full Text Available Defatted corn protein was digested using five different proteases, Alcalase, Trypsin, Neutrase, Protamex and Flavourzyme, in order to produce bile acid binding peptides. Bile acid binding capacity was analyzed in vitro using peptides from different proteases of defatted corn hydrolysate. Some crystalline bile acids like sodium glycocholate, sodium cholate and sodium deoxycholate were individually tested using HPLC to see which enzymes can release more peptides with high bile acid binding capacity. Peptides from Flavourzyme defatted corn hydrolysate exhibited significantly (p

  10. Membrane-Inspired Acidically Stable Dye-Sensitized Photocathode for Solar Fuel Production.

    Click, Kevin A; Beauchamp, Damian R; Huang, Zhongjie; Chen, Weilin; Wu, Yiying

    2016-02-01

    Tandem dye-sensitized photoelectrochemical cells (DSPECs) for water splitting are a promising method for sustainable energy conversion but so far have been limited by their lack of aqueous stability and photocurrent mismatch between the cathode and anode. In nature, membrane-enabled subcellular compartmentation is a general approach to control local chemical environments in the cell. The hydrophobic tails of the lipid make the bilayer impermeable to ions and hydrophilic molecules. Herein we report the use of an organic donor-acceptor dye that prevents both dye desorption and semiconductor degradation by mimicking the hydrophobic/hydrophilic properties of lipid bilayer membranes. The dual-functional photosensitizer (denoted as BH4) allows for efficient light harvesting while also protecting the semiconductor surface from protons and water via its hydrophobic π linker. The protection afforded by this membrane-mimicking dye gives this system excellent stability in extremely acidic (pH 0) conditions. The acidic stability also allows for the use of cubane molybdenum-sulfide cluster as the hydrogen evolution reaction (HER) catalyst. This system produces a proton-reducing current of 183 ± 36 μA/cm(2) (0 V vs NHE with 300 W Xe lamp) for an unprecedented 16 h with no degradation. These results introduce a method for developing high-current, low-pH DSPECs and are a significant move toward practical dye-sensitized solar fuel production. PMID:26744766

  11. Enhanced Sensitivity for Hydrogen Peroxide Detection: Polydiacetylene Vesicles with Phenylboronic Acid Head Group.

    Jia, Chen; Tang, Jie; Lu, Shengguo; Han, Yuwang; Huang, He

    2016-01-01

    It was recently reported that, besides UV irradiated polymerization, polymerization of diacetylene compounds could also been initiated by radicals generated from enzyme catalyzed hydrogen peroxide (H2O2) decomposition. A new optical sensing method for H2O2 was proposed based on this phenomenon. However, the sensitivity of this method is relatively lower than existed ones. In the present work, phenylboronic acid (PBA) functionalized 10, 12-pentacosadiynoic acid (PDA-PBA) was synthesized and its vesicles were formed successfully as colorimetric sensor for H2O2 detection. It was found that color change during the polymerization of vesicles composed of the PBA modified monomer is much stronger than that of the non-modified one. The response of PDA-PBA vesicles to H2O2 is 16 times more sensitive than that of the PDA. The absorption of PDA-PBA at 650 nm is linearly related to the concentration of H2O2 and a detection limit of ~5 μM could be achieved. PMID:26511954

  12. Ranking of Reactions Based on Sensitivity of Protein Noise Depends on the Choice of Noise Measure.

    Gokhale, Sucheta; Gadgil, Chetan

    2015-01-01

    Gene expression is a stochastic process. Identification of the step maximally affecting noise in the protein level is an important aspect of investigation of gene product distribution. There are numerous experimental and theoretical studies that seek to identify this important step. However, these studies have used two different measures of noise, viz. coefficient of variation and Fano factor, and have compared different processes leading to contradictory observations regarding the important step. In this study, we performed systematic global and local sensitivity analysis on two models of gene expression to investigate relative contribution of reaction rate parameters to steady state noise in the protein level using both the measures of noise. We analytically and computationally showed that the ranking of parameters based on the sensitivity of the noise to variation in a given parameter is a strong function of the choice of the noise measure. If the Fano factor is used as the noise measure, translation is the important step whereas for coefficient of variation, transcription is the important step. We derived an analytical expression for local sensitivity and used it to explain the distinct contributions of each reaction parameter to the two measures of noise. We extended the analysis to a generic linear catalysis reaction system and observed that the reaction network topology was an important factor influencing the local sensitivity of the two measures of noise. Our study suggested that, for the analysis of contributions of reactions to the noise, consideration of both the measures of noise is important. PMID:26625133

  13. Ranking of Reactions Based on Sensitivity of Protein Noise Depends on the Choice of Noise Measure.

    Sucheta Gokhale

    Full Text Available Gene expression is a stochastic process. Identification of the step maximally affecting noise in the protein level is an important aspect of investigation of gene product distribution. There are numerous experimental and theoretical studies that seek to identify this important step. However, these studies have used two different measures of noise, viz. coefficient of variation and Fano factor, and have compared different processes leading to contradictory observations regarding the important step. In this study, we performed systematic global and local sensitivity analysis on two models of gene expression to investigate relative contribution of reaction rate parameters to steady state noise in the protein level using both the measures of noise. We analytically and computationally showed that the ranking of parameters based on the sensitivity of the noise to variation in a given parameter is a strong function of the choice of the noise measure. If the Fano factor is used as the noise measure, translation is the important step whereas for coefficient of variation, transcription is the important step. We derived an analytical expression for local sensitivity and used it to explain the distinct contributions of each reaction parameter to the two measures of noise. We extended the analysis to a generic linear catalysis reaction system and observed that the reaction network topology was an important factor influencing the local sensitivity of the two measures of noise. Our study suggested that, for the analysis of contributions of reactions to the noise, consideration of both the measures of noise is important.

  14. Membrane thickness sensitivity of prestin orthologs: the evolution of a piezoelectric protein.

    Izumi, Chisako; Bird, Jonathan E; Iwasa, Kuni H

    2011-06-01

    How proteins evolve new functionality is an important question in biology; prestin (SLC26A5) is a case in point. Prestin drives outer hair cell somatic motility and amplifies mechanical vibrations in the mammalian cochlea. The motility of mammalian prestin is analogous to piezoelectricity, in which charge transfer is coupled to changes in membrane area occupied by the protein. Intriguingly, nonmammalian prestin orthologs function as anion exchangers but are apparently nonmotile. We previously found that mammalian prestin is sensitive to membrane thickness, suggesting that prestin's extended conformation has a thinner hydrophobic height in the lipid bilayer. Because prestin-based motility is a mammalian specialization, we initially hypothesized that nonmotile prestin orthologs, while functioning as anion transporters, should be much less sensitive to membrane thickness. We found the exact opposite to be true. Chicken prestin was the most sensitive to thickness changes, displaying the largest shift in voltage dependence. Platypus prestin displayed an intermediate response to membrane thickness and gerbil prestin was the least sensitive. To explain these observations, we present a theory where force production, rather than displacement, was selected for the evolution of prestin as a piezoelectric membrane motor. PMID:21641306

  15. pHuji, a pH-sensitive red fluorescent protein for imaging of exo- and endocytosis

    Shen, Yi; Rosendale, Morgane; Robert E Campbell; Perrais, David

    2014-01-01

    Fluorescent proteins with pH-sensitive fluorescence are valuable tools for the imaging of exocytosis and endocytosis. The Aequorea green fluorescent protein mutant superecliptic pHluorin (SEP) is particularly well suited to these applications. Here we describe pHuji, a red fluorescent protein with a pH sensitivity that approaches that of SEP, making it amenable for detection of single exocytosis and endocytosis events. To demonstrate the utility of the pHuji plus SEP pair, we perform simultan...

  16. Database of amino acid-nucleotide contacts in the DNA complexes with homeodomain family proteins

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

  17. Highly Sensitive Functionalized Conducting Copolypyrrole Film for DNA Sensing and Protein-resistant%Highly Sensitive Functionalized Conducting Copolypyrrole Film for DNA Sensing and Protein-resistant

    Zhang, Zhihong; Li, Guijuan; Yan, Fufeng; Zhang, Zhonghou; Fang, Shaoming

    2012-01-01

    In order to exploit the applications ofpolypyrrole (PPy) derivatives in biosensors and bioelectronics, the different immobilization mechanisms of biomolecules onto differently functionalized conducting PPy films are investigated. Pyrrole and pyrrole derivatives with carboxyl and amino groups were copolymerized with ω-(N-pyrrolyl)-octylthiol self-assembled on Au surface by the method of the chemical polymerization to form a layer of the copolymer film, i.e., poly[pyrrole-co-(N-pyrrolyl)-caproic acid] (poly(Py-co-PyCA)) and poly[pyrrole-co-(N-pyrrolyl)-hexylamine] (poly(Py-co-PyHA)), in which the carboxyl groups in poly(Py-co-PyCA) were activated to the ester groups. Based on the structure characteristics, the immobilization/hybridization of DNA molecules on PPy, poly(Py-co-PyCA) and poly(Py-co-PyHA) were surveyed by cyclic voltammograms measurements. For differently functionalized copolymers, the immobilization mechanisms of DNA are various. Besides the electrochemical properties of the composite electrodes of PPy and its copolymers being detected before and after bovine serum albumin (BSA) adsorption, the kinetic process of protein binding was determined by surface plasmon resonance of spectroscopy. Since few BSA molecules could anchor onto the PPy and its copolymers surfaces, it suggests this kind of conducting polymers can be applied as the protein-resistant material.

  18. Effects of Dietary n-3 Fatty Acids on Hepatic and Peripheral Insulin Sensitivity in Insulin-Resistant Humans

    Lalia, Antigoni Z; Johnson, Matthew L; Jensen, Michael D.; Hames, Kazanna C.; Port, John D.; Lanza, Ian R.

    2015-01-01

    OBJECTIVE Dietary n-3 polyunsaturated fatty acids, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), prevent insulin resistance and stimulate mitochondrial biogenesis in rodents, but the findings of translational studies in humans are thus far ambiguous. The aim of this study was to evaluate the influence of EPA and DHA on insulin sensitivity, insulin secretion, and muscle mitochondrial function in insulin-resistant, nondiabetic humans using a robust study design and gold-...

  19. Phosphinic acid functionalized carbon nanotubes for sensitive and selective sensing of chromium(VI)

    Deep, Akash, E-mail: dr.akashdeep@csio.res.in; Sharma, Amit L.; Tuteja, Satish K.; Paul, A.K.

    2014-08-15

    Highlights: • SWCNTs have been conjugated with bis(2,4,4-trimethylpentyl) phosphinic acid (PA/d). • SWCNT-PA/d adduct is demonstrated for electrochemical sensing of Cr(VI). • Linear response is obtained for 0.01–10 ppb Cr(VI). • Sensitivity and the limit of detection are 35 ± 4 nA/ppb and 0.01 ppb, respectively. • Proposed sensing of Cr(VI) is selective with respect to many other metals. - Abstract: Single-walled carbon nanotubes (SWCNTs) have been functionalized with a phosphinic acid derivative ‘bis(2,4,4-trimethylpentyl) phosphinic acid’ (PA/d). It has been achieved by treating the chlorinated SWCNTs with PA/d at 80 °C. Successful functionalization and different nanomaterial properties have been investigated by UV–vis–NIR, FTIR, Raman spectroscopy, AFM and FE-SEM. PA/d conjugated SWCNTs (CNT–PA) are dispersible in some common organic solvents, e.g. CH{sub 2}Cl{sub 2}, DMF, CHCl{sub 3}, and THF. The ‘CNT–PA’ complex was spin-casted on boron doped silicon wafer. Thus fabricated sensing electrode is demonstrated for sensitive and selective electrochemical sensing of chromium(VI) ions. A linear response is obtained over a wide range of Cr(VI) concentration (0.01–10 ppb). The sensor's sensitivity and the limit of detection are observed to be 35 ± 4 nA/ppb and 0.01 ppb, respectively. The practical utility of the proposed sensor is demonstrated by determining the Cr(VI) concentration in an industrial effluent sample and an underground water sample.

  20. Increasing the sensitivity of reverse phase protein arrays by antibody-mediated signal amplification

    Brase Jan C

    2010-06-01

    Full Text Available Abstract Background Reverse phase protein arrays (RPPA emerged as a useful experimental platform to analyze biological samples in a high-throughput format. Different signal detection methods have been described to generate a quantitative readout on RPPA including the use of fluorescently labeled antibodies. Increasing the sensitivity of RPPA approaches is important since many signaling proteins or posttranslational modifications are present at a low level. Results A new antibody-mediated signal amplification (AMSA strategy relying on sequential incubation steps with fluorescently-labeled secondary antibodies reactive against each other is introduced here. The signal quantification is performed in the near-infrared range. The RPPA-based analysis of 14 endogenous proteins in seven different cell lines demonstrated a strong correlation (r = 0.89 between AMSA and standard NIR detection. Probing serial dilutions of human cancer cell lines with different primary antibodies demonstrated that the new amplification approach improved the limit of detection especially for low abundant target proteins. Conclusions Antibody-mediated signal amplification is a convenient and cost-effective approach for the robust and specific quantification of low abundant proteins on RPPAs. Contrasting other amplification approaches it allows target protein detection over a large linear range.

  1. Mechanistic insight into gramicidin-based detection of protein-ligand interactions via sensitized photoinactivation

    Rokitskaya, Tatyana I; Kotova, Elena A; Antonenko, Yuri N [Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119991 (Russian Federation); Macrae, Michael X; Blake, Steven; Yang, Jerry [Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, MC 0358, La Jolla, CA 92093-0358 (United States); Egorova, Natalya S, E-mail: jerryyang@ucsd.ed, E-mail: antonen@genebee.msu.s [Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117871 Moscow (Russian Federation)

    2010-11-17

    Among the many challenges for the development of ion channel-based sensors is the poor understanding of how to engineer modified transmembrane pores with tailored functionality that can respond to external stimuli. Here, we use the method of sensitized photoinactivation of gramicidin A (gA) channels in planar bilayer lipid membranes to help elucidate the underlying mechanistic details for changes in macroscopic transmembrane ionic current observed upon interaction of C-terminally attached gA ligands with specific proteins in solution. Three different systems were studied: (i) carbonic anhydrase (CA) and gA-sulfonamide, (ii) PSD-95 protein (belonging to the 'PDZ domain-containing protein') and a gA analog carrying the KGGHRRSARYLESSV peptide sequence at the C-terminus, and (iii) an anti-biotin antibody and gA-biotin. The results challenge a previously proposed mechanistic hypothesis suggesting that protein-induced current suppression is due to steric blockage of the ion passage through gA channels, while they reveal new insight for consideration in alternative mechanistic models. Additionally, we demonstrate that the length of a linker between the ligand and the gA channel may be less important for gramicidin-based detection of monovalent compared to multivalent protein-ligand interactions. These studies collectively shed new light on the mechanism of protein-induced current alterations in bilayer recordings of gA derivatives, which may be important in the design of new gramicidin-based sensors.

  2. Mechanistic insight into gramicidin-based detection of protein-ligand interactions via sensitized photoinactivation

    Rokitskaya, Tatyana I.; Macrae, Michael X.; Blake, Steven; Egorova, Natalya S.; Kotova, Elena A.; Yang, Jerry; Antonenko, Yuri N.

    2010-11-01

    Among the many challenges for the development of ion channel-based sensors is the poor understanding of how to engineer modified transmembrane pores with tailored functionality that can respond to external stimuli. Here, we use the method of sensitized photoinactivation of gramicidin A (gA) channels in planar bilayer lipid membranes to help elucidate the underlying mechanistic details for changes in macroscopic transmembrane ionic current observed upon interaction of C-terminally attached gA ligands with specific proteins in solution. Three different systems were studied: (i) carbonic anhydrase (CA) and gA-sulfonamide, (ii) PSD-95 protein (belonging to the 'PDZ domain-containing protein') and a gA analog carrying the KGGHRRSARYLESSV peptide sequence at the C-terminus, and (iii) an anti-biotin antibody and gA-biotin. The results challenge a previously proposed mechanistic hypothesis suggesting that protein-induced current suppression is due to steric blockage of the ion passage through gA channels, while they reveal new insight for consideration in alternative mechanistic models. Additionally, we demonstrate that the length of a linker between the ligand and the gA channel may be less important for gramicidin-based detection of monovalent compared to multivalent protein-ligand interactions. These studies collectively shed new light on the mechanism of protein-induced current alterations in bilayer recordings of gA derivatives, which may be important in the design of new gramicidin-based sensors.

  3. Sensitive determination of D-amino acids in mammals and the effect of D-amino-acid oxidase activity on their amounts.

    Hamase, Kenji; Konno, Ryuichi; Morikawa, Akiko; Zaitsu, Kiyoshi

    2005-09-01

    The determination of small amounts of D-amino acids in mammalian tissues is still a challenging theme in the separation sciences. In this review, various gas-chromatographic and high-performance liquid chromatographic methods are discussed including highly selective and sensitive column-switching procedures. Based on these methods, the distributions of D-aspartic acid, D-serine, D-alanine, D-leucine and D-proline have been clarified in the mouse brain. As the regulation mechanisms of D-amino acid amounts in mammals, we focused on the D-amino-acid oxidase, which catalyzes the degradation of D-amino acids. Using the mutant mouse strain lacking D-amino-acid oxidase activity, the effects of the enzymatic activity on the amounts and distributions of various D-amino acids have been investigated. PMID:16141519

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

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

    2014-02-01

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

  5. High-sensitive C-reactive protein is associated with reduced lung function in young adults

    Rasmussen, F; Mikkelsen, D; Hancox, R J; Lambrechtsen, J; Nybo, M; Hansen, H S; Siersted, H C

    2009-01-01

    Systemic inflammation has been associated with reduced lung function. However, data on the interrelationships between lung function and inflammation are sparse, and it is not clear if low-grade inflammation leads to reduced lung function. Associations between high-sensitive C-reactive protein (CRP......, higher levels of CRP at age 20 yrs were associated with a greater reduction in both FEV(1) and forced vital capacity between ages 20 and 29 yrs. The findings show that higher levels of C-reactive protein in young adults are associated with subsequent decline in lung function, suggesting that low...... average decline was 6.2 mL.yr(-1) in the highest CRP quintile versus an increase of 1.8 mL.yr(-1) in the lowest CRP quintile. In a multiple regression analysis adjusted for sex, body mass index, cardiorespiratory fitness, smoking, asthma, airway hyperresponsiveness and serum eosinophil cationic protein...

  6. Mechanistic insight provided by glutaredoxin within a fusion to redox-sensitive yellow fluorescent protein

    Björnberg, Olof; Østergaard, Henrik; Winther, Jakob R

    2006-01-01

    Redox-sensitive yellow fluorescent protein (rxYFP) contains a dithiol disulfide pair that is thermodynamically suitable for monitoring intracellular glutathione redox potential. Glutaredoxin 1 (Grx1p) from yeast is known to catalyze the redox equilibrium between rxYFP and glutathione, and here, we...... have generated a fusion of the two proteins, rxYFP-Grx1p. In comparison to isolated subunits, intramolecular transfer of reducing equivalents made the fusion protein kinetically superior in reactions with glutathione. The rate of GSSG oxidation was thus improved by a factor of 3300. The reaction with...... separately and in the fusion. This could not be ascribed to the lack of an unproductive side reaction to glutaredoxin disulfide. Instead, slower alkylation kinetics with iodoacetamide indicates a better leaving-group capability of the remaining cysteine residue, which can explain the increased activity....

  7. Bioconjugation of therapeutic proteins and enzymes using the expanded set of genetically encoded amino acids.

    Lim, Sung In; Kwon, Inchan

    2016-10-01

    The last decade has witnessed striking progress in the development of bioorthogonal reactions that are strictly directed towards intended sites in biomolecules while avoiding interference by a number of physical and chemical factors in biological environment. Efforts to exploit bioorthogonal reactions in protein conjugation have led to the evolution of protein translational machineries and the expansion of genetic codes that systematically incorporate a range of non-natural amino acids containing bioorthogonal groups into recombinant proteins in a site-specific manner. Chemoselective conjugation of proteins has begun to find valuable applications to previously inaccessible problems. In this review, we describe bioorthogonal reactions useful for protein conjugation, and biosynthetic methods that produce proteins amenable to those reactions through an expanded genetic code. We then provide key examples in which novel protein conjugates, generated by the genetic incorporation of a non-natural amino acid and the chemoselective reactions, address unmet needs in protein therapeutics and enzyme engineering. PMID:26036278

  8. Protein absorption and fouling on poly(acrylic acid)-graft-polypropylene microfiltration membrane

    Liu, Yanjun; Ma, Huiying; Lv, Chunying; Yang, Jia; Fu, Xueqi

    2009-07-01

    A series of pH-sensitive poly (acrylic acid)-graft-polypropylene hollow fiber microfiltration membranes were prepared by UV-photo-irradiation. Bovine serum albumin (BSA) was chosen as the model protein to investigate its absorption and fouling behaviors on membranes. The results showed that the hydrophilicity of grafted membrane was improved by poly(acrylic acid) chains with parts of membrane pores blocked. The grafted membranes were markedly pH-dependent on the water permeability as pH was altered from 1 to 11. The zeta potential of grafted membranes calculated by streaming potential was negative in most pH range. Electrostatic interaction energy calculated by DLVO theory showed the electric interaction force between grafted membrane and BSA was attractive. With the rise of grafting degree, the electric attractive force between grafted membrane and BSA increased as pH=3 and decreased as pH=8, while it kept basically unchanged as pH=4.7. As a result, most serious fouling was observed as pH=4.7. Grafted membranes had a lower BSA absorption and better antifouling behavior as pH=8, while the opposite result was revealed as pH=3. In conclusion, the absorption and fouling behavior of BSA on membranes was pH-dependent due to the pH-dependence of membrane charge, and the conformation of BSA and grafting chains.

  9. Isoniazid inhibition of mycolic acid synthesis by cell extracts of sensitive and resistant strains of Mycobacterium aurum.

    Quémard, A; Lacave, C; Lanéelle, G

    1991-01-01

    Isonicotinic acid hydrazide (isoniazid; INH) inhibition of mycolic acid synthesis was studied by using cell extracts from both INH-sensitive and -resistant strains of Mycobacterium aurum. The cell extract of the INH-sensitive strain was inhibited by INH, while the preparation from the INH-resistant strain was not. This showed that the INH resistance of mycolic acid synthesis was not due to a difference in drug uptake or the level of peroxidase activity (similar in both extracts). As INH did n...

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

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

    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 differe...

  11. A comparative study of ribosomal proteins: linkage between amino acid distribution and ribosomal assembly

    Lott, Brittany Burton; Wang, Yongmei; Nakazato, Takuya

    2013-01-01

    Background Assembly of the ribosome from its protein and RNA constituents must occur quickly and efficiently in order to synthesize the proteins necessary for all cellular activity. Since the early 1960’s, certain characteristics of possible assembly pathways have been elucidated, yet the mechanisms that govern the precise recognition events remain unclear. We utilize a comparative analysis to investigate the amino acid composition of ribosomal proteins (r-proteins) with respect to their role...

  12. Dissection of the Critical Binding Determinants of Cellular Retinoic Acid Binding Protein II by Mutagenesis and Fluorescence Binding Assay

    Vasileiou, Chrysoula; Lee, Kin Sing Stephen; Crist, Rachael M.; Vaezeslami, Soheila; Goins, Sarah M.; Geiger, James H.; Borhan, Babak

    2009-01-01

    The binding of retinoic acid to mutants of Cellular Retinoic Acid Binding Protein II (CRABPII) was evaluated to better understand the importance of the direct protein/ligand interactions. The important role of Arg111 for the correct structure and function of the protein was verified and other residues that directly affect retinoic acid binding have been identified. Furthermore, retinoic acid binding to CRABPII mutants that lack all previously identified interacting amino acids was rescued by ...

  13. Accelerated protein digestion and amino acid absorption after Roux-en-Y gastric bypass

    Bojsen-Møller, Anna Kirstine; Jacobsen, Siv H; Dirksen, Carsten;

    2015-01-01

    BACKGROUND: Roux-en-Y gastric bypass (RYGB) involves exclusion of major parts of the stomach and changes in admixture of gastro-pancreatic enzymes, which could have a major impact on protein digestion and amino acid absorption. OBJECTIVE: We investigated the effect of RYGB on amino acid appearance...... RYGB. CONCLUSIONS: RYGB accelerates caseinate digestion and amino acid absorption, resulting in faster and higher but more transient postprandial elevation of plasma amino acids. Changes are likely mediated by accelerated intestinal nutrient entry and clearly demonstrate that protein digestion is not...

  14. A Single Amino Acid Change in the Newcastle Disease Virus Fusion Protein Alters the Requirement for HN Protein in Fusion

    Sergel, Theresa A.; McGinnes, Lori W.; Morrison, Trudy G

    2000-01-01

    The role of a leucine heptad repeat motif between amino acids 268 and 289 in the structure and function of the Newcastle disease virus (NDV) F protein was explored by introducing single point mutations into the F gene cDNA. The mutations affected either folding of the protein or the fusion activity of the protein. Two mutations, L275A and L282A, likely interfered with folding of the molecule since these proteins were not proteolytically cleaved, were minimally expressed at the cell surface, a...

  15. Free lysine, glycine, alanine, glutamic acid and aspartic acid reduce the glycation of human lens proteins by galactose

    The amino acids lysine, glycine, alanine, glutamate and aspartate formed adducts with galactose at physiological pH and temperature as shown by incorporation of U[14C] galactose. The percentage of galactose reacting with lysine, glycine, alanine, glutamate and aspartate was 4.5 to 7.8, 7.9 to 10.8, 3.2 to 4.6, 2.8 to 4.8 and 3 to 5.2, respectively. Studies with lysine showed that the extent of glycation of the free amino acid increased with time. Incubation of lens homogenate with galactose, effected glycation of proteins. Addition of lysine in concentrations of 5 and 10 mM to equimolar concentrations of galactose decreased the glycation of lens proteins by 64% to 71%; glycine, alanine, glutamate and aspartate decreased glycation by 23 to 68%, 32 to 61%, 35 to 56% and 26 to 61% respectively. Under similar conditions, glycine reacts to a greater extent than lysine, alanine, glutamic and aspartic acids. However, lysine was more effective than glycine, alanine, aspartic and glutamic acids in decreasing glycation of lens proteins by galactose. The decrease of glycation with added lysine increased with time. In general increase of amino acid concentration rather than that of sugar augmented the decrease of glycation of lens proteins. (author)

  16. Radiation chemical and biological examinations on sensitizers: mitomycin C, folic acid, procarbazine

    Radiochemical and radiobiological examinations of the sensitizers Mitomycin C (MMC), Folic acid (FA) and Procarbazine (PC) were performed. In the radiochemical part, absorption spectroscopy and HPLC analysis were used to follow the radiolysis of MMC, FA and PC in aqueous solutions, in presence of air, Argon and N2O. The G initial value for the substrate decomposition was found. Possible reaction mechanisms for the reaction: sensitizers with OH- radicals and solvated electrons are given. Pulse radiolysis investigations to study the kinetic of OH- attack on FA yielded K (OH + FA)=1,3.1010 dm3 mol-1 S-1. For the radical disappearance, a reaction mechanism 2nd order with 2k=3,1.108 dm3 mol-1 S-1 (short-lived radical) and 2k=6,5.107 dm3 mol-1 S-1 (long-lived radical) was found. Pulse radiolysis investigations to study the kinetic of the OH- attack on PC yielded values for the velocity constant of the radical formation as well as one short-lived and one long-lived radical disappearance. Quantum chemistry calculations through VAMP6.1 (computer program) produced electron densities from PC, enthalpy formation and total energies of possible radicals resulted from the OH attack. In the radiobiological part, the bacteria EcAB1157 was used. Toxicity tests in air and Argon, depending on pH , sensitizers concentrations and exposition time were performed. The determination of the surviving curves in presence of air, Ar and N2O with different concentrations, dose rates and two pH values (6,2 and 7,4) with MMC, FA and PC allowed to prove their efficiency and action as sensitizers. (nevyjel)

  17. Towards an understanding of Mesocestoides vogae fatty acid binding proteins' roles.

    Gabriela Alvite

    Full Text Available Two fatty acid binding proteins, MvFABPa and MvFABPb were identified in the parasite Mesocestoides vogae (Platyhelmithes, Cestoda. Fatty acid binding proteins are small intracellular proteins whose members exhibit great diversity. Proteins of this family have been identified in many organisms, of which Platyhelminthes are among the most primitive. These proteins have particular relevance in flatworms since de novo synthesis of fatty acids is absent. Fatty acids should be captured from the media needing an efficient transport system to uptake and distribute these molecules. While HLBPs could be involved in the shuttle of fatty acids to the surrounding host tissues and convey them into the parasite, FABPs could be responsible for the intracellular trafficking. In an effort to understand the role of MvFABPs in fatty acid transport of M. vogae larvae, we analysed the intracellular localization of both MvFABPs and the co-localization with in vivo uptake of fatty acid analogue BODIPY FL C16. Immunohistochemical studies on larvae sections using specific antibodies, showed a diffuse cytoplasmic distribution of each protein with some expression in nuclei and mitochondria. MvFABPs distribution was confirmed by mass spectrometry identification from 2D-electrophoresis of larvae subcellular fractions. This work is the first report showing intracellular distribution of MvFABPs as well as the co-localization of these proteins with the BODIPY FL C16 incorporated from the media. Our results suggest that fatty acid binding proteins could target fatty acids to cellular compartments including nuclei. In this sense, M. vogae FABPs could participate in several cellular processes fulfilling most of the functions attributed to vertebrate's counterparts.

  18. Natural vs. random protein sequences: Discovering combinatorics properties on amino acid words.

    Santoni, Daniele; Felici, Giovanni; Vergni, Davide

    2016-02-21

    Casual mutations and natural selection have driven the evolution of protein amino acid sequences that we observe at present in nature. The question about which is the dominant force of proteins evolution is still lacking of an unambiguous answer. Casual mutations tend to randomize protein sequences while, in order to have the correct functionality, one expects that selection mechanisms impose rigid constraints on amino acid sequences. Moreover, one also has to consider that the space of all possible amino acid sequences is so astonishingly large that it could be reasonable to have a well tuned amino acid sequence indistinguishable from a random one. In order to study the possibility to discriminate between random and natural amino acid sequences, we introduce different measures of association between pairs of amino acids in a sequence, and apply them to a dataset of 1047 natural protein sequences and 10,470 random sequences, carefully generated in order to preserve the relative length and amino acid distribution of the natural proteins. We analyze the multidimensional measures with machine learning techniques and show that, to a reasonable extent, natural protein sequences can be differentiated from random ones. PMID:26656109

  19. Single molecule DNA interaction kinetics of retroviral nucleic acid chaperone proteins

    Williams, Mark

    2010-03-01

    Retroviral nucleocapsid (NC) proteins are essential for several viral replication processes including specific genomic RNA packaging and reverse transcription. The nucleic acid chaperone activity of NC facilitates the latter process. In this study, we use single molecule biophysical methods to quantify the DNA interactions of wild type and mutant human immunodeficiency virus type 1 (HIV-1) NC and Gag and human T-cell leukemia virus type 1 (HTLV-1) NC. We find that the nucleic acid interaction properties of these proteins differ significantly, with HIV-1 NC showing rapid protein binding kinetics, significant duplex destabilization, and strong DNA aggregation, all properties that are critical components of nucleic acid chaperone activity. In contrast, HTLV-1 NC exhibits significant destabilization activity but extremely slow DNA interaction kinetics and poor aggregating capability, which explains why HTLV-1 NC is a poor nucleic acid chaperone. To understand these results, we developed a new single molecule method for quantifying protein dissociation kinetics, and applied this method to probe the DNA interactions of wild type and mutant HIV-1 and HTLV-1 NC. We find that mutations to aromatic and charged residues strongly alter the proteins' nucleic acid interaction kinetics. Finally, in contrast to HIV-1 NC, HIV-1 Gag, the nucleic acid packaging protein that contains NC as a domain, exhibits relatively slow binding kinetics, which may negatively impact its ability to act as a nucleic acid chaperone.

  20. Utilization of alimentary protein and amino acids in satisfying the nitrogen requirements of monogastric mammals

    The nitrogenous matter in the food of monogastric animals consists mainly of proteins, which are rapidly hydrolized in the intestinal tract when they have left the gastric reservoir. The digestive tube has several roles: it provides for hydrolysis of the food proteins and for a supply of endogenous nitrogen; it enables a certain digestive function to be performed by the intestinal flora and permits the transport of amino acids into the blood, selecting those which are needed for protein synthesis. The digestion products appear mainly in the form of free amino acids in the portal blood. A large proportion of these amino acids is taken up by the liver, so that intense protein synthesis takes place in the latter, coupled with a decrease in catabolism leading to a rhythmic increase in the liver content of proteins and RNA. The labile proteins retained are mainly enzymes, which catabolize the amino acids, and the liver is the site of the catabolism of most of the excess amino acids except those with chain branching. Alimentary deficiencies do not markedly reduce protein synthesis in this organ, since the rate of re-utilization of the amino acids is increased and the liver thus plays a regulatory role. The utilization of amino acids in muscle also follows a certain rhythm, partly connected with feeding, and under hormonal control. The muscle is the seat of catabolism of a large part of the branched chain amino acids, and like the liver it contributes to the energy utilization of amino acids. The rate of utilization of certain essential amino acids can be measured by metabolic criteria, including determination of blood and muscle concentrations and excretion of 14CO2 labels in the exhaled air or of 35S labels in urine. (author)

  1. One-Pot Procedure for Recovery of Gallic Acid from Wastewater and Encapsulation within Protein Particles.

    Nourbakhsh, Himan; Madadlou, Ashkan; Emam-Djomeh, Zahra; Wang, Yi-Cheng; Gunasekaran, Sundaram; Mousavi, Mohammad E

    2016-02-24

    A whey protein isolate solution was heat-denatured and treated with the enzyme transglutaminase, which cross-linked ≈26% of the amino groups and increased the magnitude of the ζ-potential value. The protein solution was microemulsified, and then the resulting water-in-oil microemulsion was dispersed within a gallic acid-rich model wastewater. Gallic acid extraction by the outlined microemulsion liquid membrane (MLM) from the exterior aqueous phase (wastewater) and accumulation within the internal aqueous nanodroplets induced protein cold-set gelation and resulted in the formation of gallic acid-enveloping nanoparticles. Measurements with a strain-controlled rheometer indicated a progressive increase in the MLM viscosity during gallic acid recovery corresponding to particle formation. The mean hydrodynamic size of the nanoparticles made from the heat-denatured and preheated enzymatically cross-linked proteins was 137 and 122 nm, respectively. The enzymatic cross-linking of whey proteins led to a higher gallic acid recovery yield and increased the glass transition enthalpy and temperature. A similar impact on glass transition indices was observed by the gallic acid-induced nanoparticulation of proteins. Scanning electron microscopy showed the existence of numerous jammed/fused nanoparticles. It was suggested on the basis of the results of Fourier transform infrared spectroscopy that the in situ nanoparticulation of proteins shifted the C-N stretching and C-H bending peaks to higher wavenumbers. X-ray diffraction results proposed a decreased β-sheet content for proteins because of the acid-induced particulation. The nanoparticles made from the enzymatically cross-linked protein were more stable against the in vitro gastrointestinal digestion and retained almost 19% of the entrapped gallic acid after 300 min sequential gastric and intestinal digestions. PMID:26862880

  2. The relationship between heat shock protein 72 expression in skeletal muscle and insulin sensitivity is dependent on adiposity

    Henstridge, Darren C; Forbes, Josephine M; Penfold, Sally A;

    2010-01-01

    Decreased gene expression of heat shock protein 72 (HSP72) in skeletal muscle is associated with insulin resistance in humans. We aimed to determine whether HSP72 protein expression in insulin-sensitive tissues is related to criterion standard measures of adiposity and insulin resistance in a young...... insulin sensitivity. Skeletal muscle and subcutaneous adipose tissue biopsies were obtained by percutaneous needle biopsy. HSP72 protein expression in skeletal muscle was inversely related to percentage body fat (r = -0.54, P <.05) and remained significant after adjustment for age and sex (P <.05......). Insulin sensitivity was also related to HSP72 protein expression in skeletal muscle (r = 0.52, P <.05); however, this relationship disappeared after adjustment for percentage body fat (P = .2). In adipose tissue, HSP72 protein expression was not related to adiposity or insulin sensitivity. Physical...

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

    Chia, Mathias Ahii, E-mail: chia28us@yahoo.com [Department of Botany, Federal University of São Carlos, Rodovia Washington Luis km 235, São Carlos, SP Cep 13565905 (Brazil); Lombardi, Ana Teresa [Department of Botany, Federal University of São Carlos, Rodovia Washington Luis km 235, São Carlos, SP Cep 13565905 (Brazil); Graça Gama Melão, Maria da [Department of Hydrobiology, Federal University of São Carlos, Rodovia Washington Luis km 235, São Carlos, SP Cep 13565905 (Brazil); Parrish, Christopher C. [Department of Ocean Sciences, Memorial University of Newfoundland, St. John’s, Newfoundland A1C 5S7 (Canada)

    2015-03-15

    Highlights: • Chlorella vulgaris was exposed to Cd under varying N concentrations. • Growth rate and cell density decreased with increasing Cd stress and N limitation. • Dry weight, chlorophyll a, total lipid, carbohydrate and protein were accumulated. • Amino acids like proline and glutamine were accumulated under N and Cd stress. • Changes in amino acid composition are sensitive biomarkers for Cd and N stress. - Abstract: 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{sup −7} and 2.0 × 10{sup −8} mol L{sup −1} Cd) under varying nitrogen (2.9 × 10{sup −6}, 1.1 × 10{sup −5} and 1.1 × 10{sup −3} mol L{sup −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.

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

    Highlights: • Chlorella vulgaris was exposed to Cd under varying N concentrations. • Growth rate and cell density decreased with increasing Cd stress and N limitation. • Dry weight, chlorophyll a, total lipid, carbohydrate and protein were accumulated. • Amino acids like proline and glutamine were accumulated under N and Cd stress. • Changes in amino acid composition are sensitive biomarkers for Cd and N stress. - Abstract: 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 mol L−1 Cd) under varying nitrogen (2.9 × 10−6, 1.1 × 10−5 and 1.1 × 10−3 mol L−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

  5. Representation of Protein-Sequence Information by Amino Acid Subalphabets

    Andersen, Claus A. F.; Brunak, Soren

    2004-01-01

    Within computational biology, algorithms are constructed with the aim of extracting knowledge from biological data, in particular, data generated by the large genome projects, where gene and protein sequences are produced in high volume. In this article, we explore new ways of representing protein-sequence information, using machine learning strategies, where the primary goal is the discovery of novel powerful representations for use in AI techniques. In the case of proteins and the 20 differ...

  6. Models of protein and amino acid requirements for cattle

    Luis Orlindo Tedeschi; Danny Gene Fox; Mozart Alves Fonseca; Luigi Francis Lima Cavalcanti

    2015-01-01

    Protein supply and requirements by ruminants have been studied for more than a century. These studies led to the accumulation of lots of scientific information about digestion and metabolism of protein by ruminants as well as the characterization of the dietary protein in order to maximize animal performance. During the 1980s and 1990s, when computers became more accessible and powerful, scientists began to conceptualize and develop mathematical nutrition models, and to program them into comp...

  7. Radioiodinated phenylalkyl malonic acid derivatives as pH-sensitive SPECT tracers.

    Matthias Bauwens

    Full Text Available INTRODUCTION: In vivo pH imaging has been a field of interest for molecular imaging for many years. This is especially important for determining tumor acidity, an important driving force of tumor invasion and metastasis formation, but also in the process of apoptosis. METHODS: 2-(4-[(123I]iodophenethyl-2-methylmalonic acid (IPMM, 2-(4-[(123I]iodophenethyl-malonic acid (IPM, 2-(4-[(123I]iodobenzyl-malonic acid (IBMM and 4-[(123I]iodophthalic acid (IP were radiolabeled via the Cu(+ isotopic nucleophilic exchange method. All tracers were tested in vitro in buffer systems to assess pH driven cell uptake. In vivo biodistribution of [(123I]IPMM and [(123I]IPM was determined in healthy mice and the pH targeting efficacy in vivo of [(123I]IPM was evaluated in an anti-Fas monoclonal antibody (mAb apoptosis model. In addition a mouse RIF-1 tumor model was explored in which tumor pH was decreased from 7.0 to 6.5 by means of induction of hyperglycemia in combination with administration of meta-iodobenzylguanidine. RESULTS: Radiosynthesis resulted in 15-20% for iodo-bromo exchange and 50-60% yield for iodo-iodo exchange while in vitro experiments showed a pH-sensitive uptake for all tracers. Shelf-life stability and in vivo stability was excellent for all tracers. [(123I]IPMM and [(123I]IPM showed a moderately fast predominantly biliary clearance while a high retention was observed in blood. The biodistribution profile of [(123I]IPM was found to be most favorable in view of pH-specific imaging. [(123I]IPM showed a clear pH-related uptake pattern in the RIF-1 tumor model. CONCLUSION: Iodine-123 labeled malonic acid derivates such as [(123I]IPM show a clearly pH dependent uptake in tumor cells both in vitro and in vivo which allows to visualize regional acidosis. However, these compounds are not suitable for detection of apoptosis due to a poor acidosis effect.

  8. Formulation of Acid-Sensitive Micelles for Delivery of Cabazitaxel into Prostate Cancer Cells.

    Aydin, Omer; Youssef, Ibrahim; Yuksel Durmaz, Yasemin; Tiruchinapally, Gopinath; ElSayed, Mohamed E H

    2016-04-01

    We report the synthesis of an amphiphilic triblock copolymer composed of a hydrophilic poly(ethylene glycol) (PEG) block, a central poly(acrylic acid) (PAA) block, and a hydrophobic poly(methyl methacrylate) (PMMA) block using atom transfer radical polymerization technique. We examined the self-assembly of PEG-b-PAA-b-PMMA copolymers in aqueous solutions forming nanosized micelles and their ability to encapsulate hydrophobic guest molecules such as Nile Red (NR) dye and cabazitaxel (CTX, an anticancer drug). We used 2,2β'-(propane-2,2-diylbis(oxy))-diethanamine to react with the carboxylic acid groups of the central PAA block forming acid-labile, shell cross-linked micelles (SCLM). We investigated the loading efficiency and release of different guest molecules from non-cross-linked micelles (NSCLM) and shell cross-linked micelles (SCLM) prepared by reacting 50% (SCLM-50) and 100% (SCLM-100) of the carboxylic acid groups in the PAA in physiologic (pH 7.4) and acidic (pH 5.0) buffer solutions as a function of time. We examined the uptake of NR-loaded NSCLM, SCLM-50, and SCLM-100 micelles into PC-3 and C4-2B prostate cancer cells and the effect of different micelle compositions on membrane fluidity of both cell lines. We also investigated the effect of CTX-loaded NSCLM, SCLM-50, and SCLM-100 micelles on the viability of PC-3 and C4-2B cancer cells compared to free CTX as a function of drug concentration. Results show that PEG-b-PAA-b-PMMA polymers form micelles at concentrations ≥11 μg/mL with an average size of 40-50 nm. CTX was encapsulated in PEG-b-PAA-b-PMMA micelles with 55% loading efficiency in NSCLM. In vitro release studies showed that 30% and 85% of the loaded CTX was released from SCLM-50 micelles in physiologic (pH 7.4) and acidic (pH 5.0) buffer solutions over 30 h, confirming micelles' sensitivity to solution pH. Results show uptake of NSCLM and SCLM into prostate cancer cells delivering their chemotherapeutic cargo, which triggered efficient cancer

  9. Proteins isolated from regenerating sciatic nerves of rats form aggregates following posttranslational amino acid modification

    Soluble proteins of regenerating sciatic nerves of rats can be posttranslationally, covalently modified by a variety of radioactive amino acids. The present study shows that once modified by a mixture of 15 amino acids, many of those proteins form aggregates that are unable to pass through a 0.45-micron filter and pellet following 20,000g centrifugation (suggesting a size of greater than 2 x 10(6) Da). Aggregation of proteins also occurs following modification by Arg or Lys alone, but does not occur following protein modification in nonregenerating nerves or in brain. Aggregates are not disrupted by treatment with 100 mM beta mercaptoethanol or by exposure to 1.0 M NaCl, but aggregates are solubilized by treatment with urea and by boiling in 1.5% SDS. Amino acid analysis of proteins modified by a mixture of [3H]amino acids shows a similar proportion of posttranslationally incorporated Ser, Pro, Val, Ala, Leu, Phe, Lys, and Arg in the soluble and pelletable fractions. Two-dimensional PAGE profiles of soluble and pelletable modified proteins show that the modified proteins in both fractions are in similar pI and molecular weight ranges, except that the soluble modified proteins include a high-molecular-weight component that is absent in the pelleted modified proteins. Kinetic studies show that while half-maximal levels of protein modification occur within 30 seconds of incubation, the appearance of the pelletable modified protein fraction is delayed significantly. These results indicate that amino acid modification of soluble proteins in regenerating sciatic nerves of rats results in physical changes in those proteins so that they form high-molecular-weight aggregates

  10. Protein phosphorylation in isolated human adipocytes - Adrenergic control of the phosphorylation of hormone-sensitive lipase

    The effect of adrenergic agents on protein phosphorylation in human adipocytes was examined. Freshly isolated human fat cells were incubated with 32PO4 in order to label intracellular ATP, then treated with a variety of adrenergic and other pharmacologic agents. Treatment with the β-adrenergic agonist isoproterenol led to a significant increase in phosphate content of at least five protein bands (Mr 52, 53, 63, 67, 84 kDa). The increase in phosphorylation was partially inhibited by the α-2 agonist clonidine. Epinephrine, a combined α and β agonist, was less effective at increasing phosphate content of the proteins than was isoproterenol. Neither insulin nor the α-1 agonist phenylephrine had any discernible effect on the pattern of protein phosphorylation. The 84 kDa phosphorylated peptide band appears to contain hormone-sensitive lipase, a key enzyme in the lipolytic pathway which is activated by phosphorylation. These results are somewhat different than previously reported results for rat adipocytes, and represent the first report of overall pattern and adrenergic modulation of protein phosphorylation in human adipocytes

  11. High temperature, drought and their interaction induced protein alterations in sensitive and tolerant wheat varieties

    Vikender Kaur, Reena Mahla And R.K.Behl

    2014-12-01

    Full Text Available Two contrasting wheat (Triticum aestivum L. cultivars WH730 (high temperature tolerant and UP2565 (high temperature sensitive were tested for differential response to combined and individually applied high temperature (HT and drought (D stress at seedling stage for peptide profile. Initial profile of the stress induced peptides was outlined via SDS electrophoresis of leaf extracts. Electrophoretic pattern of proteins revealed expression of new bands as well as disappearance of certain others in HT, D and interactive HT+D stress treated and revived samples in both wheat varieties relative to untreated control samples. Some of the bands that appeared in stress treated seedlings were also present after revival indicating their protective role, while some new peptides synthesized after stress but disappeared after revival period may be designated true stress proteins. However, all the plants from heat, drought and their interactive stress treatments continued to grow during recovery period. This suggests that these proteins and other newly synthesized proteins may have protective effects at high temperature (40°C and water scarcity and provide plants for healthy growth during the recovery period. Furthermore, elucidating the functions of proteins expressed by genes in stress tolerant and susceptible plants may provide important information for designing new strategies for crop improvement.

  12. Development of sensitive metalloporphyrin probes for chemiluminescent imaging detection of serum proteins.

    Liu, Xia; Huang, Lingyun; Baeyens, Willy R G; Ouyang, Jin; He, Dacheng; Wan, Genping; Zhang, Li

    2009-09-01

    The development of metalloporphyrin- (ferric protoporphyrin IX chloride (FePP), cobalt (III) protoporphyrin IX chloride, copper (II) protoporphyrin IX) enhanced chemiluminescent (CL) imaging detection of serum proteins after PAGE is described in this article. The detection is based on the catalytic activity of metalloporphyrins, especially FePP, in the CL reaction of the luminol-H2O2 system. Some relatively low abundant proteins such as hemopexin (Hpx) and complement C4 are sensitively detected by FePP-enhanced CL imaging. Other proteins such as haptoglobin, apolipoprotein A-1, complement C3, and alpha-1-antitrypsin are also detected and identified by MS and MS/MS techniques. Detection limit of Hpx is as low as 20 ng, without the need of expensive antibodies or tedious immunoassay procedures. The mechanism of the proposed method is discussed employing standard proteins. The application to the analysis of different protein patterns in healthy people and in Thalassemia patients is being investigated. PMID:19711378

  13. Bile Acids Trigger GLP-1 Release Predominantly by Accessing Basolaterally Located G Protein-Coupled Bile Acid Receptors

    Brighton, Cheryl A.; Rievaj, Juraj; Kuhre, Rune E.;

    2015-01-01

    -coupled bile acid transporter ASBT. The aim of this study was to identify pathways important for GLP-1 release and to determine whether bile acids target their receptors on GLP-1-secreting L-cells from the apical or basolateral compartment. Using transgenic mice expressing fluorescent sensors specifically in L......Bile acids are well-recognized stimuli of glucagon-like peptide-1 (GLP-1) secretion. This action has been attributed to activation of the G protein-coupled bile acid receptor GPBAR1 (TGR5), although other potential bile acid sensors include the nuclear farnesoid receptor and the apical sodium......-cells, we observed that taurodeoxycholate (TDCA) and taurolithocholate (TLCA) increased intracellular cAMP and Ca2+. In primary intestinal cultures, TDCA was a more potent GLP-1 secretagogue than taurocholate (TCA) and TLCA, correlating with a stronger Ca2+ response to TDCA. Using small-volume Ussing...

  14. Expression of pH-sensitive green fluorescent protein in Arabidopsis thaliana

    Moseyko, N.; Feldman, L. J.

    2001-01-01

    This is the first report on using green fluorescent protein (GFP) as a pH reporter in plants. Proton fluxes and pH regulation play important roles in plant cellular activity and therefore, it would be extremely helpful to have a plant gene reporter system for rapid, non-invasive visualization of intracellular pH changes. In order to develop such a system, we constructed three vectors for transient and stable transformation of plant cells with a pH-sensitive derivative of green fluorescent protein. Using these vectors, transgenic Arabidopsis thaliana and tobacco plants were produced. Here the application of pH-sensitive GFP technology in plants is described and, for the first time, the visualization of pH gradients between different developmental compartments in intact whole-root tissues of A. thaliana is reported. The utility of pH-sensitive GFP in revealing rapid, environmentally induced changes in cytoplasmic pH in roots is also demonstrated.

  15. Upconversion fluorescence metal-organic frameworks thermo-sensitive imprinted polymer for enrichment and sensing protein.

    Guo, Ting; Deng, Qiliang; Fang, Guozhen; Gu, Dahai; Yang, Yukun; Wang, Shuo

    2016-05-15

    A novel fluorescence material with thermo-sensitive for the enrichment and sensing of protein was successfully prepared by combining molecular imprinting technology with upconversion nanoparticles (UCNPs) and metal-organic frameworks (MOFs). Herein, the UCNPs acted as signal reporter for composite materials because of its excellent fluorescence property and chemical stability. MOFs were introduced to molecularly imprinted polymer (MIP) due to its high specific surface area which increases the rate of mass transfer relative to that of traditional bulk MIP. The thermo-sensitive imprinted material which allows for swelling and shrinking with response to temperature changes was prepared by choosing Bovine hemoglobin (BHB) as the template, N-isopropyl acrylamide (NIPAAM) as the temperature-sensitive functional monomer and N,N-methylenebisacrylamide (MBA) as the cross-linker. The recognition characterizations of imprinted material-coated UCNPs/MOFs (UCNPs/MOFs/MIP) were evaluated, and the results showed that the fluorescence intensity of UCNPs/MOFs/MIP reduced gradually with the increase of BHB concentration. The fluorescence material was response to the temperature. The adsorption capacity was as much as 167.6 mg/g at 28°C and 101.2mg/g at 44°C, which was higher than that of traditional MIP. Therefore, this new fluorescence material for enrichment and sensing protein is very promising for future applications. PMID:26722764

  16. Fish protein hydrolysates: proximate composition, amino acid composition, antioxidant activities and applications: a review.

    Chalamaiah, M; Dinesh Kumar, B; Hemalatha, R; Jyothirmayi, T

    2012-12-15

    The fish processing industry produces more than 60% by-products as waste, which includes skin, head, viscera, trimmings, liver, frames, bones, and roes. These by-product wastes contain good amount of protein rich material that are normally processed into low market-value products, such as animal feed, fish meal and fertilizer. In view of utilizing these fish industry wastes, and for increasing the value to several underutilised fish species, protein hydrolysates from fish proteins are being prepared by several researchers all over the world. Fish protein hydrolysates are breakdown products of enzymatic conversion of fish proteins into smaller peptides, which normally contain 2-20 amino acids. In recent years, fish protein hydrolysates have attracted much attention of food biotechnologists due to the availability of large quantities of raw material for the process, and presence of high protein content with good amino acid balance and bioactive peptides (antioxidant, antihypertensive, immunomodulatory and antimicrobial peptides). PMID:22980905

  17. Fish protein hydrolysate elevates plasma bile acids and reduces visceral adipose tissue mass in rats

    Liaset, Bjørn; Madsen, Lise; Hao, Qin;

    2009-01-01

    Conjugation of bile acids (BAs) to the amino acids taurine or glycine increases their solubility and promotes liver BA secretion. Supplementing diets with taurine or glycine modulates BA metabolism and enhances fecal BA excretion in rats. However, it is still unclear whether dietary proteins...... varying in taurine and glycine contents alter BA metabolism, and thereby modulate the recently discovered systemic effects of BAs. Here we show that rats fed a diet containing saithe fish protein hydrolysate (saithe FPH), rich in taurine and glycine, for 26 days had markedly elevated fasting plasma BA....../retroperitoneal adipose tissues of rats fed saithe FPH. Our results provide the first evidence that dietary protein sources with different amino acid compositions can modulate the level of plasma bile acids and our data suggest potential novel mechanisms by which dietary protein sources can affect energy metabolism....

  18. Testing for spatial clustering of amino acid replacements within protein tertiary structure

    Yu, Jiaye; Thorne, Jeffrey L

    2006-01-01

    Widely used models of protein evolution ignore protein structure. Therefore, these models do not predict spatial clustering of amino acid replacements with respect to tertiary structure. One formal and biologically implausible possibility is that there is no tendency for amino acid replacements to...... be spatially clustered during evolution. An alternative to this is that amino acid replacements are spatially clustered and this spatial clustering can be fully explained by a tendency for similar rates of amino acid replacement at sites that are nearby in protein tertiary structure. A third...... possibility is that the amount of clustering exceeds that which can be explained solely on the basis of independently evolving protein sites with spatially clustered replacement rates. We introduce two simple and not very parametric hypothesis tests that help distinguish these three possibilities. We then...

  19. Study of Stationary Phase Metabolism Via Isotopomer Analysis of Amino Acids from an Isolated Protein

    Shaikh, AfshanS.; Tang, YinjieJ.; Mukhopadhyay, Aindrila; Martin, Hector Garcia; Gin, Jennifer; Benke, Peter; Keasling, Jay D.

    2009-09-14

    Microbial production of many commercially important secondary metabolites occurs during stationary phase, and methods to measure metabolic flux during this growth phase would be valuable. Metabolic flux analysis is often based on isotopomer information from proteinogenic amino acids. As such, flux analysis primarily reflects the metabolism pertinent to the growth phase during which most proteins are synthesized. To investigate central metabolism and amino acids synthesis activity during stationary phase, addition of fully 13C-labeled glucose followed by induction of green fluorescent protein (GFP) expression during stationary phase was used. Our results indicate that Escherichia coli was able to produce new proteins (i.e., GFP) in the stationary phase, and the amino acids in GFP were mostly from degraded proteins synthesized during the exponential growth phase. Among amino acid biosynthetic pathways, only those for serine, alanine, glutamate/glutamine, and aspartate/asparagine had significant activity during the stationary phase.

  20. Use of anaerobic green fluorescent protein versus green fluorescent protein as reporter in lactic acid bacteria.

    Landete, José M; Langa, Susana; Revilla, Concepción; Margolles, Abelardo; Medina, Margarita; Arqués, Juan L

    2015-08-01

    Lactic acid bacteria (LAB) are commonly used in the production of fermented and probiotic foods. Development of molecular tools to discriminate the strains of interest from the endogenous microbiota in complex environments like food or gut is of high interest. Green fluorescent protein (GFP)-like chromophores strictly requires molecular oxygen for maturation of fluorescence, which restrict the study of microorganisms in low-oxygen environments. In this work, we have developed a noninvasive cyan-green fluorescent based reporter system for real-time tracking of LAB that is functional under anoxic conditions. The evoglow-Pp1 was cloned downstream from the promoters D-alanyl-D-alanine carboxypeptidase and elongation factor Tu of Lactobacillus reuteri CECT925 using pNZ8048 and downstream of the lactococcal P1 promoter using pT1NX. The classical gfp was also cloned in pT1NX. These recombinant expression vectors were electroporated into Lactococccus, Lactobacillus, and Enterococcus strains with biotechnological and/or probiotic interests to assess and compare their functionality under different conditions of oxygen and pH. The expression was analyzed by imaging and fluorometric methods as well as by flow cytometry. We demonstrate that reporter systems pNZ:TuR-aFP and pT1-aFP are two versatile molecular markers for monitoring LAB in food and fecal environments without the potential problems caused by oxygen and pH limitations, which could be exploited for in vivo studies. Production of the fluorescent protein did not disturb any important physiological properties of the parental strains, such as growth rate, reuterin, or bacteriocin production. PMID:26129953

  1. Analysis of the ligand binding properties of recombinant bovine liver-type fatty acid binding protein

    Rolf, B; Oudenampsen-Krüger, E; Börchers, T;

    1995-01-01

    The coding part of the cDNA for bovine liver-type fatty acid binding protein (L-FABP) has been amplified by RT-PCR, cloned and used for the construction of an Escherichia coli (E. coli) expression system. The recombinant protein made up to 25% of the soluble E. coli proteins and could be isolated...... by a simple two step protocol combining ion exchange chromatography and gel filtration. Dissociation constants for binding of oleic acid, arachidonic acid, oleoyl-CoA, lysophosphatidic acid and the peroxisomal proliferator bezafibrate to L-FABP have been determined by titration calorimetry. All ligands were...... bound in a 2:1 stoichiometry, the dissociation constants for the first ligand bound were all in the micro molar range. Oleic acid was bound with the highest affinity and a Kd of 0.26 microM. Furthermore, binding of cholesterol to L-FABP was investigated with the Lipidex assay, a liposome binding assay...

  2. Highly sensitive chemiluminescence technology for protein detection using aptamer-based rolling circle amplification platform

    Zhi-Juan Cao; Qian-Wen Peng; Xue Qiu; Cai-Yun Liu; Jian-Zhong Lu

    2011-01-01

    A robust, selective and highly sensitive chemiluminescent (CL) platform for protein assay was presented in this paper. This novel CL approach utilized rolling circle amplification (RCA) as a signal enhancement technique and the 96-well plate as the immobilization and separation carrier. Typically, the antibody immobilized on the surface of 96-well plate was sandwiched with the protein target and the aptamer-primer sequence. This aptamer-primer sequence was then employed as the primer of RCA. Based on this design, a number of the biotinylated probes and streptavidin-horseradish peroxidase (SA-HRP) were captured on the plate, and the CL signal was amplified. In summary, our results demonstrated a robust biosensor with a detection limit of 10 fM that is easy to be established and utilized, and devoid of light source. Therefore, this new technique .will broaden the perspective for future development of DNA-based biosensors for the detection of other protein biomarkers related to clinical diseases, by taking advantages of high sensitivity and selectivity.

  3. New environment-sensitive multichannel DNA fluorescent label for investigation of the protein-DNA interactions.

    Alexandra A Kuznetsova

    Full Text Available Here, we report the study of a new multichannel DNA fluorescent base analogue 3-hydroxychromone (3HC to evaluate its suitability as a fluorescent reporter probe of structural transitions during protein-DNA interactions and its comparison with the current commercially available 2-aminopurine (aPu, pyrrolocytosine (Cpy and 1,3-diaza-2-oxophenoxazine (tCO. For this purpose, fluorescent base analogues were incorporated into DNA helix on the opposite or on the 5'-side of the damaged nucleoside 5,6-dihydrouridine (DHU, which is specifically recognized and removed by Endonuclease VIII. These fluorophores demonstrated different sensitivities to the DNA helix conformational changes. The highest sensitivity and the most detailed information about the conformational changes of DNA induced by protein binding and processing were obtained using the 3HC probe. The application of this new artificial fluorescent DNA base is a very useful tool for the studies of complex mechanisms of protein-DNA interactions. Using 3HC biosensor, the kinetic mechanism of Endonuclease VIII action was specified.

  4. New environment-sensitive multichannel DNA fluorescent label for investigation of the protein-DNA interactions.

    Kuznetsova, Alexandra A; Kuznetsov, Nikita A; Vorobjev, Yuri N; Barthes, Nicolas P F; Michel, Benoît Y; Burger, Alain; Fedorova, Olga S

    2014-01-01

    Here, we report the study of a new multichannel DNA fluorescent base analogue 3-hydroxychromone (3HC) to evaluate its suitability as a fluorescent reporter probe of structural transitions during protein-DNA interactions and its comparison with the current commercially available 2-aminopurine (aPu), pyrrolocytosine (Cpy) and 1,3-diaza-2-oxophenoxazine (tCO). For this purpose, fluorescent base analogues were incorporated into DNA helix on the opposite or on the 5'-side of the damaged nucleoside 5,6-dihydrouridine (DHU), which is specifically recognized and removed by Endonuclease VIII. These fluorophores demonstrated different sensitivities to the DNA helix conformational changes. The highest sensitivity and the most detailed information about the conformational changes of DNA induced by protein binding and processing were obtained using the 3HC probe. The application of this new artificial fluorescent DNA base is a very useful tool for the studies of complex mechanisms of protein-DNA interactions. Using 3HC biosensor, the kinetic mechanism of Endonuclease VIII action was specified. PMID:24925085

  5. Hypochlorite-induced oxidation of amino acids, peptides and proteins

    Hawkins, C L; Pattison, D I; Davies, Michael Jonathan

    2003-01-01

    Activated phagocytes generate the potent oxidant hypochlorite (HOCl) via the release of the enzyme myeloperoxidase and hydrogen peroxide. HOCl is known to react with a number of biological targets including proteins, DNA, lipids and cholesterol. Proteins are likely to be major targets for reactio...

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

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

    1992-02-01

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

  7. Ruthenium(II) multi carboxylic acid complexes: chemistry and application in dye sensitized solar cells.

    Shahroosvand, Hashem; Nasouti, Fahimeh; Sousaraei, Ahmad

    2014-04-01

    Novel ruthenium multi carboxylic complexes (RMCCs) have been synthesized by using ruthenium nitrosyl nitrate, 1,2,4,5-benzenetetracarboxylic acid (H4btec) and 4,7-diphenyl-1,10-phenanthroline (BPhen) as photosensitizers for titanium dioxide semiconductor solar cells. The complexes were characterized by (1)H-NMR, FT-IR, UV-Vis, ICP and CHN analyses. The reaction details and features were then described. SEM analysis revealed that the penetration of dyes into the pores of the nanocrystalline TiO2 surface was improved by increasing the number of btec units. The solar energy to electricity conversion efficiency of complexes shows that the number of attached carboxylates on a dye has an influence on the photoelectrochemical properties of the dye-sensitized electrode. An incident photon-to-current conversion efficiency (IPCE) of 13% at 510 nm was obtained for ruthenium complexes with three btec units. PMID:24500312

  8. Redox-Sensitive and Intrinsically Fluorescent Photoclick Hyaluronic Acid Nanogels for Traceable and Targeted Delivery of Cytochrome c to Breast Tumor in Mice.

    Li, Shuai; Zhang, Jian; Deng, Chao; Meng, Fenghua; Yu, Lin; Zhong, Zhiyuan

    2016-08-24

    In spite of their high specificity and potency, few protein therapeutics are applied in clinical cancer therapy owing to a lack of safe and efficacious delivery systems. Here, we report that redox-sensitive and intrinsically fluorescent photoclick hyaluronic acid nanogels (HA-NGs) show highly efficient loading and breast tumor-targeted delivery of cytochrome c (CC). HA-NGs were obtained from hyaluronic acid-graft-oligo(ethylene glycol)-tetrazole (HA-OEG-Tet) via inverse nanoprecipitation and catalyst-free photoclick cross-linking with l-cystine dimethacrylamide (MA-Cys-MA). HA-NGs exhibited a superb CC loading content of up to 40.6 wt %, intrinsic fluorescence (λem = 510 nm), and a small size of ca. 170 nm. Notably, CC-loaded nanogels (CC-NGs) showed a fast glutathione-responsive protein release behavior. Importantly, released CC maintained its bioactivity. MTT assays revealed that CC-NGs were highly potent with a low IC50 of 3.07 μM to CD44+ MCF-7 human breast tumor cells. Confocal microscopy observed efficient and selective internalization of fluorescent HA-NGs into MCF-7 cells. Interestingly, HA-NGs exhibited also effective breast tumor penetration. The therapeutic results demonstrated that CC-NGs effectively inhibited the growth of MCF-7 breast tumor xenografts at a particularly low dose of 80 or 160 nmol CC equiv./kg. Moreover, CC-NGs did not cause any change in mice body weight, corroborating their low systemic side effects. Redox-sensitive and intrinsically fluorescent photoclick hyaluronic acid nanogels have appeared as a "smart" protein delivery nanoplatform enabling safe, efficacious, traceable, and targeted cancer protein therapy in vivo. PMID:27509045

  9. Acrylamide gel electrophoresis of proteins, acid phosphatases and RN-ases from three potato varieties

    A. Kubicz; E. Wieczorek; B. Morawiecka

    2015-01-01

    Studies on variety differences in the protein and acid phosphatase patterns as well as ribunuclease activity distribution were carried out by disc electrophoresis on saline extracts of three varieties of the potato Solanum tuberosum (L.). The protein bands varied in number, position and relative abundance. One main zone of the acid phosphatase activity was detected consisting of 2-3 electrophoretically different bands. Variety differences were concerned with the number and relative abundance ...

  10. Detergent-enabled transport of proteins and nucleic acids through hydrophobic solvents.

    Bromberg, L E; Klibanov, A M

    1994-01-01

    It is demonstrated that proteins and nucleic acids can be transported through hydrophobic organic solvents (liquid membranes) via nonspecific complex formation with detergents, whereas no macromolecule transport is observed without the latter. A protein (or a nucleic acid) first interacts with an oppositely charged detergent due to hydrophobic ion pairing in the aqueous feed phase. The resultant hydrophobic complex readily partitions into an organic solvent and then into the aqueous receiver ...

  11. Inferences from protein and nucleic acid sequences - Early molecular evolution, divergence of kingdoms and rates of change

    Dayhoff, M. O.; Barker, W. C.; Mclaughlin, P. J.

    1974-01-01

    Description of new sensitive, objective methods for establishing the probable common ancestry of very distantly related sequences and the quantitative evolutionary change which has taken place. These methods are applied to four families of proteins and nucleic acids and evolutionary trees will be derived where possible. Of the three families containing duplications of genetic material, two are nucleic acids: transfer RNA and 5S ribosomal RNA. Both of these structures are functional in the synthesis of coded proteins, and prototypes must have been present in the cell at the inception of the fundamental coding process that all living things share. There are many types of tRNA which recognize the various nucleotide triplets and the 20 amino acids. These types are thought to have arisen as a result of many gene duplications. Relationships among these types are discussed. The 5S ribosomal RNA, presently functional in both eukaryotes and prokaryotes, is very likely descended from an early form incorporating almost a complete duplication of genetic material. The amount of evolution in the various lines can again be compared. The other two families containing duplications are proteins; ferredoxin and cytochrome c.

  12. Radiation chemistry of amino acids, peptides and proteins in relation to the radiation sterilization of high-protein foods

    An important source of information on the question of whether or not toxic or other deleterious substances are formed in the radiation sterilization of foods is the chemical study of reaction products and reaction mechanisms in the radiolysis of individual food components. The present evaluation of the radiation chemistry of amino acids, peptides and proteins outlines the various radiation-induced processes which lead to amino acid degradation and to the synthesis of amino acid derivatives of higher molecular weight. Among the latter are the α,α'-diamino dicarboxylic acids which are formed as major products in the radiolysis of peptides both in aqueous solution and in the solid state. The α,α'-diamino acids are of particular interest as irradiation products because they represent a class of compounds not normally encountered in plant and animal protein sources. Such compounds have, however, been isolated from certain types of bacteria and pathogenic toxins. All of the available data strongly suggest that the α,α'-diamino acids are produced in significant yield in the radiation sterilization of high protein foods. The importance of initiating extensive chemical and biological studies of initiating extensive chemical and biological studies of these and of other high molecular weight products in irradiated food is emphasized

  13. Radiation chemistry of amino acids, peptides and proteins in relation to the radiation sterilization of high-protein foods

    An important source of information on the question of whether or not toxic or other deleterious substances are formed in the radiation sterilization of foods is the chemical study of reaction products and reaction mechanisms in the radiolysis of individual food components. The present evaluation of the radiation chemistry of amino acids, peptides, and proteins outlines the various radiation-induced processes which lead to amino acid degradation and to the synthesis of amino acid derivatives of higher molecular weight. Among the latter are the α,α'-diamino dicarboxylic acids which are formed as major products in the radiolysis of peptides both in aqueous solution and in the solid state. The α,α'-diamino acids are of particular interest as irradiation products because they represent a class of compounds not normally encountered in plant and animal protein sources. Such compounds have, however, been isolated from certain types of bacteria and bacterial products. All of the available data strongly suggest that the α,α'-diamino acids are produced in significant yield in the radiation sterilization of high protein foods. The importance of initiating extensive chemical and biological studies of these and of other high molecular weight products in irradiated food is emphasized

  14. The radiation chemistry of amino acids, peptides and proteins in relation to the radiation sterilization of high-protein foods

    An important source of information on the question of whether or not toxic or other deleterious substances are formed in the radiation sterilization of foods is the chemical study of reaction products and reaction mechanisms in the radiolysis of individual food components. The present evaluation of the radiation chemistry of amino acids, peptides and proteins outlines the various radiation-induced processes which lead to amino acid degradation and to the synthesis of amino acid derivatives of higher molecular weight. Among the latter are the α,α'-diamino dicarboxylic acids which are formed as major products in the radiolysis of peptides both in aqueous solution and in the solid state. The α,α'-diamino acids are of particular interest as irradiation products because they represent a class of compounds not normally encountered in plant and animal protein sources. Such compounds have, however, been isolated from certain types of bacteria and bacterial products. All of the available data strongly suggest that the α,α'-diamino acids are produced in significant yield in the radiation sterilization of high protein foods. The importance of initiating extensive chemical and biological studies of these and of other high molecular weight products in irradiated food is emphasised. (author)

  15. Enterocyte Fatty Acid Binding Proteins (FABPs): Different Functions of Liver- and Intestinal- FABPs in the Intestine

    Gajda, Angela M.; Storch, Judith

    2014-01-01

    SUMMARY Fatty acid binding proteins (FABP) are highly abundant cytosolic proteins that are expressed in most mammalian tissues. In the intestinal enterocyte, both Liver- (LFABP; FABP1) and Intestinal-fatty acid binding proteins (IFABP; FABP2) are expressed. These proteins display high affinity binding for long chain fatty acids (FA) and other hydrophobic ligands, thus they are believed to be involved with uptake and trafficking of lipids in the intestine. In vitro studies have identified differences in ligand binding stoichiometry and specificity, and in mechanisms of FA transfer to membranes, and it has been hypothesized that LFABP and IFABP have difference functions in the enterocyte. Studies directly comparing LFABP- and IFABP-null mice have revealed markedly different phenotypes, indicating that these proteins indeed have different functions in intestinal lipid metabolism and whole body energy homeostasis. In this review, we discuss the evolving knowledge of the functions of LFABP and IFABP in the intestinal enterocyte. PMID:25458898

  16. Cellular fatty acid composition, protein profile and antimicrobial activity of Bacillus sp., isolated from fish gut

    Pushparaj Sujith

    2014-01-01

    Full Text Available Objective: To purify and partially characterize the antimicrobial compounds from bacteria Bacillus sp., isolated from fish gut. Methods: Protein and fatty acids were isolated from the bacteria and checked for the presence of antibacterial activity. Protein has been purified to apparent homogeneity from the supernatants of culture by means of ammonium sulphate precipitation followed by dialysis. Fourier transform infrared spectroscopy analyses were performed for proteins to identify the functional groups. Results: Protein showed an apparent molecular mass 56, 47 and 39 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Fatty acids were extracted and subjected to gas chromatographic analysis. Conclusions: The antimicrobial activity of the bacteria might be due to the presence of fatty acids and proteins which holds promise for the development of new drugs.

  17. Sensitive determination of taurine, γ-aminobutyric acid and ornithine in wolfberry fruit and cortex lycii by HPLC with fluorescence detection and online mass spectrometry identification.

    Chen, Xiangming; You, Jinmao; Suo, Yourui; Fan, Baolei

    2015-04-01

    A new, simple and highly sensitive method for the determination of taurine, γ-aminobutyric acid and ornithine using high-performance liquid chromatography (HPLC) with fluorescence detection is described. Three non-protein amino acids were derivatized by a novel precolumn derivatization reagent 2-[2-(dibenzocarbazol)-ethoxy]ethyl chloroformate before injected. Optimum derivatization was obtained at 40°C for 5 min in the presence of sodium borate buffer (pH 9.0). Derivatives were sufficiently stable to be efficiently analyzed by HPLC without pretreatment. On a reversed-phase Hypersil BDS C8 column, the amino acids were separated in conjunction with a gradient elution with a good baseline resolution. The identification of derivatives was carried out by online postcolumn mass spectrometry with an electrospray ionization source in positive ion mode. Excellent linear responses were observed with the correlation coefficients of >0.9996, and instrument detection limits (at a signal to noise of 3 : 1) were in the range of 0.30-0.33 nmol/L. The proposed method is sensitive and reproducible for the precise determination of the amino acids from wolfberry fruit and cortex lycii samples. PMID:24996656

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

    Gangi Setty, Thanuja [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Cho, Christine [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Govindappa, Sowmya [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Apicella, Michael A. [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Ramaswamy, S., E-mail: ramas@instem.res.in [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India)

    2014-07-01

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

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

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

  20. Network single-walled carbon nanotube biosensors for fast and highly sensitive detection of proteins

    Hu Pingan; Zhang Jia; Wen Zhenzhong [Research Centre for Micro/Nanotechnology, Harbin Institute of Technology, No. 2 YiKuang Street, Harbin 150080 (China); Zhang Can, E-mail: hupa@hit.edu.cn [Centre for Advanced Photonics and Electronics, University of Cambridge, Cambridge CB3 0FA (United Kingdom)

    2011-08-19

    Detection of proteins is powerfully assayed in the diagnosis of diseases. A strategy for the development of an ultrahigh sensitivity biosensor based on a network single-walled carbon nanotube (SWNT) field-effect transistor (FET) has been demonstrated. Metallic SWNTs (m-SWNTs) in the network nanotube FET were selectively removed or cut via a carefully controlled procedure of electrical break-down (BD), and left non-conducting m-SWNTs which magnified the Schottky barrier (SB) area. This nanotube FET exhibited ultrahigh sensitivity and fast response to biomolecules. The lowest detection limit of 0.5 pM was achieved by exploiting streptavidin (SA) or a biotin/SA pair as the research model, and BD-treated nanotube biosensors had a 2 x 10{sup 4}-fold lower minimum detectable concentration than the device without BD treatment. The response time is in the range of 0.3-3 min.

  1. Compact, Polyvalent Mannose Quantum Dots as Sensitive, Ratiometric FRET Probes for Multivalent Protein-Ligand Interactions.

    Guo, Yuan; Sakonsinsiri, Chadamas; Nehlmeier, Inga; Fascione, Martin A; Zhang, Haiyan; Wang, Weili; Pöhlmann, Stefan; Turnbull, W Bruce; Zhou, Dejian

    2016-04-01

    A highly efficient cap-exchange approach for preparing compact, dense polyvalent mannose-capped quantum dots (QDs) has been developed. The resulting QDs have been successfully used to probe multivalent interactions of HIV/Ebola receptors DC-SIGN and DC-SIGNR (collectively termed as DC-SIGN/R) using a sensitive, ratiometric Förster resonance energy transfer (FRET) assay. The QD probes specifically bind DC-SIGN, but not its closely related receptor DC-SIGNR, which is further confirmed by its specific blocking of DC-SIGN engagement with the Ebola virus glycoprotein. Tuning the QD surface mannose valency reveals that DC-SIGN binds more efficiently to densely packed mannosides. A FRET-based thermodynamic study reveals that the binding is enthalpy-driven. This work establishes QD FRET as a rapid, sensitive technique for probing structure and thermodynamics of multivalent protein-ligand interactions. PMID:26990806

  2. pH-Sensitive Micelles Based on Double-Hydrophilic Poly(methylacrylic acid)-Poly(ethylene glycol)-Poly(methylacrylic acid) Triblock Copolymer

    Tao Youhua; Liu Ren; Liu Xiaoya; Chen Mingqing; Yang Cheng; Ni Zhongbin

    2009-01-01

    Abstract pH-sensitive micelles with hydrophilic core and hydrophilic corona were fabricated by self-assembling of triblock copolymer of poly(methylacrylic acid)-poly(ethylene glycol)-poly(methylacrylic acid) at lower solution pH. Transmission electron microscopy and laser light scattering studies showed micelles were in nano-scale with narrow size distribution. Solution pH value and the micelles concentration strongly influenced the hydrodynamic radius of the spherical micelles (48–310 ...

  3. Tunable phosphatase-sensitive stable prodrugs of 5-aminolevulinic acid for tumor fluorescence photodetection.

    Babič, Andrej; Herceg, Viktorija; Ateb, Imène; Allémann, Eric; Lange, Norbert

    2016-08-10

    5-Aminolevulinic acid (5-ALA) has been at the forefront of small molecule based fluorescence-guided tumor resection and photodynamic therapy. 5-ALA and two of its esters received marketing authorization but suffer from several major limitations, namely low stability and poor pharmacokinetic profile. Here, we present a new class of 5-ALA derivatives aiming at the stabilization of 5-ALA by incorporating a phosphatase sensitive group, with or without self-cleavable linker. Compared to 5-ALA hexyl ester (5-ALA-Hex), these compounds display an excellent stability under acidic, basic and physiological conditions. The activation and conversion into the 5-ALA is controlled and can be structure-tailored. The prodrugs display reduced acute toxicity compared to 5-ALA-Hex with superior dose response profiles of protoporphyrin IX synthesis and fluorescence intensity in human glioblastoma cells in vitro. Clinically relevant fluorescence kinetics in vivo shown in U87MG glioblastoma spheroid tumor model in chick embryos provide a solid basis for their further development and translation to clinical fluorescence guided tumor resection and photodynamic therapy. PMID:27235981

  4. A highly sensitive dual-readout assay based on gold nanoclusters for folic acid detection

    We describe a sensitive fluorometric and colorimetric dual-readout probe for folic acid (FA). It is based on the use of the gold nanoclusters (AuNCs) and cysteamine–modified gold nanoparticles (cyst-AuNPs). The bovine serum albumin stabilized AuNCs exhibit strong fluorescence emission at 652 nm. Upon addition of cyst-AuNPs, the fluorescence intensity of the AuNCs showed dramatic decrease due to the surface plasmon enhanced energy transfer process. This is due to an FA-induced aggregation of the cyst-AuNPs which shifts the absorption peaks from 530 to 670 nm. Thus, the surface plasmon enhanced energy transfer between cyst-AuNPs and AuNCs is weakened and the fluorescence intensity of AuNCs is recovered. The fluorescence intensity of the AuNCs/cyst-AuNPs system is proportional to the concentration of FA in the range from 0.11 to 2.27 μmol L−1. The dual-readout probe reported here was successfully applied to the determination of FA in spiked serum samples and folic acid tablets. (author)

  5. Repair of deoxyribonucleic acid in ultraviolet light-sensitive and -resistant Dictyostelium discoideum strains

    Some responses of the cellular slime mold Dictyostelium discoideum to ultraviolet light (uv) irradiation were investigated by analyzing two aspects of deoxyribonucleic acid (DNA) excision repair in the vegetative cells: the fate of thymine-containing dimers and the production and rejoining of single-strand breaks. Experiments were done with the parental, radiation-resistant NC-4 strain and with the radiation-sensitive γs-13 strain. The majority (greater than 85 percent) of the thymine-containing dimers produced in both strains by an energy fluence of 100 J/m2 were removed from the acid-insoluble DNA fraction within the first 3 to 4 h of reincubation in the dark. Moreover, as measured by alkaline sucrose gradients, single-strand breaks appeared in the DNA of both NC-4 and γs-13 irradiated cells very rapidly and at low temperatures. This was presumed to be a result of the incision (nicking) step of excision repair as performed by uv-specific endonuclease(s). In NC-4 the time required for dimer excision correlated with the sealing of breaks as well as with the uv-induced division delays. In γs-13 the single-strand breaks were closed at a slower rate than in NC-4. However, this was not accompanied by more extensive division delays

  6. Synthesis of Au/Graphene Oxide Composites for Selective and Sensitive Electrochemical Detection of Ascorbic Acid

    Song, Jian; Xu, Lin; Xing, Ruiqing; Li, Qingling; Zhou, Chunyang; Liu, Dali; Song, Hongwei

    2014-12-01

    In this work, we present a novel ascorbic acid (AA) sensor applied to the detection of AA in human sera and pharmaceuticals. A series of Au nanoparticles (NPs) and graphene oxide sheets (Au NP/GO) composites were successfully synthesized by reduction of gold (III) using sodium citrate. Then the Au NP/GO composites were used to construct nonenzymatic electrodes in practical AA measurement. The electrode that has the best performance presents attractive analytical features, such as a low working potential of +0.15 V, a high sensitivity of 101.86 μA mM-1 cm-2 to AA, a low detection limit of 100 nM, good reproducibility and excellent selectivity. And more,it was also employed to accurately and practically detect AA in human serum and clinical vitamin C tablet with the existence of some food additive. The enhanced AA electrochemical properties of the Au NP/GO modified electrode in our work can be attributed to the improvement of electroactive surface area of Au NPs and the synergistic effect from the combination of Au NPs and GO sheets. This work shows that the Au NP/GO/GCEs hold the prospect for sensitive and selective determination of AA in practical clinical application.

  7. Development of Anodic Titania Nanotubes for Application in High Sensitivity Amperometric Glucose and Uric Acid Biosensors

    Tai-Ping Sun

    2013-10-01

    Full Text Available The purpose of this study was to develop novel nanoscale biosensors using titania nanotubes (TNTs made by anodization. Titania nanotubes were produced on pure titanium sheets by anodization at room temperature. In this research, the electrolyte composition ethylene glycol 250 mL/NH4F 1.5 g/DI water 20 mL was found to produce the best titania nanotubes array films for application in amperometric biosensors. The amperometric results exhibit an excellent linearity for uric acid (UA concentrations in the range between 2 and 14 mg/dL, with 23.3 (µA·cm−2·(mg/dL−1 UA sensitivity, and a correlation coefficient of 0.993. The glucose biosensor presented a good linear relationship in the lower glucose concentration range between 50 and 125 mg/dL, and the corresponding sensitivity was approximately 249.6 (µA·cm−2·(100 mg/dL−1 glucose, with a correlation coefficient of 0.973.

  8. Specificity and sensitivity of noninvasive measurement of pulmonary vascular protein leak

    Noninvasive techniques employing external counting of radiolabeled protein have the potential for measuring pulmonary vascular protein permeability, but their specificity and sensitivity remain unclear. The authors tested the specificity and sensitivity of a double-radioisotope method by injecting radiolabeled albumin (131I) and erythrocytes (/sup 99m/Tc) into anesthetized dogs and measuring the counts of each isotope for 150 min after injection with an external gamma probe fixed over the lung. They calculated the rate of increase of albumin counts measured by the probe (which reflects the rate at which protein leaks into the extravascular space). To assess permeability the authors normalized the rate of increase in albumin counts for changes in labeled erythrocyte signal to minimize influence of changes in vascular surface area and thus derived an albumin leak index. They measured the albumin leak index and gravimetric lung water during hydrostatic edema (acutely elevating left atrial pressure by left atrial balloon inflation: mean pulmonary arterial wedge pressure = 22.6 Torr) and in lung injury edema induced by high- (1.0 g/kg) and low-dose (0.25 g/kg) intravenous thiourea. To test specificity hydrostatic and high-dose thiourea edema were compared. The albumin leak index increased nearly fourfold from control after thiourea injury (27.2 +/- 2.3 x 10-4 vs. 7.6 +/- 0.9 x 10-4 min-1) but did not change from control levels after elevating left atrial pressure (8.9 +/- 1.2 x 10-4 min-1) despite comparable increases in gravimetric lung water. To test sensitivity the authors compared low-dose thiourea with controls. Following low-dose thiourea, the albumin leak index nearly doubled despite the absence of a measurable increase in lung water

  9. Sequence and structural features of binding site residues in protein-protein complexes: comparison with protein-nucleic acid complexes

    Selvaraj S; Jayaram B; Saranya N; Gromiha M; Fukui Kazuhiko

    2011-01-01

    Abstract Background Protein-protein interactions are important for several cellular processes. Understanding the mechanism of protein-protein recognition and predicting the binding sites in protein-protein complexes are long standing goals in molecular and computational biology. Methods We have developed an energy based approach for identifying the binding site residues in protein–protein complexes. The binding site residues have been analyzed with sequence and structure based parameters such...

  10. A high sensitivity assay for the inflammatory marker C-Reactive protein employing acoustic biosensing

    Cooper Matthew A

    2008-04-01

    Full Text Available Abstract C-Reactive Protein (CRP is an acute phase reactant routinely used as a biomarker to assess either infection or inflammatory processes such as autoimmune diseases. CRP also has demonstrated utility as a predictive marker of future risk of cardiovascular disease. A new method of immunoassay for the detection of C-Reactive Protein has been developed using Resonant Acoustic Profiling™ (RAP™ with comparable sensitivity to a high sensitivity CRP ELISA (hsCRP but with considerable time efficiency (12 minutes turnaround time to result. In one method, standard solutions of CRP (0 to 231 ng/mL or diluted spiked horse serum sample are injected through two sensor channels of a RAP™ biosensor. One contains a surface with sheep antibody to CRP, the other a control surface containing purified Sheep IgG. At the end of a 5-minute injection the initial rate of change in resonant frequency was proportional to CRP concentration. The initial rates of a second sandwich step of anti-CRP binding were also proportional to the sample CRP concentration and provided a more sensitive method for quantification of CRP. The lower limit of detection for the direct assay and the homogenous sandwich assay were both 20 ng/mL whereas for the direct sandwich assay the lower limit was 3 ng/mL. In a step towards a rapid clinical assay, diluted horse blood spiked with human CRP was passed over one sensor channel whilst a reference standard solution at the borderline cardiovascular risk level was passed over the other. A semi-quantities ratio was thus obtained indicative of sample CRP status. Overall, the present study revealed that CRP concentrations in serum that might be expected in both normal and pathological conditions can be detected in a time-efficient, label-free immunoassay with RAP™ detection technology with determined CRP concentrations in close agreement with those determined using a commercially available high sensitivity ELISA.

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

    Hesse, Almut

    2016-01-01

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

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

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

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

    Hesse, Almut; Weller, Michael G

    2016-01-01

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

  14. Nucleic acid chaperons: a theory of an RNA-assisted protein folding

    Biro Jan C

    2005-09-01

    Full Text Available Summary Background Proteins are assumed to contain all the information necessary for unambiguous folding (Anfinsen's principle. However, ab initio structure prediction is often not successful because the amino acid sequence itself is not sufficient to guide between endless folding possibilities. It seems to be a logical to try to find the "missing" information in nucleic acids, in the redundant codon base. Results mRNA energy dot plots and protein residue contact maps were found to be rather similar. The structure of mRNA is also conserved if the protein structure is conserved, even if the sequence similarity is low. These observations led me to suppose that some similarity might exist between nucleic acid and protein folding. I found that amino acid pairs, which are co-located in the protein structure, are preferentially coded by complementary codons. This codon complementarity is not perfect; it is suboptimal where the 1st and 3rd codon residues are complementary to each other in reverse orientation, while the 2nd codon letters may be, but are not necessarily, complementary. Conclusion Partial complementary coding of co-locating amino acids in protein structures suggests that mRNA assists in protein folding and functions not only as a template but even as a chaperon during translation. This function explains the role of wobble bases and answers the mystery of why we have a redundant codon base.

  15. [The response of the blood proteins to ablation of the capsaicin-sensitive nerves].

    Kostina, N E; Spiridonov, V K

    2005-07-01

    Effects of neurotoxic doses of capsaicin (150 mg/kg) on the protein content in electrophoretic fractions (PAAG) in the Wistar rat plasma were studied. In early period (7 days) after administration of capsaicin, an increase of the alpha1-, alpha2-globulins and a decrease of the albumin, gamma-globulins, were observed. After 14-30 days, increase of the albumin and decrease of the alpha1-, gammay-globulins were detected. The ablation of the capsaicin-sensitive nerves abrogated the changes of positive and negative acute phase reactants induced by zymosan and diminished the content of gamma-globulins. PMID:16206619

  16. Skin sensitization: Modeling based on skin metabolism simulation and formation of protein conjugates

    Dimitrov, Sabcho; Low, Lawrence; Patlewicz, Grace;

    2005-01-01

    . The covalent interactions of chemicals and their metabolites with skin proteins were described by 83 reactions that fall within 39 alerting groups. The SAR/QSAR system developed was able to correctly classify about 80% of the chemicals with significant sensitizing effect and 72% of nonsensitizing...... chemicals. For some alerting groups, three-dimensional (3D)-QSARs were developed to describe the multiplicity of physicochemical, steric, and electronic parameters. These 3D-QSARs, so-called pattern recognition-type models, were applied each time a latent alerting group was identified in a parent chemical...

  17. Possible involvement of lipoic acid in binding protein-dependent transport systems in Escherichia coli.

    Richarme, G

    1985-01-01

    We describe the properties of the binding protein dependent-transport of ribose, galactose, and maltose and of the lactose permease, and the phosphoenolpyruvate-glucose phosphotransferase transport systems in a strain of Escherichia coli which is deficient in the synthesis of lipoic acid, a cofactor involved in alpha-keto acid dehydrogenation. Such a strain can grow in the absence of lipoic acid in minimal medium supplemented with acetate and succinate. Although the lactose permease and the p...

  18. Thermodynamic principles for the engineering of pH-driven conformational switches and acid insensitive proteins

    Bell-Upp, Peregrine; Robinson, Aaron C.; Whitten, Steven; Wheeler, Erika L.; Lin, Janine; Stites, Wesley E.; García-Moreno E., Bertrand

    2011-01-01

    The general thermodynamic principles behind pH driven conformational transitions of biological macromolecules are well understood. What is less obvious is how they can be used to engineer pH switches in proteins. The acid unfolding of staphylococcal nuclease (SNase) was used to illustrate different factors that can affect pH-driven conformational transitions. Acid unfolding is a structural transition driven by preferential H+ binding to the acid unfolded state (U) over the native (N) state of...

  19. Heart-type fatty-acid-binding protein (FABP3) is a lysophosphatidic acid-binding protein in human coronary artery endothelial cells

    Tsukahara, Ryoko; Haniu, Hisao; Matsuda, Yoshikazu; Tsukahara, Tamotsu

    2014-01-01

    Fatty-acid-binding protein 3, muscle and heart (FABP3), also known as heart-type FABP, is a member of the family of intracellular lipid-binding proteins. It is a small cytoplasmic protein with a molecular mass of about 15 kDa. FABPs are known to be carrier proteins for transporting fatty acids and other lipophilic substances from the cytoplasm to the nucleus, where these lipids are released to a group of nuclear receptors such as peroxisome proliferator-activated receptors (PPARs). In this study, using lysophosphatidic acid (LPA)-coated agarose beads, we have identified FABP3 as an LPA carrier protein in human coronary artery endothelial cells (HCAECs). Administration of LPA to HCAECs resulted in a dose-dependent increase in PPARγ activation. Furthermore, the LPA-induced PPARγ activation was abolished when the FABP3 expression was reduced using small interfering RNA (siRNA). We further show that the nuclear fraction of control HCAECs contained a significant amount of exogenously added LPA, whereas FABP3 siRNA-transfected HCAECs had a decreased level of LPA in the nucleus. Taken together, these results suggest that FABP3 governs the transcriptional activities of LPA by targeting them to cognate PPARγ in the nucleus. PMID:25426414

  20. Heart-type fatty-acid-binding protein (FABP3 is a lysophosphatidic acid-binding protein in human coronary artery endothelial cells

    Ryoko Tsukahara

    2014-01-01

    Full Text Available Fatty-acid-binding protein 3, muscle and heart (FABP3, also known as heart-type FABP, is a member of the family of intracellular lipid-binding proteins. It is a small cytoplasmic protein with a molecular mass of about 15 kDa. FABPs are known to be carrier proteins for transporting fatty acids and other lipophilic substances from the cytoplasm to the nucleus, where these lipids are released to a group of nuclear receptors such as peroxisome proliferator-activated receptors (PPARs. In this study, using lysophosphatidic acid (LPA-coated agarose beads, we have identified FABP3 as an LPA carrier protein in human coronary artery endothelial cells (HCAECs. Administration of LPA to HCAECs resulted in a dose-dependent increase in PPARγ activation. Furthermore, the LPA-induced PPARγ activation was abolished when the FABP3 expression was reduced using small interfering RNA (siRNA. We further show that the nuclear fraction of control HCAECs contained a significant amount of exogenously added LPA, whereas FABP3 siRNA-transfected HCAECs had a decreased level of LPA in the nucleus. Taken together, these results suggest that FABP3 governs the transcriptional activities of LPA by targeting them to cognate PPARγ in the nucleus.

  1. Electrochemistry of proteins and nucleic acids. Tools for biomedicine

    Paleček, Emil; Ostatná, Veronika; Masařík, Michal; Dorčák, Vlastimil; Trefulka, Mojmír; Havran, Luděk; Fojta, Miroslav

    Benediktbeuern, 2006. s. 1-1. [10th International Fischer Symposium. 23.07.2006-26.07.2006, Benediktbeuern] Institutional research plan: CEZ:AV0Z50040507 Keywords : electrochemistry of DNA * electroactivity of proteins Subject RIV: BO - Biophysics

  2. D-amino acid oxidase gene therapy sensitizes glioma cells to the antiglycolytic effect of 3-bromopyruvate.

    El Sayed, S M; Abou El-Magd, R M; Shishido, Y; Chung, S P; Sakai, T; Watanabe, H; Kagami, S; Fukui, K

    2012-01-01

    Glioma tumors are refractory to conventional treatment. Glioblastoma multiforme is the most aggressive type of primary brain tumors in humans. In this study, we introduce oxidative stress-energy depletion (OSED) therapy as a new suggested treatment for glioblastoma. OSED utilizes D-amino acid oxidase (DAO), which is a promising therapeutic protein that induces oxidative stress and apoptosis through generating hydrogen peroxide (H2O2). OSED combines DAO with 3-bromopyruvate (3BP), a hexokinase II (HK II) inhibitor that interferes with Warburg effect, a metabolic alteration of most tumor cells that is characterized by enhanced aerobic glycolysis. Our data revealed that 3BP induced depletion of energetic capabilities of glioma cells. 3BP induced H2O2 production as a novel mechanism of its action. C6 glioma transfected with DAO and treated with D-serine together with 3BP-sensitized glioma cells to 3BP and decreased markedly proliferation, clonogenic power and viability in a three-dimensional tumor model with lesser effect on normal astrocytes. DAO gene therapy using atelocollagen as an in vivo transfection agent proved effective in a glioma tumor model in Sprague-Dawley (SD) rats, especially after combination with 3BP. OSED treatment was safe and tolerable in SD rats. OSED therapy may be a promising therapeutic modality for glioma. PMID:21921941

  3. Effective gene delivery using stimulus-responsive catiomer designed with redox-sensitive disulfide and acid-labile imine linkers.

    Cai, Xiaojun; Dong, Chunyan; Dong, Haiqing; Wang, Gangmin; Pauletti, Giovanni M; Pan, Xiaojing; Wen, Huiyun; Mehl, Isaac; Li, Yongyong; Shi, Donglu

    2012-04-01

    A dual stimulus-responsive mPEG-SS-PLL(15)-glutaraldehyde star (mPEG-SS-PLL(15)-star) catiomer is developed and biologically evaluated. The catiomer system combines redox-sensitive removal of an external PEG shell with acid-induced escape from the endosomal compartment. The design rationale for PEG shell removal is to augment intracellular uptake of mPEG-SS-PLL(15)-star/DNA complexes in the presence of tumor-relevant glutathione (GSH) concentration, while the acid-induced dissociation is to accelerate the release of genetic payload following successful internalization into targeted cells. Size alterations of complexes in the presence of 10 mM GSH suggest stimulus-induced shedding of external PEG layers under redox conditions that intracellularly present in the tumor microenvironment. Dynamic laser light scattering experiments under endosomal pH conditions show rapid destabilization of mPEG-SS-PLL(15)-star/DNA complexes that is followed by facilitating efficient release of encapsulated DNA, as demonstrated by agarose gel electrophoresis. Biological efficacy assessment using pEGFP-C1 plasmid DNA encoding green fluorescence protein and pGL-3 plasmid DNA encoding luciferase as reporter genes indicate comparable transfection efficiency of 293T cells of the catiomer with a conventional polyethyleneimine (bPEI-25k)-based gene delivery system. These experimental results show that mPEG-SS-PLL(15)-star represents a promising design for future nonviral gene delivery applications with high DNA binding ability, low cytotoxicity, and high transfection efficiency. PMID:22443494

  4. Palmitoleic acid reduces intramuscular lipid and restores insulin sensitivity in obese sheep

    Duckett SK

    2014-11-01

    Full Text Available Susan K Duckett, Gabriela Volpi-Lagreca, Mariano Alende, Nathan M LongAnimal and Veterinary Sciences Department, Clemson University, Clemson, SC, USAAbstract: Obese sheep were used to assess the effects of palmitoleic (C16:1 cis-9 acid infusion on lipogenesis and circulating insulin levels. Infusion of 10 mg/kg body weight (BW/day C16:1 intravenously in obese sheep reduced (P<0.01 weight gain by 77%. Serum palmitoleic levels increased (P<0.05 in a linear manner with increasing levels of C16:1 infusion. Cis-11 vaccenic (C18:1 cis-11 acid, a known elongation product of palmitoleic acid, was also elevated (P<0.05 in serum after 14 days and 21 days of infusion. Plasma insulin levels were lower (P<0.05 (10 mg/kg BW/day C16:1 than controls (0 mg/kg BW/day C16:1 at 14 days and 28 days of infusion. Infusion of C16:1 resulted in linear increases in tissue concentrations of palmitoleic, cis-11 vaccenic, eicosapentaenoic, and docosapentaenoic acids in a dose-dependent manner. Total lipid content of the semitendinosus (ST muscle and mesenteric adipose tissue was reduced (P<0.01 in both 5 mg/kg and 10 mg/kg BW C16:1 dose levels. Total lipid content and mean adipocyte size in the longissimus muscle was reduced (P<0.05 in the 10 mg/kg BW C16:1 dose level only, whereas total lipid content and adipocyte size of the subcutaneous adipose tissue was not altered. Total lipid content of the liver was also unchanged with C16:1 infusion. Palmitoleic acid infusion upregulated (P<0.05 acetyl-CoA carboxylase (ACC, fatty acid elongase-6 (ELOVL6, and Protein kinase, AMP-activated, alpha 1 catalytic subunit, transcript variant 1 (AMPK mRNA expressions in liver, subcutaneous adipose, and ST muscle compared to the controls. However, mRNA expression of glucose transporter type 4 (GLUT4 and carnitine palmitoyltransferase 1b (CPT1B differed between tissues. In the subcutaneous adipose and liver, C16:1 infusion upregulated (P<0.05 GLUT4 and CPT1B, whereas these genes were

  5. Representation of protein-sequence information by amino acid subalphabets

    Andersen, C.A.F.; Brunak, Søren

    2004-01-01

    Within computational biology, algorithms are constructed with the aim of extracting knowledge from biological data, in particular, data generated by the large genome projects, where gene and protein sequences are produced in high volume. In this article, we explore new ways of representing protei......-which now are common in proteins-might have emerged from simpler selections, or alphabets, in use earlier during the evolution of living organisms....

  6. Nitrogen detected TROSY at high field yields high resolution and sensitivity for protein NMR

    Detection of 15N in multidimensional NMR experiments of proteins has sparsely been utilized because of the low gyromagnetic ratio (γ) of nitrogen and the presumed low sensitivity of such experiments. Here we show that selecting the TROSY components of proton-attached 15N nuclei (TROSY 15NH) yields high quality spectra in high field magnets (>600 MHz) by taking advantage of the slow 15N transverse relaxation and compensating for the inherently low 15N sensitivity. The 15N TROSY transverse relaxation rates increase modestly with molecular weight but the TROSY gain in peak heights depends strongly on the magnetic field strength. Theoretical simulations predict that the narrowest line width for the TROSY 15NH component can be obtained at 900 MHz, but sensitivity reaches its maximum around 1.2 GHz. Based on these considerations, a 15N-detected 2D 1H–15N TROSY-HSQC (15N-detected TROSY-HSQC) experiment was developed and high-quality 2D spectra were recorded at 800 MHz in 2 h for 1 mM maltose-binding protein at 278 K (τc ∼ 40 ns). Unlike for 1H detected TROSY, deuteration is not mandatory to benefit 15N detected TROSY due to reduced dipolar broadening, which facilitates studies of proteins that cannot be deuterated, especially in cases where production requires eukaryotic expression systems. The option of recording 15N TROSY of proteins expressed in H2O media also alleviates the problem of incomplete amide proton back exchange, which often hampers the detection of amide groups in the core of large molecular weight proteins that are expressed in D2O culture media and cannot be refolded for amide back exchange. These results illustrate the potential of 15NH-detected TROSY experiments as a means to exploit the high resolution offered by high field magnets near and above 1 GHz

  7. Nitrogen detected TROSY at high field yields high resolution and sensitivity for protein NMR

    Takeuchi, Koh [National Institute for Advanced Industrial Science and Technology, Molecular Profiling Research Center for Drug Discovery (Japan); Arthanari, Haribabu [Harvard Medical School, Department of Biochemistry and Molecular Pharmacology (United States); Shimada, Ichio, E-mail: shimada@iw-nmr.f.u-tokyo.ac.jp [National Institute for Advanced Industrial Science and Technology, Molecular Profiling Research Center for Drug Discovery (Japan); Wagner, Gerhard, E-mail: gerhard-wagner@hms.harvard.edu [Harvard Medical School, Department of Biochemistry and Molecular Pharmacology (United States)

    2015-12-15

    Detection of {sup 15}N in multidimensional NMR experiments of proteins has sparsely been utilized because of the low gyromagnetic ratio (γ) of nitrogen and the presumed low sensitivity of such experiments. Here we show that selecting the TROSY components of proton-attached {sup 15}N nuclei (TROSY {sup 15}N{sub H}) yields high quality spectra in high field magnets (>600 MHz) by taking advantage of the slow {sup 15}N transverse relaxation and compensating for the inherently low {sup 15}N sensitivity. The {sup 15}N TROSY transverse relaxation rates increase modestly with molecular weight but the TROSY gain in peak heights depends strongly on the magnetic field strength. Theoretical simulations predict that the narrowest line width for the TROSY {sup 15}N{sub H} component can be obtained at 900 MHz, but sensitivity reaches its maximum around 1.2 GHz. Based on these considerations, a {sup 15}N-detected 2D {sup 1}H–{sup 15}N TROSY-HSQC ({sup 15}N-detected TROSY-HSQC) experiment was developed and high-quality 2D spectra were recorded at 800 MHz in 2 h for 1 mM maltose-binding protein at 278 K (τ{sub c} ∼ 40 ns). Unlike for {sup 1}H detected TROSY, deuteration is not mandatory to benefit {sup 15}N detected TROSY due to reduced dipolar broadening, which facilitates studies of proteins that cannot be deuterated, especially in cases where production requires eukaryotic expression systems. The option of recording {sup 15}N TROSY of proteins expressed in H{sub 2}O media also alleviates the problem of incomplete amide proton back exchange, which often hampers the detection of amide groups in the core of large molecular weight proteins that are expressed in D{sub 2}O culture media and cannot be refolded for amide back exchange. These results illustrate the potential of {sup 15}N{sub H}-detected TROSY experiments as a means to exploit the high resolution offered by high field magnets near and above 1 GHz.

  8. Frequency of U.V. induced protein-DNA crosslinks in cell lines of different sensitivities

    U.V.-induced protein-DNA crosslinking has been measured in two cultured human cell lines of different sensitivities. Using a previously published method, involving SDS-protein precipitation, a biphasic response was obtained with an initial slope of 0.6 per cent DNA J-1 m2 up to 50 Jm-2 and a second-phase slope of 0.12 per cent DNA J-1 m2 with a background of 22 +- 13 per cent. Rigorous washing of SDS-protein precipitates reduced background binding to about 5 per cent with a linear U.V. effect up to 100 Jm-2 of 0.038 per cent DNA J-1 m2. Binding was judged to be covalent on the grounds of stability to boiling and represented 4.1 crosslinks pg DNA-1 J-1 m2 or 60 crosslinks cell-1 J-1 m2. Similar results were obtained for both cell lines. It was concluded that the differences in U.V. survival between cell lines is not related to the extent of protein-DNA crosslinking. It was impossible to detect repair of these lesions in either cell line. (author)

  9. A continuous displacement immunoassay for human heart-type fatty acid-binding protein in plasma

    van der Voort, D; Pelsers, MMAL; Korf, J; Hermens, WT; Glatz, JFC

    2004-01-01

    Human heart-type fatty acid-binding protein (FABP) is suggested as an early plasma marker of acute myocardial infarction (AMI), and several studies have proved that, for early diagnosis of AMI, FABP performs better than myoglobin, which is a more often used early marker protein. Because serial measu

  10. CONCENTRATION OF GLIAL FIBRILLARY ACIDIC PROTEIN INCREASES WITH AGE IN THE MOUSE AND RAT BRAIN

    The role of aging in the expression of the astrocyte protein, glial fibrillary acidic protein (GFAP), was examined. n both mice and rats the concentration of GFAP increased throughout the brain as a function of aging. he largest increase (2-fold) was observed in striatum for both...

  11. INCREASE IN GLIAL FIBRILLARY ACIDIC PROTEIN FOLLOWS BRAIN HYPERTHERMIA IN RATS

    Previously, the authors have demonstrated that an increase in the astrocyte-associated protein, glial fibrillary acidic protein (GFAP), accompanies brain injury induced by a variety of chemical insults. In the present study the authors examined the effects of microwave-induced hy...

  12. Insulin and amino acids stimulate whole body protein synthesis in neonates

    Insulin and amino acids (AA) stimulate muscle protein synthesis in neonatal pigs. To determine the effects of insulin and AA on whole body protein turnover, hyperinsulinemic (0 and 100 ng/(kg[0.66]/min))-euglycemic-AA clamps were performed during euaminoacidemia or hyperaminoacidemia in fasted 7-d-...

  13. Urinary excretion of fatty acid-binding proteins in idiopathic membranous nephropathy.

    Hofstra, J.M.; Deegens, J.K.J.; Steenbergen, E.J.; Wetzels, J.F.M.

    2008-01-01

    BACKGROUND: It is suggested that proteinuria contributes to progressive renal failure by inducing tubular cell injury. The site of injury is unknown. Most studies have used markers of proximal tubular cell damage. Fatty acid-binding proteins (FABPs) are intracellular carrier proteins with different

  14. Dietary proteins extend the survival of salmonella dublin in a gastric Acid environment

    Birk, Tina; Kristensen, Kim; Harboe, Anne;

    2012-01-01

    Dublin, and found that the addition of proteins such as pepsin, ovalbumin, and blended turkey meat to the simple gastric acid model significantly delayed pathogen inactivation compared with the control, for which no proteins were added. In contrast, no delay in inactivation was observed in the presence...

  15. Umami taste amino acids produced by hydrolyzing extracted protein from tomato seed meal

    Enzymatic hydrolysis was performed for extracting protein to prepare umami taste amino acids from defatted tomato seed meal (DTSM) which is a by-product of tomato processing. Papain was used as an enzyme for the hydrolysis of DTSM. The particle size distribution of DTSM, protein concentration and fr...

  16. Isoelectric Point, Electric Charge, and Nomenclature of the Acid-Base Residues of Proteins

    Maldonado, Andres A.; Ribeiro, Joao M.; Sillero, Antonio

    2010-01-01

    The main object of this work is to present the pedagogical usefulness of the theoretical methods, developed in this laboratory, for the determination of the isoelectric point (pI) and the net electric charge of proteins together with some comments on the naming of the acid-base residues of proteins. (Contains 8 figures and 4 tables.)

  17. Protein Content and Amino Acid Composition in Grains of Wheat-Related Species

    JIANG Xiao-ling; TIAN Ji-chun; HAO Zhi; ZHANG Wei-dong

    2008-01-01

    The protein content and amino acid composition for 17 wheat-related species(WRS)and three common wheats(control) were determined and analyzed,and the essential amino acids(EAAs)in WRS were evaluated according to FAO/WHO amino acid recommendations.The results showed that the mean protein content for WRS was 16.67%,which was 23.21% higher than that for the control.The mean contents(g 100 g-1 protein)of most amino acids for WRS were lysine 2.74%,threonine 2.83%,phenylalanine 4.17%,isoleucine 3.42%,valine 3.90%,histidine 2.81%,glutamic acid 29.96%,proline 9.12%,glycine 3.59%,alanine 3.37%,and cysteine 1.57%,which were higher than those for the control.The contents of the other 6 amino acids for WRS were lower than those for the control.The materials(Triticum monococcum L.,Triticum carthlicum Nevski,and Triticum turgidum L.)contained relatively high concentration of the most deficient EAAs(lysine, threonine,and methionine).Comparing with FAO/WHO amino acid recommendations,the amino acid scores(AAS)of lysine(49.8%),threonine(70.7%),and sulfur-containing amino acids(74.8%)were the lowest,which were considered as the main limiting amino acids in WRS.It was observed that the materials with Triticum urartu Tum.(AA)and Aegilops speltoides Tausch.(SS)genomes had relatively high contents of protein and EAA.The contents of protein(16.91%), phenylalanine(4.78%),isoleucine(3.53%),leucine(6.16%),and valine(4.09%)for the diploid materials were higher than those for the other materials.These results will provide some information for selecting parents in breeding about nutrient quality and utilization of fine gene in wheat.

  18. Sensitive spectrophotometric determination of ascorbic acid in drugs and foods using surface plasmon resonance band of silver nanoparticles

    Kobra Zarei

    2015-12-01

    Full Text Available A simple and sensitive procedure was proposed for spectrophotometric determination of ascorbic acid. It was found that the reduction of Ag+ to silver nanoparticles (Ag-NPs by ascorbic acid in the presence of polyvinylpyrrolidone (PVP as a stabilizing agent produce very intense surface plasmon resonance peak of Ag-NPs. The plasmon absorbance of the Ag-NPs at λ = 440 nm allows the quantitative spectrophotometric detection of the ascorbic acid. The calibration curve was linear with concentration of ascorbic acid in the range of 0.5–60 μM. The detection limit was obtained as 0.08 μM. The influence of potential interfering substances on the determination of ascorbic acid was studied. The proposed method was successfully applied for the determination of ascorbic acid in some powdered drink mixtures, commercial orange juice, natural orange juice, vitamin C injection, effervescent tablet, and multivitamin tablet.

  19. Mesoporous silica nanoparticles with bilayer coating of poly(acrylic acid-co-itaconic acid) and human serum albumin (HSA): A pH-sensitive carrier for gemcitabine delivery.

    Pourjavadi, Ali; Tehrani, Zahra Mazaheri

    2016-04-01

    Novel bilayer coated mesoporous silica nanoparticle (MCM-41) based on pH sensitive poly(acrylic acid-co-itaconic acid) and human serum albumin (HSA) was designed for controlled delivery of gemcitabine (anticancer drug) to cancer cells. The shell around the mesoporous silica has bilayer structure. Poly(acrylic acid-co-itaconic acid) was used as pH-sensitive inner shell and human serum albumin, HSA, was used as outer shell. The core-shell structure was formed due to electrostatic interaction between ammonium groups of modified MCM-41 and carboxylate groups of copolymer. Also, the albumin layer was wrapped around the copolymer coated nanoparticle by electrostatic interaction between ammonium groups from protein and carboxylate ions of copolymer shell. Moreover, the maximum release occurred at pH 5.5 (pH of endosomes) because the bilayer shell collapsed at this pH. The drug nanocarrier would be a good candidate for tumor therapy due to its biocompatibility, controlled release and pH responsive behavior. PMID:26838909

  20. Influence of Amino Acids, Dietary Protein, and Physical Activity on Muscle Mass Development in Humans

    Lars Holm

    2013-03-01

    Full Text Available Ingestion of protein is crucial for maintenance of a variety of body functions and within the scope of this review we will specifically focus on the regulation of skeletal muscle mass. A quantitative limitation exists as to how much muscle protein the body can synthesize in response to protein intake. Ingestion of excess protein exerts an unwanted load to the body and therefore, it is important to find the least amount of protein that provides the maximal hypertrophic stimulus. Hence, research has focused on revealing the relationship between protein intake (dose and its resulting stimulation of muscle protein synthesis (response. In addition to the protein amount, the protein digestibility and, hence, the availability of its constituent amino acids is decisive for the response. In this regard, recent studies have provided in-depth knowledge about the time-course of the muscle protein synthetic response dependent on the characteristics of the protein ingested. The effect of protein intake on muscle protein accretion can further be stimulated by prior exercise training. In the ageing population, physical training may counteract the development of “anabolic resistance” and restore the beneficial effect of protein feeding. Presently, our knowledge is based on measures obtained in standardized experimental settings or during long-term intervention periods. However, to improve coherence between these types of data and to further improve our knowledge of the effects of protein ingestion, other investigative approaches than those presently used are requested.

  1. Acidic preparations of platelet concentrates release bone morphogenetic protein-2.

    Wahlström, Ola; Linder, Cecilia; Kalén, Anders; Magnusson, Per

    2008-01-01

    BACKGROUND AND PURPOSE: Growth factors released from platelets have potent effects on fracture and wound healing. The acidic tide of wound healing, i.e. the pH within wounds and fractures, changes from acidic pH to neutral and alkaline pH as the healing process progresses. We investigated the influence of pH on lysed platelet concentrates regarding the release of growth factors. MATERIAL AND METHODS: Platelet concentrates free of leukocyte components were lysed and incubated in buffers with p...

  2. Crude protein, fibre and phytic acid in vitro digestibility of selected legume and buckwheat samples

    Vojtíšková, Petra; Kráčmar, Stanislav

    2013-01-01

    The aim of this study was to determine crude protein, fi bre and phytic acid in vitro digestibility of selected legumes and buckwheat products. All analyses except the phytic acid contents were performed in the line with the Commission Regulation (EC) No. 152/2009. A modifi ed version of Holt's Method was used for phytic acid (phytate) determination. None of all samples contained more than 11% of moisture. Soybeans are rich in crude protein; they contain nearly 40% of this compound. The conte...

  3. Cy5 maleimide labelling for sensitive detection of free thiols in native protein extracts: identification of seed proteins targeted by barley thioredoxin h isoforms

    Maeda, K.; Finnie, Christine; Svensson, Birte

    2004-01-01

    thioredoxin-targeted proteins based on two-dimensional electrophoresis and fluorescence labelling of thiol groups was achieved by application of a highly sensitive Cy5 maleimide dye and large-format two-dimensional gels, resulting in a 10-fold increase in the observed number of labelled protein spots. The...

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

    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.

  5. Protein and lipid deposition rates in male broiler chickens : separate responses to amino acids and protein-free energy

    Eits, R.M.; Kwakkel, R.P.; Verstegen, M.W.A.; Stoutjesdijk, P.; Greef, de K.H.

    2002-01-01

    Two experiments of similar design were conducted with male broiler chickens over two body weight ranges, 200 to 800 g in Experiment 1 and 800 to 1,600 g in Experiment 2. The data were used to test the hypothesis that protein deposition rate increases (linearly) with increasing amino acid intake, unt

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

    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 i...

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

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

    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 ...

  8. “Fuzzy oil drop” model applied to individual small proteins built of 70 amino acids

    Prymula, Katarzyna; Sałapa, Kinga; Roterman, Irena

    2010-01-01

    Abstract The proteins composed of short polypeptides (about 70 amino acid residues) representing the following functional groups (according to PDB notation): growth hormones, serine protease inhibitors, antifreeze proteins, chaperones and proteins of unknown function, were selected for structural and functional analysis. Classification based on the distribution of hydrophobicity in terms of deficiency/excess as the measure of structural and functional specificity is presented. The ...

  9. Regulation of protein degradation pathways by amino acids and insulin in skeletal muscle of neonatal pigs

    Suryawan, Agus; Davis, Teresa A.

    2014-01-01

    Background The rapid gain in lean mass in neonates requires greater rates of protein synthesis than degradation. We previously delineated the molecular mechanisms by which insulin and amino acids, especially leucine, modulate skeletal muscle protein synthesis and how this changes with development. In the current study, we identified mechanisms involved in protein degradation regulation. In experiment 1, 6- and 26-d-old pigs were studied during 1) euinsulinemic-euglycemic-euaminoacidemic, 2) e...

  10. Acid Hydrolysis of Wheat Gluten Induces Formation of New Epitopes but Does Not Enhance Sensitizing Capacity by the Oral Route: A Study in “Gluten Free” Brown Norway Rats

    Kroghsbo, Stine; Andersen, Nanna Birch; Rasmussen, Tina Frid;

    2014-01-01

    sensitizing capacity of gluten proteins per se when altered by acid or enzymatic hydrolysis relative to unmodified gluten in rats naïve to gluten. Methods High IgE-responder Brown Norway (BN) rats bred on a gluten-free diet were sensitized without the use of adjuvant to three different gluten products...... (unmodified, acid hydrolyzed and enzymatic hydrolyzed). Rats were sensitized by intraperitoneal (i.p.) immunization three times with 200 µg gluten protein/rat or by oral dosing for 35 days with 0.2, 2 or 20 mg gluten protein/rat/day. Sera were analyzed for specific IgG and IgE and IgG-binding capacity by...... ELISA. IgE functionality was measured by rat basophilic leukemia (RBL) assay. Results Regardless of the route of dosing, all products had sensitizing capacity. When sensitized i.p., all three gluten products induced a strong IgG1 response in all animals. Acid hydrolyzed gluten induced the highest level...

  11. Evaluation of restoration of sensitivities of resistant staphylococcus aureus isolates by using cefuroxime and clavulanic acid in combination

    The present study was planned to observe the activity of cefuroxime, a second generation cephalosporin after combining it with a beta-lactamase inhibitor calvulanic acid. The study was conducted to evaluate the restoration or increase in sensitivity of beta-lactamase producing isolates of Staphylococcus aureus. Staphylococcus aureus were identified by standard procedures. For beta-lactamase detection chromogenic Nitrocefin impregnated sticks were used. The sensitivity of the bacteria to the antibiotic disks was measured by disk diffusion method using standard zone diameter criteria given by National Committee of Clinical Laboratory Standards. The disks of cefuroxime with clavulanic acid had developed larger zones of inhibition. The activity of cefuroxime against Staphylococcus areus was significantly increased by clavulanic acid. Clavulanic acid if used in combination with cefuroxime, can improve the antimicrobial activity of cefuroxime against beta - lactamase producing Staphylococcus aureus. (author)

  12. Downregulation of clusterin mediates sensitivity to protein kinase inhibitors in breast cancer cells.

    Redondo, Maximino; García-Aranda, Marilina; Roldan, Maria J; Callejón, Gonzalo; Serrano, Alfonso; Jiménez, Eugenio; Téllez, Teresa

    2015-01-01

    The efficacy of protein kinase inhibitors (PKIs) has been shown in clinical assays for cancer, but as isolated agents, they only have a modest effect. One of the most important characteristics of mitogen-activated PKIs is their ability to decrease the apoptotic threshold of cancer cells, sensitizing them to the action of other antiapoptotic agents. The secretory clusterin protein is an inhibitor of apoptosis with a cytoprotective function. We describe the use of clusterin-specific antisense oligonucleotides and siRNA to sensitize breast carcinoma cells to several PKIs. MCF-7 and MDA-MB-231 cells were treated with antisense oligonucleotide or siRNA to clusterin and the following PKIs: H-89, chelerythrine and genistein. The three inhibitors used in this study upregulated clusterin expression and treatments that included antisense oligonucleotide or siRNA to clusterin reduced the number of viable cells more effectively than did treatment with the drugs alone. Therefore, treatment with such combinations may benefit patients with breast cancer. PMID:25144344

  13. A STUDY OF HIGH SENSITIVITY C-REACTIVE PROTEIN IN UNSTABLE ANGINA

    Satish

    2014-11-01

    Full Text Available BACKGROUND: Unstable angina has a wide variability in its natural history, changing concepts of Pathophysiology, and newer approaches to its management strategies. So, unstable angina still has importance and prime interest in research work. Various ongoing research works has provided newer insights in pathophysiology of unstable angina syndrome and helps in recognition of clinical variability and unpredictability of it. C - reactive protein being the most sensitive acute phase reactant currently held. A recent previous study has estimated the levels and values of high-sensitivity C - reactive protein in both stable and unstable angina pectoris. Data provided by the study indicated need for further studies in this field. With all these facts, the present study is carried out to estimated Hs CRP levels as a marker of inflammation in patient of unstable angina. AIMS AND OBJECTIVES: The present study was carried out with the following Aims and Objectives. To estimate Hs-CRP levels as a marker of inflammation in patients of unstable angina. To compare Hs-CRP levels in cases of unstable angina, with Hs-CRP levels in patients of stable angina and in healthy age and sex matched controls. MATERIAL AND METHODS: This study was carried out at Basaveshwar Teaching and General Hospital, Gulbarga, MRMC Gulbarga. Approximate duration of study was 1 ½ year from June-2008 to November, 2010. OBSERVATION: Following are the conclusions drawn from the present study.

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

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

    2016-01-01

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

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

    Hiroyuki Kato

    2016-06-01

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

  16. Mosaic protein and nucleic acid vaccines against hepatitis C virus

    Yusim, Karina; Korber, Bette T. M.; Kuiken, Carla L.; Fischer, William M.

    2013-06-11

    The invention relates to immunogenic compositions useful as HCV vaccines. Provided are HCV mosaic polypeptide and nucleic acid compositions which provide higher levels of T-cell epitope coverage while minimizing the occurrence of unnatural and rare epitopes compared to natural HCV polypeptides and consensus HCV sequences.

  17. Distribution and Variation of Ribonucleic Acid (RNA) and Protein and Its Hydrolysis Products in Lake Sediments

    梁小兵; 万国江; 黄荣贵

    2002-01-01

    Protein and RNA in lake sediments tend to be decomposed progressively with time and sedimentation depth. Their concentrations tend to decrease starting from the sedimentation depth of 17 cm and that of 19 cm, respectively. However, the products of their decomposition-amino acids and nucleotides show different rules of variation. At the depth from 27 cm to 30 cm the amino acids are most abundant in the pore waters of lake sediments. Such variation tendency seems to be related to the extent to which microbes utilize amino acids and nucleotides. Due to polymerization in the geological processes and the adsorption of protein on minerals and organic polymers, below the sedimentation depth of 17 cm there is still a certain amount of protein in the sediments. With the time passing by, protein has been well preserved in various sediment layers, indicating that its decomposition is relatively limited. The peak values of protein content in the sediments of the two lakes are produced in the surface layers at the depth of 10 cm, implicating that the surface sediments are favorable to the release of protein.The contents of amino acids in the pore waters of lake sediments are closely related to the activities of microbes. Below the depth of 27 cm, the amino acids are significantly accumulated in Lake Aha sediments, probably indicating the weakening of microbial activities.

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

    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. PMID:19006392

  19. Prediction algorithm for amino acid types with their secondary structure in proteins (PLATON) using chemical shifts

    Labudde, D.; Leitner, D.; Krueger, M.; Oschkinat, H. [Forschungsinstitut fuer Molekulare Pharmakologie (Germany)], E-mail: oschkinat@fmp-berlin.de

    2003-01-15

    The algorithm PLATON is able to assign sets of chemical shifts derived from a single residue to amino acid types with its secondary structure (amino acid species). A subsequent ranking procedure using optionally two different penalty functions yields predictions for possible amino acid species for the given set of chemical shifts. This was demonstrated in the case of the {alpha}-spectrin SH3 domain and applied to 9 further protein data sets taken from the BioMagRes database. A database consisting of reference chemical shift patterns (reference CSPs) was generated from assigned chemical shifts of proteins with known 3D-structure. This reference CSP database is used in our approach for extracting distributions of amino acid types with their most likely secondary structure elements (namely {alpha}-helix, {beta}-sheet, and coil) for single amino acids by comparison with query CSPs. Results obtained for the 10 investigated proteins indicates that the percentage of correct amino acid species in the first three positions in the ranking list, ranges from 71.4% to 93.2% for the more favorable penalty function. Where only the top result of the ranking list for these 10 proteins is considered, 36.5% to 83.1% of the amino acid species are correctly predicted. The main advantage of our approach, over other methods that rely on average chemical shift values is the ability to increase database content by incorporating newly derived CSPs, and therefore to improve PLATON's performance over time.

  20. Prediction algorithm for amino acid types with their secondary structure in proteins (PLATON) using chemical shifts

    The algorithm PLATON is able to assign sets of chemical shifts derived from a single residue to amino acid types with its secondary structure (amino acid species). A subsequent ranking procedure using optionally two different penalty functions yields predictions for possible amino acid species for the given set of chemical shifts. This was demonstrated in the case of the α-spectrin SH3 domain and applied to 9 further protein data sets taken from the BioMagRes database. A database consisting of reference chemical shift patterns (reference CSPs) was generated from assigned chemical shifts of proteins with known 3D-structure. This reference CSP database is used in our approach for extracting distributions of amino acid types with their most likely secondary structure elements (namely α-helix, β-sheet, and coil) for single amino acids by comparison with query CSPs. Results obtained for the 10 investigated proteins indicates that the percentage of correct amino acid species in the first three positions in the ranking list, ranges from 71.4% to 93.2% for the more favorable penalty function. Where only the top result of the ranking list for these 10 proteins is considered, 36.5% to 83.1% of the amino acid species are correctly predicted. The main advantage of our approach, over other methods that rely on average chemical shift values is the ability to increase database content by incorporating newly derived CSPs, and therefore to improve PLATON's performance over time

  1. Rapid and sensitive liquid chromatographic method using a conductivity detector for the determination of phytic acid in food

    Talamond, Pascale; Gallon, Georges; Trèche, Serge

    1998-01-01

    An LC method was developed for the determination of phytic acid in food. The separation was carried out by gradient elution on an anion-exchange column using a conductivity detector. Earlier reversed-phase LC procedures for the quantitation of phytic acid usually required a prepurification step. The prepurification can be avoided by the separation method described in this paper. The method is sensitive and selective, and can be rapidly and easily performed. It is therefore suitable for routin...

  2. Effect of Deoxycholic Acid on the Performance of Liquid Electrolyte Dye-Sensitized Solar Cells Using a Perylene Monoimide Derivative

    Mikroyannidis, John A.; Rajnish Kurchania; Ball, Richard J.; Sharma, Ganesh D.; Roy, Mahesh S.

    2012-01-01

    The effect of coadsorption with deoxycholic acid (DCA) on the performance of dye-sensitized solar cell based on perylene monoimide derivative (PCA) as sensitizer and liquid electrolyte had been investigated. The current-voltage characteristics under illumination and incident photon to current efficiency (IPCE) spectra of the DSSCs showed that the coadsorption of DCA with the PCA dye results in a significant improvement in short circuit photocurrent and slight increase in the open circuit phot...

  3. Synthesis and evaluation of temperature- and glucose-sensitive nanoparticles based on phenylboronic acid and N-vinylcaprolactam for insulin delivery.

    Wu, Jun-Zi; Bremner, David H; Li, He-Yu; Sun, Xiao-Zhu; Zhu, Li-Min

    2016-12-01

    Poly N-vinylcaprolactam-co-acrylamidophenylboronic acid p(NVCL-co-AAPBA) was prepared from N-vinylcaprolactam (NVCL) and 3-acrylamidophenylboronic acid (AAPBA), using 2,2-azobisisobutyronitrile (AIBN) as initiator. The synthesis and structure of the polymer were examined by Fourier Transform infrared spectroscopy (FT-IR) and (1)H-NMR. Dynamic light scattering (DLS), lower critical solution temperature (LCST) and transmission electron microscopy (TEM) were utilized to characterize the nanoparticles, CD spectroscopy was used to determine if there were any changes to the conformation of the insulin, and cell and animal toxicity were also investigated. The prepared nanoparticles were found to be monodisperse submicron particles and were glucose- and temperature-sensitive. In addition, the nanoparticles have good insulin-loading characteristics, do not affect the conformation of the insulin and show low-toxicity to cells and animals. These p(NVCL-co-AAPBA) nanoparticles may have some value for insulin or other hypoglycemic protein delivery. PMID:27612799

  4. Efficient identification of amino acid types for fast protein backbone assignments

    We describe a procedure that allows for very efficient identification of amino acid types in proteins by selective 15N-labeling. The usefulness of selective incorporation of 15N-labeled amino acids into proteins for the backbone assignment has been recognized for several years. However, widespread use of this method has been hindered by the need to purify each selectively labeled sample and by the relatively high cost of labeling with 15N-labeled amino acids. Here we demonstrate that purification of the selectively 15N-labeled samples is not necessary and that background-free HSQC spectra containing only the peaks of the overexpressed heterologous protein can be obtained in crude lysates from as little as 100 ml cultures, thus saving time and money. This method can be used for fast and automated backbone assignment of proteins

  5. From keys to bulldozers: expanding roles for winged helix domains in nucleic-acid-binding proteins.

    Harami, Gábor M; Gyimesi, Máté; Kovács, Mihály

    2013-07-01

    The winged helix domain (WHD) is a widespread nucleic-acid-binding protein structural element found in all kingdoms of life. Although the overall structure of the WHD is conserved, its functional properties and interaction profiles are extremely versatile. WHD-containing proteins can exploit nearly the full spectrum of nucleic acid structural features for recognition and even covalent modification or noncovalent rearrangement of target molecules. WHD functions range from sequence-recognizing keys in transcription factors and bulldozer-like strand-separating wedges in helicases to mediators of protein-protein interactions (PPIs). Further investigations are needed to understand the contribution of WHD structural dynamics to nucleic-acid-modifying enzymatic functions. PMID:23768997

  6. Fast and sensitive quantification of human liver cytosolic lithocholic acid sulfation using ultra-high performance liquid chromatography-tandem mass spectrometry.

    Bansal, Sumit; Lau, Aik Jiang

    2016-02-01

    Detoxification of lithocholic acid (LCA) to lithocholic acid sulfate (LCA-S) is catalyzed by sulfotransferases, mainly SULT2A1. We developed and validated an ultra-high performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method to quantify human liver cytosolic-dependent LCA sulfation. Chromatographic separation was achieved on an UPLC C18 column (2.1×50mm, 1.7μm) and a gradient elution of 0.1% formic acid in water and acetonitrile. Negative electrospray ionization with multiple reaction monitoring (MRM) mode was used to quantify LCA-S (455.3→97.0) and cholic acid (407.2→343.3; internal standard). The retention time was 3.51min for LCA-S and 3.08min for cholic acid. The lower limit of quantification of LCA-S was 0.5nM (or 0.23ng/ml in 400μl total volume) and the assay was linear from 0.2 to 200pmol. Intra-day and inter-day accuracy and precision were dithiothreitol) did not affect LCA-S formation. Human liver cytosolic LCA sulfation was linear with 20-100μg of cytosolic protein and 5-30min incubation time. This UPLC-MS/MS approach offers a specific, sensitive, fast, and direct approach for quantifying human liver cytosolic LCA sulfation. PMID:26773894

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

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

    2013-02-01

    The protection of sunflower meal (SFM) proteins by treatments with solutions of malic acid (1 M) or orthophosphoric acid (0.67 M) and heat was studied in a 3 × 3 Latin-square design using three diets and three rumen and duodenum cannulated wethers. Acid solutions were applied to SFM at a rate of 400 ml/kg under continuous mixing. Subsequently, treated meals were dried in an oven at 150°C for 6 h. Diets (ingested at 75 g/kg BW0.75) were isoproteic and included 40% Italian ryegrass hay and 60% concentrate. The ratio of untreated to treated SFM in the concentrate was 100 : 0 in the control diet and around 40 : 60 in diets including acid-treated meals. The use of acid-treated meals did not alter either ruminal fermentation or composition of rumen contents and led to moderate reductions of the rumen outflow rates of untreated SFM particles, whereas it did not affect their comminution and mixing rate. In situ effective estimates of by-pass (BP) and its intestinal effective digestibility (IED) of dry matter (DM), CP and amino acids (AAs) were obtained considering both rates and correcting the particle microbial contamination in the rumen using 15N infusion techniques. Estimates of BP and IED decreased applying microbial correction, but these variations were low in agreement with the small contamination level. Protective treatments increased on average the BP of DM (48.5%) and CP (267%), mainly decreasing both the soluble fraction and the degradation rate but also increasing the undegradable fraction, which was higher using orthophosphoric acid. Protective treatments increased the IED of DM (108%) and CP, but this increase was lower using orthophosphoric acid (11.8%) than malic acid (20.7%). Concentrations of AA were similar among all meals, except for a reduction in lysine concentrations using malic acid (16.3%) or orthophosphoric acid (20.5%). Protective treatments also increased on average the BP of all AA, as well as the IED of most of them. Evidence of higher

  8. Differential modulation of CXCR4 and CD40 protein levels by skin sensitizers and irritants in the FSDC cell line

    Neves, Bruno Miguel; Cruz, Maria Teresa; Francisco, Vera; Gonçalo, Margarida; Figueiredo, Américo; Duarte, Carlos B.; Lopes, Maria Celeste

    2008-01-01

    The development of non-animal methods for skin sensitization testing is an urgent challenge. Some of the most promising in vitro approaches are based on the analysis of phenotypical and functional modifications induced by sensitizers in dendritic cell models. In this work, we evaluated, for the first time, a fetal skin-derived dendritic cell line (FSDC) as a model to discriminate between sensitizers and irritants, through analysis of their effects on CD40 and CXCR4 protein expression. The che...

  9. Identification of steroid-sensitive gene-1/Ccdc80 as a JAK2-binding protein.

    O'Leary, Erin E; Mazurkiewicz-Muñoz, Anna M; Argetsinger, Lawrence S; Maures, Travis J; Huynh, Hung T; Carter-Su, Christin

    2013-04-01

    The tyrosine kinase Janus kinase 2 (JAK2) is activated by many cytokine receptors, including receptors for GH, leptin, and erythropoietin. However, very few proteins have been identified as binding partners for JAK2. Using a yeast 2-hybrid screen, we identified steroid-sensitive gene-1 (SSG1)/coiled-coil domain-containing protein 80 (Ccdc80) as a JAK2-binding partner. We demonstrate that Ccdc80 preferentially binds activated, tyrosyl-phosphorylated JAK2 but not kinase-inactive JAK2 (K882E) in both yeast and mammalian systems. Ccdc80 is tyrosyl phosphorylated in the presence of JAK2. The binding of Ccdc80 to JAK2 occurs via 1 or more of the 3 DUDES/SRPX (DRO1-URB-DRS-Equarin-SRPUL/sushi repeat containing protein, x-linked) domain 5 domains of Ccdc80. Mutagenesis of the second DUDES domain suggests that the N-terminal third of the DUDES domain is sufficient for JAK2 binding. Ccdc80 does not alter the kinase activity of JAK2. However, Ccdc80 increases GH-dependent phosphorylation of Stat (signal transducer and activator of transcription) 5b on Tyr699 and substantially enhances both basal and GH-dependent phosphorylation/activation of Stat3 on Tyr705. Furthermore, Ccdc80 belongs to the group of proteins that function both in the intracellular compartment and are secreted. Secreted Ccdc80 associates with the extracellular matrix and is also found in the medium. A substantial portion of the Ccdc80 detected in the medium is cleaved. Finally, consistent with the DUDES domain serving as a JAK2-binding domain, we also demonstrate that another protein that contains a DUDES domain, SRPX2, binds preferentially to the activated tyrosyl-phosphorylated form of JAK2. PMID:23449887

  10. Utility of lab-on-a-chip technology for high-throughput nucleic acid and protein analysis

    Hawtin, Paul; Hardern, Ian; Wittig, Rainer;

    2005-01-01

    On-chip electrophoresis can provide size separations of nucleic acids and proteins similar to more traditional slab gel electrophoresis. Lab-on-a-chip (LoaC) systems utilize on-chip electrophoresis in conjunction with sizing calibration, sensitive detection schemes, and sophisticated data analysis...... to achieve rapid analysis times (<120 s). This work describes the utility of LoaC systems to enable and augment systems biology investigations. RNA quality, as assessed by an RNA integrity number score, is compared to existing quality control (QC) measurements. High-throughput DNA analysis of...... multiplex PCR samples is used to stratify gene sets for disease discovery. Finally, the applicability of a high-throughput LoaC system for assessing protein purification is demonstrated. The improvements in workflow processes, speed of analysis, data accuracy and reproducibility, and automated data analysis...

  11. 3-d structure-based amino acid sequence alignment of esterases, lipases and related proteins

    Gentry, M.K.; Doctor, B.P.; Cygler, M.; Schrag, J.D.; Sussman, J.L.

    1993-05-13

    Acetylcholinesterase and butyrylcholinesterase, enzymes with potential as pretreatment drugs for organophosphate toxicity, are members of a larger family of homologous proteins that includes carboxylesterases, cholesterol esterases, lipases, and several nonhydrolytic proteins. A computer-generated alignment of 18 of the proteins, the acetylcholinesases, butyrylcholinesterases, carboxylesterases, some esterases, and the nonenzymatic proteins has been previously presented. More recently, the three-dimensional structures of two enzymes enzymes in this group, acetylcholinesterase from Torpedo californica and lipase from Geotrichum candidum, have been determined. Based on the x-ray structures and the superposition of these two enzymes, it was possible to obtain an improved amino acid sequence alignment of 32 members of this family of proteins. Examination of this alignment reveals that 24 amino acids are invariant in all of the hydrolytic proteins, and an additional 49 are well conserved. Conserved amino acids include those of the active site, the disulfide bridges, the salt bridges, in the core of the proteins, and at the edges of secondary structural elements. Comparison of the three-dimensional structures makes it possible to find a well-defined structural basis for the conservation of many of these amino acids.

  12. Hemoglobin interactions with αB crystallin: a direct test of sensitivity to protein instability.

    Tyler J W Clark

    Full Text Available As a small stress response protein, human αB crystallin, detects protein destabilization that can alter structure and function to cause self assembly of fibrils or aggregates in diseases of aging. The sensitivity of αB crystallin to protein instability was evaluated using wild-type hemoglobin (HbA and hemoglobin S (HbS, the glutamate-6-valine mutant that forms elongated, filamentous aggregates in sickling red blood cells. The progressive thermal unfolding and aggregation of HbA and HbS in solution at 37°C, 50°C and 55°C was measured as increased light scattering. UV circular dichroism (UVCD was used to evaluate conformational changes in HbA and HbS with time at the selected temperatures. The changes in interactions between αB crystallin and HbA or HbS with temperature were analyzed using differential centrifugation and SDS PAGE at 37°C, 50°C and 55°C. After only 5 minutes at the selected temperatures, differences in the aggregation or conformation of HbA and HbS were not observed, but αB crystallin bound approximately 6% and 25% more HbS than HbA at 37°C, and 50°C respectively. The results confirmed (a the remarkable sensitivity of αB crystallin to structural instabilities at the very earliest stages of thermal unfolding and (b an ability to distinguish the self assembling mutant form of HbS from the wild type HbA in solution.

  13. Sensitive electrochemical monitoring of nucleic acids coupling DNA nanostructures with hybridization chain reaction

    Zhuang, Junyang; Fu, Libing; Xu, Mingdi; Yang, Huanghao; Chen, Guonan; Tang, Dianping, E-mail: dianping.tang@fzu.edu.cn

    2013-06-14

    Graphical abstract: -- Highlights: •A new signal-on metallobioassay was developed for detection of nucleic acids. •Target-triggered long-range self-assembled DNA nanostructures are used for amplification of electronic signal. •Hybridization chain reaction is utilized for construction of long-range DNA nanostructures. -- Abstract: Methods based on metal nanotags have been developed for metallobioassay of nucleic acids, but most involve complicated labeling or stripping procedures and are unsuitable for routine use. Herein, we report the proof-of-concept of a novel and label-free metallobioassay for ultrasensitive electronic determination of human immunodeficiency virus (HIV)-related gene fragments at an ultralow concentration based on target-triggered long-range self-assembled DNA nanostructures and DNA-based hybridization chain reaction (HCR). The signal is amplified by silver nanotags on the DNA duplex. The assay mainly consists of capture probe, detection probe, and two different DNA hairpins. In the presence of target DNA, the capture probe immobilized on the sensor sandwiches target DNA with the 3′ end of detection probe. Another exposed part of detection probe at the 5′ end opens two alternating DNA hairpins in turn, and propagates a chain reaction of hybridization events to form a nicked double-helix. Finally, numerous silver nanotags are immobilized onto the long-range DNA nanostructures, each of which produces a strong electronic signal within the applied potentials. Under optimal conditions, the target-triggered long-range DNA nanostructures present good electrochemical behaviors for the detection of HIV DNA at a concentration as low as 0.5 fM. Importantly, the outstanding sensitivity can make this approach a promising scheme for development of next-generation DNA sensors without the need of enzyme labeling or fluorophore labeling.

  14. Trans fatty acid intake is associated with insulin sensitivity but independently of inflammation

    C.T. Angelieri

    2012-07-01

    Full Text Available High saturated and trans fatty acid intake, the typical dietary pattern of Western populations, favors a proinflammatory status that contributes to generating insulin resistance (IR. We examined whether the consumption of these fatty acids was associated with IR and inflammatory markers. In this cross-sectional study, 127 non-diabetic individuals were allocated to a group without IR and 56 to another with IR, defined as homeostasis model assessment-IR (HOMA-IR >2.71. Diet was assessed using 24-h food recalls. Multiple linear regression was employed to test independent associations with HOMA-IR. The IR group presented worse anthropometric, biochemical and inflammatory profiles. Energy intake was correlated with abdominal circumference and inversely with adiponectin concentrations (r = -0.227, P = 0.002, while saturated fat intake correlated with inflammatory markers and trans fat with HOMA-IR (r = 0.160, P = 0.030. Abdominal circumference was associated with HOMA-IR (r = 0.430, P < 0.001. In multiple analysis, HOMA-IR remained associated with trans fat intake (β = 1.416, P = 0.039 and body mass index (β = 0.390, P < 0.001, and was also inversely associated with adiponectin (β = -1.637, P = 0.004. Inclusion of other nutrients (saturated fat and added sugar or other inflammatory markers (IL-6 and CRP into the models did not modify these associations. Our study supports that trans fat intake impairs insulin sensitivity. The hypothesis that its effect could depend on transcription factors, resulting in expression of proinflammatory genes, was not corroborated. We speculate that trans fat interferes predominantly with insulin signaling via intracellular kinases, which alter insulin receptor substrates.

  15. Inactivation of fatty acid transport protein 1 prevents fat-induced insulin resistance in skeletal muscle

    Jason K Kim; Gimeno, Ruth E.; Higashimori, Takamasa; Kim, Hyo-Jeong; Choi, Hyejeong; Punreddy, Sandhya; Mozell, Robin L.; TAN, GUO; Stricker-Krongrad, Alain; Hirsch, David J.; Fillmore, Jonathan J.; Liu, Zhen-Xiang; Dong, Jianying; Cline, Gary; Stahl, Andreas

    2004-01-01

    Insulin resistance in skeletal muscle plays a major role in the development of type 2 diabetes and may be causally associated with increases in intramuscular fatty acid metabolites. Fatty acid transport protein 1 (FATP1) is an acyl-CoA synthetase highly expressed in skeletal muscle and modulates fatty acid uptake and metabolism by converting fatty acids into fatty acyl-CoA. To investigate the role of FATP1 in glucose homeostasis and in the pathogenesis of insulin resistance, we examined the e...

  16. Interaction of actinides with amino acids: from peptides to proteins

    Structural information on complexes of actinides with molecules of biological interest is required to better understand the mechanisms of actinides transport in living organisms, and can contribute to develop new decorporation treatments. Our study is about Th(IV), Np(IV), Pu(IV) and uranyl(VI) cations, which have a high affinity for some protein domains, and Fe(III), which is the natural cation of these biological systems. In this work, chelation of actinides has been brought to light with UV-visible-Near Infra Red spectroscopy, NMR, EPR, and ultrafiltration. Determination of the structure of the complexation site has been undertaken with Exafs measurements, and of the tertiary structure of the protein with SANS measurements. The first approach was to describe the interaction modes between actinides and essential chemical functions of proteins. Thus, the Ac-AspAspProAspAsp-NH2 peptide was studied as a possible chelate of actinides. Polynuclear species with μ-oxo or μ-hydroxo bridges were identified. The iron complex is binuclear, and the actinide ones have a higher nuclearity. The second approach was to study a real case of complexation of actinide with a protein: transferrin. Results show that around physiological ph a mononuclear complex is formed with Np(IV) and Pu(IV), while transferrin does not complex Th(IV) in the same conditions. Characteristic distances of M-transferrin complexes (M = Fe, Np, Pu) were determined. Moreover, the protein seems to be in its close conformation with Pu(IV), and in its open form with Np(IV) and UO22+. (author)

  17. Renal Liver-Type Fatty Acid Binding Protein (L-FABP) Attenuates Acute Kidney Injury in Aristolochic Acid Nephrotoxicity

    Matsui, Katsuomi; Kamijo-Ikemorif, Atsuko; Sugaya, Takeshi; Yasuda, Takashi; Kimura, Kenjiro

    2011-01-01

    Injection of aristolochic acid (AA) in mice causes AA-induced nephrotoxicity, in which oxidative stress contributes to development of tubulointerstitial damage (TID). Liver-type fatty acid binding protein (L-FABP) is expressed in human proximal tubules and has an endogenous antioxidative function. The renoprotection of renal L-FABP was examined in a model of AA-induced nephrotoxicity. Established human L-FABP (hL-FABP) transgenic (Tg) mice and wild-type (WT) mice were treated with AA for up t...

  18. Sucrose Sensitivity of Honey Bees Is Differently Affected by Dietary Protein and a Neonicotinoid Pesticide

    Démares, Fabien J.; Crous, Kendall L.; Pirk, Christian W. W.; Nicolson, Susan W.; Human, Hannelie

    2016-01-01

    Over a decade, declines in honey bee colonies have raised worldwide concerns. Several potentially contributing factors have been investigated, e.g. parasites, diseases, and pesticides. Neonicotinoid pesticides have received much attention due to their intensive use in crop protection, and their adverse effects on many levels of honey bee physiology led the European Union to ban these compounds. Due to their neuronal target, a receptor expressed throughout the insect nervous system, studies have focused mainly on neuroscience and behaviour. Through the Geometric Framework of nutrition, we investigated effects of the neonicotinoid thiamethoxam on survival, food consumption and sucrose sensitivity of honey bees (Apis mellifera). Thiamethoxam did not affect protein and carbohydrate intake, but decreased responses to high concentrations of sucrose. Interestingly, when bees ate fixed unbalanced diets, dietary protein facilitated better sucrose detection. Both thiamethoxam and dietary protein influenced survival. These findings suggest that, in the presence of a pesticide and unbalanced food, honey bee health may be severely challenged. Consequences for foraging efficiency and colony activity, cornerstones of honey bee health, are also discussed. PMID:27272274

  19. Second and third generation voltage-sensitive fluorescent proteins for monitoring membrane potential

    Thomas Knopfel

    2009-06-01

    Full Text Available Over the last decade, optical neuroimaging methods have been enriched by engineered biosensors derived from fluorescent protein (FP reporters fused to protein detectors that convert physiological signals into changes of intrinsic FP fluorescence. These FP-based indicators are genetically encoded, and hence targetable to specific cell populations within networks of heterologous cell types. Among this class of biosensors, the development of optical probes for membrane potential is both highly desirable and challenging. A suitable FP voltage sensor would indeed be a valuable tool for monitoring the activity of thousands of individual neurons simultaneously in a non-invasive manner. Previous prototypic genetically-encoded FP voltage indicators achieved a proof of principle but also highlighted several difficulties such as poor cell surface targeting and slow kinetics. Recently, we developed a new series of FRET-based Voltage-Sensitive Fluorescent Proteins (VSFPs, referred to as VSFP2s, with efficient targeting to the plasma membrane and high responsiveness to membrane potential signaling in excitable cells. In addition to these FRET-based voltage sensors, we also generated a third series of probes consisting of single FPs with response kinetics suitable for the optical imaging of fast neuronal signals. These newly available genetically-encoded reporters for membrane potential will be instrumental for future experimental approaches directed toward the understanding of neuronal network dynamics and information processing in the brain. Here, we review the development and current status of these novel fluorescent probes.

  20. Sucrose Sensitivity of Honey Bees Is Differently Affected by Dietary Protein and a Neonicotinoid Pesticide.

    Fabien J Démares

    Full Text Available Over a decade, declines in honey bee colonies have raised worldwide concerns. Several potentially contributing factors have been investigated, e.g. parasites, diseases, and pesticides. Neonicotinoid pesticides have received much attention due to their intensive use in crop protection, and their adverse effects on many levels of honey bee physiology led the European Union to ban these compounds. Due to their neuronal target, a receptor expressed throughout the insect nervous system, studies have focused mainly on neuroscience and behaviour. Through the Geometric Framework of nutrition, we investigated effects of the neonicotinoid thiamethoxam on survival, food consumption and sucrose sensitivity of honey bees (Apis mellifera. Thiamethoxam did not affect protein and carbohydrate intake, but decreased responses to high concentrations of sucrose. Interestingly, when bees ate fixed unbalanced diets, dietary protein facilitated better sucrose detection. Both thiamethoxam and dietary protein influenced survival. These findings suggest that, in the presence of a pesticide and unbalanced food, honey bee health may be severely challenged. Consequences for foraging efficiency and colony activity, cornerstones of honey bee health, are also discussed.

  1. Inhibition of Peptidoglycan, Ribonucleic Acid, and Protein Synthesis in Tolerant Strains of Streptococcus mutans

    Mychajlonka, Myron; McDowell, Thomas D.; Shockman, Gerald D.

    1980-01-01

    Exposure of exponentially growing cultures of Streptococcus mutans strains FA-1 and GS-5 to various concentrations of benzylpenicillin (Pen G) resulted in inhibition of turbidity increases at low concentrations (0.02 to 0.04 μg/ml). However, in contrast to some other streptococcal species, growth inhibition was not accompanied by cellular lysis or by a rapid loss of viability. In both strains, synthesis of insoluble cell wall peptidoglycan was very sensitive to Pen G inhibition and responded in a dose-dependent manner to concentrations of about 0.2 and 0.5 μg/ml for strains GS-5 and FA-1, respectively. Higher Pen G concentrations failed to inhibit further either growth or insoluble peptidoglycan assembly. Somewhat surprisingly, Pen G also inhibited both ribonucleic acid (RNA) and protein syntheses, each in a dose-dependent manner. Compared with inhibition of peptidoglycan synthesis, inhibition of RNA and protein syntheses by Pen G was less rapid and less extensive. Maximum amounts of radiolabeled Pen G were specifically bound to intact cells upon exposure to about 0.2 and 0.5 μg/ml of Pen G for strains GS-5 and FA-1, respectively, concentrations consistent with those that resulted in maximum or near-maximum inhibitions of the synthesis of cellular peptidoglycan, RNA, and protein. Five polypeptide bands that had a very high affinity for [14C]Pen G were detected in a crude cell envelope preparation of strain FA-1. After exposure of cultures of strain FA-1 to the effects of saturating concentrations of the drug for up to 3 h, addition of penicillinase was followed by recovery of growth after a lag. The length of the lag before regrowth depended on both Pen G concentration and time of exposure. On the basis of these and other observations, it is proposed that the secondary inhibitions of cellular RNA or protein synthesis, or both, are involved in the tolerance of these organisms to lysis and killing by Pen G and other inhibitors of insoluble peptidoglycan assembly

  2. Photo-CIDNP studies of amino acids and proteins

    Lopez, J J

    2001-01-01

    half. Chapter 6: CIDNP in Micellar Solutions The presence of detergent, below and above the critical micelle concentration, is shown to affect CIDNP intensities, due to electrostatic interactions between charged dye and detergent molecules. In the last part of this chapter, photo-CIDNP experiments with the membrane protein gramicidin A, incorporated in detergent and lipid micelles, are described. Chapter 7: CIDNP Study of the Tryptophan Radical CIDNP spectra are characteristic of the transient radical intermediates which are generated during the flash photolysis. Here, the tryptophan radicals g-factor is estimated with the help of the CIDNP dependence on the magnetic field in which the flash-photolysis takes place. The ultimate aim of the research described in this thesis is the development of methods with which ope may study the structure and function of proteins on a molecular level. This is done with the help of a combination of NMR (Nuclear Magnetic Resonance) and flash photolysis, in which light initiate...

  3. Decomposition of Intramolecular Interactions Between Amino-Acids in Globular Proteins - A Consequence for Structural Classes of Proteins and Methods of Their Classification

    Fačkovec, Boris; Vondrášek, Jiří

    Rijeka : InTech, 2011 - (Yang, N.), s. 69-82 ISBN 978-953-307-280-7 Institutional research plan: CEZ:AV0Z40550506 Keywords : proteins * amino acids * structural classes * protein architecture * protein stability Subject RIV: CF - Physical ; Theoretical Chemistry http://www.intechopen.com/books/ systems - and -computational- biology -molecular- and -cellular-experimental- systems /decomposition-of-intramolecular-interactions-between-amino-acids-in-globular-proteins-a-consequence-

  4. Search for conserved amino acid residues of the [Formula: see text]-crystallin proteins of vertebrates.

    Shiliaev, Nikita G; Selivanova, Olga M; Galzitskaya, Oxana V

    2016-04-01

    [Formula: see text]-crystallin is the major eye lens protein and a member of the small heat-shock protein (sHsp) family. [Formula: see text]-crystallins have been shown to support lens clarity by preventing the aggregation of lens proteins. We performed the bioinformatics analysis of [Formula: see text]-crystallin sequences from vertebrates to find conserved amino acid residues as the three-dimensional (3D) structure of [Formula: see text]-crystallin is not identified yet. We are the first who demonstrated that the N-terminal region is conservative along with the central domain for vertebrate organisms. We have found that there is correlation between the conserved and structured regions. Moreover, amyloidogenic regions also correspond to the structured regions. We analyzed the amino acid composition of [Formula: see text]-crystallin A and B chains. Analyzing the occurrence of each individual amino acid residue, we have found that such amino acid residues as leucine, serine, lysine, proline, phenylalanine, histidine, isoleucine, glutamic acid, and valine change their content simultaneously in A and B chains in different classes of vertebrates. Aromatic amino acids occur more often in [Formula: see text]-crystallins from vertebrates than on the average in proteins among 17 animal proteomes. We obtained that the identity between A and B chains in the mammalian group is 0.35, which is lower than the published 0.60. PMID:26972563

  5. Carboxymethyl starch cross-linked by electron beam radiation in presence of acrylic acid sensitizer

    Carboxymethyl starch (CMS) can be cross-linked by electron beam radiation to form a biocompatible and environment-friendly hydrogel at a high absorbed dose and a condensed CMS concentration. Acrylic acid (AAc) can be used as a sensitizer in order to reduce the absorbed doses to an acceptable certain level. At an absorbed dose of 3-4 kGy, the gel content of crosslinked CMS can be obtained about 50% with 5% (w/w) AAc concentration used. The compressive strength of CMS samples increased with increasing their cross-linked densities due to raising absorbed doses. The swelling ratio of cross-linked CMS was also attainable at a maximum of 50 times in the distilled water. The enzymatic degradation of cross-linked CMS was carried out in acetate buffer pH 4.6 with 0.1% α-amylase enzymatic solution incubated at 40℃ for 6 h. The crosslinked CMS samples were degraded slower than uncrosslinked CMS ones. The results indicated that the highly cross-linked CMS was almost fully degradable when the enzymatic hydrolysis was performed during 6 h. The FT IR spectra of cross-linked CMS in the presence of AAc were examined to observe the carboxyl group of AAc in the structure of cross-linked CMS. The hydrophilic of cross-linked CMS surface was determined by a contact-angle analysis. (authors)

  6. Molecularly imprinted photo-sensitive polyglutamic acid nanoparticles for electrochemical sensing of hemoglobin

    A voltammetric sensor for hemoglobin (Hb) was prepared from molecularly imprinted polymer nanoparticles (MINPs) via electrophoretic deposition. A photo-sensitive copolymer composed of poly-γ-glutamic grafted with the fluorophore 7-amino-4-methylcoumarin was converted into nanoparticles that were imprinted with Hb. The resultant MINPs were then placed on a glassy carbon electrode (GCE) via electrophoretic deposition. Subsequent photo-crosslinking locks the recognition sites. The template was removed by extraction with a mixture of acetic acid and methanol at a ratio of 1:9 (v:v) to obtain a voltammetric sensor for Hb. The current response of the sensor at a working voltage of −260 mV is linearly related to the concentration of Hb in the range from 5 to 100 μg mL−1, and recoveries range from 98.7 to 102.3 %. Compared to the respective non-imprinted nanoparticles, the sensor displays high recognition capability and affinity for Hb. (author)

  7. Free Fatty Acids Inhibit Protein Tyrosine Phosphatase 1B and Activate Akt

    Eisuke Shibata

    2013-09-01

    Full Text Available Background/Aims: Accumulating evidence has suggested that free fatty acids (FFAs interact with protein kinases and protein phosphatases. The present study examined the effect of FFAs on protein phosphatases and Akt. Methods: Activities of protein phosphatase 1 (PP1, protein phosphatase 2A (PP2A, and protein tyrosine phosphatase 1B (PTP1B were assayed under the cell-free conditions. Phosphorylation of Akt was monitored in MSTO-211H human malignant pleural mesothelioma cells without and with knocking-down phosphatidylinositol 3 kinase (PI3K or 3-phosphoinositide-dependent protein kinase-1 (PDK1. Results: In the cell-free assay, unsaturated FFAs (uFFAs such as oleic, linoleic and linolenic acid and saturated FFAs (sFFAs such as stearic, palmitic, myristic, and behenic acid markedly reduced PTP1B activity, with the potential for uFFAs greater than that for sFFAs. All the investigated sFFAs inhibited PP2A activity, but otherwise no inhibition was obtained with uFFAs. Both uFFAs and sFFAs had no effect on PP1 activity. Oleic acid phosphorylated Akt both on Thr308 and Ser473, while stearic acid phosphorylated Akt on Thr308 alone. The effects of oleic and stearic acid on Akt phosphorylation were abrogated by the PI3K inhibitor wortmannin or the PDK1 inhibitor BX912 and also by knocking-down PI3K or PDK1. Conclusion: The results of the present study indicate that uFFAs and sFFAs could activate Akt through a pathway along a PI3K/PDK1/Akt axis in association with PTP1B inhibition.

  8. Comparison of C-reactive protein and high-sensitivity C-reactive protein levels in patients on hemodialysis

    Imed Helal

    2012-01-01

    Full Text Available Chronic inflammation is highly prevalent in patients on hemodialysis (HD, as evidenced by increased levels of C-reactive protein (CRP. We compared CRP to high-sensitivity C-reactive protein (hs-CRP to determine whether it has any clinical implications and prognostic significance in terms of mortality. CRP was measured using a standard immunoturbidometric assay on the COBAS; INTEGRA system and hs-CRP was measured using the Dade Behring on the Konelab Nephelometer in 50 patients on HD. CRP (≥6 mg/L and hs-CRP (≥3 mg/L levels were elevated in 30% and 54% of the patients, respectively. A significant correlation was noted between hs-CRP and CRP levels (r = 0.98, P <0.001. Deming regression analysis showed that the slope was near one (r = 0.90; 0.83-0.94 and that the intercept was small. Multivariate regression confirmed that age above 40 years (RR = 3.69, P = 0.027 and duration on HD greater than five years (RR = 3.71, P = 0.028 remained significant independent predictors of serum hs-CRP. Thirteen patients died during follow-up (26%. Multivariate Cox regression demonstrated that hs-CRP (RR = 1.062, P = 0.03 and CRP levels (RR = 1.057, P = 0.009 and age (RR = 1.078, P = 0.001 were the most powerful predictors of mortality. The CRP standard assay presents a reasonable alternative to the hs-CRP assay in patients on HD. The advantages of the CRP standard assay are its online and real-time availability as well as lower costs, particularly in developing countries.

  9. Interface Modification of Dye-sensitized Solar Cells with Pivalic Acid to Enhance the Open-circuit Voltage

    Li, Xin

    2009-01-01

    Pivalic acid (PVA) was used as a new coadsorbent to dye-sensitized solar cells (DSCs) to modify the interface between the TiO2 films and electrolyte. The addition of PVA improved the light-to-electricity conversion efficiency of devices by 8% by enhancing the open-circuit voltage. Copyright © 2009 The Chemical Society of Japan.

  10. Physiological responses of glyphosate-resistant and glyphosate-sensitive soybean to aminomethylphosphonic acid a metabolite of glyphosate

    Aminomethylphosphonic acid (AMPA) is formed in glyphosate-treated glyphosate-resistant (GR) and glyphosate-sensitive (GS) soybean [Glycine max (L.) Merr.] plants and is known to cause yellowing in soybean. Although, AMPA is less phytotoxic than glyphosate, its mode of action is different from that o...

  11. Models of protein and amino acid requirements for cattle

    Luis Orlindo Tedeschi

    2015-03-01

    Full Text Available Protein supply and requirements by ruminants have been studied for more than a century. These studies led to the accumulation of lots of scientific information about digestion and metabolism of protein by ruminants as well as the characterization of the dietary protein in order to maximize animal performance. During the 1980s and 1990s, when computers became more accessible and powerful, scientists began to conceptualize and develop mathematical nutrition models, and to program them into computers to assist with ration balancing and formulation for domesticated ruminants, specifically dairy and beef cattle. The most commonly known nutrition models developed during this period were the National Research Council (NRC in the United States, Agricultural Research Council (ARC in the United Kingdom, Institut National de la Recherche Agronomique (INRA in France, and the Commonwealth Scientific and Industrial Research Organization (CSIRO in Australia. Others were derivative works from these models with different degrees of modifications in the supply or requirement calculations, and the modeling nature (e.g., static or dynamic, mechanistic, or deterministic. Circa 1990s, most models adopted the metabolizable protein (MP system over the crude protein (CP and digestible CP systems to estimate supply of MP and the factorial system to calculate MP required by the animal. The MP system included two portions of protein (i.e., the rumen-undegraded dietary CP - RUP - and the contributions of microbial CP - MCP as the main sources of MP for the animal. Some models would explicitly account for the impact of dry matter intake (DMI on the MP required for maintenance (MPm; e.g., Cornell Net Carbohydrate and Protein System - CNCPS, the Dutch system - DVE/OEB, while others would simply account for scurf, urinary, metabolic fecal, and endogenous contributions independently of DMI. All models included milk yield and its components in estimating MP required for lactation

  12. Amino acid starvation has opposite effects on mitochondrial and cytosolic protein synthesis.

    Mark A Johnson

    Full Text Available Amino acids are essential for cell growth and proliferation for they can serve as precursors of protein synthesis, be remodelled for nucleotide and fat biosynthesis, or be burnt as fuel. Mitochondria are energy producing organelles that additionally play a central role in amino acid homeostasis. One might expect mitochondrial metabolism to be geared towards the production and preservation of amino acids when cells are deprived of an exogenous supply. On the contrary, we find that human cells respond to amino acid starvation by upregulating the amino acid-consuming processes of respiration, protein synthesis, and amino acid catabolism in the mitochondria. The increased utilization of these nutrients in the organelle is not driven primarily by energy demand, as it occurs when glucose is plentiful. Instead it is proposed that the changes in the mitochondrial metabolism complement the repression of cytosolic protein synthesis to restrict cell growth and proliferation when amino acids are limiting. Therefore, stimulating mitochondrial function might offer a means of inhibiting nutrient-demanding anabolism that drives cellular proliferation.

  13. Calcium-myristoyl Tug is a new mechanism for intramolecular tuning of calcium sensitivity and target enzyme interaction for guanylyl cyclase-activating protein 1: dynamic connection between N-fatty acyl group and EF-hand controls calcium sensitivity.

    Peshenko, Igor V; Olshevskaya, Elena V; Lim, Sunghyuk; Ames, James B; Dizhoor, Alexander M

    2012-04-20

    Guanylyl cyclase-activating protein 1 (GCAP1), a myristoylated Ca(2+) sensor in vision, regulates retinal guanylyl cyclase (RetGC). We show that protein-myristoyl group interactions control Ca(2+) sensitivity, apparent affinity for RetGC, and maximal level of cyclase activation. Mutating residues near the myristoyl moiety affected the affinity of Ca(2+) binding to EF-hand 4. Inserting Phe residues in the cavity around the myristoyl group increased both the affinity of GCAP1 for RetGC and maximal activation of the cyclase. NMR spectra show that the myristoyl group in the L80F/L176F/V180F mutant remained sequestered inside GCAP1 in both Ca(2+)-bound and Mg(2+)-bound states. This mutant displayed much higher affinity for the cyclase but reduced Ca(2+) sensitivity of the cyclase regulation. The L176F substitution improved affinity of myristoylated and non-acylated GCAP1 for the cyclase but simultaneously reduced the affinity of Ca(2+) binding to EF-hand 4 and Ca(2+) sensitivity of the cyclase regulation by acylated GCAP1. The replacement of amino acids near both ends of the myristoyl moiety (Leu(80) and Val(180)) minimally affected regulatory properties of GCAP1. N-Lauryl- and N-myristoyl-GCAP1 activated RetGC in a similar fashion. Thus, protein interactions with the central region of the fatty acyl chain optimize GCAP1 binding to RetGC and maximize activation of the cyclase. We propose a dynamic connection (or "tug") between the fatty acyl group and EF-hand 4 via the C-terminal helix that attenuates the efficiency of RetGC activation in exchange for optimal Ca(2+) sensitivity. PMID:22383530

  14. Effect of protein/essential amino acids and resistance training on skeletal muscle hypertrophy: A case for whey protein

    Stout Jeffrey R

    2010-06-01

    Full Text Available Abstract Regardless of age or gender, resistance training or provision of adequate amounts of dietary protein (PRO or essential amino acids (EAA can increase muscle protein synthesis (MPS in healthy adults. Combined PRO or EAA ingestion proximal to resistance training, however, can augment the post-exercise MPS response and has been shown to elicit a greater anabolic effect than exercise plus carbohydrate. Unfortunately, chronic/adaptive response data comparing the effects of different protein sources is limited. A growing body of evidence does, however, suggest that dairy PRO, and whey in particular may: 1 stimulate the greatest rise in MPS, 2 result in greater muscle cross-sectional area when combined with chronic resistance training, and 3 at least in younger individuals, enhance exercise recovery. Therefore, this review will focus on whey protein supplementation and its effects on skeletal muscle mass when combined with heavy resistance training.

  15. Lactic acid induces aberrant amyloid precursor protein processing by promoting its interaction with endoplasmic reticulum chaperone proteins.

    Yiwen Xiang

    Full Text Available BACKGROUND: Lactic acid, a natural by-product of glycolysis, is produced at excess levels in response to impaired mitochondrial function, high-energy demand, and low oxygen availability. The enzyme involved in the production of β-amyloid peptide (Aβ of Alzheimer's disease, BACE1, functions optimally at lower pH, which led us to investigate a potential role of lactic acid in the processing of amyloid precursor protein (APP. METHODOLOGY/PRINCIPAL FINDINGS: Lactic acid increased levels of Aβ40 and 42, as measured by ELISA, in culture medium of human neuroblastoma cells (SH-SY5Y, whereas it decreased APP metabolites, such as sAPPα. In cell lysates, APP levels were increased and APP was found to interact with ER-chaperones in a perinuclear region, as determined by co-immunoprecipitation and fluorescence microscopy studies. Lactic acid had only a very modest effect on cellular pH, did increase the levels of ER chaperones Grp78 and Grp94 and led to APP aggregate formation reminiscent of aggresomes. CONCLUSIONS/SIGNIFICANCE: These findings suggest that sustained elevations in lactic acid levels could be a risk factor in amyloidogenesis related to Alzheimer's disease through enhanced APP interaction with ER chaperone proteins and aberrant APP processing leading to increased generation of amyloid peptides and APP aggregates.

  16. DEER Sensitivity between Iron Centers and Nitroxides in Heme-Containing Proteins Improves Dramatically Using Broadband, High-Field EPR.

    Motion, Claire L; Lovett, Janet E; Bell, Stacey; Cassidy, Scott L; Cruickshank, Paul A S; Bolton, David R; Hunter, Robert I; El Mkami, Hassane; Van Doorslaer, Sabine; Smith, Graham M

    2016-04-21

    This work demonstrates the feasibility of making sensitive nanometer distance measurements between Fe(III) heme centers and nitroxide spin labels in proteins using the double electron-electron resonance (DEER) pulsed EPR technique at 94 GHz. Techniques to measure accurately long distances in many classes of heme proteins using DEER are currently strongly limited by sensitivity. In this paper we demonstrate sensitivity gains of more than 30 times compared with previous lower frequency (X-band) DEER measurements on both human neuroglobin and sperm whale myoglobin. This is achieved by taking advantage of recent instrumental advances, employing wideband excitation techniques based on composite pulses and exploiting more favorable relaxation properties of low-spin Fe(III) in high magnetic fields. This gain in sensitivity potentially allows the DEER technique to be routinely used as a sensitive probe of structure and conformation in the large number of heme and many other metalloproteins. PMID:27035368

  17. Nucleic acid labeling with [3H]orotic acid and nucleotide profile in rats in protein deprivation, enteral and parenteral essential amino acid administration, and 5-fluorouracil treatment

    Rats were fed a 0% casein diet for 1 week, with or without enteral or parenteral administration of essential amino acids, or a 25% casein diet, in one group supplemented with 5-fluorouracil treatment. Ninety minutes before sacrifice the rats were given a tracer of [3H]orotic acid. Incorporation into the acid soluble fraction, RNA, and DNA was determined in liver, small intestine, bone marrow, and kidney. Nucleotide profile was examined in liver and intestine. Protein deficiency caused inter alia a decrease in body weight; a decrease in RNA/DNA ratio and an increase in the specific RNA labeling in liver and kidney; an altered nucleotide profile in the liver; an increase in the nucleotide/DNA and RNA/DNA ratios and a decrease in the specific labeling of the acid soluble fraction, RNA, and DNA in the bone marrow. These changes were prevented to the same extent by giving essential amino acids, either orally or intravenously. The minor changes in intestinal nucleotide profile in protein deprivation were prevented to a slightly larger extent by amino acids orally than parenterally. 5-Fluorouracil treatment gave a decrease in the RNA/DNA ratio in the liver and kidney but an increase in the nucleotide/DNA and RNA/DNA ratios in the bone marrow. Nucleotide profiles were unaltered. The amount of DNA per gram of tissue decreased in bone marrow and increased in kidney. Parenteral administration per se resulted in almost no changes

  18. Stimuli-Sensitive Hydrogel Based on N-Isopropylacrylamide and Itaconic Acid for Entrapment and Controlled Release of Candida rugosa Lipase under Mild Conditions

    Nikola Milašinović

    2014-01-01

    Full Text Available Stimuli responsive pH- and temperature-sensitive hydrogel drug delivery systems, as those based on N-isopropylacrylamide (NiPAAm and itaconic acid (IA, have been attracting much of the attention of the scientific community nowadays, especially in the field of drug release. By adjusting comonomer composition, the matrix is enabled to protect the incorporated protein in the highly acidic environment of upper gastrointestinal tract and deliver it in the neutral or slightly basic region of the lower intestine. The protein/poly(NiPAAm-co-IA hydrogels were synthetized by free radical crosslinking copolymerization and were characterized concerning their swelling capability, mechanical properties, and morphology. The pore structure and sizes up to 1.90 nm allowed good entrapment of lipase molecules. Model protein, lipase from Candida rugosa, was entrapped within hydrogels upon mild conditions that provided its protection from harmful environmental influences. The efficiency of the lipase entrapment reached 96.7%, and was dependent on the initial concentration of lipase solution. The swelling of the obtained hydrogels in simulated pH and temperature of gastrointestinal tract, the lipase entrapment efficiency, and its release profiles from hydrogels were investigated as well.

  19. A highly sensitive and selective viral protein detection method based on RNA oligonucleotide nanoparticle

    Changhyun Roh

    2010-04-01

    Full Text Available Changhyun Roh1, Ho-Young Lee2, Sang-Eun Kim2, Sung-Kee Jo11Radiation Research Division for Biotechnology, Advanced Radiation Technology Institute (ARTI, Korea Atomic Energy Research Institute (KAERI, Sinjeong-dong, Jeongeup, Jeonbuk, South Korea; 2Department of Nuclear Medicine, College of Medicine, Seoul National University, South KoreaAbstract: Globally, approximately 170 million people (representing approximately 3% of the population worldwide, are infected with hepatitis C virus (HCV and at risk of serious liver disease, including chronic hepatitis. We propose a new quantum dots (QDs-supported RNA oligonucleotide approach for the specific and sensitive detection of viral protein using a biochip. This method was developed by immobilizing a HCV nonstructural protein 5B (NS5B on the surface of a glass chip via the formation of a covalent bond between an amine protein group and a ProLinkerTM glass chip. The QDs-supported RNA oligonucleotide was conjugated via an amide formation reaction from coupling of a 5′-end-amine-modified RNA oligonucleotide on the surface of QDs displaying carboxyl groups via standard EDC coupling. The QDs-conjugated RNA oligonucleotide was interacted to immobilized viral protein NS5B on the biochip. The detection is based on the variation of signal of QDs-supported RNA oligonucleotide bound on an immobilized biochip. It was demonstrated that the value of the signal has a linear relationship with concentrations of the HCV NS5B viral protein in the 1 μg mL-1 to 1 ng mL-1 range with a detection limit of 1 ng mL-1. The major advantages of this RNA-oligonucleotide nanoparticle assay are its good specificity, ease of performance, and ability to perform one-spot monitoring. The proposed method could be used as a general method of HCV detection and is expected to be applicable to other types of diseases as well.Keywords: hepatitis C virus, viral protein, RNA oligonucleotide, quantum dots, biochip

  20. Aqueous amino acids and proteins near solid surfaces: ZnO, ZnS, Au, and mica

    Cieplak, Marek

    2015-03-01

    We calculate potentials of the mean force for 20 amino acids in the vicinity of the (111) surface of Au, four surfaces of ZnO, and the (110) surface of ZnS using molecular dynamics simulations combined with the umbrella sampling method. In the case of Au, we compare results obtained within three different force fields: one hydrophobic (for a contaminated surface) and two hydrophilic - with and without polarization of the solid. The properties of water near the surface sensitively depend on the force field. All of these fields lead to good binding with very different specificities and to unlike patterns in the density and polarization of water. We demonstrate that binding energies of dipeptides are distinct from the combined binding energies of their amino acidic components. We show that ZnS is more more hydrophobic than ZnO and that the density profile of water is quite different than that forming near ZnO - it has only a minor articulation into layers. Furthermore, the first layer of water is disordered and mobile. In the case of ZnS, not all amino acids can attach to the surface and when they do, the binding energies are comparable to those found for the surfaces of ZnO (and to hydrogen bonds in proteins) but the nature of the specificity is distinct. The covalent bond with the sulfur atom on cysteine is modeled by the Morse potential. For the hydrophobic Au, adsorption events of a small protein (the tryptophan cage) are driven by attraction to the strongest binding amino acids. This is not so for ZnO, ZnS and for the hydrophilic models of Au - a result of smaller specificities combined with the difficulty for proteins, but sometimes not for single amino acids, to penetrate the first layer of water. Molecular dynamics studies of several proteins near mica with a net charge on its surface indicate existence of two types of states: deformed and unfolded. Using a coarse-grained model, we also study a glassy behavior of protein layers at air-water interfaces. Polish

  1. Whey Protein Delays Gastric Emptying and Suppresses Plasma Fatty Acids and Their Metabolites Compared to Casein, Gluten, and Fish Protein

    Stanstrup, Jan; Schou, Simon S; Holmer-Jensen, Jens;

    2014-01-01

    studies, the WI meal caused a decreased rate of gastric emptying compared to the other test meals. The WI meal also caused elevated levels of a number of amino acids, possibly stimulating insulin release leading to reduced plasma glucose. The WI meal also caused decreased levels of a number of fatty acids......Whey protein has been demonstrated to improve fasting lipid and insulin response in overweight and obese individuals. To establish new hypotheses for this effect and to investigate the impact of stomach emptying, we compared plasma profiles after intake of whey isolate (WI), casein, gluten (GLU...

  2. A comparison of chemical shift sensitivity of trifluoromethyl tags: optimizing resolution in {sup 19}F NMR studies of proteins

    Ye, Libin; Larda, Sacha Thierry; Frank Li, Yi Feng [University of Toronto, UTM, Department of Chemistry (Canada); Manglik, Aashish [Stanford University School of Medicine, Department of Molecular and Cellular Physiology (United States); Prosser, R. Scott, E-mail: scott.prosser@utoronto.ca [University of Toronto, UTM, Department of Chemistry (Canada)

    2015-05-15

    The elucidation of distinct protein conformers or states by fluorine ({sup 19}F) NMR requires fluorinated moieties whose chemical shifts are most sensitive to subtle changes in the local dielectric and magnetic shielding environment. In this study we evaluate the effective chemical shift dispersion of a number of thiol-reactive trifluoromethyl probes [i.e. 2-bromo-N-(4-(trifluoromethyl)phenyl)acetamide (BTFMA), N-(4-bromo-3-(trifluoromethyl)phenyl)acetamide (3-BTFMA), 3-bromo-1,1,1-trifluoropropan-2-ol (BTFP), 1-bromo-3,3,4,4,4-pentafluorobutan-2-one (BPFB), 3-bromo-1,1,1-trifluoropropan-2-one (BTFA), and 2,2,2-trifluoroethyl-1-thiol (TFET)] under conditions of varying polarity. In considering the sensitivity of the {sup 19}F NMR chemical shift to the local environment, a series of methanol/water mixtures were prepared, ranging from relatively non-polar (MeOH:H{sub 2}O = 4) to polar (MeOH:H{sub 2}O = 0.25). {sup 19}F NMR spectra of the tripeptide, glutathione ((2S)-2-amino-4-{[(1R)-1-[(carboxymethyl)carbamoyl] -2-sulfanylethyl]carbamoyl}butanoic acid), conjugated to each of the above trifluoromethyl probes, revealed that the BTFMA tag exhibited a significantly greater range of chemical shift as a function of solvent polarity than did either BTFA or TFET. DFT calculations using the B3LYP hybrid functional and the 6-31G(d,p) basis set, confirmed the observed trend in chemical shift dispersion with solvent polarity.

  3. Highly sensitive C-reactive protein (CRP) assay using metal-enhanced fluorescence (MEF)

    Zhang, Yi; Keegan, Gemma L., E-mail: gemmakeegan@gmail.com [Dublin City University, School of Physical Sciences, Biomedical Diagnostics Institute (Ireland); Stranik, Ondrej [Leibniz Institute of Photonic Technology, Department of NanoBiophotonics (Germany); Brennan-Fournet, Margaret E. [CMP-EMSE, MOC, Department of Bioelectronics, Ecole Nationale Superieure des Mines (France); McDonagh, Colette [Dublin City University, School of Physical Sciences, Biomedical Diagnostics Institute (Ireland)

    2015-07-15

    Fluorescence has been extensively employed in the area of diagnostic immunoassays. A significant enhancement of fluorescence can be achieved when noble metal nanoparticles are placed in close proximity to fluorophores. This effect, referred to as metal-enhanced fluorescence (MEF), has the potential to produce immunoassays with a high sensitivity and a low limit of detection (LOD). In this study, we investigate the fluorescence enhancement effect of two different nanoparticle systems, large spherical silver nanoparticles (AgNPs) and gold edge-coated triangular silver nanoplates, and both systems were evaluated for MEF. The extinction properties and electric field enhancement of both systems were modeled, and the optimum system, spherical AgNPs, was used in a sandwich immunoassay for human C-reactive protein with a red fluorescent dye label. A significant enhancement in the fluorescence was observed, which corresponded to an LOD improvement of ∼19-fold compared to a control assay without AgNPs.

  4. On the sensitivity of protein data bank normal mode analysis: an application to GH10 xylanases

    Tirion, Monique M.

    2015-12-01

    Protein data bank entries obtain distinct, reproducible flexibility characteristics determined by normal mode analyses of their three dimensional coordinate files. We study the effectiveness and sensitivity of this technique by analyzing the results on one class of glycosidases: family 10 xylanases. A conserved tryptophan that appears to affect access to the active site can be in one of two conformations according to x-ray crystallographic electron density data. The two alternate orientations of this active site tryptophan lead to distinct flexibility spectra, with one orientation thwarting the oscillations seen in the other. The particular orientation of this sidechain furthermore affects the appearance of the motility of a distant, C terminal region we term the mallet. The mallet region is known to separate members of this family of enzymes into two classes.

  5. Relationship between high-sensitivity C-reactive protein level and angiographical characteristics of coronary atherosclerosis

    JIA En-zhi; HUANG Jun; MA Wen-zhu; YANG Zhi-jian; YUAN Biao; ZANG Xiao-ling; WANG Rong-hu; ZHU Tie-bing; WANG Lian-sheng; CHEN Bo; CAO Ke-jiang

    2006-01-01

    @@ Arole for inflammation has become well established over the past decade or more in theories describing the atherosclerotic disease process.1,2 From a pathological viewpoint, all stages, ie, initiation, growth, and complication of the atherosclerotic plaque,3,4 might be considered to be an inflammatory response to injury. Several prospective studies 5-7 recently showed that plasma high sensitivity C-reactive protein (hsCRP) levels, which are one of the markers of systemic inflammation, are a powerful predictor of future myocardial infarction and cardiac death among apparently healthy individuals. However, the association between the plasma hsCRP levels and the extent of coronary stenosis in subjects remains controversial. Some studies previously demon- strated such associations,8,9 whereas other could not found.10,11 Gensini's score assigns a severity score for a stenosed vessel depending on the degree of luminal narrowing and the importance of its location.12

  6. Highly sensitive C-reactive protein (CRP) assay using metal-enhanced fluorescence (MEF)

    Fluorescence has been extensively employed in the area of diagnostic immunoassays. A significant enhancement of fluorescence can be achieved when noble metal nanoparticles are placed in close proximity to fluorophores. This effect, referred to as metal-enhanced fluorescence (MEF), has the potential to produce immunoassays with a high sensitivity and a low limit of detection (LOD). In this study, we investigate the fluorescence enhancement effect of two different nanoparticle systems, large spherical silver nanoparticles (AgNPs) and gold edge-coated triangular silver nanoplates, and both systems were evaluated for MEF. The extinction properties and electric field enhancement of both systems were modeled, and the optimum system, spherical AgNPs, was used in a sandwich immunoassay for human C-reactive protein with a red fluorescent dye label. A significant enhancement in the fluorescence was observed, which corresponded to an LOD improvement of ∼19-fold compared to a control assay without AgNPs

  7. Better prediction of protein contact number using a support vector regression analysis of amino acid sequence

    Yuan Zheng

    2005-10-01

    Full Text Available Abstract Background Protein tertiary structure can be partly characterized via each amino acid's contact number measuring how residues are spatially arranged. The contact number of a residue in a folded protein is a measure of its exposure to the local environment, and is defined as the number of Cβ atoms in other residues within a sphere around the Cβ atom of the residue of interest. Contact number is partly conserved between protein folds and thus is useful for protein fold and structure prediction. In turn, each residue's contact number can be partially predicted from primary amino acid sequence, assisting tertiary fold analysis from sequence data. In this study, we provide a more accurate contact number prediction method from protein primary sequence. Results We predict contact number from protein sequence using a novel support vector regression algorithm. Using protein local sequences with multiple sequence alignments (PSI-BLAST profiles, we demonstrate a correlation coefficient between predicted and observed contact numbers of 0.70, which outperforms previously achieved accuracies. Including additional information about sequence weight and amino acid composition further improves prediction accuracies significantly with the correlation coefficient reaching 0.73. If residues are classified as being either "contacted" or "non-contacted", the prediction accuracies are all greater than 77%, regardless of the choice of classification thresholds. Conclusion The successful application of support vector regression to the prediction of protein contact number reported here, together with previous applications of this approach to the prediction of protein accessible surface area and B-factor profile, suggests that a support vector regression approach may be very useful for determining the structure-function relation between primary protein sequence and higher order consecutive protein structural and functional properties.

  8. Effect of increased protein intake on renal acid load and renal hemodynamic responses.

    Teunissen-Beekman, Karianna F M; Dopheide, Janneke; Geleijnse, Johanna M; Bakker, Stephan J L; Brink, Elizabeth J; de Leeuw, Peter W; van Baak, Marleen A

    2016-03-01

    Increased protein intake versus maltodextrin intake for 4 weeks lowers blood pressure. Concerns exist that high-protein diets reduce renal function. Effects of acute and 4-week protein intake versus maltodextrin intake on renal acid load, glomerular filtration rate and related parameters were compared in this study. Seventy-nine overweight individuals with untreated elevated blood pressure and normal kidney function were randomized to consume a mix of protein isolates (60 g/day) or maltodextrin (60 g/day) for 4 weeks in energy balance. Twenty-four-hour urinary potential renal acid load (uPRAL) was compared between groups. A subgroup (maltodextrin N = 27, protein mix N = 25) participated in extra test days investigating fasting levels and postprandial effects of meals supplemented with a moderate protein- or maltodextrin-load on glomerular filtration rate, effective renal plasma flow, plasma renin, aldosterone, pH, and bicarbonate. uPRAL was significantly higher in the protein group after 4 weeks (P ≤ 0.001). Postprandial filtration fraction decreased further after the protein-supplemented breakfast than after the maltodextrin-supplemented breakfast after 4 weeks of supplementation (P ≤ 0.001). Fasting and postprandial levels of glomerular filtration rate, effective renal plasma flow, renin, aldosterone, angiotensin-converting enzyme, pH and bicarbonate did not differ between groups. In conclusion, 4 weeks on an increased protein diet (25% of energy intake) increased renal acid load, but did not affect renal function. Postprandial changes, except for filtration fraction, also did not differ between groups. These data suggest that a moderate increase in protein intake by consumption of a protein mix for 4 weeks causes no (undesirable) effects on kidney function in overweight and obese individuals with normal kidney function. PMID:26997623

  9. Alterations in proteins and amino acids of the Nile cyanobacteria Pseudanabaena limnetica and Anabaena wisconsinense in response to industrial wastewater pollution

    Mostafa Mohamed El-Sheekh; Ahmed Mohamed El-Otify; Hani Saber

    2011-01-01

    The effect of industrial wastewater on the Nile cyanobacteria Pseudanabaena limnetica and Anabaena wisconsinense was investigated. The data showed that P. limnetica was more sensitive to pollution than A. wisconsinense. The treatments with different levels of wastewater exerted pronounced reductions in protein and amino acids content. SDS-PAGE analysis revealed that the cyanobacteria grown in the industrial wastewater showed induction in the synthesis of certain polypeptides and repression of...

  10. Rumen Degradability and Small Intestinal Digestibility of the Amino Acids in Four Protein Supplements.

    Wang, Y; Jin, L; Wen, Q N; Kopparapu, N K; Liu, J; Liu, X L; Zhang, Y G

    2016-02-01

    The supplementation of livestock feed with animal protein is a present cause for public concern, and plant protein shortages have become increasingly prominent in China. This conflict may be resolved by fully utilizing currently available sources of plant protein. We estimated the rumen degradability and the small intestinal digestibility of the amino acids (AA) in rapeseed meal (RSM), soybean meal (SBM), sunflower seed meal (SFM) and sesame meal (SSM) using the mobile nylon bag method to determine the absorbable AA content of these protein supplements as a guide towards dietary formulations for the dairy industry. Overall, this study aimed to utilize protein supplements effectively to guide dietary formulations to increase milk yield and save plant protein resources. To this end, we studied four cows with a permanent rumen fistula and duodenal T-shape fistula in a 4×4 Latin square experimental design. The results showed that the total small intestine absorbable amino acids and small intestine absorbable essential amino acids were higher in the SBM (26.34% and 13.11% dry matter [DM], respectively) than in the SFM (13.97% and 6.89% DM, respectively). The small intestine absorbable Lys contents of the SFM, SSM, RSM and SBM were 0.86%, 0.88%, 1.43%, and 2.12% (DM basis), respectively, and the absorbable Met contents of these meals were 0.28%, 1.03%, 0.52%, and 0.47% (DM basis), respectively. Among the examined food sources, the milk protein score of the SBM (0.181) was highest followed by those of the RSM (0.136), SSM (0.108) and SFM (0.106). The absorbable amino acid contents of the protein supplements accurately reflected protein availability, which is an important indicator of the balance of feed formulation. Therefore, a database detailing the absorbable AA should be established. PMID:26732449

  11. Analysis of protein-protein interactions in MCF-7 and MDA-MB-231 cell lines using phthalic acid chemical probes.

    Liang, Shih-Shin; Wang, Tsu-Nai; Tsai, Eing-Mei

    2014-01-01

    Phthalates are a class of plasticizers that have been characterized as endocrine disrupters, and are associated with genital diseases, cardiotoxicity, hepatotoxicity, and nephrotoxicity in the GeneOntology gene/protein database. In this study, we synthesized phthalic acid chemical probes and demonstrated differing protein-protein interactions between MCF-7 cells and MDA-MB-231 breast cancer cell lines. Phthalic acid chemical probes were synthesized using silicon dioxide particle carriers, which were modified using the silanized linker 3-aminopropyl triethoxyslane (APTES). Incubation with cell lysates from breast cancer cell lines revealed interactions between phthalic acid and cellular proteins in MCF-7 and MDA-MB-231 cells. Subsequent proteomics analyses indicated 22 phthalic acid-binding proteins in both cell types, including heat shock cognate 71-kDa protein, ATP synthase subunit beta, and heat shock protein HSP 90-beta. In addition, 21 MCF-7-specific and 32 MDA-MB-231 specific phthalic acid-binding proteins were identified, including related proteasome proteins, heat shock 70-kDa protein, and NADPH dehydrogenase and ribosomal correlated proteins, ras-related proteins, and members of the heat shock protein family, respectively. PMID:25402641

  12. Effect of protein kinase C inhibitor (PKCI) on radiation sensitivity and c-fos transcription activity

    The human genetic disorder ataxia-telangiectasia (AT) is a multisystem disease characterized by extreme radiosensitivity. The recent identification of the gene mutated in AT, ATM, and the demonstration that it encodes a homologous domain of phosphatidylinositol 3-kinase (PI3-K), the catalytic subunit of an enzyme involved in transmitting signals from the cell surface to the nucleus, provide support for a role of this gene in signal transduction. Although ionizing radiation was known to induce c-fos transcription, nothing is known about how ATM or PKCI mediated signal transduction pathway modulates the c-fos gene transcription and gene expression. Here we have studied the effect of PKCI on radiation sensitivity and c-fos transcription in normal and AT cells. Normal (LM217) and AT (AT58IVA) cells were transfected with PKCI expression plasmid and the overexpression and integration of PKCI was evaluated by northern blotting and polymerase chain reaction, respectively. 5 Gy of radiation was exposed to LM and AT cells transfected with PKCI expression plasmid and cells were harvested 48 hours after radiation and investigated apoptosis with TUNEL method. The c-fos transcription activity was studied by performing CAT assay of reporter gene after transfection of c-fos CAT plasmid into AT and LM cells. Our results demonstrate for the first time a role of PKCI on. the radiation sensitivity and c-fos expression in LM and AT cells. PKCI increased radiation induced apoptosis in LM cells but reduced apoptosis in AT cells. The basal c-fos transcription activity is 70 times lower in AT cells than that in LM cells. The c-fos transcription activity was repressed by overexpression of PKCI in LM cells but not in AT cells. After induction of c-fos by Ras protein, overexpression of PKCI repressed c-fos transcription in LM cells but not in AT cells. Overexpression of PKCI increased radiation sensitivity and repressed c-fos transcription in LM cells but not in AT cells. The results may be a

  13. l-Arginine grafted alginate hydrogel beads: A novel pH-sensitive system for specific protein delivery

    Mohamed S. Mohy Eldin

    2015-05-01

    Full Text Available Novel pH-sensitive hydrogels based on l-arginine grafted alginate (Arg-g-Alg hydrogel beads were synthesized and utilized as a new carrier for protein delivery (BSA in specific pH media. l-arginine was grafted onto the polysaccharide backbone of virgin alginate via amine functions. Evidences of grafting of alginate were extracted from FT-IR and thermal analysis, while the morphological structure of Arg-g-Alg hydrogel beads was investigated by SEM photographs. Factors affecting on the grafting process e.g. l-arginine concentration, reaction time, reaction temperature, reaction pH, and crosslinking conditions, have been studied. Whereas, grafting efficiency of each factor was evaluated. Grafting of alginate has improved both thermal and morphological properties of Arg-g-Alg hydrogel beads. The swelling behavior of Arg-g-Alg beads was determined as a function of pH and compared with virgin calcium alginate beads. The cumulative in vitro release profiles of BSA loaded beads were studied at different pHs for simulating the physiological environments of the gastrointestinal tract. The amount of BSA released from neat alginate beads at pH 2 was almost 15% after 5 h, while the Arg-g-Alg beads at the same conditions were clearly higher than 45%, then it increased to 90% at pH 7.2. Accordingly, grafting of alginate has improved its release profile behavior particularly in acidic media. The preliminary results clearly suggested that the Arg-g-Alg hydrogel may be a potential candidate for polymeric carrier for oral delivery of protein or drugs.

  14. Protein content and amino acids profile of pseudocereals.

    Mota, Carla; Santos, Mariana; Mauro, Raul; Samman, Norma; Matos, Ana Sofia; Torres, Duarte; Castanheira, Isabel

    2016-02-15

    Quinoa (Chenopodium quinoa), amaranth (Amaranthus caudatus) and buckwheat (Fagopyrum esculentum) represent the main protein source in several diets, although these pseudocereals are not currently present in the FCDB nutrient profile information. The aim of this work is to characterise the AA profile of these pseudocereals and compare them with rice. Total protein content revealed to vary from 16.3g/100g (quinoa Salta) to 13.1g/100g (buckwheat) and lower values were found in rice samples (6.7g/100g). For pseudocereals the most abundant essential AA was leucine. Quinoa-Salta evidences the highest leucine content (1013mg/100g) and the minor methionine content (199mg/100g). Buckwheat was the cereal with the highest phenylalanine content (862mg/100g). Rice (Oryza sativa) presents the lowest content for all AA. Results showed pseudocereals as the best source of AA. EuroFIR guidelines where strictly followed and proved to be a crucial tool to guarantee data interchangeability and comparability. PMID:26433287

  15. D-amino acids indirectly inhibit biofilm formation in Bacillus subtilis by interfering with protein synthesis.

    Leiman, Sara A; May, Janine M; Lebar, Matthew D; Kahne, Daniel; Kolter, Roberto; Losick, Richard

    2013-12-01

    The soil bacterium Bacillus subtilis forms biofilms on surfaces and at air-liquid interfaces. It was previously reported that these biofilms disassemble late in their life cycle and that conditioned medium from late-stage biofilms inhibits biofilm formation. Such medium contained a mixture of D-leucine, D-methionine, D-tryptophan, and D-tyrosine and was reported to inhibit biofilm formation via the incorporation of these D-amino acids into the cell wall. Here, we show that L-amino acids were able to specifically reverse the inhibitory effects of their cognate D-amino acids. We also show that D-amino acids inhibited growth and the expression of biofilm matrix genes at concentrations that inhibit biofilm formation. Finally, we report that the strain routinely used to study biofilm formation has a mutation in the gene (dtd) encoding D-tyrosyl-tRNA deacylase, an enzyme that prevents the misincorporation of D-amino acids into protein in B. subtilis. When we repaired the dtd gene, B. subtilis became resistant to the biofilm-inhibitory effects of D-amino acids without losing the ability to incorporate at least one noncanonical D-amino acid, D-tryptophan, into the peptidoglycan peptide side chain. We conclude that the susceptibility of B. subtilis to the biofilm-inhibitory effects of D-amino acids is largely, if not entirely, due to their toxic effects on protein synthesis. PMID:24097941

  16. Cytosolic fatty acid-binding proteins: subjects and tools in metabolic research

    Binas, B. [Max Delbrueck Center for Molecular Medicine, Berlin-Buch (Germany)

    1998-12-31

    Fatty acid-binding proteins (FABPs) are major targets for specific binding of fatty acids in vivo. They constitute a widely expressed family of genetically related, small cytosolic proteins which very likely mediate intracellular transport of free long chain fatty acids. Genetic inhibition of FABP expression in vivo should therefore provide a useful tool to investigate and engineer fatty acid metabolism. (orig.) [Deutsch] Fettsaeurebindungsproteine (FABPs) sind wichtige Bindungsstellen fuer Fettsaeuren in vivo; sie bilden eine breit exprimierte Familie genetisch verwandter kleiner Zytosoleiweisse, die sehr wahrscheinlich den intrazellulaeren Transport unveresterter langkettiger Fettsaeuren vermitteln. Die genetische Hemmung der FABP-Expanssion in vivo bietet sich deshalb als Werkzeug zur Erforschung und gezielten Veraenderung des Fettsaeurestoffwechsels an. (orig.)

  17. Fatty acid binding protein 7 and n-3 poly unsaturated fatty acid supply in early rat brain development.

    Maximin, Elise; Langelier, Bénédicte; Aïoun, Josiane; Al-Gubory, Kaïs H; Bordat, Christian; Lavialle, Monique; Heberden, Christine

    2016-03-01

    Fatty acid binding protein 7 (FABP7), abundant in the embryonic brain, binds with the highest affinity to docosahexaenoic acid (DHA) and is expressed in the early stages of embryogenesis. Here, we have examined the consequences of the exposure to different DHA levels and of the in utero depletion of FABP7 on early rat brain development. Neurodevelopment was evaluated through the contents of two proteins, connexin 43 (Cx43) and cyclin-dependent kinase 5 (CDK5), both involved in neuroblast proliferation, differentiation, and migration. The dams were fed with diets presenting different DHA contents, from deficiency to supplementation. DHA brain embryos contents already differed at embryonic day 11.5 and the differences kept increasing with time. Cx43 and CDK5 contents were positively associated with the brain DHA levels. When FABP7 was depleted in vivo by injections of siRNA in the telencephalon, the enhancement of the contents of both proteins was lost in supplemented animals, but FABP7 depletion did not modify phospholipid compositions regardless of the diets. Thus, FABP7 is a necessary mediator of the effect of DHA on these proteins synthesis, but its role in DHA uptake is not critical, although FABP7 is localized in phospholipid-rich areas. Our study shows that high contents of DHA associated with FABP7 are necessary to promote early brain development, which prompted us to recommend DHA supplementation early in pregnancy. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 287-297, 2016. PMID:26037116

  18. Ruminal protein metabolism and intestinal amino acid utilization as affected by dietary protein and carbohydrate sources in sheep.

    Hussein, H S; Jordan, R M; Stern, M D

    1991-05-01

    Eight wether lambs fitted with ruminal, duodenal, and ileal cannulas were used in a replicated 4 x 4 Latin square design to study the effects of carbohydrate and protein sources on ruminal protein metabolism and carbohydrate fermentation and intestinal amino acid (AA) absorption. Treatments were arranged as a 2 x 2 factorial. Carbohydrate sources were corn and barley; protein sources were soybean meal (SBM) and fish meal (FM). Diets contained 15.5% CP, of which 40% was supplied by SBM or FM. Corn or barley provided 39% of dietary DM that contained equal amounts of grass hay and wheat straw. Fish meal diets produced a lower (P less than .05) ruminal NH3 concentration and resulted in less CP degradation and bacterial protein flow to the duodenum than did SBM diets. Replacing SBM with FM increased (P less than .05) ruminal digestion of all fiber fractions. In addition, cellulose and hemicellulose digestibilities in the rumen tended to increase (P greater than .05) when barley replaced corn in the FM diets. Carbohydrate x protein interactions (P less than .05) were observed for OM digestion in the rumen and AA absorption in the small intestine (percentage of AA entering); these interactions were highest for the barley-FM diet. These results suggest that feeding FM with barley, which is high in both degradable carbohydrate and protein, might benefit ruminants more than feeding FM with corn, which is high in degradable carbohydrate but relatively low in degradable protein. PMID:1648551

  19. Recognizing protein–protein interfaces with empirical potentials and reduced amino acid alphabets

    Wodak Shoshana

    2007-07-01

    Full Text Available Abstract Background In structural genomics, an important goal is the detection and classification of protein–protein interactions, given the structures of the interacting partners. We have developed empirical energy functions to identify native structures of protein–protein complexes among sets of decoy structures. To understand the role of amino acid diversity, we parameterized a series of functions, using a hierarchy of amino acid alphabets of increasing complexity, with 2, 3, 4, 6, and 20 amino acid groups. Compared to previous work, we used the simplest possible functional form, with residue–residue interactions and a stepwise distance-dependence. We used increased computational ressources, however, constructing 290,000 decoys for 219 protein–protein complexes, with a realistic docking protocol where the protein partners are flexible and interact through a molecular mechanics energy function. The energy parameters were optimized to correctly assign as many native complexes as possible. To resolve the multiple minimum problem in parameter space, over 64000 starting parameter guesses were tried for each energy function. The optimized functions were tested by cross validation on subsets of our native and decoy structures, by blind tests on series of native and decoy structures available on the Web, and on models for 13 complexes submitted to the CAPRI structure prediction experiment. Results Performance is similar to several other statistical potentials of the same complexity. For example, the CAPRI target structure is correctly ranked ahead of 90% of its decoys in 6 cases out of 13. The hierarchy of amino acid alphabets leads to a coherent hierarchy of energy functions, with qualitatively similar parameters for similar amino acid types at all levels. Most remarkably, the performance with six amino acid classes is equivalent to that of the most detailed, 20-class energy function. Conclusion This suggests that six carefully chosen amino

  20. Biochemical Roles for Conserved Residues in the Bacterial Fatty Acid-binding Protein Family.

    Broussard, Tyler C; Miller, Darcie J; Jackson, Pamela; Nourse, Amanda; White, Stephen W; Rock, Charles O

    2016-03-18

    Fatty acid kinase (Fak) is a ubiquitous Gram-positive bacterial enzyme consisting of an ATP-binding protein (FakA) that phosphorylates the fatty acid bound to FakB. In Staphylococcus aureus, Fak is a global regulator of virulence factor transcription and is essential for the activation of exogenous fatty acids for incorporation into phospholipids. The 1.2-Å x-ray structure of S. aureus FakB2, activity assays, solution studies, site-directed mutagenesis, and in vivo complementation were used to define the functions of the five conserved residues that define the FakB protein family (Pfam02645). The fatty acid tail is buried within the protein, and the exposed carboxyl group is bound by a Ser-93-fatty acid carboxyl-Thr-61-His-266 hydrogen bond network. The guanidinium of the invariant Arg-170 is positioned to potentially interact with a bound acylphosphate. The reduced thermal denaturation temperatures of the T61A, S93A, and H266A FakB2 mutants illustrate the importance of the hydrogen bond network in protein stability. The FakB2 T61A, S93A, and H266A mutants are 1000-fold less active in the Fak assay, and the R170A mutant is completely inactive. All FakB2 mutants form FakA(FakB2)2 complexes except FakB2(R202A), which is deficient in FakA binding. Allelic replacement shows that strains expressing FakB2 mutants are defective in fatty acid incorporation into phospholipids and virulence gene transcription. These conserved residues are likely to perform the same critical functions in all bacterial fatty acid-binding proteins. PMID:26774272