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Sample records for aldh1a2 protein levels

  1. Retinoic acid homeostasis through aldh1a2 and cyp26a1 mediates meiotic entry in Nile tilapia (Oreochromis niloticus)

    PubMed Central

    Feng, Ruijuan; Fang, Lingling; Cheng, Yunying; He, Xue; Jiang, Wentao; Dong, Ranran; Shi, Hongjuan; Jiang, Dongneng; Sun, Lina; Wang, Deshou

    2015-01-01

    Meiosis is a process unique to the differentiation of germ cells. Retinoic acid (RA) is the key factor controlling the sex-specific timing of meiotic initiation in tetrapods; however, the role of RA in meiotic initiation in teleosts has remained unclear. In this study, the genes encoding RA synthase aldh1a2, and catabolic enzyme cyp26a1 were isolated from Nile tilapia (Oreochromis niloticus), a species without stra8. The expression of aldh1a2 was up-regulated and expression of cyp26a1 was down-regulated before the meiotic initiation in ovaries and in testes. Treatment with RA synthase inhibitor or disruption of Aldh1a2 by CRISPR/Cas9 resulted in delayed meiotic initiation, with simultaneous down-regulation of cyp26a1 and up-regulation of sycp3. By contrast, treatment with an inhibitor of RA catabolic enzyme and disruption of cyp26a1 resulted in earlier meiotic initiation, with increased expression of aldh1a2 and sycp3. Additionally, treatment of XY fish with estrogen (E2) and XX fish with fadrozole led to sex reversal and reversion of meiotic initiation. These results indicate that RA is indispensable for meiotic initiation in teleosts via a stra8 independent signaling pathway where both aldh1a2 and cyp26a1 are critical. In contrast to mammals, E2 is a major regulator of sex determination and meiotic initiation in teleosts. PMID:25976364

  2. prep1.2 and aldh1a2 participate to a positive loop required for branchial arches development in zebrafish.

    PubMed

    Vaccari, Enrico; Deflorian, Gianluca; Bernardi, Elisa; Pauls, Stefan; Tiso, Natascia; Bortolussi, Marino; Argenton, Francesco

    2010-07-01

    Segmentation is a key step in embryonic development. Acting in all germ layers, it is responsible for the generation of antero-posterior asymmetries. Hox genes, with their diverse expression in individual segments, are fundamental players in the determination of different segmental fates. In vertebrates, Hox gene products gain specificity for DNA sequences by interacting with Pbx, Prep and Meis homeodomain transcription factors. In this work we cloned and analysed prep1.2 in zebrafish. In-situ hybridization experiments show that prep1.2 is maternally and ubiquitously expressed up to early somitogenesis when its expression pattern becomes more restricted to the head and trunk mesenchyme. Experiments of loss of function with prep1.2 morpholinos change the shape of the hyoid and third pharyngeal cartilages while arches 4-7 and pectoral fins are absent, a phenotype strikingly similar to that caused by loss of retinoic acid (RA). In fact, we show that prep1.2 is positively regulated by RA and required for the normal expression of aldh1a2 at later stages, particularly in tissues involved in the development of the branchial arches and pectoral fins. Thus, prep1.2 and aldh1a2 are members of an indirect positive feedback loop required for pharyngeal endoderm and posterior branchial arches development. As the paralogue gene prep1.1 is more important in hindbrain patterning and neural crest chondrogenesis, we provide evidence of a functional specialization of prep genes in zebrafish head segmentation and morphogenesis. PMID:20423710

  3. Atomic-level analysis of membrane-protein structure.

    PubMed

    Hendrickson, Wayne A

    2016-06-01

    Membrane proteins are substantially more challenging than natively soluble proteins as subjects for structural analysis. Thus, membrane proteins are greatly underrepresented in structural databases. Recently, focused consortium efforts and advances in methodology for protein production, crystallographic analysis and cryo-EM analysis have accelerated the pace of atomic-level structure determination of membrane proteins. PMID:27273628

  4. Ethanol Disrupts Chondrification of the Neurocranial Cartilages in Medaka Embryos without Affecting Aldehyde Dehydrogenase 1A2 (Aldh1A2) Promoter Methylation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Medaka (Oryzias latipes) embryos at different developmental stages were exposed to ethanol for 48 h, then allowed to hatch. Teratogenic effects were evaluated in hatchlings after examining chondrocranial cartilage deformities. Ethanol disrupted cartilage development in medaka in a dose and developme...

  5. Yeast prions: Paramutation at the protein level?

    PubMed

    Tuite, Mick F

    2015-08-01

    Prions are proteins that have the potential to refold into a novel conformation that templates the conversion of like molecules to the altered infectious form. In the yeast Saccharomyces cerevisiae, trans-generational epigenetic inheritance can be mediated by a number of structurally and functionally diverse prions. Prionogenesis can confer both loss-of-function and gain-of-function properties to the prion protein and this in turn can have a major impact on host phenotype, short-term adaptation and evolution of new traits. Prionogenesis shares a number of properties in common with paramutation and can be considered as a mitotically and meiotically heritable change in protein conformation induced by trans-interactions between homologous proteins. PMID:26386407

  6. High levels of plasma protein C in nephrotic syndrome.

    PubMed

    Pabinger-Fasching, I; Lechner, K; Niessner, H; Schmidt, P; Balzar, E; Mannhalter, C

    1985-02-18

    In patients with severe nephrotic syndrome determinations of plasma protein C: Ag levels (8 patients: 5 adults, 3 children) and protein C activity (3 out of 8 patients) revealed significantly elevated plasma protein C concentrations. Furthermore we observed a significant inverse correlation of protein C: Ag to AT III: Ag levels. No protein C: Ag could be detected in the urine of two patients studied. We conclude from our data, that changes of plasma protein C do not contribute to the high thrombotic tendency in nephrotic syndrome. PMID:3838827

  7. Dietary protein source and level alters growth in neon tetras.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nutritional studies for aquarium fish like the neon tetra are sparse in comparison with those for food fish. To determine the optimum dietary protein level and source for growth of neon tetras, diets were formulated to contain 25, 35, 45 and 55% dietary protein from either marine animal protein or ...

  8. Synaptic protein levels altered in vascular dementia

    PubMed Central

    Sinclair, Lindsey I; Tayler, Hannah M; Love, Seth

    2015-01-01

    Introduction Cerebral ischaemia is the defining pathophysiological abnormality in most forms of vascular dementia (VAD), but the pathogenesis of the dementia remains poorly understood. In Alzheimer's disease (AD), there is early loss of synaptic proteins, but these have been little studied in VAD. Materials and Methods We measured synaptophysin, postsynaptic density protein 95 (PSD-95), drebrin, synaptosomal-associated protein 25 (SNAP-25) and vascular endothelial growth factor (VEGF) by enzyme-linked immunosorbent assays in superior temporal cortex from 11 patients with VAD and, initially, 11 non-dementia controls. We corrected for neuronal content by measurement of neuron-specific enolase. A further 11 controls were subsequently used in a validation study. Simulation of post-mortem delay found that PSD-95 was stable at 4°C but declined slightly at RT. SNAP-25 and drebrin showed good post-mortem stability. Previous studies had shown good post-mortem preservation of synaptophysin and VEGF. Results The VAD cases had lower synaptophysin (but P > 0.05 in initial study), significantly lower SNAP-25 (P = 0.024) and significantly higher drebrin (P = 0.020). On comparison with the second control group, the reduction in synaptophysin was significant (P = 0.008), and the other results were confirmed. Conclusion There is probably a reduction in presynaptic proteins in the temporal cortex in VAD, although not as marked as in AD. In VAD, there is also an increase in drebrin, which may be a response to reduced synaptic input. PMID:25559750

  9. Translation Levels Control Multi-Spanning Membrane Protein Expression

    PubMed Central

    Brown, Cecilia; Bostrom, Jenny; Fuh, Germaine; Lee, Chingwei V.; Huang, Arthur; Vandlen, Richard L.; Yansura, Daniel G.

    2012-01-01

    Attempts to express eukaryotic multi-spanning membrane proteins at high-levels have been generally unsuccessful. In order to investigate the cause of this limitation and gain insight into the rate limiting processes involved, we have analyzed the effect of translation levels on the expression of several human membrane proteins in Escherichia coli (E. coli). These results demonstrate that excessive translation initiation rates of membrane proteins cause a block in protein synthesis and ultimately prevent the high-level accumulation of these proteins. Moderate translation rates allow coupling of peptide synthesis and membrane targeting, resulting in a significant increase in protein expression and accumulation over time. The current study evaluates four membrane proteins, CD20 (4-transmembrane (TM) helixes), the G-protein coupled receptors (GPCRs, 7-TMs) RA1c and EG-VEGFR1, and Patched 1 (12-TMs), and demonstrates the critical role of translation initiation rates in the targeting, insertion and folding of integral membrane proteins in the E. coli membrane. PMID:22563408

  10. Dietary protein level and performance of growing Baladi kids

    PubMed Central

    Abdelrahman, M. M.; Aljumaah, R. S.

    2014-01-01

    A study was conducted to evaluate the effect of feeding different levels of protein to black Baladi breed kids. Weanling Baladi kids (n=18; 75 to 90 days old) were selected and individually housed at our experimental farm. Kids were divided randomly to one of the three treatments for 12 weeks. The three dietary treatments were: T1: control ration, formulated according to NRC to cover the protein (level 1) and other nutrients requirements. T2: ration formulated to cover only 75% of protein (level 2) recommended by NRC. T3: control diet + 2.4 g undegradable methionine (Smartamine®)/day/kid (level 3). Feed intake, initial and monthly body weights were recorded. Blood samples were collected monthly and analyzed for metabolites and Co, Zn and Cu levels. Decreasing the dietary level of protein (T2) negatively affected (P<0.05) the total live weight gain, average daily gain and feed conversion ratio when compared with the control and T3 groups. Moreover, treatment, time and time × treatment caused a significant change on Co concentration in blood serum with higher value at the end of the experiment. Treatments had a significant effect (P<0.05) on blood serum cholesterol and protein levels. Undegradable methionine supplementation (T3) significantly increased longissimus dorsi weight, fat thickness and omental fat%. In conclusion, feeding Baladi kids below the NRC requirements of protein negatively affect the growth performance and feed efficiency. The recommended protein level by NRC for growing kids cover the requirements of growing black Baladi kids for maximum growth and productivity. PMID:27175130

  11. Protein engineering strategies with potential applications for altering clinically relevant cellular pathways at the protein level.

    PubMed

    Regan, Lynne; Hinrichsen, Michael R; Oi, Curran

    2016-05-01

    All diseases can be fundamentally viewed as the result of malfunctioning cellular pathways. Protein engineering offers the potential to develop new tools that will allow these dysfunctional pathways to be better understood, in addition to potentially providing new routes to restore proper function. Here we discuss different approaches that can be used to change the intracellular activity of a protein by intervening at the protein level: targeted protein sequestration, protein recruitment, protein degradation, and selective inhibition of binding interfaces. The potential of each of these tools to be developed into effective therapeutic treatments will also be discussed, along with any major barriers that currently block their translation into the clinic. PMID:27031866

  12. Protein expression analyses at the single cell level.

    PubMed

    Ohno, Masae; Karagiannis, Peter; Taniguchi, Yuichi

    2014-01-01

    The central dogma of molecular biology explains how genetic information is converted into its end product, proteins, which are responsible for the phenotypic state of the cell. Along with the protein type, the phenotypic state depends on the protein copy number. Therefore, quantification of the protein expression in a single cell is critical for quantitative characterization of the phenotypic states. Protein expression is typically a dynamic and stochastic phenomenon that cannot be well described by standard experimental methods. As an alternative, fluorescence imaging is being explored for the study of protein expression, because of its high sensitivity and high throughput. Here we review key recent progresses in fluorescence imaging-based methods and discuss their application to proteome analysis at the single cell level. PMID:25197931

  13. Source and level of supplemental protein for growing lambs.

    PubMed

    Dabiri, N; Thonney, M L

    2004-11-01

    Two 3 x 2 factorial growth trials and a companion metabolism trial with 13, 15, or 17% dietary CP (DM basis), with or without 3% of the DM replaced with slowly degraded menhaden fish meal, were conducted to determine if level of dietary protein influences whether slowly degraded protein improves lamb growth and protein use. The growth trials included 32 and 34 pens of two weanling lambs initially weighing 23 to 26 kg and fed for 42 d. The metabolism trial included 12 additional lambs fed in metabolism cages with a 2-wk adjustment period, a 1-wk preliminary period, and a 7-d collection period. Plasma urea N (PUN) was measured in all lambs at the conclusion of the second growth trial and at the end of the metabolism trial. There was a protein level x protein source interaction (P = 0.05) for PUN of the 12 lambs in the metabolism trial but not for the 68 lambs in the second growth trial. Replacement of part of the soybean meal protein with protein from fish meal did not affect ADG or G:F at any protein level, but it lowered (P = 0.08) PUN in the second growth trial. Plasma urea N values were higher (P = 0.002) in lambs fed diets with 15 or 17% CP; however, ADG (P = 0.037 in Exp. 1 and P = 0.055 in Exp. 2), and G:F (P = 0.094 in Exp. 1 and P = 0.003 in Exp. 2) were lower for lambs fed the diets with 13% CP. There was little difference in ADG or G:F between lambs fed the diets with 15 or 17% CP, suggesting that a CP level of 15% with supplemental protein from soybean meal would be optimal for 25- to 40-kg growing Finnsheep x Dorset lambs. PMID:15542470

  14. Soy protein isolate molecular level contributions to bulk adhesive properties

    NASA Astrophysics Data System (ADS)

    Shera, Jeanne Norton

    Increasing environmental awareness and the recognized health hazards of formaldehyde-based resins has prompted a strong demand for environmentally-responsible adhesives for wood composites. Soy protein-based adhesives have been shown to be commercially viable with 90-day shelf stability and composite physical properties comparable to those of commercial formaldehyde-based particleboards. The main research focus is to isolate and characterize the molecular level features in soy protein isolate responsible for providing mechanical properties, storage stability, and water resistance during adhesive formulation, processing, and wood composite fabrication. Commercial composite board will be reviewed to enhance our understanding of the individual components and processes required for particleboard production. The levels of protein structure will be defined and an overview of current bio-based technology will be presented. In the process, the logic for utilizing soy protein as a sole binder in the adhesive will be reinforced. Variables such as adhesive components, pH, divalent ions, blend aging, protein molecular weight, formulation solids content, and soy protein functionalization will relate the bulk properties of soy protein adhesives to the molecular configuration of the soybean protein. This work has demonstrated that when intermolecular beta-sheet interactions and protein long-range order is disrupted, viscosity and mechanical properties decrease. Storage stability can be maintained through the stabilization of intermolecular beta-sheet interactions. When molecular weight is reduced through enzymatic digestion, long-range order is disrupted and viscosity and mechanical properties decrease accordingly. Processibility and physical properties must be balanced to increase solids while maintaining low viscosity, desirable mechanical properties, and adequate storage stability. The structure of the soybean protein must be related to the particleboard bulk mechanical

  15. A residue level protein-protein interaction model in electrolyte solutions

    NASA Astrophysics Data System (ADS)

    Song, Xueyu

    2014-03-01

    The osmotic second virial coefficients B2 are directly related to the solubility of protein molecules in electrolyte solutions and can be useful to narrow down the search parameter space of protein crystallization conditions. Using a residue level model of protein-protein interaction in electrolyte solutions B2 of bovine pancreatic trypsin inhibitor and lysozyme in various solution conditions such as salt concentration, pH and temperature are calculated using an extended Fast Multipole Methods in combination with the boundary element formulation. Overall, the calculated B2 are well correlated with the experimental observations for various solution conditions. In combination with our previous work on the binding affinity calculations of protein complexes it is demonstrated that our residue level model can be used as a reliable model to describe protein-protein interaction in solutions.

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

    PubMed

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

    2015-01-01

    Accurate identification of protein-protein interactions (PPI) is the key step in understanding proteins' biological functions, which are typically context-dependent. Many existing PPI predictors rely on aggregated features from protein sequences, however only a few methods exploit local information about specific residue contacts. In this work we present a two-stage machine learning approach for prediction of protein-protein interactions. We start with the carefully filtered data on protein complexes available for Saccharomyces cerevisiae in the Protein Data Bank (PDB) database. First, we build linear descriptions of interacting and non-interacting sequence segment pairs based on their inter-residue distances. Secondly, we train machine learning classifiers to predict binary segment interactions for any two short sequence fragments. The final prediction of the protein-protein interaction is done using the 2D matrix representation of all-against-all possible interacting sequence segments of both analysed proteins. The level-I predictor achieves 0.88 AUC for micro-scale, i.e., residue-level prediction. The level-II predictor improves the results further by a more complex learning paradigm. We perform 30-fold macro-scale, i.e., protein-level cross-validation experiment. The level-II predictor using PSIPRED-predicted secondary structure reaches 0.70 precision, 0.68 recall, and 0.70 AUC, whereas other popular methods provide results below 0.6 threshold (recall, precision, AUC). Our results demonstrate that multi-scale sequence features aggregation procedure is able to improve the machine learning results by more than 10% as compared to other sequence representations. Prepared datasets and source code for our experimental pipeline are freely available for download from: http://zubekj.github.io/mlppi/ (open source Python implementation, OS independent). PMID:26157620

  17. DPF2 regulates OCT4 protein level and nuclear distribution.

    PubMed

    Liu, Chao; Zhang, Dijuan; Shen, Yuxian; Tao, Xiaofang; Liu, Lihua; Zhong, Yongwang; Fang, Shengyun

    2015-12-01

    The amount of transcription factor OCT4 is strictly regulated. A tight regulation of OCT4 levels is crucial for mammalian embryonic development and oncogenesis. However, the mechanisms underlying regulation of OCT4 protein expression and nuclear distribution are largely unknown. Here, we report that DPF2, a plant homeodomain (PHD) finger protein, is upregulated during H9 cell differentiation induced by retinoic acid. Endogenous interaction between DPF2 and OCT4 in P19 cells was revealed by an immunoprecipitation assay. GST-pull down assay proved that OCT4 protein in H9 cells and recombinant OCT4 can precipitate with DPF2 in vitro. In vitro ubiquitination assay demonstrated DPF2 might serve as an E3 ligase. Knock down of dpf2 using siRNA increased OCT4 protein level and stability in P19 cells. DPF2 siRNAs also up-regulates OCT4 but not NANOG in H9 cells. However, RA fails to downregulates OCT4 protein level in cells infected by lenitviruses containing DPF2 siRNA. Moreover, overexpression of both DPF2 and OCT4 in 293 cells proved the DPF2-OCT4 interaction. DPF2 but not PHD2 mutant DPF2 enhanced ubiquitination and degradation of OCT4 in 293 cells co-expressed DPF2 and OCT4. Both wild type DPF2 and PHD2 mutant DPF2 redistributes nuclear OCT4 without affecting DPF2-OCT4 interaction. Further analysis indicated that DPF2 decreases monomeric and mono-ubiquitinated OCT4, assembles poly-ubiquitin chains on OCT4 mainly through Ub-K48 linkage. These findings contribute to an understanding of how OCT4 protein level and nuclear distribution is regulated by its associated protein. PMID:26417682

  18. Smoking, COPD and 3-Nitrotyrosine Levels of Plasma Proteins

    SciTech Connect

    Jin, Hongjun; Webb-Robertson, Bobbie-Jo M.; Peterson, Elena S.; Tan, Ruimin; Bigelow, Diana J.; Scholand, Mary Beth; Hoidal, John R.; Pounds, Joel G.; Zangar, Richard C.

    2011-09-01

    BACKGROUND: Nitric oxide is a physiologically regulator of endothelial function and hemodynamics. Oxidized products of nitric oxide can form nitrotyrosine, which is a marker of nitrative stress. Cigarette smoking decreases exhaled nitric oxide, and the underlying mechanism may be important in the cardiovascular toxicity of cigarette smoke, although it is not clear if this effect results from decreased nitric oxide production or oxidation of nitric oxide to reactive, nitrating, species. These processes would be expected to have opposite effects on nitrotyrosine levels, a marker of nitrative stress. OBJECTIVE: In this study, we determine the effects of smoking and chronic obstructive pulmonary disease (COPD) on circulating levels of nitrotyrosine, and thereby gain insight into the processes regulating nitrotyrosine formation. METHODS: A custom antibody microarray platform was used to analyze the levels of 3-nitrotyrosine modifications on 24 proteins in plasma. Plasma samples from 458 individuals were analyzed. RESULTS: Nitrotyrosine levels in circulating proteins were uniformly reduced in smokers but increased in COPD patients. We also observed a persistent suppression of nitrotyrosine in former smokers. CONCLUSIONS: Smoking broadly suppresses the levels of 3-nitrotyrosine in plasma proteins, suggesting that cigarette smoke suppresses endothelial nitric oxide production. In contrast, the increase in nitrotyrosine levels in COPD patients most likely results from inflammatory processes. This study provides the first evidence that smoking has irreversible effects on endothelial production of nitric oxide, and provides insight into how smoking could induce a loss of elasticity in the vasculature and a long-term increase in the risk of cardiovascular disease.

  19. Predicting Abdominal Aortic Aneurysm Target Genes by Level-2 Protein-Protein Interaction

    PubMed Central

    Fu, Yi; Cui, Qinghua; Kong, Wei

    2015-01-01

    Abdominal aortic aneurysm (AAA) is frequently lethal and has no effective pharmaceutical treatment, posing a great threat to human health. Previous bioinformatics studies of the mechanisms underlying AAA relied largely on the detection of direct protein-protein interactions (level-1 PPI) between the products of reported AAA-related genes. Thus, some proteins not suspected to be directly linked to previously reported genes of pivotal importance to AAA might have been missed. In this study, we constructed an indirect protein-protein interaction (level-2 PPI) network based on common interacting proteins encoded by known AAA-related genes and successfully predicted previously unreported AAA-related genes using this network. We used four methods to test and verify the performance of this level-2 PPI network: cross validation, human AAA mRNA chip array comparison, literature mining, and verification in a mouse CaPO4 AAA model. We confirmed that the new level-2 PPI network is superior to the original level-1 PPI network and proved that the top 100 candidate genes predicted by the level-2 PPI network shared similar GO functions and KEGG pathways compared with positive genes. PMID:26496478

  20. ORMDL proteins regulate ceramide levels during sterile inflammation.

    PubMed

    Cai, Lin; Oyeniran, Clement; Biswas, Debolina D; Allegood, Jeremy; Milstien, Sheldon; Kordula, Tomasz; Maceyka, Michael; Spiegel, Sarah

    2016-08-01

    The bioactive sphingolipid metabolite, ceramide, regulates physiological processes important for inflammation and elevated levels of ceramide have been implicated in IL-1-mediated events. Although much has been learned about ceramide generation by activation of sphingomyelinases in response to IL-1, the contribution of the de novo pathway is not completely understood. Because yeast ORM1 and ORM2 proteins negatively regulate ceramide levels through inhibition of serine palmitoyltransferase, the first committed step in ceramide biosynthesis, we examined the functions of individual mammalian ORM orthologs, ORM (yeast)-like (ORMDL)1-3, in regulation of ceramide levels. In HepG2 liver cells, downregulation of ORMDL3 markedly increased the ceramide precursors, dihydrosphingosine and dihydroceramide, primarily from de novo biosynthesis based on [U-(13)C]palmitate incorporation into base-labeled and dual-labeled dihydroceramides, whereas downregulation of each isoform increased dihydroceramides [(13)C]labeled in only the amide-linked fatty acid. IL-1 and the IL-6 family cytokine, oncostatin M, increased dihydroceramide and ceramide levels in HepG2 cells and concomitantly decreased ORMDL proteins. Moreover, during irritant-induced sterile inflammation in mice leading to induction of the acute-phase response, which is dependent on IL-1, expression of ORMDL proteins in the liver was strongly downregulated and accompanied by increased ceramide levels in the liver and accumulation in the blood. Together, our results suggest that ORMDLs may be involved in regulation of ceramides during IL-1-mediated sterile inflammation. PMID:27313060

  1. HPV-related methylation signature predicts survival in oropharyngeal squamous cell carcinomas.

    PubMed

    Kostareli, Efterpi; Holzinger, Dana; Bogatyrova, Olga; Hielscher, Thomas; Wichmann, Gunnar; Keck, Michaela; Lahrmann, Bernd; Grabe, Niels; Flechtenmacher, Christa; Schmidt, Christopher R; Seiwert, Tanguy; Dyckhoff, Gerhard; Dietz, Andreas; Höfler, Daniela; Pawlita, Michael; Benner, Axel; Bosch, Franz X; Plinkert, Peter; Plass, Christoph; Weichenhan, Dieter; Hess, Jochen

    2013-06-01

    High-risk types of human papilloma virus (HPV) are increasingly associated with oropharyngeal squamous cell carcinoma (OPSCC). Strikingly, patients with HPV-positive OPSCC are highly curable with ionizing radiation and have better survival compared with HPV-negative patients, but the underlying molecular mechanisms remain poorly understood. We applied an array-based approach to monitor global changes in CpG island hypermethylation between HPV-negative and HPV-positive OPSCCs and identified a specific pattern of differentially methylated regions that critically depends on the presence of viral transcripts. HPV-related alterations were confirmed for the majority of candidate gene promoters by mass spectrometric, quantitative methylation analysis. There was a significant inverse correlation between promoter hypermethylation of ALDH1A2, OSR2, GATA4, GRIA4, and IRX4 and transcript levels. Interestingly, Kaplan-Meier analysis revealed that a combined promoter methylation pattern of low methylation levels in ALDH1A2 and OSR2 promoters and high methylation levels in GATA4, GRIA4, and IRX4 promoters was significantly correlated with improved survival in 3 independent patient cohorts. ALDH1A2 protein levels, determined by immunohistochemistry on tissue microarrays, confirmed the association with clinical outcome. In summary, our study highlights specific alterations in global gene promoter methylation in HPV-driven OPSCCs and identifies a signature that predicts the clinical outcome in OPSCCs. PMID:23635773

  2. Predicting Protein-Protein Interactions from the Molecular to the Proteome Level.

    PubMed

    Keskin, Ozlem; Tuncbag, Nurcan; Gursoy, Attila

    2016-04-27

    Identification of protein-protein interactions (PPIs) is at the center of molecular biology considering the unquestionable role of proteins in cells. Combinatorial interactions result in a repertoire of multiple functions; hence, knowledge of PPI and binding regions naturally serve to functional proteomics and drug discovery. Given experimental limitations to find all interactions in a proteome, computational prediction/modeling of protein interactions is a prerequisite to proceed on the way to complete interactions at the proteome level. This review aims to provide a background on PPIs and their types. Computational methods for PPI predictions can use a variety of biological data including sequence-, evolution-, expression-, and structure-based data. Physical and statistical modeling are commonly used to integrate these data and infer PPI predictions. We review and list the state-of-the-art methods, servers, databases, and tools for protein-protein interaction prediction. PMID:27074302

  3. Importance of ALDH1A enzymes in determining human testicular retinoic acid concentrations

    PubMed Central

    Arnold, Samuel L.; Kent, Travis; Hogarth, Cathryn A.; Schlatt, Stefan; Prasad, Bhagwat; Haenisch, Michael; Walsh, Thomas; Muller, Charles H.; Griswold, Michael D.; Amory, John K.; Isoherranen, Nina

    2015-01-01

    Retinoic acid (RA), the active metabolite of vitamin A, is required for spermatogenesis and many other biological processes. RA formation requires irreversible oxidation of retinal to RA by aldehyde dehydrogenase enzymes of the 1A family (ALDH1A). While ALDH1A1, ALDH1A2, and ALDH1A3 all form RA, the expression pattern and relative contribution of these enzymes to RA formation in the testis is unknown. In this study, novel methods to measure ALDH1A protein levels and intrinsic RA formation were used to accurately predict RA formation velocities in individual human testis samples and an association between RA formation and intratesticular RA concentrations was observed. The distinct localization of ALDH1A in the testis suggests a specific role for each enzyme in controlling RA formation. ALDH1A1 was found in Sertoli cells, while only ALDH1A2 was found in spermatogonia, spermatids, and spermatocytes. In the absence of cellular retinol binding protein (CRBP)1, ALDH1A1 was predicted to be the main contributor to intratesticular RA formation, but when CRBP1 was present, ALDH1A2 was predicted to be equally important in RA formation as ALDH1A1. This study provides a comprehensive novel methodology to evaluate RA homeostasis in human tissues and provides insight to how the individual ALDH1A enzymes mediate RA concentrations in specific cell types. PMID:25502770

  4. Low copper and high manganese levels in prion protein plaques

    USGS Publications Warehouse

    Johnson, Christopher J.; Gilbert, P.U.P.A.; Abrecth, Mike; Baldwin, Katherine L.; Russell, Robin E.; Pedersen, Joel A.; McKenzie, Debbie

    2013-01-01

    Accumulation of aggregates rich in an abnormally folded form of the prion protein characterize the neurodegeneration caused by transmissible spongiform encephalopathies (TSEs). The molecular triggers of plaque formation and neurodegeneration remain unknown, but analyses of TSE-infected brain homogenates and preparations enriched for abnormal prion protein suggest that reduced levels of copper and increased levels of manganese are associated with disease. The objectives of this study were to: (1) assess copper and manganese levels in healthy and TSE-infected Syrian hamster brain homogenates; (2) determine if the distribution of these metals can be mapped in TSE-infected brain tissue using X-ray photoelectron emission microscopy (X-PEEM) with synchrotron radiation; and (3) use X-PEEM to assess the relative amounts of copper and manganese in prion plaques in situ. In agreement with studies of other TSEs and species, we found reduced brain levels of copper and increased levels of manganese associated with disease in our hamster model. We also found that the in situ levels of these metals in brainstem were sufficient to image by X-PEEM. Using immunolabeled prion plaques in directly adjacent tissue sections to identify regions to image by X-PEEM, we found a statistically significant relationship of copper-manganese dysregulation in prion plaques: copper was depleted whereas manganese was enriched. These data provide evidence for prion plaques altering local transition metal distribution in the TSE-infected central nervous system.

  5. Ascertaining effects of nanoscale polymeric interfaces on competitive protein adsorption at the individual protein level.

    PubMed

    Song, Sheng; Xie, Tian; Ravensbergen, Kristina; Hahm, Jong-in

    2016-02-14

    With the recent development of biomaterials and biodevices with reduced dimensionality, it is critical to comprehend protein adhesion processes to nanoscale solid surfaces, especially those occurring in a competitive adsorption environment. Complex sequences of adhesion events in competitive adsorption involving multicomponent protein systems have been extensively investigated, but our understanding is still limited primarily to macroscopic adhesion onto chemically simple surfaces. We examine the competitive adsorption behavior from a binary protein mixture containing bovine serum albumin and fibrinogen at the single protein level. We subsequently evaluate a series of adsorption and displacement processes occurring on both the macroscopic homopolymer and nanoscopic diblock copolymer surfaces, while systematically varying the protein concentration and incubation time. We identify the similarities and dissimilarities in competitive protein adsorption behavior between the two polymeric surfaces, the former presenting chemical uniformity at macroscale versus the latter exhibiting periodic nanointerfaces of chemically alternating polymeric segments. We then present our novel experimental finding of a large increase in the nanointerface-engaged residence time of the initially bound proteins and further explain the origin of this phenomenon manifested on nanoscale diblock copolymer surfaces. The outcomes of this study may provide timely insight into nanoscale competitive protein adsorption that is much needed in designing bioimplant and tissue engineering materials. In addition, the fundamental understanding gained from this study can be beneficial for the development of highly miniaturized biodevices and biomaterials fabricated by using nanoscale polymeric materials and interfaces. PMID:26794230

  6. Effect of Crude Protein Levels in Concentrate and Concentrate Levels in Diet on In vitro Fermentation

    PubMed Central

    Van Dung, Dinh; Shang, Weiwei; Yao, Wen

    2014-01-01

    The effect of concentrate mixtures with crude protein (CP) levels 10%, 13%, 16%, and 19% and diets with roughage to concentrate ratios 80:20, 60:40, 40:60, and 20:80 (w/w) were determined on dry matter (DM) and organic matter (OM) digestibility, and fermentation metabolites using an in vitro fermentation technique. In vitro fermented attributes were measured after 4, 24, and 48 h of incubation respectively. The digestibility of DM and OM, and total volatile fatty acid (VFA) increased whereas pH decreased with the increased amount of concentrate in the diet (p<0.001), however CP levels of concentrate did not have any influence on these attributes. Gas production reduced with increased CP levels, while it increased with increasing concentrate levels. Ammonia nitrogen (NH3-N) concentration and microbial CP production increased significantly (p<0.05) by increasing CP levels and with increasing concentrate levels in diet as well, however, no significant difference was found between 16% and 19% CP levels. Therefore, 16% CP in concentrate and increasing proportion of concentrate up to 80% in diet all had improved digestibility of DM and organic matter, and higher microbial protein production, with improved fermentation characteristics. PMID:25050017

  7. Ascertaining effects of nanoscale polymeric interfaces on competitive protein adsorption at the individual protein level

    NASA Astrophysics Data System (ADS)

    Song, Sheng; Xie, Tian; Ravensbergen, Kristina; Hahm, Jong-In

    2016-02-01

    With the recent development of biomaterials and biodevices with reduced dimensionality, it is critical to comprehend protein adhesion processes to nanoscale solid surfaces, especially those occurring in a competitive adsorption environment. Complex sequences of adhesion events in competitive adsorption involving multicomponent protein systems have been extensively investigated, but our understanding is still limited primarily to macroscopic adhesion onto chemically simple surfaces. We examine the competitive adsorption behavior from a binary protein mixture containing bovine serum albumin and fibrinogen at the single protein level. We subsequently evaluate a series of adsorption and displacement processes occurring on both the macroscopic homopolymer and nanoscopic diblock copolymer surfaces, while systematically varying the protein concentration and incubation time. We identify the similarities and dissimilarities in competitive protein adsorption behavior between the two polymeric surfaces, the former presenting chemical uniformity at macroscale versus the latter exhibiting periodic nanointerfaces of chemically alternating polymeric segments. We then present our novel experimental finding of a large increase in the nanointerface-engaged residence time of the initially bound proteins and further explain the origin of this phenomenon manifested on nanoscale diblock copolymer surfaces. The outcomes of this study may provide timely insight into nanoscale competitive protein adsorption that is much needed in designing bioimplant and tissue engineering materials. In addition, the fundamental understanding gained from this study can be beneficial for the development of highly miniaturized biodevices and biomaterials fabricated by using nanoscale polymeric materials and interfaces.With the recent development of biomaterials and biodevices with reduced dimensionality, it is critical to comprehend protein adhesion processes to nanoscale solid surfaces, especially those

  8. Characterization of protein expression levels with label-free detected reverse phase protein arrays.

    PubMed

    Guo, Xuexue; Deng, Yihong; Zhu, Chenggang; Cai, Junlong; Zhu, Xiangdong; Landry, James P; Zheng, Fengyun; Cheng, Xunjia; Fei, Yiyan

    2016-09-15

    In reverse-phase protein arrays (RPPA), one immobilizes complex samples (e.g., cellular lysate, tissue lysate or serum etc.) on solid supports and performs parallel reactions of antibodies with immobilized protein targets from the complex samples. In this work, we describe a label-free detection of RPPA that enables quantification of RPPA data and thus facilitates comparison of studies performed on different samples and on different solid supports. We applied this detection platform to characterization of phosphoserine aminotransferase (PSAT) expression levels in Acanthamoeba lysates treated with artemether and the results were confirmed by Western blot studies. PMID:27372609

  9. Effects of soy protein and calcium levels on mineral bioaccessibility and protein digestibility from enteral formulas.

    PubMed

    Galán, María Gimena; Drago, Silvina Rosa

    2014-09-01

    Enteral formulas (EF) are complex food systems which have all the nutrients in their matrix for the complete human nourishment. However, there are components in EF which can interact with minerals, reducing their absorption, and thereof the EF nutritional quality. The effect of soy protein (SP) and Ca content on Fe, Zn, and Ca bioaccessibility and protein digestibility (%DP) was assessed using a response surface design in EF. Tested SP levels were 2.5-5.0 g/100 mL of total protein. Ca levels were adjusted with Ca citrate within a range between 50 and 100 mg/100 mL. SP content negatively influenced %DP and Fe, Zn and Ca bioaccessibility. As SP content increased, mineral bioaccessibility and %DP decreased, probably due to the increased levels of phytic acid and trypsin inhibitors from SP. Ca content only affected %DCa, which had a direct relationship with Ca levels, while did not affect Fe and Zn bioaccessibility or %DP. Since Ca citrate did not impair Fe and Zn bioaccessibility, it could be an appropriate Ca source for EF fortification. PMID:25079612

  10. C-reactive protein levels in hereditary angioedema.

    PubMed

    Hofman, Z L M; Relan, A; Hack, C E

    2014-07-01

    Hereditary angioedema (HAE) patients experience recurrent episodes of angioedema attacks that can be painful, disfiguring and even life-threatening. The disorder results from a mutation in the gene that controls the synthesis of C1-inhibitor (C1INH). C1INH is a major regulator of activation of the contact system. It is often assumed that attacks results from uncontrolled local activation of the contact system with subsequent formation of bradykinin. To evaluate the involvement of inflammatory reactions in HAE, we analysed C-reactive protein (CRP) levels. HAE patients included in a clinical database of recombinant human C1-inhibitor (rhC1INH) studies were evaluated. For the current study we analysed CRP levels when patients were asymptomatic, during a clinical attack and in a follow-up period, and correlated these with the clinical manifestations of the attack. Data from 68 HAE patients were analysed and included CRP levels on 273 occasions. While asymptomatic, 20% of the patients analysed had increased CRP. At the onset of the attack (P = 0·049) and during the next 24 h CRP rose significantly (P = 0·002) in patients with an abdominal location, and post-attack levels were significantly higher in these patients than in patients with attacks at other locations (P = 0·034). In conclusion, CRP levels are elevated in a substantial proportion of asymptomatic HAE patients. Levels of CRP increase significantly during an abdominal attack. These data suggest low-grade systemic inflammatory reactions in HAE patients as well as a triggering event for attacks that starts prior to symptom onset. PMID:24588117

  11. PCDq: human protein complex database with quality index which summarizes different levels of evidences of protein complexes predicted from H-Invitational protein-protein interactions integrative dataset

    PubMed Central

    2012-01-01

    Background Proteins interact with other proteins or biomolecules in complexes to perform cellular functions. Existing protein-protein interaction (PPI) databases and protein complex databases for human proteins are not organized to provide protein complex information or facilitate the discovery of novel subunits. Data integration of PPIs focused specifically on protein complexes, subunits, and their functions. Predicted candidate complexes or subunits are also important for experimental biologists. Description Based on integrated PPI data and literature, we have developed a human protein complex database with a complex quality index (PCDq), which includes both known and predicted complexes and subunits. We integrated six PPI data (BIND, DIP, MINT, HPRD, IntAct, and GNP_Y2H), and predicted human protein complexes by finding densely connected regions in the PPI networks. They were curated with the literature so that missing proteins were complemented and some complexes were merged, resulting in 1,264 complexes comprising 9,268 proteins with 32,198 PPIs. The evidence level of each subunit was assigned as a categorical variable. This indicated whether it was a known subunit, and a specific function was inferable from sequence or network analysis. To summarize the categories of all the subunits in a complex, we devised a complex quality index (CQI) and assigned it to each complex. We examined the proportion of consistency of Gene Ontology (GO) terms among protein subunits of a complex. Next, we compared the expression profiles of the corresponding genes and found that many proteins in larger complexes tend to be expressed cooperatively at the transcript level. The proportion of duplicated genes in a complex was evaluated. Finally, we identified 78 hypothetical proteins that were annotated as subunits of 82 complexes, which included known complexes. Of these hypothetical proteins, after our prediction had been made, four were reported to be actual subunits of the

  12. Sputum and BAL Clara cell secretory protein and surfactant protein D levels in asthma.

    PubMed

    Emmanouil, P; Loukides, S; Kostikas, K; Papatheodorou, G; Papaporfyriou, A; Hillas, G; Vamvakaris, I; Triggidou, R; Katafigiotis, P; Kokkini, A; Papiris, S; Koulouris, N; Bakakos, P

    2015-06-01

    Clara cell secretory protein (CC16) is associated with Th2 modulation. Surfactant protein D (SPD) plays an important role in surfactant homeostasis and eosinophil chemotaxis. We measured CC16 and SPD in sputum supernatants of 84 asthmatic patients and 12 healthy controls. In 22 asthmatics, we additionally measured CC16 and SPD levels in BAL and assessed smooth muscle area (SMA), reticular basement membrane (RBM) thickness, and epithelial detachment (ED) in bronchial biopsies. Induced sputum CC16 and SPD were significantly higher in patients with severe asthma (SRA) compared to mild-moderate and healthy controls. BAL CC16 and SPD levels were also higher in SRA compared to mild-moderate asthma. CC16 BAL levels correlated with ED, while SPD BAL levels correlated with SMA and RBM. Severity represented a significant covariate for these associations. CC16 and SPD levels are upregulated in SRA and correlate with remodeling indices, suggesting a possible role of these biomarkers in the remodeling process. PMID:25728058

  13. Honey bee protein atlas at organ-level resolution.

    PubMed

    Chan, Queenie W T; Chan, Man Yi; Logan, Michelle; Fang, Yuan; Higo, Heather; Foster, Leonard J

    2013-11-01

    Genome sequencing has provided us with gene lists but cannot tell us where and how their encoded products work together to support life. Complex organisms rely on differential expression of subsets of genes/proteins in organs and tissues, and, in concert, evolved to their present state as they function together to improve an organism's overall reproductive fitness. Proteomics studies of individual organs help us understand their basic functions, but this reductionist approach misses the larger context of the whole organism. This problem could be circumvented if all the organs in an organism were comprehensively studied by the same methodology and analyzed together. Using honey bees (Apis mellifera L.) as a model system, we report here an initial whole proteome of a complex organism, measuring 29 different organ/tissue types among the three honey bee castes: queen, drone, and worker. The data reveal that, e.g., workers have a heightened capacity to deal with environmental toxins and queens have a far more robust pheromone detection system than their nestmates. The data also suggest that workers altruistically sacrifice not only their own reproductive capacity but also their immune potential in favor of their queen. Finally, organ-level resolution of protein expression offers a systematic insight into how organs may have developed. PMID:23878156

  14. Honey bee protein atlas at organ-level resolution

    PubMed Central

    Chan, Queenie W.T.; Chan, Man Yi; Logan, Michelle; Fang, Yuan; Higo, Heather; Foster, Leonard J.

    2013-01-01

    Genome sequencing has provided us with gene lists but cannot tell us where and how their encoded products work together to support life. Complex organisms rely on differential expression of subsets of genes/proteins in organs and tissues, and, in concert, evolved to their present state as they function together to improve an organism's overall reproductive fitness. Proteomics studies of individual organs help us understand their basic functions, but this reductionist approach misses the larger context of the whole organism. This problem could be circumvented if all the organs in an organism were comprehensively studied by the same methodology and analyzed together. Using honey bees (Apis mellifera L.) as a model system, we report here an initial whole proteome of a complex organism, measuring 29 different organ/tissue types among the three honey bee castes: queen, drone, and worker. The data reveal that, e.g., workers have a heightened capacity to deal with environmental toxins and queens have a far more robust pheromone detection system than their nestmates. The data also suggest that workers altruistically sacrifice not only their own reproductive capacity but also their immune potential in favor of their queen. Finally, organ-level resolution of protein expression offers a systematic insight into how organs may have developed. PMID:23878156

  15. Auxin acts independently of DELLA proteins in regulating gibberellin levels.

    PubMed

    Reid, James B; Davidson, Sandra E; Ross, John J

    2011-03-01

    Shoot elongation is a vital process for plant development and productivity, in both ecological and economic contexts. Auxin and bioactive gibberellins (GAs), such as GA1, play critical roles in the control of elongation, along with environmental and endogenous factors, including other hormones such as the brassinosteroids. The effect of auxins, such as indole-3-acetic acid (IAA), is at least in part mediated by its effect on GA metabolism, since auxin up-regulates biosynthesis genes such as GA 3-oxidase and GA 20-oxidase and down regulates GA catabolism genes such as GA 2-oxidases, leading to elevated levels of bioactive GA 1. In our recent paper, we have provided evidence that this action of IAA is largely independent of DELLA proteins, the negative regulators of GA action, since the auxin effects are still present in the DELLA-deficient la cry-s genotype of pea. This was a crucial issue to resolve, since like auxin, the DELLAs also promote GA 1 synthesis and inhibit its deactivation. DELLAs are deactivated by GA, and thereby mediate a feedback system by which bioactive GA regulates its own level. However, our recent results, in themselves, do not show the generality of the auxin-GA relationship across species and phylogenetic groups or across different tissue types and responses. Further, they do not touch on the ecological benefits of the auxin-GA interaction. These issues are discussed below as well as the need for the development of suitable experimental systems to allow this process to be examined. PMID:21358281

  16. Development, Characterization, and Optimization of Protein Level in Date Bars Using Response Surface Methodology

    PubMed Central

    Nadeem, Muhammad; Salim-ur-Rehman; Muhammad Anjum, Faqir; Murtaza, Mian Anjum; Mueen-ud-Din, Ghulam

    2012-01-01

    This project was designed to produce a nourishing date bar with commercial value especially for school going children to meet their body development requirements. Protein level of date bars was optimized using response surface methodology (RSM). Economical and underutilized sources, that is, whey protein concentrate and vetch protein isolates, were explored for protein supplementation. Fourteen date bar treatments were produced using a central composite design (CCD) with 2 variables and 3 levels for each variable. Date bars were then analyzed for nutritional profile. Proximate composition revealed that addition of whey protein concentrate and vetch protein isolates improved the nutritional profile of date bars. Protein level, texture, and taste were considerably improved by incorporating 6.05% whey protein concentrate and 4.35% vetch protein isolates in date bar without affecting any sensory characteristics during storage. Response surface methodology was observed as an economical and effective tool to optimize the ingredient level and to discriminate the interactive effects of independent variables. PMID:22792044

  17. Development, characterization, and optimization of protein level in date bars using response surface methodology.

    PubMed

    Nadeem, Muhammad; Salim-ur-Rehman; Muhammad Anjum, Faqir; Murtaza, Mian Anjum; Mueen-ud-Din, Ghulam

    2012-01-01

    This project was designed to produce a nourishing date bar with commercial value especially for school going children to meet their body development requirements. Protein level of date bars was optimized using response surface methodology (RSM). Economical and underutilized sources, that is, whey protein concentrate and vetch protein isolates, were explored for protein supplementation. Fourteen date bar treatments were produced using a central composite design (CCD) with 2 variables and 3 levels for each variable. Date bars were then analyzed for nutritional profile. Proximate composition revealed that addition of whey protein concentrate and vetch protein isolates improved the nutritional profile of date bars. Protein level, texture, and taste were considerably improved by incorporating 6.05% whey protein concentrate and 4.35% vetch protein isolates in date bar without affecting any sensory characteristics during storage. Response surface methodology was observed as an economical and effective tool to optimize the ingredient level and to discriminate the interactive effects of independent variables. PMID:22792044

  18. Microsecond protein dynamics observed at the single-molecule level

    PubMed Central

    Otosu, Takuhiro; Ishii, Kunihiko; Tahara, Tahei

    2015-01-01

    How polypeptide chains acquire specific conformations to realize unique biological functions is a central problem of protein science. Single-molecule spectroscopy, combined with fluorescence resonance energy transfer, is utilized to study the conformational heterogeneity and the state-to-state transition dynamics of proteins on the submillisecond to second timescales. However, observation of the dynamics on the microsecond timescale is still very challenging. This timescale is important because the elementary processes of protein dynamics take place and direct comparison between experiment and simulation is possible. Here we report a new single-molecule technique to reveal the microsecond structural dynamics of proteins through correlation of the fluorescence lifetime. This method, two-dimensional fluorescence lifetime correlation spectroscopy, is applied to clarify the conformational dynamics of cytochrome c. Three conformational ensembles and the microsecond transitions in each ensemble are indicated from the correlation signal, demonstrating the importance of quantifying microsecond dynamics of proteins on the folding free energy landscape. PMID:26151767

  19. Atomic-level Snapshot Catches Protein Motor in Action

    SciTech Connect

    2009-01-01

    Using a state-of-the-art protein crystallography beamline at Berkeley Labs Advanced Light Source, researchers have captured a critical action shapshot of an enzyme that is vital to the survival of all biological cells.

  20. Nrf2 reduces levels of phosphorylated tau protein by inducing autophagy adaptor protein NDP52

    NASA Astrophysics Data System (ADS)

    Jo, Chulman; Gundemir, Soner; Pritchard, Susanne; Jin, Youngnam N.; Rahman, Irfan; Johnson, Gail V. W.

    2014-03-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is a pivotal transcription factor in the defence against oxidative stress. Here we provide evidence that activation of the Nrf2 pathway reduces the levels of phosphorylated tau by induction of an autophagy adaptor protein NDP52 (also known as CALCOCO2) in neurons. The expression of NDP52, which we show has three antioxidant response elements (AREs) in its promoter region, is strongly induced by Nrf2, and its overexpression facilitates clearance of phosphorylated tau in the presence of an autophagy stimulator. In Nrf2-knockout mice, phosphorylated and sarkosyl-insoluble tau accumulates in the brains concurrent with decreased levels of NDP52. Moreover, NDP52 associates with phosphorylated tau from brain cortical samples of Alzheimer disease cases, and the amount of phosphorylated tau in sarkosyl-insoluble fractions is inversely proportional to that of NDP52. These results suggest that NDP52 plays a key role in autophagy-mediated degradation of phosphorylated tau in vivo.

  1. Methods to alter levels of a DNA repair protein

    DOEpatents

    Petrini, John H.; Morgan, William Francis; Maser, Richard Scott; Carney, James Patrick

    2006-10-17

    An isolated and purified DNA molecule encoding a DNA repair protein, p95, is provided, as is isolated and purified p95. Also provided are methods of detecting p95 and DNA encoding p95. The invention further provides p95 knock-out mice.

  2. Effect of N-acetylcysteine administration on homocysteine level, oxidative damage to proteins, and levels of iron (Fe) and Fe-related proteins in lead-exposed workers.

    PubMed

    Kasperczyk, Sławomir; Dobrakowski, Michał; Kasperczyk, Aleksandra; Romuk, Ewa; Rykaczewska-Czerwińska, Monika; Pawlas, Natalia; Birkner, Ewa

    2016-09-01

    N-Acetylcysteine (NAC) could be included in protocols designed for the treatment of lead toxicity. Therefore, in this study, we decided to investigate the influence of NAC administration on homocysteine (Hcy) levels, oxidative damage to proteins, and the levels of iron (Fe), transferrin (TRF), and haptoglobin (HPG) in lead (Pb)-exposed workers. The examined population (n = 171) was composed of male employees who worked with Pb. They were randomized into four groups. Workers who were not administered any antioxidants, drugs, vitamins, or dietary supplements were classified as the reference group (n = 49). The remaining three groups consisted of workers who were treated orally with NAC at three different doses (1 × 200, 2 × 200, or 2 × 400 mg) for 12 weeks. After the treatment, blood Pb levels significantly decreased in the groups receiving NAC compared with the reference group. The protein concentration was not affected by NAC administration. In contrast, Hcy levels significantly decreased or showed a strong tendency toward lower values depending on the NAC dose. Levels of the protein carbonyl groups were significantly decreased in all of the groups receiving NAC. Conversely, glutamate dehydrogenase activity was significantly elevated in all of the groups receiving NAC, while the level of protein thiol groups was significantly elevated only in the group receiving 200 mg of NAC. Treatment with NAC did not significantly affect Fe and TRF levels, whereas HPG levels showed a tendency toward lower values. Treatment with NAC normalized the level of Hcy and decreased oxidative stress as measured by the protein carbonyl content; this effect occurred in a dose-dependent manner. Moreover, small doses of NAC elevated the levels of protein thiol groups. Therefore, NAC could be introduced as an alternative therapy for chronic Pb toxicity in humans. PMID:25731901

  3. GUN1 Controls Accumulation of the Plastid Ribosomal Protein S1 at the Protein Level and Interacts with Proteins Involved in Plastid Protein Homeostasis.

    PubMed

    Tadini, Luca; Pesaresi, Paolo; Kleine, Tatjana; Rossi, Fabio; Guljamow, Arthur; Sommer, Frederik; Mühlhaus, Timo; Schroda, Michael; Masiero, Simona; Pribil, Mathias; Rothbart, Maxi; Hedtke, Boris; Grimm, Bernhard; Leister, Dario

    2016-03-01

    Developmental or metabolic changes in chloroplasts can have profound effects on the rest of the plant cell. Such intracellular responses are associated with signals that originate in chloroplasts and convey information on their physiological status to the nucleus, which leads to large-scale changes in gene expression (retrograde signaling). A screen designed to identify components of retrograde signaling resulted in the discovery of the so-called genomes uncoupled (gun) mutants. Genetic evidence suggests that the chloroplast protein GUN1 integrates signals derived from perturbations in plastid redox state, plastid gene expression, and tetrapyrrole biosynthesis (TPB) in Arabidopsis (Arabidopsis thaliana) seedlings, exerting biogenic control of chloroplast functions. However, the molecular mechanism by which GUN1 integrates retrograde signaling in the chloroplast is unclear. Here we show that GUN1 also operates in adult plants, contributing to operational control of chloroplasts. The gun1 mutation genetically interacts with mutations of genes for the chloroplast ribosomal proteins S1 (PRPS1) and L11. Analysis of gun1 prps1 lines indicates that GUN1 controls PRPS1 accumulation at the protein level. The GUN1 protein physically interacts with proteins involved in chloroplast protein homeostasis based on coimmunoprecipitation experiments. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation experiments suggest that GUN1 might transiently interact with several TPB enzymes, including Mg-chelatase subunit D (CHLD) and two other TPB enzymes known to activate retrograde signaling. Moreover, the association of PRPS1 and CHLD with protein complexes is modulated by GUN1. These findings allow us to speculate that retrograde signaling might involve GUN1-dependent formation of protein complexes. PMID:26823545

  4. Effect of phosphorus levels on the protein profiles of secreted protein and root surface protein of rice.

    PubMed

    Shinano, Takuro; Yoshimura, Tomoko; Watanabe, Toshihiro; Unno, Yusuke; Osaki, Mitsuru; Nanjo, Yohei; Komatsu, Setsuko

    2013-11-01

    Plant roots are complicated organs that absorb water and nutrients from the soil. Roots also play an essential role in protecting plants from attack by soil pathogens and develop a beneficial role with some soil microorganisms. Plant-derived rhizosphere proteins (e.g., root secretory proteins and root surface binding proteins) are considered to play important roles in developing mutual relationships in the rhizosphere. In the rhizosphere, where plant roots meet the surrounding environment, it has been suggested that root secretory protein and root surface binding protein are important factors. Furthermore, it is not known how the physiological status of the plant affects the profile of these proteins. In this study, rice plants were grown aseptically, with or without phosphorus nutrition, and proteins were obtained from root bathing solution (designated as root secretory proteins) and obtained using 0.2 M CaCl2 solution (designated as root surface binding proteins). The total number of identified proteins in the root bathing solution was 458, and the number of root surface binding proteins was 256. More than half of the proteins were observed in both fractions. Most of the proteins were categorized as either having signal peptides or no membrane transport helix sites. The functional categorization suggested that most of the proteins seemed to have secretory pathways and were involved in defense/disease-related functions. These characteristics seem to be unique to rhizosphere proteins, and the latter might be part of the plants strategy to defeat pathogens in the soil. The low phosphorus treatment significantly increased the number of pathogenesis-related proteins in the root secretory proteins, whereas the change was small in the case of the root surface binding proteins. The results suggested that the roots are actively and selectively secreting protein into the rhizosphere. PMID:24083427

  5. Protein metabolism in growing pigs fed corn or cassava peel based diets containing graded protein levels.

    PubMed

    Tewe, O O

    1985-05-01

    Sixty-four Large White cross Landrace weanling pigs were randomly allotted to eight treatments in a two by four factorial arrangement. The two dietary variables were cassava peel (0 and 40 per cent) and crude protein (20, 15, 10 and 5 per cent). Total serum protein concentration was significantly (P less than 0.01) reduced by protein deficiency and by its interaction with cassava peel. The multiple coefficient of determination (R2) showed that protein intake was the primary factor determining changes in serum protein. R2 values for cyanide intake (independent variable) on serum protein (dependent variable) increased from day 30 to 90 of the trial. Serum urea was increased on the 5 per cent protein diets on days 60 and 90 of the trial. The R2 values for cyanide and protein intake on serum urea concentration increased from day 30 to day 90 of the trial. Serum creatinine increased (P less than 0.05) on the 5 per cent protein diet on day 90 of the trial. The R2 value for the effects of protein intake on serum creatinine was higher than for cyanide intake on days 30 and 90. The results confirm the progressive and pronounced effects of long term cyanide intake on serum nitrogenous metabolites in pigs consuming between 110 and 120 ppm hydrocyanic acid, especially in diets containing 10 per cent or less protein. PMID:2989987

  6. Real-time quantification of protein expression at the single-cell level via dynamic protein synthesis translocation reporters

    PubMed Central

    Aymoz, Delphine; Wosika, Victoria; Durandau, Eric; Pelet, Serge

    2016-01-01

    Protein expression is a dynamic process, which can be rapidly induced by extracellular signals. It is widely appreciated that single cells can display large variations in the level of gene induction. However, the variability in the dynamics of this process in individual cells is difficult to quantify using standard fluorescent protein (FP) expression assays, due to the slow maturation of their fluorophore. Here we have developed expression reporters that accurately measure both the levels and dynamics of protein synthesis in live single cells with a temporal resolution under a minute. Our system relies on the quantification of the translocation of a constitutively expressed FP into the nucleus. As a proof of concept, we used these reporters to measure the transient protein synthesis arising from two promoters responding to the yeast hyper osmolarity glycerol mitogen-activated protein kinase pathway (pSTL1 and pGPD1). They display distinct expression dynamics giving rise to strikingly different instantaneous expression noise. PMID:27098003

  7. High level expression of mammalian protein farnesyltransferase in a baculovirus system. The purified protein contains zinc.

    PubMed

    Chen, W J; Moomaw, J F; Overton, L; Kost, T A; Casey, P J

    1993-05-01

    The mammalian enzyme protein farnesyltransferase is a heterodimeric protein that catalyzes the addition of a farnesyl isoprenoid to a cysteine in ras proteins. Since oncogenic forms of ras proteins require the farnesyl group for transforming activity, the structure and mechanism of this enzyme are important to define. However, such studies have been difficult to approach because of the low abundance of the enzyme in mammalian tissues and hence the problems of obtaining large quantities of the protein. We report here the co-expression of the two subunits of protein farnesyltransferase by Sf9 cells infected with a recombinant baculovirus containing the coding sequences of both polypeptides. This results in the production of milligram quantities of enzyme which can be readily purified by conventional chromatographic methods. The individual subunits of the enzyme can also be expressed in the Sf9 cells, but the ability to reconstitute active enzyme from extracts containing individual subunits is quite low. In contrast, the enzyme produced by co-expression of the two subunits is fully active and retains the properties of the mammalian form, including the specificity for the COOH-terminal amino acid of substrate proteins and the ability to bind short peptides encompassing the prenylation site of a ras protein. Furthermore, through atomic absorption analysis of the purified protein, we have confirmed the previous tentative assignment of protein farnesyltransferase as a zinc metalloenzyme by demonstrating that it contains an essentially stoichiometric amount of zinc. The ability to produce and purify milligram quantities of protein farnesyltransferase readily will allow detailed mechanistic and structural studies on this enzyme. PMID:8486655

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  10. Progranulin protein levels are differently regulated in plasma and CSF

    PubMed Central

    Nicholson, Alexandra M.; Finch, NiCole A.; Thomas, Colleen S.; Wojtas, Aleksandra; Rutherford, Nicola J.; Mielke, Michelle M.; Roberts, Rosebud O.; Boeve, Bradley F.; Knopman, David S.; Petersen, Ronald C.

    2014-01-01

    Objective: We aimed to investigate the relationship between plasma and CSF progranulin (PGRN) levels. Methods: Plasma and CSF PGRN were measured in a cohort of 345 subjects from the Mayo Clinic Study of Aging by ELISA. Single nucleotide polymorphism genotyping was performed using TaqMan assays. Associations between PGRN and sex, age at sample collection, diagnosis, single nucleotide polymorphism genotypes (GRN, SORT1, and APOE), and Pittsburgh compound B score were explored separately in CSF and plasma using single variable linear regression models. Pearson partial correlation coefficient was used to estimate the correlation of PGRN in CSF and plasma. Results: Plasma (p = 0.0031) and CSF (p = 0.0044) PGRN significantly increased with age, whereas plasma PGRN levels were 7% lower (p = 0.0025) and CSF PGRN levels 5% higher (p = 0.0024) in male compared with female participants. Correcting for age and sex, higher plasma PGRN was associated with higher CSF PGRN (partial r = 0.17, p = 0.004). In plasma, both rs5848 (GRN; p = 0.002) and rs646776 (SORT1; p = 3.56E-7) were associated with PGRN, while only rs5848 showed highly significant association in CSF (p = 5.59E-14). Age, sex, rs5848 genotype, and plasma PGRN together accounted for only 18% of the variability observed in CSF PGRN. Conclusions: While some correlation exists between plasma and CSF PGRN, age, sex, and genetic factors differently affect PGRN levels. Therefore, caution should be taken when using plasma PGRN to predict PGRN changes in the brain. These findings further highlight that plasma PGRN levels may not accurately predict clinical features or response to future frontotemporal lobar degeneration therapies. PMID:24771538

  11. Calcium, phosphorus and protein levels as factors in the distribution of the pheasant

    USGS Publications Warehouse

    Dale, F.H.; DeWitt, J.B.

    1958-01-01

    Summary of work on pheasant nutrition conducted since 1949 at the Patuxent Research Refuge. Pheasant chicks fed experimental diets failed to develop normally on protein levels of 15 and 18%. With 22% protein they grew at a reduced rate as compared to those on 28%. Protein level of the reproductive diet was shown to be important; low production of eggs and young resulted from levels below 25%. Calcium was found to be even more critical than protein level for reproduction; birds on a winter diet that furnished 145 mg./kg. per day had poor reproductive success the following spring. About 600 mg./kg. of Ca per day was necessary in the reproduction diet. Birds on an intermediate level of Ca (about 0.5% of diet) showed evidence of cumulative deficiency. It was concluded that pheasants receiving levels of Ca no higher than 0.5% in nature might display 'straggling failure' such as has been observed in several midwestern areas.

  12. Importin-β facilitates nuclear import of human GW proteins and balances cytoplasmic gene silencing protein levels

    PubMed Central

    Schraivogel, Daniel; Schindler, Susann G.; Danner, Johannes; Kremmer, Elisabeth; Pfaff, Janina; Hannus, Stefan; Depping, Reinhard; Meister, Gunter

    2015-01-01

    MicroRNAs (miRNAs) guide Argonaute (Ago) proteins to distinct target mRNAs leading to translational repression and mRNA decay. Ago proteins interact with a member of the GW protein family, referred to as TNRC6A-C in mammals, which coordinate downstream gene-silencing processes. The cytoplasmic functions of TNRC6 and Ago proteins are reasonably well established. Both protein families are found in the nucleus as well. Their detailed nuclear functions, however, remain elusive. Furthermore, it is not clear which import routes Ago and TNRC6 proteins take into the nucleus. Using different nuclear transport assays, we find that Ago as well as TNRC6 proteins shuttle between the cytoplasm and the nucleus. While import receptors might function redundantly to transport Ago2, we demonstrate that TNRC6 proteins are imported by the Importin-β pathway. Finally, we show that nuclear localization of both Ago2 and TNRC6 proteins can depend on each other suggesting actively balanced cytoplasmic Ago – TNRC6 levels. PMID:26170235

  13. A local average connectivity-based method for identifying essential proteins from the network level.

    PubMed

    Li, Min; Wang, Jianxin; Chen, Xiang; Wang, Huan; Pan, Yi

    2011-06-01

    Identifying essential proteins is very important for understanding the minimal requirements of cellular survival and development. Fast growth in the amount of available protein-protein interactions has produced unprecedented opportunities for detecting protein essentiality from the network level. Essential proteins have been found to be more abundant among those highly connected proteins. However, there exist a number of highly connected proteins which are not essential. By analyzing these proteins, we find that few of their neighbors interact with each other. Thus, we propose a new local method, named LAC, to determine a protein's essentiality by evaluating the relationship between a protein and its neighbors. The performance of LAC is validated based on the yeast protein interaction networks obtained from two different databases: DIP and BioGRID. The experimental results of the two networks show that the number of essential proteins predicted by LAC clearly exceeds that explored by Degree Centrality (DC). More over, LAC is also compared with other seven measures of protein centrality (Neighborhood Component (DMNC), Betweenness Centrality (BC), Closeness Centrality (CC), Bottle Neck (BN), Information Centrality (IC), Eigenvector Centrality (EC), and Subgraph Centrality (SC)) in identifying essential proteins. The comparison results based on the validations of sensitivity, specificity, F-measure, positive predictive value, negative predictive value, and accuracy consistently show that LAC outweighs these seven previous methods. PMID:21704260

  14. A Bayesian framework for cell-level protein network analysis for multivariate proteomics image data

    NASA Astrophysics Data System (ADS)

    Kovacheva, Violet N.; Sirinukunwattana, Korsuk; Rajpoot, Nasir M.

    2014-03-01

    The recent development of multivariate imaging techniques, such as the Toponome Imaging System (TIS), has facilitated the analysis of multiple co-localisation of proteins. This could hold the key to understanding complex phenomena such as protein-protein interaction in cancer. In this paper, we propose a Bayesian framework for cell level network analysis allowing the identification of several protein pairs having significantly higher co-expression levels in cancerous tissue samples when compared to normal colon tissue. It involves segmenting the DAPI-labeled image into cells and determining the cell phenotypes according to their protein-protein dependence profile. The cells are phenotyped using Gaussian Bayesian hierarchical clustering (GBHC) after feature selection is performed. The phenotypes are then analysed using Difference in Sums of Weighted cO-dependence Profiles (DiSWOP), which detects differences in the co-expression patterns of protein pairs. We demonstrate that the pairs highlighted by the proposed framework have high concordance with recent results using a different phenotyping method. This demonstrates that the results are independent of the clustering method used. In addition, the highlighted protein pairs are further analysed via protein interaction pathway databases and by considering the localization of high protein-protein dependence within individual samples. This suggests that the proposed approach could identify potentially functional protein complexes active in cancer progression and cell differentiation.

  15. Multi-level learning: improving the prediction of protein, domain and residue interactions by allowing information flow between levels

    PubMed Central

    Yip, Kevin Y; Kim, Philip M; McDermott, Drew; Gerstein, Mark

    2009-01-01

    Background Proteins interact through specific binding interfaces that contain many residues in domains. Protein interactions thus occur on three different levels of a concept hierarchy: whole-proteins, domains, and residues. Each level offers a distinct and complementary set of features for computationally predicting interactions, including functional genomic features of whole proteins, evolutionary features of domain families and physical-chemical features of individual residues. The predictions at each level could benefit from using the features at all three levels. However, it is not trivial as the features are provided at different granularity. Results To link up the predictions at the three levels, we propose a multi-level machine-learning framework that allows for explicit information flow between the levels. We demonstrate, using representative yeast interaction networks, that our algorithm is able to utilize complementary feature sets to make more accurate predictions at the three levels than when the three problems are approached independently. To facilitate application of our multi-level learning framework, we discuss three key aspects of multi-level learning and the corresponding design choices that we have made in the implementation of a concrete learning algorithm. 1) Architecture of information flow: we show the greater flexibility of bidirectional flow over independent levels and unidirectional flow; 2) Coupling mechanism of the different levels: We show how this can be accomplished via augmenting the training sets at each level, and discuss the prevention of error propagation between different levels by means of soft coupling; 3) Sparseness of data: We show that the multi-level framework compounds data sparsity issues, and discuss how this can be dealt with by building local models in information-rich parts of the data. Our proof-of-concept learning algorithm demonstrates the advantage of combining levels, and opens up opportunities for further

  16. Effects of dietary protein level on growth and utilization of protein and energy by juvenile mangrove red snapper (Lutjanus argentimaculatus)

    NASA Astrophysics Data System (ADS)

    Ghulam, Abbas; Khalid, Jamil; Rukhsana, Akhtar; Lin, Hong

    2005-01-01

    A feeding trial was conducted in a recirculating water system to investigate the effects of dietary protein levels on growth, feed utilization, hepatosomatic index and liver lipid deposition of juvenile red snapper, Lutjanus argentimaculatus (average initial wet weight 8.0 ± 0.39 g and total length 3.14 ± 0.3 cm). In the experiment, six fishmeal-based diets were formulated to contain various protein levels (20% to 45% in 5% increments), with dietary energy ranging from 2210.7kJ lOOg to 2250.2kJlOOg dry matter. The protein to energy ratios of diets ranged from 8.58 mg protein kJ-1 to 20.03 mg protein kJ-1. Diets were fed for 90d to triplicate groups of fish stocked in 0.128m3 seawater tanks, 25 individuals each. The daily ration of 2% wet body weight was offered to the fish thrice a day. The fish at the end of the study had more than ten-fold (77.0g) increase in weight compared to the initial (8.0g). Fish fed diets of 40% and 45% protein produced significantly (P<0.05) higher weight gain of 77.2g and 76.5g, and specific growth rate (SGR) of 2.65% and 2.62% than those of 67.0 g and 68.3g, and 2.49% and 2.51% of the other diets. The broken-line regression of SGR against dietary protein level yielded an optimum dietary protein requirement of 42.6% (Y=-1.6295 + 0.1114 X 2,P<0.05). Survival remained 100% among groups. Feed conversion ratio decreased from 0.45 for fish fed 20% dietary protein to 0.35 for fish fed 45% dietary protein. Nitrogen intake increased with an increase in dietary protein, which in turn resulted in an increase in nitrogen gain of fish whole body. Fish fed 40% and 45% protein diets showed higher (P<0.05) nitrogen gain (0.27g and 0.26g) than those (0.23g and 025g) fed all other diets. Gross energy intake (GEI) in fish fed 45% protein was lower (600.67kJ) than that (607.97 kJ) of 40% protein diet, though the differences were not statistically significant (P>0.05); GEI ranging from 677.31 kJ to 663.20 kJ at remaining four diets (20% to 35% protein

  17. Influence of protein level and supplemental methionine in practical rations for young endangered masked bobwhite quail

    USGS Publications Warehouse

    Serafin, J.A.

    1982-01-01

    A study was conducted to examine the protein requirement of young endangered masked Bobwhite quail (Colinus virginianus ridgwayi). Five practical starting rations containing 24 to 32% protein were fed alone and supplemented with methionine for 5 weeks. Supplemental methionine significantly improved growth of quail fed diets containing 24 and 26% protein. Increasing the protein level improved growth of quail fed unsupplemented diets but did not do so when diets contained supplemental methionine. A methionine-supplemented ration containing 24% protein appeared adequate for supporting rapid growth of masked Bobwhite quail.

  18. Systematic Protein Level Regulation via Degradation Machinery Induced by Genotoxic Drugs.

    PubMed

    Kume, Kohei; Ishida, Kazushige; Ikeda, Miyuki; Takemoto, Kazuhiro; Shimura, Tsutomu; Young, Lynn; Nishizuka, Satoshi S

    2016-01-01

    In this study we monitored protein dynamics in response to cisplatin, 5-fluorouracil, and irinotecan with different concentrations and administration modes using "reverse-phase" protein arrays (RPPAs) in order to gain comprehensive insight into the protein dynamics induced by genotoxic drugs. Among 666 protein time-courses, 38% exhibited an increasing trend, 32% exhibited a steady decrease, and 30% fluctuated within 24 h after drug exposure. We analyzed almost 12,000 time-course pairs of protein levels based on the geometrical similarity by correlation distance (dCor). Twenty-two percent of the pairs showed dCor > 0.8, which indicates that each protein of the pair had similar dynamics. These trends were disrupted by a proteasome inhibitor, MG132, suggesting that the protein degradation system was activated in response to the drugs. Among the pairs with high dCor, the average dCor of pairs with apoptosis-related protein was significantly higher than those without, indicating that regulation of protein levels was induced by the drugs. These results suggest that the levels of numerous functionally distinct proteins may be regulated by common degradation machinery induced by genotoxic drugs. PMID:26625007

  19. Synergistic Control of Kinetochore Protein Levels by Psh1 and Ubr2

    PubMed Central

    Herrero, Eva; Thorpe, Peter H.

    2016-01-01

    The accurate segregation of chromosomes during cell division is achieved by attachment of chromosomes to the mitotic spindle via the kinetochore, a large multi-protein complex that assembles on centromeres. The budding yeast kinetochore comprises more than 60 different proteins. Although the structure and function of many of these proteins has been investigated, we have little understanding of the steady state regulation of kinetochores. The primary model of kinetochore homeostasis suggests that kinetochores assemble hierarchically from the centromeric DNA via the inclusion of a centromere-specific histone into chromatin. We tested this model by trying to perturb kinetochore protein levels by overexpressing an outer kinetochore gene, MTW1. This increase in protein failed to change protein recruitment, consistent with the hierarchical assembly model. However, we find that deletion of Psh1, a key ubiquitin ligase that is known to restrict inner kinetochore protein loading, does not increase levels of outer kinetochore proteins, thus breaking the normal kinetochore stoichiometry. This perturbation leads to chromosome segregation defects, which can be partially suppressed by mutation of Ubr2, a second ubiquitin ligase that normally restricts protein levels at the outer kinetochore. Together these data show that Psh1 and Ubr2 synergistically control the amount of proteins at the kinetochore. PMID:26891228

  20. Efficient monitoring of protein ubiquitylation levels using TUBEs-based microarrays.

    PubMed

    Serna, Sonia; Xolalpa, Wendy; Lang, Valérie; Aillet, Fabienne; England, Patrick; Reichardt, Niels; Rodriguez, Manuel S

    2016-08-01

    Analyzing protein ubiquitylation changes during physiological or pathological processes is challenging due to its high reversibility and dynamic turnover of modified targets. We have developed a protein microarray to assess endogenous ubiquitylation levels from cell cultures, employing tandem ubiquitin-binding entities (TUBEs) with three or four ubiquitin-associated (UBA) domains as capture probes. Adriamycin (ADR)-stimulated MCF7 cells were used to differentiate protein ubiquitylation levels between cells that are sensitive or resistant to ADR treatment. We show that TUBEs-based microarrays can be used for the analysis of cellular processes regulated by ubiquitylation and for the detection of pathologies with aberrant ubiquitylation levels. PMID:27410252

  1. A Cul-3-BTB ubiquitylation pathway regulates junctional levels and asymmetry of core planar polarity proteins

    PubMed Central

    Strutt, Helen; Searle, Elizabeth; Thomas-MacArthur, Victoria; Brookfield, Rosalind; Strutt, David

    2013-01-01

    The asymmetric localisation of core planar polarity proteins at apicolateral junctions is required to specify cell polarity in the plane of epithelia. This asymmetric distribution of the core proteins is proposed to require amplification of an initial asymmetry by feedback loops. In addition, generation of asymmetry appears to require the regulation of core protein levels, but the importance of such regulation and the underlying mechanisms is unknown. Here we show that ubiquitylation acts through more than one mechanism to control core protein levels in Drosophila, and that without this regulation cellular asymmetry is compromised. Levels of Dishevelled at junctions are regulated by a Cullin-3-Diablo/Kelch ubiquitin ligase complex, the activity of which is most likely controlled by neddylation. Furthermore, activity of the deubiquitylating enzyme Fat facets is required to maintain Flamingo levels at junctions. Notably, ubiquitylation does not alter the total cellular levels of Dishevelled or Flamingo, but only that of the junctional population. When junctional core protein levels are either increased or decreased by disruption of the ubiquitylation machinery, their asymmetric localisation is reduced and this leads to disruption of planar polarity at the tissue level. Loss of asymmetry by altered core protein levels can be explained by reference to feedback models for amplification of asymmetry. PMID:23487316

  2. GTP Cyclohydrolase I Expression, Protein, and Activity Determine Intracellular Tetrahydrobiopterin Levels, Independent of GTP Cyclohydrolase Feedback Regulatory Protein Expression

    PubMed Central

    Tatham, Amy L.; Crabtree, Mark J.; Warrick, Nicholas; Cai, Shijie; Alp, Nicholas J.; Channon, Keith M.

    2009-01-01

    GTP cyclohydrolase I (GTPCH) is a key enzyme in the synthesis of tetrahydrobiopterin (BH4), a required cofactor for nitricoxide synthases and aromatic amino acid hydroxylases. Alterations of GTPCH activity and BH4 availability play an important role in human disease. GTPCH expression is regulated by inflammatory stimuli, in association with reduced expression of GTP cyclohydrolase feedback regulatory protein (GFRP). However, the relative importance of GTPCH expression versus GTPCH activity and the role of GFRP in relation to BH4 bioavailability remain uncertain. We investigated these relationships in a cell line with tet-regulated GTPCH expression and in the hph-1 mouse model of GTPCH deficiency. Doxycycline exposure resulted in a dose-dependent decrease in GTPCH protein and activity, with a strong correlation between GTPCH expression and BH4 levels (r2 = 0.85, p < 0.0001). These changes in GTPCH and BH4 had no effect on GFRP expression or protein levels. GFRP overexpression and knockdown in tet-GCH cells did not alter GTPCH activity or BH4 levels, and GTPCH-specific knockdown in sEnd.1 endothelial cells had no effect on GFRP protein. In mouse liver we observed a graded reduction of GTPCH expression, protein, and activity, from wild type, heterozygote, to homozygote littermates, with a striking linear correlation between GTPCH expression and BH4 levels (r2 = 0.82, p < 0.0001). Neither GFRP expression nor protein differed between wild type, heterozygote, nor homozygote mice, despite the substantial differences in BH4. We suggest that GTPCH expression is the primary regulator of BH4 levels, and changes in GTPCH or BH4 are not necessarily accompanied by changes in GFRP expression. PMID:19286659

  3. GTP cyclohydrolase I expression, protein, and activity determine intracellular tetrahydrobiopterin levels, independent of GTP cyclohydrolase feedback regulatory protein expression.

    PubMed

    Tatham, Amy L; Crabtree, Mark J; Warrick, Nicholas; Cai, Shijie; Alp, Nicholas J; Channon, Keith M

    2009-05-15

    GTP cyclohydrolase I (GTPCH) is a key enzyme in the synthesis of tetrahydrobiopterin (BH4), a required cofactor for nitricoxide synthases and aromatic amino acid hydroxylases. Alterations of GTPCH activity and BH4 availability play an important role in human disease. GTPCH expression is regulated by inflammatory stimuli, in association with reduced expression of GTP cyclohydrolase feedback regulatory protein (GFRP). However, the relative importance of GTPCH expression versus GTPCH activity and the role of GFRP in relation to BH4 bioavailability remain uncertain. We investigated these relationships in a cell line with tet-regulated GTPCH expression and in the hph-1 mouse model of GTPCH deficiency. Doxycycline exposure resulted in a dose-dependent decrease in GTPCH protein and activity, with a strong correlation between GTPCH expression and BH4 levels (r(2) = 0.85, p < 0.0001). These changes in GTPCH and BH4 had no effect on GFRP expression or protein levels. GFRP overexpression and knockdown in tet-GCH cells did not alter GTPCH activity or BH4 levels, and GTPCH-specific knockdown in sEnd.1 endothelial cells had no effect on GFRP protein. In mouse liver we observed a graded reduction of GTPCH expression, protein, and activity, from wild type, heterozygote, to homozygote littermates, with a striking linear correlation between GTPCH expression and BH4 levels (r(2) = 0.82, p < 0.0001). Neither GFRP expression nor protein differed between wild type, heterozygote, nor homozygote mice, despite the substantial differences in BH4. We suggest that GTPCH expression is the primary regulator of BH4 levels, and changes in GTPCH or BH4 are not necessarily accompanied by changes in GFRP expression. PMID:19286659

  4. Calpain expression in lymphoid cells. Increased mRNA and protein levels after cell activation.

    PubMed

    Deshpande, R V; Goust, J M; Chakrabarti, A K; Barbosa, E; Hogan, E L; Banik, N L

    1995-02-10

    Although calpain is ubiquitously present in human tissues and is thought to play a role in demyelination, its activity is very low in resting normal lymphocytes. To determine the nature of calpain expression at the mRNA and protein levels in human lymphoid cells, we studied human T lymphocytic, B lymphocytic, and monocytic lines as well as peripheral blood mononuclear cells. Stimulation of cells with the phorbol ester phorbol myristate acetate and the calcium ionophore A23187 resulted in increased calpain mRNA and protein expression. Calpain mRNA expression is also increased in human T cells stimulated with anti-CD3. A dissociation between the increases of RNA and protein suggested that calpain could be released from the cells; the subsequent experiments showed its presence in the extracellular environment. 5,6-Dichloro-1b-D-ribofuranosylbenzimidazole, a reversible inhibitor of mRNA synthesis, reduced calpain mRNA levels by 50-67% and protein levels by 72-91%. Its removal resulted in resumption of both calpain mRNA and protein synthesis. Cycloheximide, a translational inhibitor, reduced calpain protein levels by 77-81% and calpain mRNA levels by 96% in activated THP-1 cells. Interferon-gamma induced calpain mRNA and protein in U-937 and THP-1 cells. Dexamethasone increased mRNA expression in THP-1 cells. Our results indicate that activation of lymphoid cells results in de novo synthesis and secretion of calpain. PMID:7852311

  5. Identification and Validation of Genetic Variants that Influence Transcription Factor and Cell Signaling Protein Levels

    PubMed Central

    Hause, Ronald J.; Stark, Amy L.; Antao, Nirav N.; Gorsic, Lidija K.; Chung, Sophie H.; Brown, Christopher D.; Wong, Shan S.; Gill, Daniel F.; Myers, Jamie L.; To, Lida Anita; White, Kevin P.; Dolan, M. Eileen; Jones, Richard Baker

    2014-01-01

    Many genetic variants associated with human disease have been found to be associated with alterations in mRNA expression. Although it is commonly assumed that mRNA expression changes will lead to consequent changes in protein levels, methodological challenges have limited our ability to test the degree to which this assumption holds true. Here, we further developed the micro-western array approach and globally examined relationships between human genetic variation and cellular protein levels. We collected more than 250,000 protein level measurements comprising 441 transcription factor and signaling protein isoforms across 68 Yoruba (YRI) HapMap lymphoblastoid cell lines (LCLs) and identified 12 cis and 160 trans protein level QTLs (pQTLs) at a false discovery rate (FDR) of 20%. Whereas up to two thirds of cis mRNA expression QTLs (eQTLs) were also pQTLs, many pQTLs were not associated with mRNA expression. Notably, we replicated and functionally validated a trans pQTL relationship between the KARS lysyl-tRNA synthetase locus and levels of the DIDO1 protein. This study demonstrates proof of concept in applying an antibody-based microarray approach to iteratively measure the levels of human proteins and relate these levels to human genome variation and other genomic data sets. Our results suggest that protein-based mechanisms might functionally buffer genetic alterations that influence mRNA expression levels and that pQTLs might contribute phenotypic diversity to a human population independently of influences on mRNA expression. PMID:25087611

  6. Relationships between cell cycle regulator gene copy numbers and protein expression levels in Schizosaccharomyces pombe.

    PubMed

    Chino, Ayako; Makanae, Koji; Moriya, Hisao

    2013-01-01

    We previously determined the copy number limits of overexpression for cell division cycle (cdc) regulatory genes in the fission yeast Schizosaccharomyces pombe using the "genetic tug-of-war" (gTOW) method. In this study, we measured the levels of tandem affinity purification (TAP)-tagged target proteins when their copy numbers are increased in gTOW. Twenty analyzed genes showed roughly linear correlations between increased protein levels and gene copy numbers, which suggested a general lack of compensation for gene dosage in S. pombe. Cdc16 and Sid2 protein levels but not their mRNA levels were much lower than that expected by their copy numbers, which suggested the existence of a post-transcriptional down regulation of these genes. The cyclin Cig1 protein level and its mRNA level were much higher than that expected by its copy numbers, which suggested a positive feedback mechanism for its expression. A higher Cdc10 protein level and its mRNA level, probably due to cloning its gene into a plasmid, indicated that Cdc10 regulation was more robust than that previously predicted. PMID:24019917

  7. Optimality and evolutionary tuning of the expression level of a protein.

    PubMed

    Dekel, Erez; Alon, Uri

    2005-07-28

    Different proteins have different expression levels. It is unclear to what extent these expression levels are optimized to their environment. Evolutionary theories suggest that protein expression levels maximize fitness, but the fitness as a function of protein level has seldom been directly measured. To address this, we studied the lac system of Escherichia coli, which allows the cell to use the sugar lactose for growth. We experimentally measured the growth burden due to production and maintenance of the Lac proteins (cost), as well as the growth advantage (benefit) conferred by the Lac proteins when lactose is present. The fitness function, given by the difference between the benefit and the cost, predicts that for each lactose environment there exists an optimal Lac expression level that maximizes growth rate. We then performed serial dilution evolution experiments at different lactose concentrations. In a few hundred generations, cells evolved to reach the predicted optimal expression levels. Thus, protein expression from the lac operon seems to be a solution of a cost-benefit optimization problem, and can be rapidly tuned by evolution to function optimally in new environments. PMID:16049495

  8. High level expression of peptides and proteins using cytochrome b5 as a fusion host.

    PubMed

    Mitra, Ashima; Chakrabarti, Kalyan Sundar; Shahul Hameed, M S; Srinivas, Kalyan V; Senthil Kumar, Ganesan; Sarma, Siddhartha P

    2005-05-01

    A novel fusion protein system based on the highly soluble heme-binding domain of cytochrome b5 has been designed. The ability of cytochrome b5 to increase the levels of expression and solubility of target proteins has been tested by expressing several proteins and peptides, viz., alpha hemoglobin stabilizing protein, the regulatory subunits of acetohydroxy acid synthase I (ilvM) and II (ilvN), the carboxy terminal domains of mouse neuronal kinesin and pantothenate synthatase, two peptide toxins from cone snails, and the inactivation gate from the brain voltage gated sodium channel, NaV1.2. The fusion protein system has been designed to incorporate protease cleavage sites for commonly used proteases, viz., enterokinase, Factor Xa, and Tobacco etch virus protease. Accumulation of expressed protein as a function of time may be visually ascertained by the fact that the cells take on a bright red color during the course of induction. In all the cases tested so far, the fusion protein accumulates in the soluble fraction to high levels. A novel purification protocol has been designed to purify the fusion proteins using metal affinity chromatography, without the need of a hexahistidine-tag. Mass spectral analysis has shown that the fusion proteins are of full length. CD studies have shown that the solubilized fusion proteins are structured. The proteins of interest may be cleaved from the parent protein by either chemical or enzymatic means. The results presented here demonstrate the versatility of the cytochrome b5 based fusion system for the production of peptides and small proteins (<15 kDa). PMID:15802225

  9. Integrative analysis of RNA, translation, and protein levels reveals distinct regulatory variation across humans

    PubMed Central

    Cenik, Can; Cenik, Elif Sarinay; Byeon, Gun W.; Grubert, Fabian; Candille, Sophie I.; Spacek, Damek; Alsallakh, Bilal; Tilgner, Hagen; Araya, Carlos L.; Tang, Hua; Ricci, Emiliano; Snyder, Michael P.

    2015-01-01

    Elucidating the consequences of genetic differences between humans is essential for understanding phenotypic diversity and personalized medicine. Although variation in RNA levels, transcription factor binding, and chromatin have been explored, little is known about global variation in translation and its genetic determinants. We used ribosome profiling, RNA sequencing, and mass spectrometry to perform an integrated analysis in lymphoblastoid cell lines from a diverse group of individuals. We find significant differences in RNA, translation, and protein levels suggesting diverse mechanisms of personalized gene expression control. Combined analysis of RNA expression and ribosome occupancy improves the identification of individual protein level differences. Finally, we identify genetic differences that specifically modulate ribosome occupancy—many of these differences lie close to start codons and upstream ORFs. Our results reveal a new level of gene expression variation among humans and indicate that genetic variants can cause changes in protein levels through effects on translation. PMID:26297486

  10. Monitoring protein phosphatase 1 isoform levels as a marker for cellular stress.

    PubMed

    Amador, Fátima Camões; Henriques, Ana Gabriela; da Cruz E Silva, Odete A B; da Cruz E Silva, Edgar F

    2004-01-01

    Reversible protein phosphorylation is a central mechanism regulating many biological functions, and abnormal protein phosphorylation can have a devastating impact on cellular control mechanisms, including a contributing role in neurodegenerative processes. Hence, many promising novel drug development strategies involve targeting protein phosphorylation systems. In this study, we demonstrate that various cellular stresses relevant to neurodegeneration can specifically affect the protein expression levels of protein phosphatase 1 (PP1). PP1 levels were altered upon exposure of PC12 and COS-1 cells to aluminium, Abeta peptides, sodium azide, and even heat shock. Particularly interesting, given PP1's involvement in aging and neurodegeneration, was the consistent decrease in PP1gamma(1) levels in response to stress agents. In fact, alterations in the expression levels of PP1 appear to correspond to an early response of stress induction, that is, before alterations in heat shock proteins can be detected. Our data suggest that monitoring PP1 isoform expression could constitute a useful diagnostic tool for cellular stress, possibly even neurodegeneration. Additionally, our results strengthen the rationale for signal transduction therapeutics and indicate that altering the specific activity of PP1 either directly or by targeting its regulatory proteins may be a useful therapeutic development strategy for the future. PMID:15113600

  11. Increased levels of hyper-stable protein aggregates in plasma of older adults.

    PubMed

    Xia, Ke; Trasatti, Hannah; Wymer, James P; Colón, Wilfredo

    2016-06-01

    Proteins that misfold into hyper-stable/degradation-resistant species during aging may accumulate and disrupt protein homeostasis (i.e., proteostasis), thereby posing a survival risk to any organism. Using the method diagonal two-dimensional (D2D) SDS-PAGE, which separates hyper-stable SDS-resistant proteins at a proteomics level, we analyzed the plasma of healthy young (<30 years) and older (60-80 years) adults. We discovered the presence of soluble SDS-resistant protein aggregates in the plasma of older adults, but found significantly lower levels in the plasma of young adults. We identified the inflammation-related chaperone protein haptoglobin as the main component of the hyper-stable aggregates. This observation is consistent with the growing link between accumulations of protein aggregates and aging across many organisms. It is plausible higher amounts of SDS-resistant protein aggregates in the plasma of older adults may reflect a compromise in proteostasis that may potentially indicate cellular aging and/or disease risk. The results of this study have implications for further understanding the link between aging and the accumulation of protein aggregates, as well as potential for the development of aging-related biomarkers. More broadly, this novel application of D2D SDS-PAGE may be used to identify, quantify, and characterize the degradation-resistant protein aggregates in human plasma or any biological system. PMID:27179971

  12. System-level comparison of protein-protein interactions between viruses and the human type I interferon system network.

    PubMed

    Navratil, V; de Chassey, B; Meyniel, L; Pradezynski, F; André, P; Rabourdin-Combe, C; Lotteau, V

    2010-07-01

    Innate immunity has evolved complex molecular pathways to protect organisms from viral infections. One pivotal line of cellular defense is the induction of the antiviral effect of interferon. To circumvent this primary response and achieve their own replication, viruses have developed complex molecular strategies. Here, we provide a systems-level study of the human type I interferon system subversion by the viral proteome, by reconstructing the underlying protein-protein interaction network. At this network level, viruses establish a massive and a gradual attack, from receptors to transcription factors, by interacting preferentially with highly connected and central proteins as well as interferon-induced proteins. We also demonstrate that viruses significantly target 22% of the proteins directly interacting with the type I interferon system network, suggesting the relevance of our network-based method to identify new candidates involved in the regulation of the antiviral response. Finally, based on the comparative analysis of interactome profiles across four viral families, we provide evidence of common and differential targeting strategies. PMID:20459142

  13. Protein level affects the relative lysine requirement of growing rainbow trout (Oncorhynchus mykiss) fry.

    PubMed

    Bodin, Noelie; Govaerts, Bernadette; Abboudi, Tarik; Detavernier, Christel; De Saeger, Sarah; Larondelle, Yvan; Rollin, Xavier

    2009-07-01

    The effect of two digestible protein levels (310 and 469 g/kg DM) on the relative lysine (Lys; g Lys/kg DM or g Lys/100 g protein) and the absolute Lys (g Lys intake/kg 0.75 per d) requirements was studied in rainbow trout fry using a dose-response trial. At each protein level, sixteen isoenergetic (22-23 MJ digestible energy/kg DM) diets were tested, involving a full range (2-70 g/kg DM) of sixteen Lys levels. Each diet was given to one group of sixty rainbow trout fry (mean initial body weight 0.78 g) reared at 15 degrees C for 31 feeding d. The Lys requirements were estimated based on the relationships between weight, protein, and Lys gains (g/kg 0.75 per d) and Lys concentration (g/kg DM or g/100 g protein) or Lys intake (g/kg 0.75 per d), using the broken-line model (BLM) and the non-linear four-parameter saturation kinetics model (SKM-4). Both the model and the response criterion chosen markedly impacted the relative Lys requirement. The relative Lys requirement for Lys gain of rainbow trout estimated with the BLM (and SKM-4 at 90 % of the maximum response) increased from 16.8 (19.6) g/kg DM at a low protein level to 23.4 (24.5) g/kg DM at a high protein level. However, the dietary protein content affected neither the absolute Lys requirement nor the relative Lys requirement expressed as g Lys/100 g protein nor the Lys requirement for maintenance (21 mg Lys/kg 0.75 per d). PMID:19138439

  14. C-reactive protein gene variants: independent association with late-life depression and circulating protein levels.

    PubMed

    Ancelin, M-L; Farré, A; Carrière, I; Ritchie, K; Chaudieu, I; Ryan, J

    2015-01-01

    C-reactive protein (CRP) is a heritable biomarker of systemic inflammation that is commonly elevated in depressed patients. Variants in the CRP gene that influence protein levels could thus be associated with depression but this has seldom been examined, especially in the elderly. Depression was assessed in 990 people aged at least 65 years as part of the ESPRIT study. A clinical level of depression (DEP) was defined as having a score of ⩾16 on The Center for Epidemiologic Studies Depression scale or a diagnosis of current major depression based on the Mini-International Neuropsychiatric Interview and according to Diagnostic and Statistical Manual of Mental Disorders-IV criteria. Five single-nucleotide polymorphisms spanning the CRP gene were genotyped, and circulating levels of high-sensitivity CRP were determined. Multivariable analyses adjusted for socio-demographic characteristics, smoking, ischemic pathologies, cognitive impairment and inflammation-related chronic pathologies. The minor alleles of rs1130864 and rs1417938 were associated with a decreased risk of depression in women at Bonferroni-corrected significance levels (P=0.002). CRP gene variants were associated with serum levels in a gender-specific manner, but only rs1205 was found to be nominally associated with both an increased risk of DEP and lower circulating CRP levels in women. Variants of the CRP gene thus influence circulating CRP levels and appear as independent susceptibility factors for late-life depression. PMID:25603415

  15. EFFECTS OF VARYING DIETARY PROTEIN AND ENERGY LEVELS ON THE PRODUCTION OF LACTATING DAIRY COWS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Forty-five multiparous and 18 primiparous Holstein cows averaging 41 kg/d of milk were fed three levels of crude protein (CP), each at three levels of neutral detergent fiber (NDF), to identify optimal dietary CP and energy. Cows were blocked by lactation and days in milk into seven groups of nine a...

  16. Burial Level Change Defines a High Energetic Relevance for Protein Binding Interfaces.

    PubMed

    Li, Zhenhua; He, Ying; Wong, Limsoon; Li, Jinyan

    2015-01-01

    Protein-protein interfaces defined through atomic contact or solvent accessibility change are widely adopted in structural biology studies. But, these definitions cannot precisely capture energetically important regions at protein interfaces. The burial depth of an atom in a protein is related to the atom's energy. This work investigates how closely the change in burial level of an atom/residue upon complexation is related to the binding. Burial level change is different from burial level itself. An atom deeply buried in a monomer with a high burial level may not change its burial level after an interaction and it may have little burial level change. We hypothesize that an interface is a region of residues all undergoing burial level changes after interaction. By this definition, an interface can be decomposed into an onion-like structure according to the burial level change extent. We found that our defined interfaces cover energetically important residues more precisely, and that the binding free energy of an interface is distributed progressively from the outermost layer to the core. These observations are used to predict binding hot spots. Our approach's F-measure performance on a benchmark dataset of alanine mutagenesis residues is much superior or similar to those by complicated energy modeling or machine learning approaches. PMID:26357227

  17. Decreased melatonin levels and increased levels of advanced oxidation protein products in the seminal plasma are related to male infertility.

    PubMed

    Kratz, Ewa Maria; Piwowar, Agnieszka; Zeman, Michal; Stebelová, Katarína; Thalhammer, Theresia

    2016-03-01

    Melatonin, an indolamine secreted by the pineal gland, is known as a powerful free-radical scavenger and wide-spectrum antioxidant. Therefore, the aim of this study was to correlate markers of oxidative protein damage (advanced oxidation protein products, AOPPs) and the total antioxidant capacity (TAC) with melatonin levels in the seminal plasma of men with azoospermia (n=37), theratozoospermia (n=29) and fertile controls (normozoospermia, n=37). Melatonin concentration was measured by radioimmunoassay. The levels of AOPP as well as TAC efficiency (determined by the ferric reducing antioxidant power, FRAP) were estimated by spectrophotometric methods. The concentration of melatonin and AOPP significantly differed in azoospermic (P<0.0001) and theratozoospermic (P<0.0001) patients versus fertile men, and correlated negatively (r=-0.33, P=0.0016). The TAC levels were significantly higher in azoospermia than in theratozoospermia (P=0.0022) and the control group (P=0.00016). In azoospermia, the AOPP concentration was also significantly higher than that observed in theratozoospermia (P=0.00029). Decreased levels of melatonin together with elevated AOPP altered the oxidative-antioxidative balance in the ejaculate, thereby reducing fertility. Therefore, melatonin and AOPP levels may serve as additional diagnostic markers of semen quality and male reproductive potential. PMID:25218686

  18. Targeting of a histone acetyltransferase domain to a promoter enhances protein expression levels in mammalian cells.

    PubMed

    Kwaks, T H J; Sewalt, R G A B; van Blokland, R; Siersma, T J; Kasiem, M; Kelder, A; Otte, A P

    2005-01-12

    Silencing of transfected genes in mammalian cells is a fundamental problem that probably involves the (in)accessibility status of chromatin. A potential solution to this problem is to provide a cell with protein factors that make the chromatin of a promoter more open or accessible for transcription. We tested this by targeting such proteins to different promoters. We found that targeting the p300 histone acetyltransferase (HAT) domain to strong viral or cellular promoters is sufficient to result in higher expression levels of a reporter protein. In contrast, targeting the chromatin-remodeling factor Brahma does not result in stable, higher protein expression levels. The long-term effects of the targeted p300HAT domain on protein expression levels are positively reinforced, when also anti-repressor elements are applied to flank the reporter construct. These elements were previously shown to be potent blockers of chromatin-associated repressors. The simultaneous application of the targeted p300HAT domain and anti-repressor elements conveys long-term stability to protein expression. Whereas no copy number dependency is achieved by targeting of the p300HAT domain alone, copy number dependency is improved when anti-repressor elements are included. We conclude that targeting of protein domains such as HAT domains helps to facilitate expression of transfected genes in mammalian cells. However, the simultaneous application of other genomic elements such as the anti-repressor elements prevents silencing more efficiently. PMID:15607223

  19. Exceptionally high heterologous protein levels in transgenic dicotyledonous seeds using Phaseolus vulgaris regulatory sequences.

    PubMed

    De Jaeger, Geert; Angenon, Geert; Depicker, Ann

    2003-01-01

    Seeds are concentrated sources of protein and thus may be ideal 'bioreactors' for the production of heterologous proteins. For this application, strong seed-specific expression signals are required. A set of expression cassettes were designed using 5' and 3' regulatory sequences of the seed storage protein gene arcelin 5-I (arc5-I) from Phaseolus vulgaris, and evaluated for the production of heterologous proteins in dicotyledonous plant species. A murine single-chain variable fragment (scFv) was chosen as model protein because of the current industrial interest to produce antibodies and derived fragments in crops. Because the highest scFv accumulation in seed had previously been achieved in the endoplasmic reticulum (ER), the scFv-encoding sequence was provided with signal sequences for accumulation in the ER. Transgenic Arabidopsis seed stocks, expressing the scFv under control of the 35S promoter, contained scFv accumulation levels in the range of 1% of total soluble protein (TSP). However, the seed storage promoter constructs boosted the scFv to exceptionally high levels. Maximum scFv levels were obtained in homozygous seed stocks, being 12.5% of TSP under control of the arc5-I regulatory sequences and even up to 36.5% of TSP upon replacing the arc5-I promoter by the beta-phaseolin promoter of Phaseolus vulgaris. Even at such very high levels, the scFv proteins retain their full antigen-binding activity. Moreover, the presence of very high scFv levels has only minory effects on seed germination and no effect on seed production. These results demonstrate that the expression levels of arcelin 5-I and beta-phaseolin seed storage protein genes can be transferred to heterologous proteins, giving exceptionally high levels of heterologous proteins, which can be of great value for the molecular farming industry by raising production yield and lowering bio-mass production and purification costs. Finally, the feasibility of heterologous protein production using the

  20. Intercellular Variability in Protein Levels from Stochastic Expression and Noisy Cell Cycle Processes

    PubMed Central

    Soltani, Mohammad; Vargas-Garcia, Cesar A.; Antunes, Duarte; Singh, Abhyudai

    2016-01-01

    Inside individual cells, expression of genes is inherently stochastic and manifests as cell-to-cell variability or noise in protein copy numbers. Since proteins half-lives can be comparable to the cell-cycle length, randomness in cell-division times generates additional intercellular variability in protein levels. Moreover, as many mRNA/protein species are expressed at low-copy numbers, errors incurred in partitioning of molecules between two daughter cells are significant. We derive analytical formulas for the total noise in protein levels when the cell-cycle duration follows a general class of probability distributions. Using a novel hybrid approach the total noise is decomposed into components arising from i) stochastic expression; ii) partitioning errors at the time of cell division and iii) random cell-division events. These formulas reveal that random cell-division times not only generate additional extrinsic noise, but also critically affect the mean protein copy numbers and intrinsic noise components. Counter intuitively, in some parameter regimes, noise in protein levels can decrease as cell-division times become more stochastic. Computations are extended to consider genome duplication, where transcription rate is increased at a random point in the cell cycle. We systematically investigate how the timing of genome duplication influences different protein noise components. Intriguingly, results show that noise contribution from stochastic expression is minimized at an optimal genome-duplication time. Our theoretical results motivate new experimental methods for decomposing protein noise levels from synchronized and asynchronized single-cell expression data. Characterizing the contributions of individual noise mechanisms will lead to precise estimates of gene expression parameters and techniques for altering stochasticity to change phenotype of individual cells. PMID:27536771

  1. Intercellular Variability in Protein Levels from Stochastic Expression and Noisy Cell Cycle Processes.

    PubMed

    Soltani, Mohammad; Vargas-Garcia, Cesar A; Antunes, Duarte; Singh, Abhyudai

    2016-08-01

    Inside individual cells, expression of genes is inherently stochastic and manifests as cell-to-cell variability or noise in protein copy numbers. Since proteins half-lives can be comparable to the cell-cycle length, randomness in cell-division times generates additional intercellular variability in protein levels. Moreover, as many mRNA/protein species are expressed at low-copy numbers, errors incurred in partitioning of molecules between two daughter cells are significant. We derive analytical formulas for the total noise in protein levels when the cell-cycle duration follows a general class of probability distributions. Using a novel hybrid approach the total noise is decomposed into components arising from i) stochastic expression; ii) partitioning errors at the time of cell division and iii) random cell-division events. These formulas reveal that random cell-division times not only generate additional extrinsic noise, but also critically affect the mean protein copy numbers and intrinsic noise components. Counter intuitively, in some parameter regimes, noise in protein levels can decrease as cell-division times become more stochastic. Computations are extended to consider genome duplication, where transcription rate is increased at a random point in the cell cycle. We systematically investigate how the timing of genome duplication influences different protein noise components. Intriguingly, results show that noise contribution from stochastic expression is minimized at an optimal genome-duplication time. Our theoretical results motivate new experimental methods for decomposing protein noise levels from synchronized and asynchronized single-cell expression data. Characterizing the contributions of individual noise mechanisms will lead to precise estimates of gene expression parameters and techniques for altering stochasticity to change phenotype of individual cells. PMID:27536771

  2. Controlled levels of protein modification through a chromatography-mediated bioconjugation

    DOE PAGESBeta

    Kwant, Richard L.; Jaffe, Jake; Palmere, Peter J.; Francis, Matthew B.

    2015-02-27

    Synthetically modified proteins are increasingly finding applications as well-defined scaffolds for materials. In practice it remains difficult to construct bioconjugates with precise levels of modification because of the limited number of repeated functional groups on proteins. This article describes a method to control the level of protein modification in cases where there exist multiple potential modification sites. A protein is first tagged with a handle using any of a variety of modification chemistries. This handle is used to isolate proteins with a particular number of modifications via affinity chromatography, and then the handle is elaborated with a desired moiety usingmore » an oxidative coupling reaction. This method results in a sample of protein with a well-defined number of modifications, and we find it particularly applicable to systems like protein homomultimers in which there is no way to discern between chemically identical subunits. We demonstrate the use of this method in the construction of a protein-templated light-harvesting mimic, a type of system which has historically been difficult to make in a well-defined manner.« less

  3. Controlled levels of protein modification through a chromatography-mediated bioconjugation

    SciTech Connect

    Kwant, Richard L.; Jaffe, Jake; Palmere, Peter J.; Francis, Matthew B.

    2015-02-27

    Synthetically modified proteins are increasingly finding applications as well-defined scaffolds for materials. In practice it remains difficult to construct bioconjugates with precise levels of modification because of the limited number of repeated functional groups on proteins. This article describes a method to control the level of protein modification in cases where there exist multiple potential modification sites. A protein is first tagged with a handle using any of a variety of modification chemistries. This handle is used to isolate proteins with a particular number of modifications via affinity chromatography, and then the handle is elaborated with a desired moiety using an oxidative coupling reaction. This method results in a sample of protein with a well-defined number of modifications, and we find it particularly applicable to systems like protein homomultimers in which there is no way to discern between chemically identical subunits. We demonstrate the use of this method in the construction of a protein-templated light-harvesting mimic, a type of system which has historically been difficult to make in a well-defined manner.

  4. A two level hierarchical model of protein retention in ion exchange chromatography.

    PubMed

    Salvalaglio, Matteo; Paloni, Matteo; Guelat, Bertrand; Morbidelli, Massimo; Cavallotti, Carlo

    2015-09-11

    Predicting protein retention in ion exchange chromatography (IEX) from first principles is a fascinating perspective. In this work a two level hierarchical modeling strategy is proposed in order to calculate protein retention factors. Model predictions are tested against experimental data measured for Lysozyme and Chymotrypsinogen A in IEX columns as a function of ionic strength and pH. At the highest level of accuracy Molecular Dynamics (MD) simulations in explicit water are used to determine the interaction free energy between each of the two proteins and the IEX stationary phase for a reference pH and ionic strength. At a lower level of accuracy a linear response model based on an implicit treatment of solvation and adopting a static protein structure is used to calculate interaction free energies for the full range of pHs and ionic strengths considered. A scaling coefficient, determined comparing MD and implicit solvent simulations, is then introduced in order to correct the linear response model for errors induced by the adoption of a static protein structure. The calculated free energies are then used to compute protein retention factors, which can be directly compared with experimental data. The possibility to introduce a third level of accuracy is explored testing the predictions of a semiempirical model. A quantitative agreement between the predicted and measured protein retention factors is obtained using the coupled MD-linear response models, supporting the reliability of the proposed approach. The model allows quantifying the electrostatic, van der Waals, and conformational contributions to the interaction free energies. A good agreement between experiments and model is obtained also using the semiempirical model that, although requiring parameterization over higher level models or experimental data, proves to be useful in order to rapidly determine protein retention factors across wide pH and ionic strength ranges as it is computationally inexpensive

  5. BCRP protein levels do not differ regionally in adult human livers, but decline in the elderly.

    PubMed

    Riches, Zoe; Abanda, Ngu; Collier, Abby C

    2015-12-01

    The aim of this study was to characterize the ontogeny and variability of the BCRP (ABCG2) transporter in healthy human liver. Levels of BCRP mRNA and protein were determined with q-RT-PCR and western blot in a cohort of 87 human livers aged from 7 days to 87 years. A study of the regional expression of BCRP within adult livers was also performed in a nested cohort of 14 individuals with multiple samples per person collected from pre-selected sites. Levels of BCRP mRNA were not significantly different at any age, but protein levels for BCRP were lower in the elderly compared with adults (p < 0.001) and children (p < 0.05). The intra-liver levels of BCRP protein ranged approximately 6.5-fold and inter-liver BCRP protein varied 8.5-fold in the cohort. No differences in BCRP mRNA or protein were observed with sex or ethnicity, although higher levels of BCRP mRNA were observed in livers from overweight individuals (Body Mass Index ≥ 25-29.9) as compared to underweight or ideal weight individuals. There were no differences in the levels of BCRP mRNA or protein in different regions of the large lobe (n = 3 regions), small lobe (n = 3 regions), directly adjacent to the portal vein or directly adjacent to the common bile duct. This indicates that BCRP researchers can source tissue from all parts of the adult liver without artificial bias in their results. Lower BCRP protein expression in the elderly may be associated with compromised xeno- and endobiotic transport. PMID:26462791

  6. Reduced activity-dependent protein levels in a mouse model of the fragile X premutation.

    PubMed

    von Leden, Ramona E; Curley, Lindsey C; Greenberg, Gian D; Hunsaker, Michael R; Willemsen, Rob; Berman, Robert F

    2014-03-01

    Environmental enrichment results in increased levels of Fmrp in brain and increased dendritic complexity. The present experiment evaluated activity-dependent increases in Fmrp levels in the motor cortex in response to training on a skilled forelimb reaching task in the CGG KI mouse model of the fragile X premutation. Fmrp, Arc, and c-Fos protein levels were quantified by Western blot in the contralateral motor cortex of mice following training to reach for sucrose pellets with a non-preferred paw and compared to levels in the ipsilateral motor cortex. After training, all mice showed increases in Fmrp, Arc, and c-Fos protein levels in the contralateral compared to the ipsilateral hemisphere; however, the increase in CGG KI mice was less than wildtype mice. Increases in Fmrp and Arc proteins scaled with learning, whereas this relationship was not observed with the c-Fos levels. These data suggest the possibility that reduced levels of activity-dependent proteins associated with synaptic plasticity such as Fmrp and Arc may contribute to the neurocognitive phenotype reported in the CGG KI mice and the fragile X premutation. PMID:24462720

  7. Alcohol-Binding Sites in Distinct Brain Proteins: The Quest for Atomic Level Resolution

    PubMed Central

    Howard, Rebecca J.; Slesinger, Paul A.; Davies, Daryl L.; Das, Joydip; Trudell, James R.; Harris, R. Adron

    2011-01-01

    Defining the sites of action of ethanol on brain proteins is a major prerequisite to understanding the molecular pharmacology of this drug. The main barrier to reaching an atomic-level understanding of alcohol action is the low potency of alcohols, ethanol in particular, which is a reflection of transient, low-affinity interactions with their targets. These mechanisms are difficult or impossible to study with traditional techniques such as radioligand binding or spectroscopy. However, there has been considerable recent progress in combining X-ray crystallography, structural modeling, and site-directed mutagenesis to define the sites and mechanisms of action of ethanol and related alcohols on key brain proteins. We review such insights for several diverse classes of proteins including inwardly rectifying potassium, transient receptor potential, and neurotransmit-ter-gated ion channels, as well as protein kinase C epsilon. Some common themes are beginning to emerge from these proteins, including hydrogen bonding of the hydroxyl group and van der Waals interactions of the methylene groups of ethanol with specific amino acid residues. The resulting binding energy is proposed to facilitate or stabilize low-energy state transitions in the bound proteins, allowing ethanol to act as a “molecular lubricant” for protein function. We discuss evidence for characteristic, discrete alcohol-binding sites on protein targets, as well as evidence that binding to some proteins is better characterized by an interaction region that can accommodate multiple molecules of ethanol. PMID:21676006

  8. AFM visualization at a single-molecule level of denaturated states of proteins on graphite.

    PubMed

    Barinov, Nikolay A; Prokhorov, Valery V; Dubrovin, Evgeniy V; Klinov, Dmitry V

    2016-10-01

    Different graphitic materials are either already used or believed to be advantageous in biomedical and biotechnological applications, e.g., as biomaterials or substrates for sensors. Most of these applications or associated important issues, such as biocompatibility, address the problem of adsorption of protein molecules and, in particular the conformational state of the adsorbed protein molecule on graphite. High-resolution AFM demonstrates highly oriented pyrolytic graphite (HOPG) induced denaturation of four proteins of blood plasma, such as ferritin, fibrinogen, human serum albumin (HSA) and immunoglobulin G (IgG), at a single molecule level. Protein denaturation is accompanied by the decrease of the heights of protein globules and spreading of the denatured protein fraction on the surface. In contrast, the modification of HOPG with the amphiphilic oligoglycine-hydrocarbon derivative monolayer preserves the native-like conformation and provides even more mild conditions for the protein adsorption than typically used mica. Protein unfolding on HOPG may have universal character for "soft" globular proteins. PMID:27451365

  9. Hydrophobic carboxy-terminal residues dramatically reduce protein levels in the haloarchaeon Haloferax volcanii

    PubMed Central

    Reuter, Christopher J.; Uthandi, Sivakumar; Puentes, Jose A.; Maupin-Furlow, Julie A.

    2010-01-01

    Proteolysis is important not only to cell physiology but also to the successful development of biocatalysts. While a wide-variety of signals are known to trigger protein degradation in bacteria and eukaryotes, these mechanisms are poorly understood in archaea, known for their ability to withstand harsh conditions. Here we present a systematic study in which single C-terminal amino acid residues were added to a reporter protein and shown to influence its levels in an archaeal cell. All 20 amino acid residues were examined for their impact on protein levels, using the reporter protein soluble modified red-shifted GFP (smRS-GFP) expressed in the haloarchaeon Haloferax volcanii as a model system. Addition of hydrophobic residues, including Leu, Cys, Met, Phe, Ala, Tyr, Ile and Val, gave the most pronounced reduction in smRS-GFP levels compared with the addition of either neutral or charged hydrophilic residues. In contrast to the altered protein levels, the C-terminal alterations had no influence on smRS-GFP-specific transcript levels, thus revealing that the effect is post-transcriptional. PMID:19850616

  10. Protein Consumption and the Elderly: What Is the Optimal Level of Intake?

    PubMed Central

    Baum, Jamie I.; Kim, Il-Young; Wolfe, Robert R.

    2016-01-01

    Maintaining independence, quality of life, and health is crucial for elderly adults. One of the major threats to living independently is the loss of muscle mass, strength, and function that progressively occurs with aging, known as sarcopenia. Several studies have identified protein (especially the essential amino acids) as a key nutrient for muscle health in elderly adults. Elderly adults are less responsive to the anabolic stimulus of low doses of amino acid intake compared to younger individuals. However, this lack of responsiveness in elderly adults can be overcome with higher levels of protein (or essential amino acid) consumption. The requirement for a larger dose of protein to generate responses in elderly adults similar to the responses in younger adults provides the support for a beneficial effect of increased protein in older populations. The purpose of this review is to present the current evidence related to dietary protein intake and muscle health in elderly adults. PMID:27338461

  11. Effect of dietary protein levels on sex hormones in growing male rats kept under constant darkness.

    PubMed

    Hanai, Miho; Esashi, Takatoshi

    2012-01-01

    The purpose of this experiment was to clarify the effects of dietary protein levels on the gonadal development and sex hormones in male rats kept under constant darkness as a model of disturbed daily rhythm. Four-week-old male rats (Fischer 344 strain) were kept under constant darkness or normal lighting (12-h light/dark cycle). Two kinds of experimental diet were prepared, one with low dietary protein levels (9% casein) and one with normal levels (18% casein). Harper mineral mixture and Panvitan were used as mineral and vitamin sources, respectively. After 4 weeks, gonadal weight, serum testosterone, and other hormone contents were evaluated. The gonadal weight in the constant darkness groups (D-groups) was lower than that in the normal lighting groups (N-groups). Although the low-protein diet in the D-groups significantly reduced gonadal weight, the normal-protein diet mitigated the reduction of gonadal weight in rats kept under constant darkness. Serum testosterone and androstenedione concentrations were lower in D-group rats fed the low-protein diet. There were no effects of lighting condition or protein levels on serum luteinizing hormone (LH), follicle- stimulating hormone (FSH), or progesterone concentrations. These results indicate that the suppression of gonadal development in D-group rats fed the low-protein diet was caused by low testosterone, which we attribute to the inhibition of synthesized androstenedione, a precursor of testosterone. The present study showed that constant darkness and the low- protein diet inhibited the synthetic pathway from progesterone to androstenedione. PMID:23095819

  12. Expression of fas protein on CD4+T cells irradiated by low level He-Ne

    NASA Astrophysics Data System (ADS)

    Nie, Fan; Zhu, Jing; Zhang, Hui-Guo

    2005-07-01

    Objective: To investigate the influence on the Expression of Fas protein on CD4+ T cells irradiated by low level He-Ne laser in the cases of psoriasis. Methods:the expression of CD4+ T Fas protein was determined in the casee of psoriasis(n=5) pre and post-low level laser irradiation(30 min、60min and 120min)by flow cytometry as compared withthe control(n=5). Results:In the cases of psoriasis,the expression of CD4+T FAS protein 21.4+/-3.1% was increased significantly than that of control group 16.8+/-2.1% pre-irradiation, p<0.05in the control,there is no difference between pre and post- irradiation,p>0.05in the cases , the expression of CD4+T Fas protein wae positively corelated to the irradiation times, when the energy density arrived to 22.92J/cm2(60 minutes)and 45.84J/cm2(120minutes), the expression of CD4+ T Fas protein was increased significantly as compared with pre-irradiation,p<0.05.Conclusion: The expression of CD4+T Fas protein may be increased by low level He-Ne laser irradiation ,the uncontrolled status of apoptosis could be corrected.

  13. Acute effect of protein or carbohydrate breakfasts on human cerebrospinal fluid monoamine precursor and metabolite levels.

    PubMed

    Teff, K L; Young, S N; Marchand, L; Botez, M I

    1989-01-01

    Patients with normal pressure hydrocephalus who had three lumbar punctures during 1 week ingested either water, a protein breakfast, or a carbohydrate breakfast 2.5 h before each of the lumbar punctures. The CSF was analyzed for biogenic amine precursors and metabolites. The protein meal raised CSF tyrosine levels, a finding consistent with animal data, but did not alter those of tryptophan or any of the biogenic amine metabolites. The carbohydrate meal increased CSF 3-methoxy-4-hydroxyphenylethylene glycol, an unexplained finding. The carbohydrate meal did not affect CSF tryptophan, tyrosine, 5-hydroxyindoleacetic acid, or homovanillic acid. Our results support the idea that in humans protein or carbohydrate meals do not alter plasma amino acid levels sufficiently to cause appreciable changes in CNS tryptophan levels or 5-hydroxytryptamine synthesis. PMID:2462018

  14. Differential Acute and Chronic Effects of Leptin on Hypothalamic Astrocyte Morphology and Synaptic Protein Levels

    PubMed Central

    García-Cáceres, Cristina; Fuente-Martín, Esther; Burgos-Ramos, Emma; Granado, Miriam; Frago, Laura M.; Barrios, Vicente; Horvath, Tamas

    2011-01-01

    Astrocytes participate in neuroendocrine functions partially through modulation of synaptic input density in the hypothalamus. Indeed, glial ensheathing of neurons is modified by specific hormones, thus determining the availability of neuronal membrane space for synaptic inputs, with the loss of this plasticity possibly being involved in pathological processes. Leptin modulates synaptic inputs in the hypothalamus, but whether astrocytes participate in this action is unknown. Here we report that astrocyte structural proteins, such as glial fibrillary acidic protein (GFAP) and vimentin, are induced and astrocyte morphology modified by chronic leptin administration (intracerebroventricular, 2 wk), with these changes being inversely related to modifications in synaptic protein densities. Similar changes in glial structural proteins were observed in adult male rats that had increased body weight and circulating leptin levels due to neonatal overnutrition (overnutrition: four pups/litter vs. control: 12 pups/litter). However, acute leptin treatment reduced hypothalamic GFAP levels and induced synaptic protein levels 1 h after administration, with no effect on vimentin. In primary hypothalamic astrocyte cultures leptin also reduced GFAP levels at 1 h, with an induction at 24 h, indicating a possible direct effect of leptin. Hence, one mechanism by which leptin may affect metabolism is by modifying hypothalamic astrocyte morphology, which in turn could alter synaptic inputs to hypothalamic neurons. Furthermore, the responses to acute and chronic leptin exposure are inverse, raising the possibility that increased glial activation in response to chronic leptin exposure could be involved in central leptin resistance. PMID:21343257

  15. Physiological enzymology: The next frontier in understanding protein structure and function at the cellular level.

    PubMed

    Lee, Irene; Berdis, Anthony J

    2016-01-01

    Historically, the study of proteins has relied heavily on characterizing the activity of a single purified protein isolated from other cellular components. This classic approach allowed scientists to unambiguously define the intrinsic kinetic and chemical properties of that protein. The ultimate hope was to extrapolate this information toward understanding how the enzyme or receptor behaves within its native cellular context. These types of detailed in vitro analyses were necessary to reduce the innate complexities of measuring the singular activity and biochemical properties of a specific enzyme without interference from other enzymes and potential competing substrates. However, recent developments in fields encompassing cell biology, molecular imaging, and chemical biology now provide the unique chemical tools and instrumentation to study protein structure, function, and regulation in their native cellular environment. These advancements provide the foundation for a new field, coined physiological enzymology, which quantifies the function and regulation of enzymes and proteins at the cellular level. In this Special Edition, we explore the area of Physiological Enzymology and Protein Function through a series of review articles that focus on the tools and techniques used to measure the cellular activity of proteins inside living cells. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions. PMID:26277093

  16. Luciferase NanoLuc as a reporter for gene expression and protein levels in Saccharomyces cerevisiae.

    PubMed

    Masser, Anna E; Kandasamy, Ganapathi; Kaimal, Jayasankar Mohanakrishnan; Andréasson, Claes

    2016-05-01

    Reporter proteins are essential tools in the study of biological processes and are employed to monitor changes in gene expression and protein levels. Luciferases are reporter proteins that enable rapid and highly sensitive detection with an outstanding dynamic range. Here we evaluated the usefulness of the 19 kDa luciferase NanoLuc (Nluc), derived from the deep sea shrimp Oplophorus gracilirostris, as a reporter protein in yeast. Cassettes with codon-optimized genes expressing yeast Nluc (yNluc) or its destabilized derivative yNlucPEST have been assembled in the context of the dominant drug resistance marker kanMX. The reporter proteins do not impair the growth of yeast cells and exhibit half-lives of 40 and 5 min, respectively. The commercial substrate Nano-Glo® is compatible with detection of yNluc bioluminescence in < 50 cells. Using the unstable yNlucPEST to report on the rapid and transient expression of a heat-shock promoter (PCYC1-HSE ), we found a close match between the intensity of the bioluminescent signal and mRNA levels during both induction and decay. We demonstrated that the bioluminescence of yNluc fused to the C-terminus of a temperature-sensitive protein reports on its protein levels. In conclusion, yNluc and yNlucPEST are valuable new reporter proteins suitable for experiments with yeast using standard commercial substrate. © 2016 The Authors. Yeast published by John Wiley & Sons Ltd. PMID:26860732

  17. Ground level environmental protein concentrations in various ecuadorian environments: potential uses of aerosolized protein for ecological research

    USGS Publications Warehouse

    Staton, Sarah J.R.; Woodward, Andrea; Castillo, Josemar A.; Swing, Kelly; Hayes, Mark A.

    2014-01-01

    Large quantities of free protein in the environment and other bioaerosols are ubiquitous throughout terrestrial ground level environments and may be integrative indicators of ecosystem status. Samples of ground level bioaerosols were collected from various ecosystems throughout Ecuador, including pristine humid tropical forest (pristine), highly altered secondary humid tropical forest (highly altered), secondary transitional very humid forest (regrowth transitional), and suburban dry montane deforested (suburban deforested). The results explored the sensitivity of localized aerosol protein concentrations to spatial and temporal variations within ecosystems, and their value for assessing environmental change. Ecosystem specific variations in environmental protein concentrations were observed: pristine 0.32 ± 0.09 μg/m3, highly altered 0.07 ± 0.05 μg/m3, regrowth transitional 0.17 ± 0.06 μg/m3, and suburban deforested 0.09 ± 0.04 μg/m3. Additionally, comparisons of intra-environmental differences in seasonal/daily weather (dry season 0.08 ± 0.03 μg/m3 and wet season 0.10 ± 0.04 μg/m3), environmental fragmentation (buffered 0.19 ± 0.06 μg/m3 and edge 0.15 ± 0.06 μg/m3), and sampling height (ground level 0.32 ± 0.09 μg/m3 and 10 m 0.24 ± 0.04 μg/m3) demonstrated the sensitivity of protein concentrations to environmental conditions. Local protein concentrations in altered environments correlated well with satellite-based spectral indices describing vegetation productivity: normalized difference vegetation index (NDVI) (r2 = 0.801), net primary production (NPP) (r2 = 0.827), leaf area index (LAI) (r2 = 0.410). Moreover, protein concentrations distinguished the pristine site, which was not differentiated in spectral indices, potentially due to spectral saturation typical of highly vegetated environments. Bioaerosol concentrations represent an inexpensive method to increase understanding of environmental changes, especially in densely vegetated

  18. Determination of protein levels in soy and peanut oils by colorimetric assay and ELISA.

    PubMed

    Jablonski, Joseph E; Fu, Tong-Jen; Jackson, Lauren S; Gendel, Steven M

    2010-01-01

    Analytical methods are needed for measuring the levels of protein from allergenic food transferred into cooking oil. A simple method for determination of total protein in cooking oils was developed. Oil was extracted with phosphate-buffered saline with 0.05% Tween (PBST) and the extracts were partitioned with hexane to remove residual oil. Total protein in the PBST extracts was assayed with bicinchoninic acid (BCA), micro-BCA, reducing-agent compatible BCA and CB-XT kits. These methods were used to measure recovery of protein from peanut butter spikes of soy and peanut oil in the range of 50-1000 ppm. Recoveries were generally above 70%. However, the BCA and micro-BCA assays were subject to interference and enhanced color formation which were probably due to co-extracted antioxidants present in oil. The reducing agent-compatible BCA and CB-X protein assays reduced interference and gave lower protein values in crude, cold-pressed, and refined peanut oils. Heating oil to 180 degrees C before extraction also reduced interference-induced color enhancement. A commercial ELISA test kit was also used to measure peanut protein in oil spiked with peanut butter. Recovery of peanut residues measured by ELISA was significantly decreased when the peanut butter-spiked oil was heated to 180 degrees C compared to unheated oil. Recovery of spiked peanut butter protein measured by the buffer extraction-colorimetric method was not decreased in heated oil. The method developed here could be used to determine protein levels in crude and refined oil, and to assess the potential for allergen cross-contact from reused cooking oil. PMID:20334183

  19. mRNA and Protein levels of rat pancreas specific protein disulphide isomerase are downregulated during Hyperglycemia.

    PubMed

    Gupta, Rajani; Bhar, Kaushik; Sen, Nandini; Bhowmick, Debajit; Mukhopadhyay, Satinath; Panda, Koustubh; Siddhanta, Anirban

    2016-02-01

    Diabetes (Type I and Type II) which affects nearly every organ in the body is a multi-factorial non-communicable disorder. Hyperglycemia is the most characteristic feature of this disease. Loss of beta cells is common in both types of diabetes whose detailed cellular and molecular mechanisms are yet to be elucidated. As this disease is complex, identification of specific biomarkers for its early detection, management and devising new therapies is challenging. Based on the fact that functionally defective proteins provide the biochemical basis for many diseases, in this study, we tried to identify differentially expressed proteins during hyperglycemia. For that, hyperglycemia was induced in overnight fasted rats by intra-peritoneal injection of streptozotocin (STZ). The pancreas was isolated from control and treated rats for subsequent analyses. The 2D-gel electrophoresis followed by MALDI-TOF-MS-MS analyses revealed several up- and down-regulated proteins in hyperglycemic rat pancreas including the downregulation of a pancreas specific isoform of protein disulphide isomerase a2 (Pdia2).This observation was validated by western blot. Quantitative PCR experiments showed that the level of Pdia2 mRNA is also proportionally reduced in hyperglycemic pancreas. PMID:26934777

  20. Codon influence on protein expression in E. coli correlates with mRNA levels.

    PubMed

    Boël, Grégory; Letso, Reka; Neely, Helen; Price, W Nicholson; Wong, Kam-Ho; Su, Min; Luff, Jon D; Valecha, Mayank; Everett, John K; Acton, Thomas B; Xiao, Rong; Montelione, Gaetano T; Aalberts, Daniel P; Hunt, John F

    2016-01-21

    Degeneracy in the genetic code, which enables a single protein to be encoded by a multitude of synonymous gene sequences, has an important role in regulating protein expression, but substantial uncertainty exists concerning the details of this phenomenon. Here we analyse the sequence features influencing protein expression levels in 6,348 experiments using bacteriophage T7 polymerase to synthesize messenger RNA in Escherichia coli. Logistic regression yields a new codon-influence metric that correlates only weakly with genomic codon-usage frequency, but strongly with global physiological protein concentrations and also mRNA concentrations and lifetimes in vivo. Overall, the codon content influences protein expression more strongly than mRNA-folding parameters, although the latter dominate in the initial ~16 codons. Genes redesigned based on our analyses are transcribed with unaltered efficiency but translated with higher efficiency in vitro. The less efficiently translated native sequences show greatly reduced mRNA levels in vivo. Our results suggest that codon content modulates a kinetic competition between protein elongation and mRNA degradation that is a central feature of the physiology and also possibly the regulation of translation in E. coli. PMID:26760206

  1. Levels of major proteins of Escherichia coli during growth at different temperatures.

    PubMed Central

    Herendeen, S L; VanBogelen, R A; Neidhardt, F C

    1979-01-01

    The adaptation of Escherichia coli B/r to temperature was studied by measuring the levels of 133 proteins (comprising 70% of the cell's protein mass) during balanced growth in rich medium at seven temperatures from 13.5 to 46 degrees C. The growth rate of this strain in either rich or minimal medium varies as a simple function of temperature with an Arrhenius constant of approximately 13,500 cal (ca. 56,500 J) per mol from 23 to 37 degrees C, the so-called normal range; above and below this range the growth rate decreases sharply. Analysis of the detailed results indicates that (i) metabolic coordination within the normal (Arrhenius) range is largely achieved by modulation of enzyme activity rather than amount; (ii) the restricted growth that occurs outside this range is accompanied by marked changes in the levels of most of these proteins; (iii) a few proteins are thermometer-like in varying simply with temperature over the whole temperature range irrespective of the influence of temperature on cell growth; and (iv) the temperature response of half of the proteins can be predicted from current information on their metabolic role or from their variation in level in different media at 37 degrees C. PMID:156716

  2. Protein Fibrillar Nanopolymers: Molecular-Level Insights into Their Structural, Physical and Mechanical Properties

    NASA Astrophysics Data System (ADS)

    Trusova, Valeriya M.

    2015-09-01

    Amyloid fibrils represent a generic class of mechanically strong and stable biomaterials with extremely advantageous properties. Although amyloids were initially associated only with severe neurological disorders, the role of these structures nowadays is shifting from health debilitating to highly beneficial both in biomedical and technological aspects. Intensive involvement of fibrillar assemblies into the wide range of pathogenic and functional processes strongly necessitate the molecular level characterization of the structural, physical and elastic features of protein nanofibrils. In the present contribution, we made an attempt to highlight the up-to-date progress in the understanding of amyloid properties from the polymer physics standpoint. The fundamental insights into protein fibril behavior are essential not only for development of therapeutic strategies to combat the protein misfolding disorders but also for rational and precise design of novel biodegradable protein-based nanopolymers.

  3. Insulin regulates milk protein synthesis at multiple levels in the bovine mammary gland.

    PubMed

    Menzies, Karensa K; Lefèvre, Christophe; Macmillan, Keith L; Nicholas, Kevin R

    2009-05-01

    The role of insulin in milk protein synthesis is unresolved in the bovine mammary gland. This study examined the potential role of insulin in the presence of two lactogenic hormones, hydrocortisone and prolactin, in milk protein synthesis. Insulin was shown to stimulate milk protein gene expression, casein synthesis and (14)C-lysine uptake in mammary explants from late pregnant cows. A global assessment of changes in gene expression in mammary explants in response to insulin was undertaken using Affymetrix microarray. The resulting data provided insight into the molecular mechanisms stimulated by insulin and showed that the hormone stimulated the expression of 28 genes directly involved in protein synthesis. These genes included the milk protein transcription factor, ELF5, translation factors, the folate metabolism genes, FOLR1 and MTHFR, as well as several genes encoding enzymes involved in catabolism of essential amino acids and biosynthesis of non-essential amino acids. These data show that insulin is not only essential for milk protein gene expression, but stimulates milk protein synthesis at multiple levels within bovine mammary epithelial cells. PMID:19107532

  4. Transcriptional Bursting Explains the Noise Versus Mean Relationship in mRNA and Protein Levels

    SciTech Connect

    Dar, Dr. Roy; Shaffer, S; Singh, A; Razooky, B; Simpson, Michael L; Raj, A; Weinberger, Dr. Leor

    2016-01-01

    Recent analysis demonstrates that the HIV-1 Long Terminal Repeat (HIV LTR) promoter exhibits a range of possible transcriptional burst sizes and frequencies for any mean-expression level. However, these results have also been interpreted as demonstrating that cell-tocell expression variability (noise) and mean are uncorrelated, a significant deviation from previous results. Here, we re-examine the available mRNA and protein abundance data for the HIV LTR and find that noise in mRNA and protein expression scales inversely with the mean along analytically predicted transcriptional burst-size manifolds. We then experimentally perturb transcriptional activity to test a prediction of the multiple burst-size model: that increasing burst frequency will cause mRNA noise to decrease along given burst-size lines as mRNA levels increase. The data show that mRNA and protein noise decrease as mean expression increases, supporting the canonical inverse correlation between noise and mean.

  5. Nucleoside diphosphate regulation of overall rates of protein biosynthesis acting at the level of initiation.

    PubMed

    Hucul, J A; Henshaw, E C; Young, D A

    1985-12-15

    A sensitive assay method developed to examine the effects of subtle, physiologically relevant, changes in the levels of adenine and guanine mono-, di-, and triphosphorylated nucleotides specifically on the initiation of protein synthesis is described. Initiation rates are quantified by measuring the amount of protein synthesis resulting from the run-off of ribosomes which have initiated during defined intervals in a modified in vitro protein-synthesizing system developed from Ehrlich ascites tumor cell lysates (Henshaw, E.C., and Panniers, R. (1983) Methods Enzymol. 101, 616-629). The modifications include the attenuation of the ATP-regenerating system so that the relative nucleotide levels more nearly reflect actual intracellular conditions. With this system the rate of initiation is highly sensitive to changes in the ADP:ATP and GDP:GTP ratios, but indifferent to the absolute levels of either diphosphate. While the tight coupling of these two ratios by endogenous nucleoside diphosphate kinase activity prevents the independent manipulation of either ratio, the data do eliminate both AMP and GMP per se as inhibitory species. The close agreement of our data calculated in terms of energy charge to previously published results on overall rates of protein synthesis in rat thymocytes (Mendelsohn, S.K., Nordeen, S.K., and Young, D.A. (1977) Biochem. Biophys. Res. Commun. 79, 53-60) continues to suggest a physiologically relevant regulatory influence of subtle changes in nucleotides acting at the level of the initiation reaction. PMID:2999123

  6. Systemic Glucose Level Changes with a Carbohydrate-Restricted and Higher Protein Diet Combined with Exercise

    ERIC Educational Resources Information Center

    Bowden, Rodney G.; Lanning, Beth A.; Doyle, Eva I.; Slonaker, Becky; Johnston, Holly M.; Scanes, Georgene

    2007-01-01

    Objective: The authors' purpose in this study was to compare the effects of macronutrient intake on systemic glucose levels in previously sedentary participants who followed 1 of 4 diets that were either higher protein or high carbohydrate, while initiating an exercise program. Participants and Methods: The authors randomly assigned 94 sedentary…

  7. Low levels of aflatoxin B1, ricin and milk enhance recombinant protein production in mammalian cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Changing the optimal tissue culture medium by adding low levels of environmental stress such as 1 µM of the fungal toxin aflatoxin B1 (AFB1), 1 ng of the castor bean protein toxin ricin in transduced mammalian cells or 1% reconstituted milk enhances transcription and increases production of the foll...

  8. Effects of prebiotic, protein level, and stocking density on performance, immunity, and stress indicators of broilers.

    PubMed

    Houshmand, M; Azhar, K; Zulkifli, I; Bejo, M H; Kamyab, A

    2012-02-01

    An experiment was conducted to determine the effects of period on the performance, immunity, and some stress indicators of broilers fed 2 levels of protein and stocked at a normal or high stocking density. Experimental treatments consisted of a 2 × 2 × 2 factorial arrangement with 2 levels of prebiotic (with or without prebiotic), 2 levels of dietary CP [NRC-recommended or low CP level (85% of NRC-recommended level)], and 2 levels of stocking density (10 birds/m(2) as the normal density or 16 birds/m(2) as the high density), for a total of 8 treatments. Each treatment had 5 replicates (cages). Birds were reared in 3-tiered battery cages with wire floors in an open-sided housing system under natural tropical conditions. Housing and general management practices were similar for all treatment groups. Starter and finisher diets in mash form were fed from 1 to 21 d and 22 to 42 d of age, respectively. Supplementation with a prebiotic had no significant effect on performance, immunity, and stress indicators (blood glucose, cholesterol, corticosterone, and heterophil:lymphocyte ratio). Protein level significantly influenced broiler performance but did not affect immunity or stress indicators (except for cholesterol level). The normal stocking density resulted in better FCR and also higher antibody titer against Newcastle disease compared with the high stocking density. However, density had no significant effect on blood levels of glucose, cholesterol, corticosterone, and the heterophil:lymphocyte ratio. Significant interactions between protein level and stocking density were observed for BW gain and final BW. The results indicated that, under the conditions of this experiment, dietary addition of a prebiotic had no significant effect on the performance, immunity, and stress indicators of broilers. PMID:22252353

  9. Relationship between C-reactive protein levels and obstructive sleep apnea syndrome.

    PubMed

    Tie, Y X; Fu, Y Y; Xu, Z; Peng, Y

    2016-01-01

    This study aims to determine the relationship between C-reactive protein levels and obstructive sleep apnea syndrome (OSAS). We recruited 30 OSAS patients into the observation group (OSAS group), and subdivided them into mild, moderate and severe groups according to the apnea hypopnea index. In addition, 20 normal individuals were included in the control group. Plasma CRP levels of two groups were measured. As compared with the control group, the CRP levels in the OSAS group were significantly increased (P < 0.05). ANOVA showed that CRP levels in the three subgroups differ; statistically significant differences between the mild and severe OSA patients were observed (P < 0.05). It was hypothesized that OSAS patients show elevated serum CRP levels, and that serum CRP levels are associated with OSAS severity. PMID:27323094

  10. Influence of thyroid dysfunction on serum levels of angiopoietin-like protein 6.

    PubMed

    Lim, Jung Ah; Kim, Hyo Jeong; Ahn, Hwa Young; Park, Kyoung Un; Yi, Ka Hee; Park, Do Joon; Jang, Hak Chul; Park, Young Joo

    2015-10-01

    Angiopoietin-like protein 6 (ANGPTL6) is a novel metabolic regulator that modulates energy expenditure as well as glucose and lipid metabolism. Thyroid hormone can induce metabolic changes that are similar to those induced by ANGPTL6. Herein, we investigated whether circulating ANGPTL6 levels change according to thyroid hormone status in humans. We measured the serum levels of ANGPTL6 and metabolic parameters in 150 drug-naïve subjects with overt hyperthyroid, subclinical hyperthyroid, euthyroid, subclinical hypothyroid, or overt hypothyroid status (n=30 in each group). Serum ANGPTL6 levels were significantly higher in patients with overt hypothyroidism than in the other subjects. Women had significantly higher serum levels of ANGPTL6 than men. ANGPTL6 levels correlated positively with thyroid stimulating hormone (TSH), total cholesterol, aspartate aminotransferase, and alanine aminotransferase (ALT) and negatively with serum free thyroxine (T4) level. Multiple stepwise linear regression analysis revealed that sex, TSH, free T4, and ALT were independent predictors of serum ANGPTL6 levels. In summary, serum ANGPTL6 levels increased in patients with a hypothyroid status, and both TSH and free T4 levels are associated with ANGPLT6 levels, suggesting a possible association between thyroid function and ANGPTL6 levels. Whether the upregulated ANGPTL6 level in the hypothyroid status is primarily owing to a direct association or a compensatory mechanism remains to be determined. PMID:26189599

  11. Protein Domain-Level Landscape of Cancer-Type-Specific Somatic Mutations

    PubMed Central

    Yang, Fan; Petsalaki, Evangelia; Rolland, Thomas; Hill, David E.; Vidal, Marc; Roth, Frederick P.

    2015-01-01

    Identifying driver mutations and their functional consequences is critical to our understanding of cancer. Towards this goal, and because domains are the functional units of a protein, we explored the protein domain-level landscape of cancer-type-specific somatic mutations. Specifically, we systematically examined tumor genomes from 21 cancer types to identify domains with high mutational density in specific tissues, the positions of mutational hotspots within these domains, and the functional and structural context where possible. While hotspots corresponding to specific gain-of-function mutations are expected for oncoproteins, we found that tumor suppressor proteins also exhibit strong biases toward being mutated in particular domains. Within domains, however, we observed the expected patterns of mutation, with recurrently mutated positions for oncogenes and evenly distributed mutations for tumor suppressors. For example, we identified both known and new endometrial cancer hotspots in the tyrosine kinase domain of the FGFR2 protein, one of which is also a hotspot in breast cancer, and found new two hotspots in the Immunoglobulin I-set domain in colon cancer. Thus, to prioritize cancer mutations for further functional studies aimed at more precise cancer treatments, we have systematically correlated mutations and cancer types at the protein domain level. PMID:25794154

  12. Elevated levels of an exocrine pancreatic secretory protein in Alzheimer disease brain.

    PubMed Central

    Ozturk, M; de la Monte, S M; Gross, J; Wands, J R

    1989-01-01

    We identified by a monoclonal antibody-based immunoradiometric assay high concentrations of an exocrine pancreatic protein called pancreatic thread protein (PTP) in several areas of Alzheimer disease (AD) brain. The saline-extractable soluble immunoreactivity shares at least three epitopes in common with the native pancreatic form of the protein; the Mr varies from approximately 17,000 to 20,000. Quantitative measurements of PTP immunoreactivity in various regions of several AD brains revealed levels varying from 12 to 295 ng/g of tissue (mean, 116 ng/g) compared with 1-11 ng/g of tissue (mean, 5 ng/g) found in comparable areas of control brains. Immunocytochemistry performed with the anti-PTP monoclonal antibodies demonstrate PTP immunoreactivity within large pyramidal neurons--many of which contain neurofibrillary tangles in both AD and Down syndrome. Less accumulation was observed in astrocytes, and some PTP immunoreactivity was found extracellularly. The highest number of labeled cells in AD and Down syndrome was seen in the hippocampal formation. Fewer positive-staining cells were noted in normal and disease control brains. We conclude, therefore, that an exocrine pancreatic protein is present in the central nervous system of normal individuals at low levels; in AD brain concentrations of this protein are much higher. Images PMID:2463628

  13. Understanding epigenetic regulation: Tracking protein levels across multiple generations of cells

    NASA Astrophysics Data System (ADS)

    Rowat, A. C.; Weitz, D. A.

    2009-11-01

    Cells and organisms are remarkably robust: they alter the variety and levels of expressed genes and proteins in response to environmental stimuli, including temperature, chemicals, and the stiffness of their surroundings. Ultimately changes in gene and protein expression can result in a distinct phenotypic state, which in some cases is maintained over multiple generations; the ability to pass on a particular phenotypic state to progeny cells is critical for differentiation. Moreover, epigenetic regulation of phenotype is also thought to provide an evolutionary advantage for a population of cells adapting to a fluctuating environment on faster timescales than the occurrence of genetic mutations. However, simple methods to study patterns of gene and protein expression on multi-generational timescales are sparse. Here we describe a technique to study lineages of single cells over multiple generations using a microfluidic device; this reveals patterns of expression where protein levels are correlated across multiple generations. Such quantitative information of protein expression in the context of pedigree remains hidden when studying the population as an ensemble.

  14. SPX proteins regulate Pi homeostasis and signaling in different subcellular level

    PubMed Central

    Zhou, Zhipeng; Wang, Zhiye; Lv, Qundan; Shi, Jing; Zhong, Yongjia; Wu, Ping; Mao, Chuanzao

    2015-01-01

    To cope with low phosphate (Pi) availability, plants have to adjust its gene expression profile to facilitate Pi acquisition and remobilization. Sensing the levels of Pi is essential for reprogramming the gene expression profile to adapt to the fluctuating Pi environment. AtPHR1 in Arabidopsis and OsPHR2 in rice are central regulators of Pi signaling, which regulates the expression of phosphate starvation-induced (PSI) genes by binding to the P1BS elements in the promoter of PSI genes. However, how the Pi level affects the central regulator to regulate the PSI genes have puzzled us for a decade. Recent progress in SPX proteins indicated that the SPX proteins play important role in regulating the activity of central regulator AtPHR1/OsPHR2 in a Pi dependent manner at different subcellular levels. PMID:26224365

  15. Estimation of Levels of Salivary Mucin, Amylase and Total Protein in Gingivitis and Chronic Periodontitis Patients

    PubMed Central

    Bhandary, Rahul; Thomas, Biju; Kumari, Suchetha

    2014-01-01

    Background: Periodontal diseases are a group of inflammatory conditions resulting from interaction between a pathogenic bacterial biofilm and susceptible host’s inflammatory response eventually leading to the destruction of periodontal structures and subsequent tooth loss. Hence, investigation of salivary proteins in individuals with periodontal diseases may be useful to enhance the knowledge of their roles in these diseases. Materials and Methods: This case-control study was conducted at A.B. Shetty Memorial Institute of Dental Sciences, Mangalore. The study comprised of 90 patients of age between 25-60 years who were clinically examined and divided into three groups of 30 each: namely clinically healthy, gingivitis and chronic periodontitis. These were classified according to the values of gingival index score, clinical attachment loss and probing pocket depth. Unstimulated saliva was collected and salivary mucin, amylase and total protein levels were determined. Statistical analysis: Results obtained were tabulated and statistically analyzed using ANOVA test and Karl pearson’s correlation test. Results: The results of the study showed an increased concentration of salivary mucin, amylase and total protein in gingivitis patients and increased levels of amylase and total protein in saliva of chronic periodontitis patients compared to healthy individuals which were statistically significant. A decrease in mucin concentration was observed in the periodontitis group compared to gingivitis group. A positive correlation was present between salivary mucin, amylase and total protein levels in the three groups. Conclusion: Salivary mucin, amylase and total protein may serve as an important biochemical parameter of inflammation of the periodontium. Also, it can be hypothesized that various enzyme inhibitors might be useful as a part of host modulation therapy in the treatment of periodontal diseases. PMID:25478449

  16. Alternatively Spliced Homologous Exons Have Ancient Origins and Are Highly Expressed at the Protein Level.

    PubMed

    Abascal, Federico; Ezkurdia, Iakes; Rodriguez-Rivas, Juan; Rodriguez, Jose Manuel; del Pozo, Angela; Vázquez, Jesús; Valencia, Alfonso; Tress, Michael L

    2015-06-01

    Alternative splicing of messenger RNA can generate a wide variety of mature RNA transcripts, and these transcripts may produce protein isoforms with diverse cellular functions. While there is much supporting evidence for the expression of alternative transcripts, the same is not true for the alternatively spliced protein products. Large-scale mass spectroscopy experiments have identified evidence of alternative splicing at the protein level, but with conflicting results. Here we carried out a rigorous analysis of the peptide evidence from eight large-scale proteomics experiments to assess the scale of alternative splicing that is detectable by high-resolution mass spectroscopy. We find fewer splice events than would be expected: we identified peptides for almost 64% of human protein coding genes, but detected just 282 splice events. This data suggests that most genes have a single dominant isoform at the protein level. Many of the alternative isoforms that we could identify were only subtly different from the main splice isoform. Very few of the splice events identified at the protein level disrupted functional domains, in stark contrast to the two thirds of splice events annotated in the human genome that would lead to the loss or damage of functional domains. The most striking result was that more than 20% of the splice isoforms we identified were generated by substituting one homologous exon for another. This is significantly more than would be expected from the frequency of these events in the genome. These homologous exon substitution events were remarkably conserved--all the homologous exons we identified evolved over 460 million years ago--and eight of the fourteen tissue-specific splice isoforms we identified were generated from homologous exons. The combination of proteomics evidence, ancient origin and tissue-specific splicing indicates that isoforms generated from homologous exons may have important cellular roles. PMID:26061177

  17. Alternatively Spliced Homologous Exons Have Ancient Origins and Are Highly Expressed at the Protein Level

    PubMed Central

    Abascal, Federico; Ezkurdia, Iakes; Rodriguez-Rivas, Juan; Rodriguez, Jose Manuel; del Pozo, Angela; Vázquez, Jesús; Valencia, Alfonso; Tress, Michael L.

    2015-01-01

    Alternative splicing of messenger RNA can generate a wide variety of mature RNA transcripts, and these transcripts may produce protein isoforms with diverse cellular functions. While there is much supporting evidence for the expression of alternative transcripts, the same is not true for the alternatively spliced protein products. Large-scale mass spectroscopy experiments have identified evidence of alternative splicing at the protein level, but with conflicting results. Here we carried out a rigorous analysis of the peptide evidence from eight large-scale proteomics experiments to assess the scale of alternative splicing that is detectable by high-resolution mass spectroscopy. We find fewer splice events than would be expected: we identified peptides for almost 64% of human protein coding genes, but detected just 282 splice events. This data suggests that most genes have a single dominant isoform at the protein level. Many of the alternative isoforms that we could identify were only subtly different from the main splice isoform. Very few of the splice events identified at the protein level disrupted functional domains, in stark contrast to the two thirds of splice events annotated in the human genome that would lead to the loss or damage of functional domains. The most striking result was that more than 20% of the splice isoforms we identified were generated by substituting one homologous exon for another. This is significantly more than would be expected from the frequency of these events in the genome. These homologous exon substitution events were remarkably conserved—all the homologous exons we identified evolved over 460 million years ago—and eight of the fourteen tissue-specific splice isoforms we identified were generated from homologous exons. The combination of proteomics evidence, ancient origin and tissue-specific splicing indicates that isoforms generated from homologous exons may have important cellular roles. PMID:26061177

  18. Effects crude protein levels on female Nile tilapia (Oreochromis niloticus) reproductive performance parameters.

    PubMed

    de Oliveira, Marinez Moraes; Ribeiro, Tainá; Orlando, Tamira Maria; de Oliveira, Dênio Garcia Silva; Drumond, Mariana Martins; de Freitas, Rilke Tadeu Fonseca; Rosa, Priscila Vieira

    2014-11-10

    The goal of the present work was to study the reproductive performance of Nile Tilapia (Oreochromis niloticus) female broodstock fed diets containing different levels of crude protein (CP). Two hundred and forty Nile tilapia (O. niloticus) were used at an average age of 30 months, with 180 females and 60 males. The broodstock were lodged separately in masonry tanks with continuous water flow. The females were stocked in thirty tanks with dimensions of 8 m(3) in a completely randomized design consisting of five treatments and six replications. The treatments consisted of five diets with different levels of CP (32, 34, 36, 38 and 40%) and with digestible energy per gram of protein of 9.5 kg of feed. The crude protein (CP) levels positively influenced (p<0.05) reproductive parameters (female relative and absolute fecundity, egg diameter, fasting larvae survival capacity), the somatic indexes (gonadosomatic (GSI), hepatosomatic (HIS), viscerosomatic (VSI)), total plasma protein, albumin and triglycerides. There were no significant differences (p>0.05) observed with regard to spawning weight and female weight. The reproductive parameters studied in the present research indicate that diets formulated with 38% CP with digestible energy per gram of CP of 9.5 were the best diets for tilapia females during the reproductive period. PMID:25201770

  19. Expression level tuning for optimal heterologous protein secretion in Saccharomyces cerevisiae.

    PubMed

    Parekh, R N; Wittrup, K D

    1997-01-01

    The relationship between expression level and secretion of bovine pancreatic trypsin inhibitor (BPTI) was determined in Saccharomyces cerevisiae using a tunable amplifiable delta integration vector. Optimal secretory productivity of 15 mg of BPTI/g cell dry weight yields 180 mg/L secreted active BPTI in test-tube cultures, an order of magnitude increase over 2 mu plasmid-directed secretion. Maximum productivity is determined by the protein folding capacity of the endoplasmic reticulum (ER). Unfolded protein accumulates in the ER as synthesis increases, until a physiological instability is reached and secretion decreases precipitously despite high BPTI mRNA levels. Optimal specific productivity of a standard laboratory strain of S. cerevisiae is double that reported for secretion of BPTI by Pichia pastoris, indicating that efficient utilization of S. cerevisiae's available secretory capacity can eliminate apparent differences among yeast species in their capacity for heterologous protein secretion. Although not generally recognized, the existence of an optimum synthesis level for secretion is apparently a general feature of eucaryotic expression systems and could be of substantial significance for maximization of protein secretion in mammalian and insect cell culture. PMID:9104035

  20. Stable Plastid Transformation for High-Level Recombinant Protein Expression: Promises and Challenges

    PubMed Central

    Gao, Meili; Li, Yongfei; Xue, Xiaochang; Wang, Xianfeng; Long, Jiangang

    2012-01-01

    Plants are a promising expression system for the production of recombinant proteins. However, low protein productivity remains a major obstacle that limits extensive commercialization of whole plant and plant cell bioproduction platform. Plastid genetic engineering offers several advantages, including high levels of transgenic expression, transgenic containment via maternal inheritance, and multigene expression in a single transformation event. In recent years, the development of optimized expression strategies has given a huge boost to the exploitation of plastids in molecular farming. The driving forces behind the high expression level of plastid bioreactors include codon optimization, promoters and UTRs, genotypic modifications, endogenous enhancer and regulatory elements, posttranslational modification, and proteolysis. Exciting progress of the high expression level has been made with the plastid-based production of two particularly important classes of pharmaceuticals: vaccine antigens, therapeutic proteins, and antibiotics and enzymes. Approaches to overcome and solve the associated challenges of this culture system that include low transformation frequencies, the formation of inclusion bodies, and purification of recombinant proteins will also be discussed. PMID:23093835

  1. Codon optimization for high level expression of human bone morphogenetic protein-2 in Escherichia coli.

    PubMed

    Retnoningrum, Debbie S; Pramesti, H T; Santika, P Y; Valerius, O; Asjarie, S; Suciati, T

    2012-08-01

    Codons in the open reading frame (ORF) encoding for human bone morphogenetic protein-2 (hBMP-2) were optimized to reach high level expression in Escherichia coli. The optimization was done by the computer programs DNA works and DNA Star according to Thermodynamically Balanced Inside Out (TBIO) approach. The ORF consisting of 342 base pairs (bp) was assembled using two-steps Polymerase Chain Reaction, cloned into a pGEM-T vector with a mutation rate of 6.38 bp per kb and transformed into E. coli JM109. After a DNA sequence confirmation, mutation-free ORF was subcloned into pET32b and transformed into E. coli BL21(DE3). The rhBMP-2 was produced as a thioredoxin-his-tag fusion protein at relatively high level, approximately 60% of total intracellular proteins as inclusion bodies (IB), with a yield of 1.39 g per liter culture. Solubilization of IB gave soluble monomer rhBMP-2 with a recovery of 13.6% and refolding of soluble rhBMP-2 produced dimeric forms with a yield of 8.7%. The size and identity of the purified rhBMP-2 was confirmed by nano-LC-MS/MS2 analysis. Our work demonstrates for the first time that by using TBIO approach, a codon-optimized ORF encoding for rhBMP-2 protein can be expressed at high level in E. coli expression system. PMID:22691543

  2. High level protein expression in mammalian cells using a safe viral vector: modified vaccinia virus Ankara.

    PubMed

    Hebben, Matthias; Brants, Jan; Birck, Catherine; Samama, Jean-Pierre; Wasylyk, Bohdan; Spehner, Danièle; Pradeau, Karine; Domi, Arban; Moss, Bernard; Schultz, Patrick; Drillien, Robert

    2007-12-01

    Vaccinia virus vectors are attractive tools to direct high level protein synthesis in mammalian cells. In one of the most efficient strategies developed so far, the gene to be expressed is positioned downstream of a bacteriophage T7 promoter within the vaccinia genome and transcribed by the T7 RNA polymerase, also encoded by the vaccinia virus genome. Tight regulation of transcription and efficient translation are ensured by control elements of the Escherichia coli lactose operon and the encephalomyocarditis virus leader sequence, respectively. We have integrated such a stringently controlled expression system, previously used successfully in a standard vaccinia virus backbone, into the modified vaccinia virus Ankara strain (MVA). In this manner, proteins of interest can be produced in mammalian cells under standard laboratory conditions because of the inherent safety of the MVA strain. Using this system for expression of beta-galactosidase, about 15 mg protein could be produced from 10(8) BHK21 cells over a 24-h period, a value 4-fold higher than the amount produced from an identical expression system based on a standard vaccinia virus strain. In another application, we employed the MVA vector to produce human tubulin tyrosine ligase and demonstrate that this protein becomes a major cellular protein upon induction conditions and displays its characteristic enzymatic activity. The MVA vector should prove useful for many other applications in which mammalian cells are required for protein production. PMID:17892951

  3. Tumor redox metabolism correlation with the expression level of red fluorescent protein

    NASA Astrophysics Data System (ADS)

    Sha, Shuang; Wang, Anle; Lin, Qiaoya; Zhang, Zhihong

    2015-03-01

    The redox metabolism is variable and complicated with the progress of tumor development. Whether the tumor redox state will affect the exogenous gene expression or not, are still not clear now . To investigate the relationship between tumor endogenous redox state and the exogenous gene expression level, a far red fluorescent protein fRFP was used to monitor tumor cells proliferation and as an exogenous protein expression in tumors. NADH (nicotinamide adenine dinucleotide) and Fp (flavin protein) are two important coenzymes in the mitochondria respiratory chain, which can be as a standard representation for redox metabolism state. Three tumor subcutaneous models (melanoma, human pancreatic carcinoma and nasopharyngeal carcinoma) were used to observe their redox state and protein expression by our home-made redox scanner. The results showed that the distribution of fRFP fluorescent protein expression in the inner tumor regions are heterogeneous, and the fluorescent intensity of fRFP and the fluorescent intensity of NADH have high correlation. In addition, we also found the linear coefficient in three tumors are different, the value of coefficient is (R2 = 0.966 and R2 = 0.943) in melanoma, (R2 = 0.701 and R2 = 0.942) in human pancreatic carcinoma, and (R2 = 0.994) in nasopharyngeal carcinoma, respectively. From these results, we consider that the exogenous protein expression of fRFP in tumor had some relationship with the tumor redox state of NADH.

  4. Low plasma protein nitrotyrosine levels distinguish primary Raynaud's phenomenon from scleroderma

    PubMed Central

    Kingdon, E J; Mani, A R; Frost, M T; Denton, C P; Powis, S H; Black, C M; Moore, K P

    2006-01-01

    Objective To investigate the hypothesis that increased formation of reactive nitrogen species may contribute to the vascular pathology that develops in patients with connective tissue disease such as scleroderma. Patients and methods The level of protein‐bound nitrotyrosine in plasma was measured by stable isotope dilution gas chromatography/negative ion chemical ionisation mass spectrometry in 11 patients with primary Raynaud's phenomenon, 37 with scleroderma, 13 with chronic renal impairment, and in 23 healthy controls. Results Plasma protein‐bound nitrotyrosine was markedly decreased in patients with primary Raynaud's phenomenon (mean (SEM) 0.60 (0.06) ng/mg dry protein) compared with patients with scleroderma (1.78 (0.21) ng/mg protein), chronic renal impairment (1.42 (0.17) ng/mg protein) or healthy controls (1.63±0.15 ng/mg protein, ANOVA p<0.001). Conclusion These data suggest that there is decreased nitration of plasma proteins, or increased degradation of nitrated proteins from the circulation of patients with primary but not secondary Raynaud's phenomenon. PMID:16308344

  5. High Level Expression and Purification of Recombinant Proteins from Escherichia coli with AK-TAG

    PubMed Central

    Luo, Dan; Wen, Caixia; Zhao, Rongchuan; Liu, Xinyu; Liu, Xinxin; Cui, Jingjing; Liang, Joshua G.; Liang, Peng

    2016-01-01

    Adenylate kinase (AK) from Escherichia coli was used as both solubility and affinity tag for recombinant protein production. When fused to the N-terminus of a target protein, an AK fusion protein could be expressed in soluble form and purified to near homogeneity in a single step from Blue-Sepherose via affinity elution with micromolar concentration of P1, P5- di (adenosine—5’) pentaphosphate (Ap5A), a transition-state substrate analog of AK. Unlike any other affinity tags, the level of a recombinant protein expression in soluble form and its yield of recovery during each purification step could be readily assessed by AK enzyme activity in near real time. Coupled to a His-Tag installed at the N-terminus and a thrombin cleavage site at the C terminus of AK, the streamlined method, here we dubbed AK-TAG, could also allow convenient expression and retrieval of a cleaved recombinant protein in high yield and purity via dual affinity purification steps. Thus AK-TAG is a new addition to the arsenal of existing affinity tags for recombinant protein expression and purification, and is particularly useful where soluble expression and high degree of purification are at stake. PMID:27214237

  6. Targeted quantification of low ng/mL level proteins in human serum without immunoaffinity depletion

    SciTech Connect

    Shi, Tujin; Sun, Xuefei; Gao, Yuqian; Fillmore, Thomas L.; Schepmoes, Athena A.; Zhao, Rui; He, Jintang; Moore, Ronald J.; Kagan, Jacob; Rodland, Karin D.; Liu, Tao; Liu, Alvin Y.; Smith, Richard D.; Tang, Keqi; Camp, David G.; Qian, Weijun

    2013-07-05

    We recently reported an antibody-free targeted protein quantification strategy, termed high-pressure, high-resolution separations with intelligent selection and multiplexing (PRISM) for achieving significantly enhanced sensitivity using selected reaction monitoring (SRM) mass spectrometry. Integrating PRISM with front-end IgY14 immunoaffinity depletion, sensitive detection of targeted proteins at 50-100 pg/mL levels in human blood plasma/serum was demonstrated. However, immunoaffinity depletion is often associated with undesired losses of target proteins of interest. Herein we report further evaluation of PRISM-SRM quantification of low-abundance serum proteins without immunoaffinity depletion and the multiplexing potential of this technique. Limits of quantification (LOQs) at low ng/mL levels with a median CV of ~12% were achieved for proteins spiked into human female serum using as little as 2 µL serum. PRISM-SRM provided up to ~1000-fold improvement in the LOQ when compared to conventional SRM measurements. Multiplexing capability of PRISM-SRM was also evaluated by two sets of serum samples with 6 and 21 target peptides spiked at the low attomole/µL levels. The results from SRM measurements for pooled or post-concatenated samples were comparable to those obtained from individual peptide fractions in terms of signal-to-noise ratios and SRM peak area ratios of light to heavy peptides. PRISM-SRM was applied to measure several ng/mL-level endogenous plasma proteins, including prostate-specific antigen, in clinical patient sera where correlation coefficients > 0.99 were observed between the results from PRISM-SRM and ELISA assays. Our results demonstrate that PRISM-SRM can be successfully used for quantification of low-abundance endogenous proteins in highly complex samples. Moderate throughput (50 samples/week) can be achieved by applying the post-concatenation or fraction multiplexing strategies. We anticipate broad applications for targeted PRISM

  7. Modulation of Protein Levels in Chromosomal Dosage Series of Maize: The Biochemical Basis of Aneuploid Syndromes

    PubMed Central

    Birchler, James A.; Newton, Kathleen J.

    1981-01-01

    Genetically defined dosage series of chromosome arms 1L, 3L, 4S, 5L, 7L, 9S, 10L and combinations of 1L–3L, collectively spanning approximately one-third of the maize genome, were examined for alterations in the expression of total protein profiles in scutellar tissue. The major effects found were negative correlations of specific proteins with the dosage of particular regions in a manner similar to that previously described for enzyme activity levels (Birchler 1979). Chromosome arms 1L, 4S and 5L produced the most severe negative effects, with 3L and 7L exhibiting this phenomenon to a lesser degree. Positive correlations of certain proteins were observed with the dosage of the 1L, 3L, 5L and 7L regions. The structural locus of one of the major scutellar proteins (PRO) is present in the long arm of chromosome 1 (Schwartz 1979), but exhibits compensation in a dosage series involving whole-arm comparisons. Multiple factors in 1L affect the level of the protein. The compound TB-1La-3L4759-3 (1L 0.20–0.39) has a slight negative effect on PRO, while TB-1La-3Le (1L 0.20–0.58) and TB-1La-3L5267 (1L 0.20–0.72) have a more pronounced negative influence. The level of this protein is not altered by the dosage of 3L. These observations suggest that compensation is brought about by the cancellation of a positive structural gene dosage effect by the negative inverse effect. Other regions of the genome that contribute to the control of PRO levels are 4S and 5L. Total protein profiles were also compared in haploid, diploid and tetraploid maize as a comparison to the aneuploid series. Most proteins exhibit structural-gene-dosage effects through the ploidy series, but others show a positive effect greater than expected from varying the structural genes. Still others are negatively affected by ploidy changes. In general, the ploidy alterations are not as great as predicted from the cumulative action of the aneuploid effects. The bearing of these observations on the

  8. Elevation of serum heat-shock protein levels in amyotrophic lateral sclerosis.

    PubMed

    Miyazaki, Daigo; Nakamura, Akinori; Hineno, Akiyo; Kobayashi, Chinatsu; Kinoshita, Tomomi; Yoshida, Kunihiro; Ikeda, Shu-Ichi

    2016-08-01

    Heat-shock proteins (HSPs) have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). In this study, we aimed to examine whether the serum levels of HSPs (HSP27, HSP70, and HSP90) are altered in patients with ALS. We included 58 patients diagnosed with ALS and 85 control individuals. Serum HSP levels of patients and controls were determined using enzyme-linked immunosorbent assay. The serum levels of HSP70 and HSP90 were significantly higher in patients than in controls. In contrast, serum levels of HSP27 did not differ significantly between the patient and control groups. Moreover, serum levels of HSP70 and HSP90 in patients remained high throughout the duration of the disease. Taken together, our findings suggest that HSPs might have a role in ALS progression throughout the course of the disease. Further studies are needed to clarify the role of HSPs in the pathogenesis of ALS. PMID:27112486

  9. Protein levels in beef cattle finishing diets: industry application, university research, and systems results.

    PubMed

    Galyean, M L

    1996-11-01

    Consulting nutritionists were surveyed to determine current formulation and management practices for finishing beef cattle. Among the six consultants surveyed, percentage of CP in finishing diets ranged from 12.5 to 14.4%, with urea levels ranging from .5 to 1.5% of DM. Finishing diets were based primarily on highly processed, rapidly fermented grains (steam-flaked and high-moisture grain), with roughage levels ranging from 3 to 11% of DM. All six consultants considered feed bunk management to be a critical factor affecting feed intake and performance; five of the six consultants used aggressive implant programs based on estrogen + trenbolone acetate. Recent university research was reviewed with respect to CP level and source in finishing diets. Finishing cattle managed on aggressive implant programs seem to respond to higher levels of CP better than would be expected from the factorial calculation approach. Moreover, improvements in performance noted in recent research seemed to be more consistent when supplemental CP was derived from ruminally degraded vs undegraded sources. Calculation of protein requirements with a metabolizable protein (MP) system yielded estimates of protein needs by finishing cattle that agreed more closely with current industry practices than did calculation based on the factorial method. The difference between the MP system and the factorial method was primarily a result of accounting for microbial N needs in the MP system. Reasons for production responses to CP levels that are greater than those calculated by the factorial method include increased accretion of protein by rapidly growing, implanted cattle, particularly during the initial phase of the finishing period, alleviation of a microbial N deficiency, and ruminal and systemic effects of ruminally degraded N on acid-base balance of beef cattle fed rapidly fermented, high-grain diets. Reasons for production responses to supplemental CP need further research. PMID:8923202

  10. Study of Serum Levels of Leptin, C-Reactive Protein and Nutritional Status in Hemodialysis Patients

    PubMed Central

    Montazerifar, Farzaneh; Karajibani, Mansour; Hassanpour, Zahra; Pourmofatteh, Mahla

    2015-01-01

    Background: Leptin is secreted by adipose tissue and decreases appetite. However, the role of leptin in the pathogenesis of hemodialysis (HD)-related malnutrition has not been fully evaluated. Objectives: The aim of study was to investigate the association between the serum leptin levels, serum C-reactive protein (CRP) levels, and nutritional status in hemodialysis patients. Patients and Methods: This analytical descriptive study included 45 hemodialysis patients and 40 healthy subjects. Biochemical parameters and serum leptin levels were measured. The nutritional status was evaluated using a food frequency questionnaire (FFQ) and the calculation of the body mass index (BMI). Results: Serum leptin (P < 0.05) and albumin (P < 0.0001) levels and BMI (P < 0.001) of HD patients were significantly lower, while CRP levels were significantly higher than those of controls (P < 0.0001). HD patients consumed the lower daily servings of the food groups compared to the control subjects (P < 0.0001). A significant positive correlation between serum levels of leptin and albumin and BMI was demonstrated. No significant correlations were identified between leptin level, CRP level, and other variables. Conclusions: The findings suggest that low levels of leptin may be a contributory factor for malnutrition in HD patients. Further studies are required to ascertain the significance of leptin levels in relation to nutritional factors in hemodialysis patients. PMID:26430525

  11. Ultrasensitive detection of proteins and sugars at single-cell level

    PubMed Central

    Watabe, Satoshi; Morikawa, Mika; Kaneda, Mugiho; Nakaishi, Kazunari; Nakatsuma, Akira; Ninomiya, Masaki; Yoshimura, Teruki; Miura, Toshiaki; Ito, Etsuro

    2016-01-01

    ABSTRACT Each cell produces its own responses even if it appears identical to other cells. To analyze these individual cell characteristics, we need to measure trace amounts of molecules in a single cell. Nucleic acids in a single cell can be easily amplified by polymerase chain reaction, but single-cell measurement of proteins and sugars will require de novo techniques. In the present study, we outline the techniques we have developed toward this end. For proteins, our ultrasensitive enzyme-linked immunosorbent assay (ELISA) coupled with thionicotinamide-adenine dinucleotide cycling can detect proteins at subattomoles per assay. For sugars, fluorescence correlation spectroscopy coupled with glucose oxidase-catalyzed reaction allows us to measure glucose at tens of nM. Our methods thus offer versatile techniques for single-cell-level analyses, and they are hoped to strongly promote single-cell biology as well as to develop noninvasive tests in clinical medicine. PMID:27064305

  12. Amyloid beta protein levels in cerebrospinal fluid are elevated in early-onset Alzheimer's disease.

    PubMed

    Nakamura, T; Shoji, M; Harigaya, Y; Watanabe, M; Hosoda, K; Cheung, T T; Shaffer, L M; Golde, T E; Younkin, L H; Younkin, S G

    1994-12-01

    The 4-kd amyloid beta protein (A beta) deposited as amyloid in Alzheimer's disease (AD) is produced and released by normal proteolytic processing of the amyloid beta protein precursor (beta APP) and is readily detected in cerebrospinal fluid (CSF). Here, we present the levels of A beta in CSF from a total of 95 subjects, including 38 patients with AD, 14 with early-onset AD and 24 with late-onset AD, 25 normal control subjects, and 32 patients with other neurological diseases. The level of A beta decreased with normal aging, and there was a significant elevation in the level of A beta in the CSF of early-onset AD patients (4.14 +/- 1.37 pmol/ml, p < 0.01). Neither Mini-Mental State nor Functional Assessment Staging were correlated with the amount of A beta in the CSF. The A beta/secreted form of beta APP ratio was elevated, but the level of alpha 1-antichymotrypsin in the CSF did not correlate with the level of CSF A beta in early-onset AD patients. Thus, the level of A beta in the CSF is elevated in early-onset AD patients and is suggested to be correlated with the pathology in the brain that characterizes AD. PMID:7998778

  13. Correlating Flavivirus virulence and levels of intrinsic disorder in shell proteins: protective roles vs. immune evasion.

    PubMed

    Goh, Gerard Kian-Meng; Dunker, A Keith; Uversky, Vladimir N

    2016-05-24

    Computational analyses revealed correlations between the intrinsic disorder propensity of shell proteins and case fatality rates (CFRs) among Flaviviruses and within at least two Flavivirus species, such as tick-borne encephalitis virus (TBEV) and dengue virus (DENV). The shell proteins analyzed in this study are capsid (C) and membrane (PrM, Pr, and M) proteins. The highest correlations can be found when regression analyses were conducted using Pr (Flavivirus: r(2) = 0.78, p < 0.01) or M (Flavivirus: r(2) = 0.91, p < 0.01) as an independent variable with C and CFR as co-explanatory and dependent variables, respectively. Interestingly, while predicted intrinsic disorder levels (PIDs) of both C and M are positively correlated with the virulence, the PIDs of Pr and CFR are negatively correlated. This is likely due to the fact that the Pr portion of PrM plays various roles in protecting the virion from damage, whereas M and C are assisted by greater potential in binding promiscuity as a result of greater disorder. The C protein of yellow fever virus (YFV), which is the most virulent virus in the sample, has the highest PID levels, whereas the second most virulent TBEV FE subtype has the second highest PID score due to its C protein, and the least virulent West Nile virus (WNV) has the least disordered C protein. This knowledge can be used while working on the development and identification of attenuated strains for vaccine. Curiously, unlike Flaviviruses, a disordered outer shell was described for hepatitis C virus (HCV), human immunodeficiency virus (HIV), and human simplex virus 2 (HSV-2), which currently have no effective vaccine. PMID:27102744

  14. Ultraviolet-ozone treatment reduces levels of disease-associated prion protein and prion infectivity

    USGS Publications Warehouse

    Johnson, C.J.; Gilbert, P.; McKenzie, D.; Pedersen, J.A.; Aiken, Judd M.

    2009-01-01

    Background. Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative diseases caused by novel infectious agents referred to as prions. Prions appear to be composed primarily, if not exclusively, of a misfolded isoform of the cellular prion protein. TSE infectivity is remarkably stable and can resist many aggressive decontamination procedures, increasing human, livestock and wildlife exposure to TSEs. Findings. We tested the hypothesis that UV-ozone treatment reduces levels of the pathogenic prion protein and inactivates the infectious agent. We found that UV-ozone treatment decreased the carbon and prion protein content in infected brain homogenate to levels undetectable by dry-ashing carbon analysis or immunoblotting, respectively. After 8 weeks of ashing, UV-ozone treatment reduced the infectious titer of treated material by a factor of at least 105. A small amount of infectivity, however, persisted despite UV-ozone treatment. When bound to either montmorillonite clay or quartz surfaces, PrPTSE was still susceptible to degradation by UV-ozone. Conclusion. Our findings strongly suggest that UV-ozone treatment can degrade pathogenic prion protein and inactivate prions, even when the agent is associated with surfaces. Using larger UV-ozone doses or combining UV-ozone treatment with other decontaminant methods may allow the sterilization of TSE-contaminated materials. ?? 2009 Aiken et al; licensee BioMed Central Ltd.

  15. Change in the protein level of mevalonate pyrophosphate decarboxylase in tissues of mouse by pravastatin.

    PubMed

    Michihara, Akihiro; Akasaki, Kenji; Yamori, Yukio; Tsuji, Hiroshi

    2003-08-01

    We previously reported that treatment of rats with a diet containing 0.1% pravastatin and 5% cholestyramine markedly increased mevalonate pyrophosphate decarboxylase (MPD) activity in liver crude extracts compared with nontreated rats. In this study, we examined the change in the protein level of MPD in the tissues of mice administered pravastatin. When MPD content in the tissues of nontreated mice was analyzed by quantitative immunoblotting, a single protein band with an apparent molecular weight of 46 kDa was detected in all tissues and the specific protein content of MPD in liver and kidney was markedly higher than that in other tissues. When MPD content in the tissues of pravastatin-treated mice was analyzed by immunoblotting, MPD was markedly increased (9-fold) only in the liver compared with nontreated mice. Next, when MPD activity was measured in the liver between nontreated and pravastatin-treated mice, MPD activity as well as protein levels were markedly increased (11-fold) in the liver of pravastatin-treated mice compared with nontreated mice. These data suggest that a marked induction of MPD in the liver by pravastatin is responsible for the tissue-specific effect of pravastatin. PMID:12913254

  16. Visualizing and quantifying protein polySUMOylation at the single-molecule level.

    PubMed

    Yang, Yong; Zhang, Chun-yang

    2014-01-21

    Protein polySUMOylation, the attachment of small ubiquitin-like modifier (SUMO) chains to the target protein, is associated with a variety of physiological processes. However, the analysis of protein polySUMOylation is often complicated by the heterogeneity of SUMO-target conjugates. Here, we develop a new strategy to visualize and quantify polySUMOylation at the single-molecule level by integrating the tetracysteine (TC) tag labeling technology and total internal reflection fluorescence (TIRF)-based single-molecule imaging. As a proof-of-concept, we employ the human SUMO-2 as the model. The addition of TC tag to SUMO-2 can specifically translate the SUMO-mediated modification into visible fluorescence signal without disturbing the function of SUMO-2. The SUMO monomers display homogeneous fluorescence spots at the single-molecule level, whereas the mixed SUMO chains exhibit nonuniform fluorescence spots with a wide range of intensities. Analysis of the number and the brightness of fluorescence spots enable quantitative measurement of the polySUMOylation degree inside the cells under different physiological conditions. Due to the frequent occurrence of posttranslational modification by polymeric chains in cells, this single-molecule strategy has the potential to be broadly applied for studying protein posttranslational modification in normal cellular physiology and disease etiology. PMID:24383460

  17. Quantitation of tyrosine hydroxylase, protein levels: Spot immunolabeling with an affinity-purified antibody

    SciTech Connect

    Haycock, J.W. )

    1989-09-01

    Tyrosine hydroxylase was purified from bovine adrenal chromaffin cells and rat pheochromocytoma using a rapid (less than 2 days) procedure performed at room temperature. Rabbits were immunized with purified enzyme that was denatured with sodium dodecylsulfate, and antibodies to tyrosine hydroxylase were affinity-purified from immune sera. A Western blot procedure using the affinity-purified antibodies and {sup 125}I-protein A demonstrated a selective labeling of a single Mr approximately 62,000 band in samples from a number of different tissues. The relative lack of background {sup 125}I-protein A binding permitted the development of a quantitative spot immunolabeling procedure for tyrosine hydroxylase protein. The sensitivity of the assay is 1-2 ng of enzyme. Essentially identical standard curves were obtained with tyrosine hydroxylase purified from rat pheochromocytoma, rat corpus striatum, and bovine adrenal medulla. An extract of PC 12 cells (clonal rat pheochromocytoma cells) was calibrated against purified rat pheochromocytoma tyrosine hydroxylase and used as an external standard against which levels of tyrosine hydroxylase in PC12 cells and other tissue were quantified. With this procedure, qualitative assessment of tyrosine hydroxylase protein levels can be obtained in a few hours and quantitative assessment can be obtained in less than a day.

  18. The Genomic Landscape of Position Effects on Protein Expression Level and Noise in Yeast.

    PubMed

    Chen, Xiaoshu; Zhang, Jianzhi

    2016-05-25

    Position effect, the influence of the chromosomal location of a gene on its activity, is a fundamental property of the genome. By placing a GFP gene cassette at 482 different locations across all chromosomes in budding yeast, we quantified the position effects on protein expression level and noise at the genomic scale. The position effects are significant, altering the mean protein expression level by up to 15 times and expression noise by up to 20 times. DNA replication timing, 3D chromosomal conformation, and several histone modifications are major covariates of position effects. Essential genes are enriched in genomic regions with inherently low expression noise, supporting the hypothesis that chromosomal clustering of essential genes results from selection against their expressional stochasticity. Position effects exhibit significant interactions with promoters. Together, our results suggest that position effects have shaped the evolution of chromosome organization and should inform future genome engineering efforts. PMID:27185547

  19. Association of androgen with gender difference in serum adipocyte fatty acid binding protein levels

    PubMed Central

    Hu, Xiang; Ma, Xiaojing; Pan, Xiaoping; Luo, Yuqi; Xu, Yiting; Xiong, Qin; Bao, Yuqian; Jia, Weiping

    2016-01-01

    Clinical investigations have indicated women have higher levels of adipocyte fatty acid binding protein (A-FABP) than men. The present study aimed to identify factors related to gender difference in serum A-FABP levels. A total of 507 participants (194 men, 132 premenopausal women, and 181 postmenopausal women) were enrolled in the present study. Serum A-FABP levels increased in the order from men to premenopausal women to postmenopausal women in both body mass index categories (<25.0 and ≥25.0 kg/m2; all P < 0.05). Multiple stepwise regression analyses showed that after adjustment for factors related to serum A-FABP levels, the trunk fat mass was an independent and positive factor of serum A-FABP levels. For men, total testosterone was associated independently and inversely with serum A-FABP levels. For pre- and postmenopausal women, bioavailable testosterone and total testosterone were independent and positive factors associated with serum A-FABP levels, respectively. The present study demonstrated that the androgen was correlated with the serum A-FABP levels negatively in men, but positively in women. With these effects on the fat content, especially trunk fat, androgen might contribute to the gender difference in serum A-FABP levels. PMID:27270834

  20. Association of androgen with gender difference in serum adipocyte fatty acid binding protein levels.

    PubMed

    Hu, Xiang; Ma, Xiaojing; Pan, Xiaoping; Luo, Yuqi; Xu, Yiting; Xiong, Qin; Bao, Yuqian; Jia, Weiping

    2016-01-01

    Clinical investigations have indicated women have higher levels of adipocyte fatty acid binding protein (A-FABP) than men. The present study aimed to identify factors related to gender difference in serum A-FABP levels. A total of 507 participants (194 men, 132 premenopausal women, and 181 postmenopausal women) were enrolled in the present study. Serum A-FABP levels increased in the order from men to premenopausal women to postmenopausal women in both body mass index categories (<25.0 and ≥25.0 kg/m(2); all P < 0.05). Multiple stepwise regression analyses showed that after adjustment for factors related to serum A-FABP levels, the trunk fat mass was an independent and positive factor of serum A-FABP levels. For men, total testosterone was associated independently and inversely with serum A-FABP levels. For pre- and postmenopausal women, bioavailable testosterone and total testosterone were independent and positive factors associated with serum A-FABP levels, respectively. The present study demonstrated that the androgen was correlated with the serum A-FABP levels negatively in men, but positively in women. With these effects on the fat content, especially trunk fat, androgen might contribute to the gender difference in serum A-FABP levels. PMID:27270834

  1. The human phenolsulphotransferase polymorphism is determined by the level of expression of the enzyme protein.

    PubMed Central

    Jones, A L; Roberts, R C; Coughtrie, M W

    1993-01-01

    We have examined the expression of platelet phenolsulphotransferase (PST) in 60 individuals. Using an antibody which recognizes both forms of PST present in man (P-PST and M-PST), we determined that the polymorphism of platelet P-PST activity is determined by the level of expression of the enzyme protein. The implications for susceptibility to adverse drug reactions and chemical carcinogenesis are discussed. Images Figure 1 Figure 2 Figure 3 PMID:8257413

  2. Ileal apical sodium-dependent bile acid transporter protein levels are down-regulated through ubiquitin-dependent protein degradation induced by bile acids.

    PubMed

    Miyata, Masaaki; Yamakawa, Hiroki; Hayashi, Kenjiro; Kuribayashi, Hideaki; Yamazoe, Yasushi; Yoshinari, Kouichi

    2013-08-15

    The ileal apical sodium-dependent bile acid transporter (ASBT or SLC10A2) has a crucial role in intestinal bile acid absorption. We previously reported that enterobacteria-mediated bile acid conversion was involved in the alteration of ileal ASBT expression levels. In the present study, to investigate the hypothesis that ileal ASBT protein levels are post-translationally regulated by enterobacteria-associated bile acids, alteration of ileal ASBT protein levels was analysed in mice 12 h and 24 h after anti-bacterial drug ampicillin (ABPC) treatment (100 mg/kg, single shot) that altered bile acid composition in the intestinal lumen. In ABPC-treated mice, enterobacteria-biotransformed bile acid, taurodeoxycholic acid (TDCA) and cholic acid (CA) levels were decreased, whereas taurocholic acid (TCA) and tauro-β-muricholic acid levels were increased in the intestinal lumen. Ileal ASBT protein levels in brush-border membrane vesicles (BBMVs), but not ileal Asbt mRNA levels, were significantly increased in the ABPC-treated mice, and the extent of ubiquitination of the ileal ASBT protein was reduced in the ABPC-treated mice. Treatment of ABPC-pretreated mice with CA or TDCA, but not TCA, significantly decreased ileal ASBT protein levels and increased the extent of ubiquitination of ileal ASBT protein. Treatment of mice with the lysosome inhibitor, chloroquine, or the proteasome inhibitor, MG132, increased ileal ASBT protein levels in BBMVs. CA-mediated reduction of ASBT protein levels in the ABPC-pretreated mice was attenuated by co-treatment with chloroquine or MG132. These results suggest that ileal ASBT protein is degraded by a ubiquitin-dependent pathway in response to enterobacteria-associated bile acids. PMID:23872411

  3. Melamine Impairs Female Fertility via Suppressing Protein Level of Juno in Mouse Eggs

    PubMed Central

    Dai, Xiaoxin; Zhang, Mianqun; Lu, Yajuan; Miao, Yilong; Zhou, Changyin; Sun, Shaochen; Xiong, Bo

    2015-01-01

    Melamine is an organic nitrogenous compound widely used as an industrial chemical, and it has been recently reported by us that melamine has a toxic effect on the female reproductive system in mice, and renders females subfertile; the molecular basis, however, has not been adequately assessed. In the present study, we explore the underlying mechanism regarding how melamine compromises fertility in the mouse. The data showed that melamine exposure significantly impaired the fertilization capability of the egg during in vitro fertilization. To further figure out the cause, we analyzed ovastacin localization and protein level, the sperm binding ability of zona pellucida, and ZP2 cleavage status in unfertilized eggs from melamine fed mice, and no obvious differences were found between control and treatment groups. However, the protein level of Juno on the egg plasma membrane in the high-dose feeding group indeed significantly decreased compared to the control group. Thus, these data suggest that melamine compromises female fertility via suppressing Juno protein level on the egg membrane. PMID:26633308

  4. Melamine Impairs Female Fertility via Suppressing Protein Level of Juno in Mouse Eggs.

    PubMed

    Dai, Xiaoxin; Zhang, Mianqun; Lu, Yajuan; Miao, Yilong; Zhou, Changyin; Sun, Shaochen; Xiong, Bo

    2015-01-01

    Melamine is an organic nitrogenous compound widely used as an industrial chemical, and it has been recently reported by us that melamine has a toxic effect on the female reproductive system in mice, and renders females subfertile; the molecular basis, however, has not been adequately assessed. In the present study, we explore the underlying mechanism regarding how melamine compromises fertility in the mouse. The data showed that melamine exposure significantly impaired the fertilization capability of the egg during in vitro fertilization. To further figure out the cause, we analyzed ovastacin localization and protein level, the sperm binding ability of zona pellucida, and ZP2 cleavage status in unfertilized eggs from melamine fed mice, and no obvious differences were found between control and treatment groups. However, the protein level of Juno on the egg plasma membrane in the high-dose feeding group indeed significantly decreased compared to the control group. Thus, these data suggest that melamine compromises female fertility via suppressing Juno protein level on the egg membrane. PMID:26633308

  5. A Compendium of Caenorhabditis elegans RNA Binding Proteins Predicts Extensive Regulation at Multiple Levels

    PubMed Central

    Tamburino, Alex M.; Ryder, Sean P.; Walhout, Albertha J. M.

    2013-01-01

    Gene expression is regulated at multiple levels, including transcription and translation, as well as mRNA and protein stability. Although systems-level functions of transcription factors and microRNAs are rapidly being characterized, few studies have focused on the posttranscriptional gene regulation by RNA binding proteins (RBPs). RBPs are important to many aspects of gene regulation. Thus, it is essential to know which genes encode RBPs, which RBPs regulate which gene(s), and how RBP genes are themselves regulated. Here we provide a comprehensive compendium of RBPs from the nematode Caenorhabditis elegans (wRBP1.0). We predict that as many as 887 (4.4%) of C. elegans genes may encode RBPs ~250 of which likely function in a gene-specific manner. In addition, we find that RBPs, and most notably gene-specific RBPs, are themselves enriched for binding and modification by regulatory proteins, indicating the potential for extensive regulation of RBPs at many different levels. wRBP1.0 will provide a significant contribution toward the comprehensive delineation of posttranscriptional regulatory networks and will provide a resource for further studies regulation by RBPs. PMID:23390605

  6. Dietary whey protein hydrolysates increase skeletal muscle glycogen levels via activation of glycogen synthase in mice.

    PubMed

    Kanda, Atsushi; Morifuji, Masashi; Fukasawa, Tomoyuki; Koga, Jinichiro; Kanegae, Minoru; Kawanaka, Kentaro; Higuchi, Mitsuru

    2012-11-14

    Previously, we have shown that consuming carbohydrate plus whey protein hydrolysates (WPHs) replenished muscle glycogen after exercise more effectively than consuming intact whey protein or branched-chain amino acids (BCAAs). The mechanism leading to superior glycogen replenishment after consuming WPH is unclear. In this 5 week intervention, ddY mice were fed experimental diets containing WPH, a mixture of whey amino acids (WAAs), or casein (control). After the intervention, gastrocnemius muscle glycogen levels were significantly higher in the WPH group (4.35 mg/g) than in the WAA (3.15 mg/g) or control (2.51 mg/g) groups. In addition, total glycogen synthase (GS) protein levels were significantly higher in the WPH group (153%) than in the WAA (89.2%) or control groups, and phosphorylated GS levels were significantly decreased in the WPH group (51.4%). These results indicate that dietary WPH may increase the muscle glycogen content through increased GS activity. PMID:23113736

  7. Inclusion of Yucca schidigera extract in diets with different protein levels for dogs.

    PubMed

    Dos Reis, Jéssica S; Zangerônimo, Márcio G; Ogoshi, Rosana C S; França, Janine; Costa, Adriano C; Almeida, Thomás N; Dos Santos, João P F; Pires, Carolina P; Chizzotti, Ana F; Leite, Carlos A L; Saad, Flávia M O B

    2016-08-01

    This study evaluated the effects of inclusion of Yucca schidigera extract (YSE) in two diets with different levels of crude protein (CP) for dogs on facal odour, nutrient digestibility, ammonia concentration in feces and hematological and serum biochemical profiles. Twenty adults Beagles were used, distributed in a randomized block design in a 2 × 4 factorial design (two diets, 25% and 34% CP, and four YSE levels: 0, 250, 500 and 750 mg/kg) with five replicates, obtained during two experimental periods. The fecal odour reduced (P < 0.05) when 500 mg/kg of YSE was used in diets with higher CP. The inclusion of YSE reduced (P < 0.05) fecal ammonia, and the inclusion of 250 and 500 mg/kg YSE reduced intestinal gas. The inclusion of 750 mg/kg YSE increased the mean corpuscular hemoglobin (MCH), alanine aminotransferase (ALT) activity and tended to increase the serum cholesterol concentration, regardless of the protein level of the diets. There was no effect on the digestibility of nutrients, fecal consistency, nitrogen balance and thickness of the intestinal wall. The inclusion of 500 mg/kg YSE is effective in reducing fecal odour in dogs receiving diets with 34% of CP. Regardless of the protein content, YSE reduces fecal ammonia, but may cause adverse effects if included at higher doses. PMID:26800023

  8. Oxidative stress and hippocampal synaptic protein levels in elderly cognitively intact individuals with Alzheimer's disease pathology.

    PubMed

    Scheff, Stephen W; Ansari, Mubeen A; Mufson, Elliott J

    2016-06-01

    Neuritic amyloid plaques and neurofibrillary tangles are hallmarks of Alzheimer's disease (AD) and are major components used for the clinical diagnosis of this disorder. However, many individuals with no cognitive impairment (NCI) also present at autopsy with high levels of these AD pathologic hallmarks. In this study, we evaluated 15 autopsy cases from NCI individuals with high levels of AD-like pathology (high pathology no cognitive impairment) and compared them to age- and postmortem-matched cohorts of individuals with amnestic mild cognitive impairment and NCI cases with low AD-like pathology (low pathology no cognitive impairment [LPNCI]). Individuals classified as high pathology no cognitive impairment or amnestic mild cognitive impairment had a significant loss of both presynaptic and postsynaptic proteins in the hippocampus compared with those in the LPNCI cohort. In addition, these 2 groups had a significant increase in 3 different markers of oxidative stress compared with that in the LPNCI group. The changes in levels of synaptic proteins are strongly associated with levels of oxidative stress. These data suggest that cognitively older subjects without dementia but with increased levels of AD-like pathology may represent a very early preclinical stage of AD. PMID:27143416

  9. Studies of composition and major protein level in milk and colostrum of mares.

    PubMed

    Pecka, Ewa; Dobrzański, Zbigniew; Zachwieja, Andrzej; Szulc, Tadeusz; Czyż, Katarzyna

    2012-02-01

    The aim of the study was to determine the changes in composition and physicochemical features (pH, density, thermostability and acidity) of mare colostrum and milk, and of protein fraction contribution (serum albumin, β-casein, γ-casein, α-lactalbumin, G class immunoglobulins) depending on lactation stage. The research material was colostrum and milk samples from 12 Arabian mares. Colostrum samples were collected within 2 h after parturition and milk samples were collected twice, in the 3rd and 6th weeks of lactation. The level of basic milk components decreased significantly (only lactose content increased) as compared to colostrum. Total bacteria count and somatic cell count decreased significantly with an increase in resistance and urea level. The changes observed were connected to differentiated contribution of particular protein fractions and their relative proportions. Lower levels of γ-casein (P ≤ 0.05), β-casein, serum albumin as well as α-lactalbumin were observed in colostrum as compared to those in milk. Any relationship between lactation stage and β-casein content was observed. Serum albumin and α-lactalbumin content increased in subsequent milkings. The level of G class immunoglobulins decreased significantly and its highest level was noted in colostrum. Any significant differences between the 3rd and 6th lactation weeks were obtained. PMID:22339698

  10. Birdsong decreases protein levels of FoxP2, a molecule required for human speech.

    PubMed

    Miller, Julie E; Spiteri, Elizabeth; Condro, Michael C; Dosumu-Johnson, Ryan T; Geschwind, Daniel H; White, Stephanie A

    2008-10-01

    Cognitive and motor deficits associated with language and speech are seen in humans harboring FOXP2 mutations. The neural bases for FOXP2 mutation-related deficits are thought to reside in structural abnormalities distributed across systems important for language and motor learning including the cerebral cortex, basal ganglia, and cerebellum. In these brain regions, our prior research showed that FoxP2 mRNA expression patterns are strikingly similar between developing humans and songbirds. Within the songbird brain, this pattern persists throughout life and includes the striatal subregion, Area X, that is dedicated to song development and maintenance. The persistent mRNA expression suggests a role for FoxP2 that extends beyond the formation of vocal learning circuits to their ongoing use. Because FoxP2 is a transcription factor, a role in shaping circuits likely depends on FoxP2 protein levels which might not always parallel mRNA levels. Indeed our current study shows that FoxP2 protein, like its mRNA, is acutely downregulated in mature Area X when adult males sing with some differences. Total corticosterone levels associated with the different behavioral contexts did not vary, indicating that differences in FoxP2 levels are not likely attributable to stress. Our data, together with recent reports on FoxP2's target genes, suggest that lowered FoxP2 levels may allow for expression of genes important for circuit modification and thus vocal variability. PMID:18701760

  11. Birdsong Decreases Protein Levels of FoxP2, a Molecule Required for Human Speech

    PubMed Central

    Miller, Julie E.; Spiteri, Elizabeth; Condro, Michael C.; Dosumu-Johnson, Ryan T.; Geschwind, Daniel H.; White, Stephanie A.

    2008-01-01

    Cognitive and motor deficits associated with language and speech are seen in humans harboring FOXP2 mutations. The neural bases for FOXP2 mutation-related deficits are thought to reside in structural abnormalities distributed across systems important for language and motor learning including the cerebral cortex, basal ganglia, and cerebellum. In these brain regions, our prior research showed that FoxP2 mRNA expression patterns are strikingly similar between developing humans and songbirds. Within the songbird brain, this pattern persists throughout life and includes the striatal subregion, Area X, that is dedicated to song development and maintenance. The persistent mRNA expression suggests a role for FoxP2 that extends beyond the formation of vocal learning circuits to their ongoing use. Because FoxP2 is a transcription factor, a role in shaping circuits likely depends on FoxP2 protein levels which might not always parallel mRNA levels. Indeed our current study shows that FoxP2 protein, like its mRNA, is acutely downregulated in mature Area X when adult males sing with some differences. Total corticosterone levels associated with the different behavioral contexts did not vary, indicating that differences in FoxP2 levels are not likely attributable to stress. Our data, together with recent reports on FoxP2's target genes, suggest that lowered FoxP2 levels may allow for expression of genes important for circuit modification and thus vocal variability. PMID:18701760

  12. Increased Levels of Antinutritional and/or Defense Proteins Reduced the Protein Quality of a Disease-Resistant Soybean Cultivar

    PubMed Central

    Sousa, Daniele O. B.; Carvalho, Ana F. U.; Oliveira, José Tadeu A.; Farias, Davi F.; Castelar, Ivan; Oliveira, Henrique P.; Vasconcelos, Ilka M.

    2015-01-01

    The biochemical and nutritional attributes of two soybean (Glycine max (L.) Merr.) cultivars, one susceptible (Seridó) and the other resistant (Seridó-RCH) to stem canker, were examined to assess whether the resistance to pathogens was related to levels of antinutritional and/or defense proteins in the plant and subsequently affected the nutritional quality. Lectin, urease, trypsin inhibitor, peroxidase and chitinase activities were higher in the resistant cultivar. Growing rats were fed with isocaloric and isoproteic diets prepared with defatted raw soybean meals. Those on the Seridó-RCH diet showed the worst performance in terms of protein quality indicators. Based on regression analysis, lectin, trypsin inhibitor, peroxidase and chitinase appear to be involved in the resistance trait but also in the poorer nutritional quality of Seridó-RCH. Thus, the development of cultivars for disease resistance may lead to higher concentrations of antinutritional compounds, affecting the quality of soybean seeds. Further research that includes the assessment of more cultivars/genotypes is needed. PMID:26205163

  13. Increased Levels of Antinutritional and/or Defense Proteins Reduced the Protein Quality of a Disease-Resistant Soybean Cultivar.

    PubMed

    Sousa, Daniele O B; Carvalho, Ana F U; Oliveira, José Tadeu A; Farias, Davi F; Castelar, Ivan; Oliveira, Henrique P; Vasconcelos, Ilka M

    2015-07-01

    The biochemical and nutritional attributes of two soybean (Glycine max (L.) Merr.) cultivars, one susceptible (Seridó) and the other resistant (Seridó-RCH) to stem canker, were examined to assess whether the resistance to pathogens was related to levels of antinutritional and/or defense proteins in the plant and subsequently affected the nutritional quality. Lectin, urease, trypsin inhibitor, peroxidase and chitinase activities were higher in the resistant cultivar. Growing rats were fed with isocaloric and isoproteic diets prepared with defatted raw soybean meals. Those on the Seridó-RCH diet showed the worst performance in terms of protein quality indicators. Based on regression analysis, lectin, trypsin inhibitor, peroxidase and chitinase appear to be involved in the resistance trait but also in the poorer nutritional quality of Seridó-RCH. Thus, the development of cultivars for disease resistance may lead to higher concentrations of antinutritional compounds, affecting the quality of soybean seeds. Further research that includes the assessment of more cultivars/genotypes is needed. PMID:26205163

  14. Level of dietary protein does not impact whole body protein turnover during an exercise induced energy deficit

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Introduction: This study examined the effect of a high protein diet on whole body protein turnover during an exercise-induced energy deficit. A sustained energy deficit induced by energy intake restriction increases protein catabolism which can cause lean-body mass loss. A high-protein diet has be...

  15. Optimal dietary protein level improved growth, disease resistance, intestinal immune and physical barrier function of young grass carp (Ctenopharyngodon idella).

    PubMed

    Xu, Jing; Wu, Pei; Jiang, Wei-Dan; Liu, Yang; Jiang, Jun; Kuang, Sheng-Yao; Tang, Ling; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu; Feng, Lin

    2016-08-01

    This study investigated the effects of dietary proteins on the growth, disease resistance, intestinal immune and physical barrier functions of young grass carp (Ctenopharyngodon idella). A total of 540 young grass carp (264.11 ± 0.76 g) were fed six diets containing graded levels of protein (143.1, 176.7, 217.2, 257.5, 292.2 and 322.8 g digestible protein kg(-1) diet) for 8 weeks. After the growth trial, fish were challenged with Aeromonas hydrophila and mortalities were recorded for 14 days. The results indicated that optimal dietary protein levels: increased the production of antibacterial components, up-regulated anti-inflammatory cytokines, inhibitor of κBα, target of rapamycin and ribosomal protein S6 kinases 1 mRNA levels, whereas down-regulated pro-inflammatory cytokines, nuclear factor kappa B (NF-κB) P65, NF-κB P52, c-Rel, IκB kinase β, IκB kinase γ and eIF4E-binding proteins 2 mRNA levels in three intestinal segments of young grass carp (P < 0.05), suggesting that optimal dietary protein level could enhance fish intestinal immune barrier function; up-regulated the mRNA levels of tight junction complexes, B-cell lymphoma protein-2, inhibitor of apoptosis proteins, myeloid cell leukemia-1 and NF-E2-related factor 2, and increased the activities and mRNA levels of antioxidant enzymes, whereas down-regulated myosin light chain kinase, cysteinyl aspartic acid-protease 2, 3, 7, 8, 9, fatty acid synthetase ligand, apoptotic protease activating factor-1, Bcl-2 associated X protein, p38 mitogen-activated protein kinase, c-Jun N-terminal protein kinase and Kelch-like-ECH-associated protein 1b mRNA levels, and decreased reactive oxygen species, malondialdehyde and protein carbonyl contents in three intestinal segments of young grass carp (P < 0.05), indicating that optimal dietary protein level could improve fish intestinal physical barrier function. Finally, the optimal dietary protein levels for the growth performance (PWG) and against enteritis

  16. Protein level identification of the Listeria monocytogenes Sigma H, Sigma L, and Sigma C regulons

    PubMed Central

    2013-01-01

    Background Transcriptional regulation by alternative sigma (σ) factors represents an important mechanism that allows bacteria to rapidly regulate transcript and protein levels in response to changing environmental conditions. While the role of the alternative σ factor σB has been comparatively well characterized in L. monocytogenes, our understanding of the roles of the three other L. monocytogenes alternative σ factors is still limited. In this study, we employed a quantitative proteomics approach using Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) to characterize the L. monocytogenes σL, σH, and σC protein regulons. Proteomic comparisons used a quadruple alternative σ factor mutant strain (ΔBCHL) and strains expressing a single alternative σ factor (i.e., σL, σH, and σC; strains ΔBCH, ΔBCL, and ΔBHL) to eliminate potential redundancies between σ factors. Results Among the three alternative σ factors studied here, σH provides positive regulation for the largest number of proteins, consistent with previous transcriptomic studies, while σL appears to contribute to negative regulation of a number of proteins. σC was found to regulate a small number of proteins in L. monocytogenes grown to stationary phase at 37°C. Proteins identified as being regulated by multiple alternative σ factors include MptA, which is a component of a PTS system with a potential role in regulation of PrfA activity. Conclusions This study provides initial insights into global regulation of protein production by the L. monocytogenes alternative σ factors σL, σH, and σC. While, among these σ factors, σH appears to positively regulate the largest number of proteins, we also identified PTS systems that appear to be co-regulated by multiple alternative σ factors. Future studies should not only explore potential roles of alternative σ factors in activating a “cascade” of PTS systems that potentially regulate PrfA, but also may want to explore the

  17. Effect of pumpkin seed (Cucurbita pepo) protein isolate on the activity levels of certain plasma enzymes in CCl4-induced liver injury in low-protein fed rats.

    PubMed

    Nkosi, C Z; Opoku, A R; Terblanche, S E

    2005-04-01

    The effects of pumpkin seed (Cucurbita pepo) protein isolate on the activity levels of lactate dehydrogenase (LD), alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) against carbon tetrachloride (CCl4)-induced acute liver injury in low-protein fed rats were investigated. A group of male Sprague-Dawley rats maintained on a low-protein diet for 5 days were divided into three subgroups. Two subgroups were injected with carbon tetrachloride and the other group with an equivalent amount of olive oil. Two hours after CCl4 intoxication one of the two subgroups was administered with pumpkin seed protein isolate. All three subgroups of rats were maintained on the low-protein diet for the duration of the investigation. Groups of rats from the different subgroups were killed at 24, 48 and 72 h after their respective treatments. After 5 days on the low-protein diet the activity levels of all four enzymes were significantly higher than their counterparts on a normal balanced diet. CCl4 intoxication resulted in significant increases in the activity levels of all four enzymes investigated. The administration of pumpkin seed protein isolate after CCl4 intoxication resulted in significantly reduced activity levels of all four enzymes. It is concluded that pumpkin seed protein isolate administration was effective in alleviating the detrimental effects associated with protein malnutrition. PMID:16041732

  18. Wolbachia Endosymbionts Modify Drosophila Ovary Protein Levels in a Context-Dependent Manner

    PubMed Central

    Christensen, Steen; Pérez Dulzaides, Ricardo; Hedrick, Victoria E.; Momtaz, A. J. M. Zehadee; Nakayasu, Ernesto S.; Paul, Lake N.

    2016-01-01

    ABSTRACT Endosymbiosis is a unique form of interaction between organisms, with one organism dwelling inside the other. One of the most widespread endosymbionts is Wolbachia pipientis, a maternally transmitted bacterium carried by insects, crustaceans, mites, and filarial nematodes. Although candidate proteins that contribute to maternal transmission have been identified, the molecular basis for maternal Wolbachia transmission remains largely unknown. To investigate transmission-related processes in response to Wolbachia infection, ovarian proteomes were analyzed from Wolbachia-infected Drosophila melanogaster and D. simulans. Endogenous and variant host-strain combinations were investigated. Significant and differentially abundant ovarian proteins were detected, indicating substantial regulatory changes in response to Wolbachia. Variant Wolbachia strains were associated with a broader impact on the ovary proteome than endogenous Wolbachia strains. The D. melanogaster ovarian environment also exhibited a higher level of diversity of proteomic responses to Wolbachia than D. simulans. Overall, many Wolbachia-responsive ovarian proteins detected in this study were consistent with expectations from the experimental literature. This suggests that context-specific changes in protein abundance contribute to Wolbachia manipulation of transmission-related mechanisms in oogenesis. IMPORTANCE Millions of insect species naturally carry bacterial endosymbionts called Wolbachia. Wolbachia bacteria are transmitted by females to their offspring through a robust egg-loading mechanism. The molecular basis for Wolbachia transmission remains poorly understood at this time, however. This proteomic study identified specific fruit fly ovarian proteins as being upregulated or downregulated in response to Wolbachia infection. The majority of these protein responses correlated specifically with the type of host and Wolbachia strain involved. This work corroborates previously identified

  19. Rapid Dietary Protein Changes and Relationships among Enzyme Activities, Their RNA's and Plasma Hormone Levels in the Broiler Chicken

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ross 708 broiler chickens were fed one of three levels of crude protein (12, 21 or 30%) from 7 to 28 days of age. Birds were then switched to either higher (12 to 30%, 21 to 30%) or lower levels of crude protein (30-12%, 21-12%) and sampled three days following the switch. The purpose of these treat...

  20. [Relation between serum eosinophil cationic protein (ECP) level and asthma attack in children].

    PubMed

    Ishigaki, N; Masuhara, C; Sakamaki, K; Ishikawa, Y; Ohta, K; Koike, R; Mikuni, K; Haruna, H; Awa, S

    2000-11-01

    Serum eosinophil cationic protein (sECP) levels were measured in 339 patients with childhood asthma, and the clinical courses of these patients were followed for 57 weeks. While considering the history and characteristics of each patient, we examined the correlation between asthma attack frequency and sECP, blood eosinophil count, and serum total IgE (tIgE) to determine their usefulness in predicting asthma attacks. Among patients with no other allergic diseases, sECP levels in patients who had no asthma attacks two weeks before or after the measurement were significantly lower than those of patients who had attacks during the same four-week period. Among patients who had attacks, those patients with no attack for a year after the measurement were also found to have low sECP levels. Similarly, even among patients with asthma attacks and high sECP levels, there were cases where attacks were well controlled using nebulizer treatments with DSCG or BDP. The incident rate of attacks for patients with other allergic diseases and a low sECP was low. Yet, there was no common trend in patients with high sECP levels. Moreover, this study detected a significant correlation between sECP level and blood eosinophil count as well as between sECP level and serum tIgE. The most significant correlation with asthma attack frequency was sECP level. Thus, sECP level seems to reflect the allergy activity level, especially two weeks prior to and after the measurement. For patients without other allergic diseases, asthma attack prediction during the two weeks period after the measurement of sECP also seems possible. Therefore, periodic measurement of sECP level is useful in objectively monitoring the improvement of symptoms and establishing the treatment plan, including treatment with DSCG or BDP. PMID:11193461

  1. Serum levels of hypersensitive-C-reactive protein in moderate and severe acne

    PubMed Central

    Namazi, M. R.; Parhizkar, A. R.; Jowkar, F.

    2015-01-01

    Background: Elevation of C-reactive protein (CRP) has been reported to occur in psoriasis, urticaria, acne, rosacea and many other dermatological and nondermatological conditions. Chronic systemic inflammation has been implicated in the development of neuropsychiatric/degenerative disorders, atherosclerosis, coronary artery disease, diabetes mellitus and even carcinogenesis. The present study is designed to determine whether the level of inflammation created by acne vulgaris could be high enough to raise the serum levels of high-sensitive CRP. Materials and Methods: Forty-two patients with moderate and severe acne vulgaris were enrolled, along with 44 age and sex matched healthy blood donors as controls. Hypersensitive-CRP (Hs-CRP) was measured in both groups. Results: Hypersensitive-C-reactive protein levels in the case group varied between 0 and 28.1 μg/ml with an average of 2.24 ± 4.87 μg/ml (mean ± standard deviation) and a median of 0.6 μg/ml (interquartile range [IQR] =0.3, 1.4 μg/ml). Hs-CRP levels of the control group varied between 0 and 14 μg/ml with an average of 3.12 ± 3.67 μg/ml and a median of 1.5 μg/ml (IQR = 0.55, 5.0 μg/ml). No significant difference of Hs-CRP level between the two groups was seen (t = –0.961, 95% confidence interval: Lower = –2.6942, upper = 0.9377; P = 0.339). Additionally, no significant difference in the level of Hs-CRP was noted between the moderate and severe acne groups (95% confidence interval: Lower = –5.2495, upper = 1.6711; P = 0.165). Conclusion: Acne vulgaris, even in its severe grades (excluding acne fulminans and acne conglobata), does not induce significant inflammation at the systemic level. PMID:26225329

  2. Circulating growth arrest-specific protein 6 levels are associated with erythropoietin resistance in hemodialysis patients.

    PubMed

    Chen, Miao-Pei; Chen, Chien-Wen; Chen, Jin-Shuen; Mao, Hung-Chung; Chou, Chu-Lin

    2016-01-01

    Growth arrest-specific protein 6 (Gas6) works synergistically with erythropoietin (EPO) to increase the proliferation and maturation of erythroblasts. However, the role of Gas 6 levels on EPO resistance in hemodialysis (HD) patients remains unclear. Therefore, the objective of this study was the first to examine the correlation between plasma Gas6 levels and EPO resistance in HD patients. We enrolled 134 HD patients and 85 healthy individuals. The HD patients were divided into 2 groups: 98 non-EPO-resistant patients and 36 EPO-resistant patients. Plasma levels of Gas6, interleukin 6 (IL-6), high-sensitivity C-reactive protein (hs-CRP), and albumin were quantified. Compared with non-EPO-resistant patients, EPO-resistant patients had elevated plasma concentrations of Gas6 (15.4 ± 3.3 vs. 13.7 ± 3.2 ng/mL, P = 0.006), IL-6 (3.1 ± 3.1 vs. 2.1 ± 1.5 pg/mL, P = 0.009), and hs-CRP (12.7 ± 25.2 vs. 4.5 ± 5.5 mg/L, P = 0.002). In EPO-resistant HD patients, plasma Gas6 levels were negatively correlated with albumin levels (r = -0.388, P < 0.021). Elevated Gas6 levels are associated with EPO resistance in HD patients. Also, EPO resistance is related to inflammation and malnutrition. Thus, circulating Gas6 levels could be used as the potential marker in HD patients with EPO resistance. PMID:26788441

  3. Correlation between radiation dose and p53 protein expression levels in human lymphocytes.

    PubMed

    Cavalcanti, Mariana B; Fernandes, Thiago S; Silva, Edvane B; Amaral, Ademir

    2015-09-01

    The aim of this research was to evaluate the relationship between p53 protein levels and absorbed doses from in vitro irradiated human lymphocytes. For this, samples of blood from 23 donors were irradiated with 0.5; 1; 2; and 4 Gy from a Cobalt-60 source, and the percentages of lymphocytes expressing p53 were scored using Flow Cytometry. The subjects were divided into 3 groups, in accordance with the p53 levels expressed per radiation dose: low (Group I), high (Group II), and excessive levels (Group III). For all groups, the analyses showed that the p53 expression levels increase with the absorbed dose. Particularly for groups I and II, the correlation between this protein expression and the dose follows the linear-quadratic model, such as for radioinduced chromosomal aberrations. In conclusion, our findings indicate possible applications of this approach in evaluating individual radiosensitivity prior to radiotherapeutical procedures as well as in medical surveillance of occupationally exposed workers. Furthermore, due to the rapidity of flow-cytometric analyses, the methodology here employed would play an important role in emergency responses to a large-scale radiation incident where many people may have been exposed. PMID:26312422

  4. Effect of Dietary Protein Levels on Composition of Odorous Compounds and Bacterial Ecology in Pig Manure.

    PubMed

    Cho, Sungback; Hwang, Okhwa; Park, Sungkwon

    2015-09-01

    This study was performed to investigate the effect of different levels of dietary crude protein (CP) on composition of odorous compounds and bacterial communities in pig manure. A total of 48 male pigs (average initial body weight 45 kg) fed diets containing three levels of dietary CP (20%, 17.5%, and 15%) and their slurry samples were collected from the pits under the floor every week for one month. Changes in composition of odorous compounds and bacterial communities were analyzed by gas chromatography and 454 FLX titanium pyrosequencing systems, respectively. Levels of phenols, indoles, short chain fatty acid and branched chain fatty acid were lowest (p<0.05) in CP 15% group among three CP levels. Relative abundance of Bacteroidetes phylum and bacterial genera including Leuconostoc, Bacillus, Atopostipes, Peptonphilus, Ruminococcaceae_uc, Bacteroides, and Pseudomonas was lower (p<0.05) in CP 15% than in CP 20% group. There was a positive correlation (p<0.05) between odorous compounds and bacterial genera: phenol, indole, iso-butyric acid, and iso-valeric acid with Atopostipes, p-cresol and skatole with Bacteroides, acetic acid and butyric acid with AM982595_g of Porphyromonadaceae family, and propionic acid with Tissierella. Taken together, administration of 15% CP showed less production of odorous compounds than 20% CP group and this result might be associated with the changes in bacterial communities especially whose roles in protein metabolism. PMID:26194219

  5. Effect of Dietary Protein Levels on Composition of Odorous Compounds and Bacterial Ecology in Pig Manure

    PubMed Central

    Cho, Sungback; Hwang, Okhwa; Park, Sungkwon

    2015-01-01

    This study was performed to investigate the effect of different levels of dietary crude protein (CP) on composition of odorous compounds and bacterial communities in pig manure. A total of 48 male pigs (average initial body weight 45 kg) fed diets containing three levels of dietary CP (20%, 17.5%, and 15%) and their slurry samples were collected from the pits under the floor every week for one month. Changes in composition of odorous compounds and bacterial communities were analyzed by gas chromatography and 454 FLX titanium pyrosequencing systems, respectively. Levels of phenols, indoles, short chain fatty acid and branched chain fatty acid were lowest (p<0.05) in CP 15% group among three CP levels. Relative abundance of Bacteroidetes phylum and bacterial genera including Leuconostoc, Bacillus, Atopostipes, Peptonphilus, Ruminococcaceae_uc, Bacteroides, and Pseudomonas was lower (p<0.05) in CP 15% than in CP 20% group. There was a positive correlation (p<0.05) between odorous compounds and bacterial genera: phenol, indole, iso-butyric acid, and iso-valeric acid with Atopostipes, p-cresol and skatole with Bacteroides, acetic acid and butyric acid with AM982595_g of Porphyromonadaceae family, and propionic acid with Tissierella. Taken together, administration of 15% CP showed less production of odorous compounds than 20% CP group and this result might be associated with the changes in bacterial communities especially whose roles in protein metabolism. PMID:26194219

  6. Development of Multigene Expression Signature Maps at the Protein Level from Digitized Immunohistochemistry Slides

    PubMed Central

    Metzger, Gregory J.; Dankbar, Stephen C.; Henriksen, Jonathan; Rizzardi, Anthony E.; Rosener, Nikolaus K.; Schmechel, Stephen C.

    2012-01-01

    Molecular classification of diseases based on multigene expression signatures is increasingly used for diagnosis, prognosis, and prediction of response to therapy. Immunohistochemistry (IHC) is an optimal method for validating expression signatures obtained using high-throughput genomics techniques since IHC allows a pathologist to examine gene expression at the protein level within the context of histologically interpretable tissue sections. Additionally, validated IHC assays may be readily implemented as clinical tests since IHC is performed on routinely processed clinical tissue samples. However, methods have not been available for automated n-gene expression profiling at the protein level using IHC data. We have developed methods to compute expression level maps (signature maps) of multiple genes from IHC data digitized on a commercial whole slide imaging system. Areas of cancer for these expression level maps are defined by a pathologist on adjacent, co-registered H&E slides, allowing assessment of IHC statistics and heterogeneity within the diseased tissue. This novel way of representing multiple IHC assays as signature maps will allow the development of n-gene expression profiling databases in three dimensions throughout virtual whole organ reconstructions. PMID:22438942

  7. Identification of proteins whose synthesis is preferentially enhanced by polyamines at the level of translation in mammalian cells.

    PubMed

    Nishimura, Kazuhiro; Okudaira, Hiroyuki; Ochiai, Eriko; Higashi, Kyohei; Kaneko, Mayumi; Ishii, Itsuko; Nishimura, Tomoe; Dohmae, Naoshi; Kashiwagi, Keiko; Igarashi, Kazuei

    2009-11-01

    In Escherichia coli, several proteins whose synthesis is enhanced by polyamines at the level of translation have been identified. We looked for proteins that are similarly regulated in eukaryotes using a mouse mammary carcinoma FM3A cell culture system. Polyamine deficiency was induced by adding an inhibitor of ornithine decarboxylase, alpha-difluoromethylornithine, to the medium. Proteins enhanced by polyamines were determined by comparison of protein levels in control and polyamine-deficient cells using two-dimensional gel electrophoresis, and were identified by Edman degradation and/or LC/MALDI-TOF/TOF tandem mass spectrometry. Polyamine stimulation of the synthesis of these proteins at the level of translation was confirmed by measuring levels of the corresponding mRNAs and proteins, and levels of the [(35)S]methionine pulse-labeled proteins. The proteins identified in this way were T-complex protein 1, beta subunit (Cct2); heterogeneous nuclear ribonucleoprotein L (Hnrpl); and phosphoglycerate mutase 1 (Pgam1). Since Cct2 was most strongly enhanced by polyamines among three proteins, the mechanism of polyamine stimulation of Cct2 synthesis was studied using NIH3T3 cells transiently transfected with genes encoding Cct2-EGFP fusion mRNA with normal or mutated 5'-untranslated region (5'-UTR) of Cct2 mRNA. Polyamines most likely enhanced ribosome shunting on the 5'-UTR of Cct2 mRNA. PMID:19427401

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

    PubMed Central

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

    2014-01-01

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

  9. Correlation of diacylglycerol level and protein kinase C activity in rat retina to retinal circulation.

    PubMed

    Shiba, T; Inoguchi, T; Sportsman, J R; Heath, W F; Bursell, S; King, G L

    1993-11-01

    The increases in diacylglycerol (DAG) level and protein kinase C (PKC) activity have been characterized biochemically and functionally in the retina and the brain of diabetic rats as well as in cultured vascular cells. PKC specific activities were increased in the membraneous fraction of retina from streptozotocin (STZ)-induced diabetic rats and the genetically determined diabetic BB rats, respectively, after 1 or 2 wk of diabetes, compared with control. The ratio of total PKC activities from membraneous and cytosol fractions was also increased in the retina of diabetic rats. With diabetes, all the isoenzymes and the total DAG level were increased in the rat retina, whereas no changes were found in the rat brain. Insulin treatment normalized plasma glucose levels and partially prevented the increases in the membraneous PKC activity and all the isoenzymes in the retina. In the retinal endothelial cells, the total DAG level and PKC specific activities are increased by 36 and 22%, respectively, in the membraneous pool when the glucose levels are changed from 5.5 to 22 mM. Activation of PKC activity and isoform beta II by the vitreal injection of phorbol dibutyrate mimicked the abnormal retinal blood circulation observed in diabetic rats (2.22 +/- 0.24 vs. 1.83 +/- 0.40 s). Thus diabetes and elevated glucose levels will increase DAG level and PKC activities and its isoenzyme specifically in vascular cells and may affect retinal hemodynamics. PMID:8238505

  10. Intermediate divergence levels maximize the strength of structure-sequence correlations in enzymes and viral proteins.

    PubMed

    Jackson, Eleisha L; Shahmoradi, Amir; Spielman, Stephanie J; Jack, Benjamin R; Wilke, Claus O

    2016-07-01

    Structural properties such as solvent accessibility and contact number predict site-specific sequence variability in many proteins. However, the strength and significance of these structure-sequence relationships vary widely among different proteins, with absolute correlation strengths ranging from 0 to 0.8. In particular, two recent works have made contradictory observations. Yeh et al. (Mol. Biol. Evol. 31:135-139, 2014) found that both relative solvent accessibility (RSA) and weighted contact number (WCN) are good predictors of sitewise evolutionary rate in enzymes, with WCN clearly out-performing RSA. Shahmoradi et al. (J. Mol. Evol. 79:130-142, 2014) considered these same predictors (as well as others) in viral proteins and found much weaker correlations and no clear advantage of WCN over RSA. Because these two studies had substantial methodological differences, however, a direct comparison of their results is not possible. Here, we reanalyze the datasets of the two studies with one uniform analysis pipeline, and we find that many apparent discrepancies between the two analyses can be attributed to the extent of sequence divergence in individual alignments. Specifically, the alignments of the enzyme dataset are much more diverged than those of the virus dataset, and proteins with higher divergence exhibit, on average, stronger structure-sequence correlations. However, the highest structure-sequence correlations are observed at intermediate divergence levels, where both highly conserved and highly variable sites are present in the same alignment. PMID:26971720

  11. Global Analysis of Protein Expression and Phosphorylation Levels in Nicotine-Treated Pancreatic Stellate Cells.

    PubMed

    Paulo, Joao A; Gaun, Aleksandr; Gygi, Steven P

    2015-10-01

    Smoking is a risk factor in pancreatic disease; however, the biochemical mechanisms correlating smoking with pancreatic dysfunction remain poorly understood. Strategies using multiplexed isobaric tag-based mass spectrometry facilitate the study of drug-induced perturbations on biological systems. Here, we present the first large-scale analysis of the proteomic and phosphoproteomic alterations in pancreatic stellate cells following treatment with two nicotinic acetylcholine receptor (nAChR) ligands: nicotine and α-bungarotoxin. We treated cells with nicotine or α-bungarotoxin for 12 h in triplicate and compared alterations in protein expression and phosphorylation levels to mock-treated cells using a tandem mass tag (TMT9plex)-based approach. Over 8100 proteins were quantified across all nine samples, of which 46 were altered in abundance upon treatment with nicotine. Proteins with increased abundance included those associated with neurons, defense mechanisms, indicators of pancreatic disease, and lysosomal proteins. In addition, we measured differences for ∼16 000 phosphorylation sites across all nine samples using a titanium dioxide-based strategy, of which 132 sites were altered with nicotine and 451 with α-bungarotoxin treatment. Many altered phosphorylation sites were involved in nuclear function and transcriptional events. This study supports the development of future targeted investigations to establish a better understanding for the role of nicotine and associated receptors in pancreatic disease. PMID:26265067

  12. The RNA-binding protein gene, hermes, is expressed at high levels in the developing heart.

    PubMed

    Gerber, W V; Yatskievych, T A; Antin, P B; Correia, K M; Conlon, R A; Krieg, P A

    1999-01-01

    In a screen for novel sequences expressed during embryonic heart development we have isolated a gene which encodes a putative RNA-binding protein. This protein is a member of one of the largest families of RNA-binding proteins, the RRM (RNA Recognition Motif) family. The gene has been named hermes (for HEart, RRM Expressed Sequence). The hermes protein is 197-amino acids long and contains a single RRM domain. In situ hybridization analysis indicates that hermes is expressed at highest levels in the myocardium of the heart and to a lesser extent in the ganglion layer of the retina, the pronephros and epiphysis. Expression of hermes in each of these tissues begins at approximately the time of differentiation and is maintained throughout development. Analysis of the RNA expression of the hermes orthologues from chicken and mouse reveals that, like Xenopus, the most prominent tissue of expression is the developing heart. The sequence and expression pattern of hermes suggests a role in post-transcriptional regulation of heart development. PMID:10096065

  13. Global analysis of protein expression and phosphorylation levels in nicotine-treated pancreatic stellate cells

    PubMed Central

    Paulo, Joao A.; Gaun, Aleksandr; Gygi, Steven P.

    2016-01-01

    Smoking is a risk factor in pancreatic disease, however, the biochemical mechanisms correlating smoking with pancreatic dysfunction remain poorly understood. Strategies using multiplexed isobaric tag-based mass spectrometry facilitate the study of drug-induced perturbations on biological systems. Here, we present the first large scale analysis of the proteomic and phosphoproteomic alterations in pancreatic stellate cells following treatment with two nicotinic acetylcholine receptor (nAChR) ligands: nicotine and α-bungarotoxin. We treated cells with nicotine or α-bungarotoxin for 12hr in triplicate and compared alterations in protein expression and phosphorylation levels to mock treated cells using a tandem mass tag (TMT9plex)-based approach. Over 8,100 proteins were quantified across all nine samples of which 46 were altered in abundance upon treatment with nicotine. Proteins with increased abundance included those associated with neurons, defense mechanisms, indicators of pancreatic disease and lysosomal proteins. In addition, we measured differences for ∼16,000 phosphorylation sites across all nine samples using a titanium dioxide-based strategy, of which 132 sites were altered with nicotine and 451 with α-bungarotoxin treatment. Many altered phosphorylation sites were involved in nuclear function and transcriptional events. This study supports the development of future targeted investigations to establish a better understanding for the role of nicotine and associated receptors in pancreatic disease. PMID:26265067

  14. Lung surfactant levels are regulated by Ig-Hepta/GPR116 by monitoring surfactant protein D.

    PubMed

    Fukuzawa, Taku; Ishida, Junji; Kato, Akira; Ichinose, Taro; Ariestanti, Donna Maretta; Takahashi, Tomoya; Ito, Kunitoshi; Abe, Jumpei; Suzuki, Tomohiro; Wakana, Shigeharu; Fukamizu, Akiyoshi; Nakamura, Nobuhiro; Hirose, Shigehisa

    2013-01-01

    Lung surfactant is a complex mixture of lipids and proteins, which is secreted from the alveolar type II epithelial cell and coats the surface of alveoli as a thin layer. It plays a crucial role in the prevention of alveolar collapse through its ability to reduce surface tension. Under normal conditions, surfactant homeostasis is maintained by balancing its release and the uptake by the type II cell for recycling and the internalization by alveolar macrophages for degradation. Little is known about how the surfactant pool is monitored and regulated. Here we show, by an analysis of gene-targeted mice exhibiting massive accumulation of surfactant, that Ig-Hepta/GPR116, an orphan receptor, is expressed on the type II cell and sensing the amount of surfactant by monitoring one of its protein components, surfactant protein D, and its deletion results in a pulmonary alveolar proteinosis and emphysema-like pathology. By a coexpression experiment with Sp-D and the extracellular region of Ig-Hepta/GPR116 followed by immunoprecipitation, we identified Sp-D as the ligand of Ig-Hepta/GPR116. Analyses of surfactant metabolism in Ig-Hepta(+/+) and Ig-Hepta(-/-) mice by using radioactive tracers indicated that the Ig-Hepta/GPR116 signaling system exerts attenuating effects on (i) balanced synthesis of surfactant lipids and proteins and (ii) surfactant secretion, and (iii) a stimulating effect on recycling (uptake) in response to elevated levels of Sp-D in alveolar space. PMID:23922714

  15. Levels of the E2 interacting protein TopBP1 modulate papillomavirus maintenance stage replication

    SciTech Connect

    Kanginakudru, Sriramana; DeSmet, Marsha; Thomas, Yanique; Morgan, Iain M.; Androphy, Elliot J.

    2015-04-15

    The evolutionarily conserved DNA topoisomerase II beta-binding protein 1 (TopBP1) functions in DNA replication, DNA damage response, and cell survival. We analyzed the role of TopBP1 in human and bovine papillomavirus genome replication. Consistent with prior reports, TopBP1 co-localized in discrete nuclear foci and was in complex with papillomavirus E2 protein. Similar to E2, TopBP1 is recruited to the region of the viral origin of replication during G1/S and early S phase. TopBP1 knockdown increased, while over-expression decreased transient virus replication, without affecting cell cycle. Similarly, using cell lines harboring HPV-16 or HPV-31 genome, TopBP1 knockdown increased while over-expression reduced viral copy number relative to genomic DNA. We propose a model in which TopBP1 serves dual roles in viral replication: it is essential for initiation of replication yet it restricts viral copy number. - Highlights: • Protein interaction study confirmed In-situ interaction between TopBP1 and E2. • TopBP1 present at papillomavirus ori in G1/S and early S phase of cell cycle. • TopBP1 knockdown increased, over-expression reduced virus replication. • TopBP1 protein level change did not influence cell survival or cell cycle. • TopBP1 displaced from papillomavirus ori after initiation of replication.

  16. Transcriptional Bursting Explains the Noise–Versus–Mean Relationship in mRNA and Protein Levels

    PubMed Central

    Dar, Roy D.; Shaffer, Sydney M.; Singh, Abhyudai; Razooky, Brandon S.; Simpson, Michael L.; Raj, Arjun; Weinberger, Leor S.

    2016-01-01

    Recent analysis demonstrates that the HIV-1 Long Terminal Repeat (HIV LTR) promoter exhibits a range of possible transcriptional burst sizes and frequencies for any mean-expression level. However, these results have also been interpreted as demonstrating that cell-to-cell expression variability (noise) and mean are uncorrelated, a significant deviation from previous results. Here, we re-examine the available mRNA and protein abundance data for the HIV LTR and find that noise in mRNA and protein expression scales inversely with the mean along analytically predicted transcriptional burst-size manifolds. We then experimentally perturb transcriptional activity to test a prediction of the multiple burst-size model: that increasing burst frequency will cause mRNA noise to decrease along given burst-size lines as mRNA levels increase. The data show that mRNA and protein noise decrease as mean expression increases, supporting the canonical inverse correlation between noise and mean. PMID:27467384

  17. Transcriptional Bursting Explains the Noise Versus Mean Relationship in mRNA and Protein Levels

    DOE PAGESBeta

    Dar, Dr. Roy; Shaffer, S; Singh, A; Razooky, B; Simpson, Michael L; Raj, A; Weinberger, Dr. Leor

    2016-01-01

    Recent analysis demonstrates that the HIV-1 Long Terminal Repeat (HIV LTR) promoter exhibits a range of possible transcriptional burst sizes and frequencies for any mean-expression level. However, these results have also been interpreted as demonstrating that cell-tocell expression variability (noise) and mean are uncorrelated, a significant deviation from previous results. Here, we re-examine the available mRNA and protein abundance data for the HIV LTR and find that noise in mRNA and protein expression scales inversely with the mean along analytically predicted transcriptional burst-size manifolds. We then experimentally perturb transcriptional activity to test a prediction of the multiple burst-size model: thatmore » increasing burst frequency will cause mRNA noise to decrease along given burst-size lines as mRNA levels increase. The data show that mRNA and protein noise decrease as mean expression increases, supporting the canonical inverse correlation between noise and mean.« less

  18. No association between lck gene polymorphisms and protein level in type 1 diabetes.

    PubMed

    Nervi, Solange; Nicodeme, Sandra; Gartioux, Corinne; Atlan, Catherine; Lathrop, Marc; Reviron, Denis; Naquet, Philippe; Matsuda, Fumihiko; Imbert, Jean; Vialettes, Bernard

    2002-11-01

    We previously described a reduced expression of the protein tyrosine kinase Lck in T-cells from type 1 diabetic patients, the origin of which is still unknown. The human lck gene, located on chromosome 1p35-34.3, was evaluated as a candidate susceptibility gene for type 1 diabetes. A molecular scan of the sequence variations in the coding, the relevant promoter, and most of the intronic sequences of the lck gene (representing a total of 10.5 kb fragment) was performed in 187 Caucasian subjects including 91 type 1 diabetic patients and 96 normoglycemic control subjects. We identified 35 sequence variations, including one deletion and 34 single nucleotide polymorphisms (SNPs), 33 of them being new. Four variants were frequent but not significantly associated with diabetes or Lck protein level. Of the SNP variants, 11 were only found within the diabetic population and some were associated with low Lck protein levels. The low frequency of these polymorphisms did not permit any statistically significant correlations with the disease status, suggesting that the lck gene probably does not contribute to genetic susceptibility to type 1 diabetes. PMID:12401726

  19. Transcriptional bursting explains the noise–versus–mean relationship in mRNA and protein levels

    DOE PAGESBeta

    Dar, Roy; Shaffer, Sydney M.; Singh, Abhyudai; Razooky, Brandon S.; Simpson, Michael L.; Raj, Arjun; Weinberger, Leor S.

    2016-07-28

    Recent analysis demonstrates that the HIV-1 Long Terminal Repeat (HIV LTR) promoter exhibits a range of possible transcriptional burst sizes and frequencies for any mean-expression level. However, these results have also been interpreted as demonstrating that cell-tocell expression variability (noise) and mean are uncorrelated, a significant deviation from previous results. Here, we re-examine the available mRNA and protein abundance data for the HIV LTR and find that noise in mRNA and protein expression scales inversely with the mean along analytically predicted transcriptional burst-size manifolds. We then experimentally perturb transcriptional activity to test a prediction of the multiple burst-size model: thatmore » increasing burst frequency will cause mRNA noise to decrease along given burst-size lines as mRNA levels increase. In conclusion, the data show that mRNA and protein noise decrease as mean expression increases, supporting the canonical inverse correlation between noise and mean.« less

  20. Telomere protein RAP1 levels are affected by cellular aging and oxidative stress

    PubMed Central

    Swanson, Mark J.; Baribault, Michelle E.; Israel, Joanna N.; Bae, Nancy S.

    2016-01-01

    Telomeres are important for maintaining the integrity of the genome through the action of the shelterin complex. Previous studies indicted that the length of the telomere did not have an effect on the amount of the shelterin subunits; however, those experiments were performed using immortalized cells with stable telomere lengths. The interest of the present study was to observe how decreasing telomere lengths over successive generations would affect the shelterin subunits. As neonatal human dermal fibroblasts aged and their telomeres became shorter, the levels of the telomere-binding protein telomeric repeat factor 2 (TRF2) decreased significantly. By contrast, the levels of one of its binding partners, repressor/activator protein 1 (RAP1), decreased to a lesser extent than would be expected from the decrease in TRF2. Other subunits, TERF1-interacting nuclear factor 2 and protection of telomeres protein 1, remained stable. The decrease in RAP1 in the older cells occurred in the nuclear and cytoplasmic fractions. Hydrogen peroxide (H2O2) stress was used as an artificial means of aging in the cells, and this resulted in RAP1 levels decreasing, but the effect was only observed in the nuclear portion. Similar results were obtained using U251 glioblastoma cells treated with H2O2 or grown in serum-depleted medium. The present findings indicate that TRF2 and RAP1 levels decrease as fibroblasts naturally age. RAP1 remains more stable compared to TRF2. RAP1 also responds to oxidative stress, but the response is different to that observed in aging. PMID:27446538

  1. Genome-Wide Tuning of Protein Expression Levels to Rapidly Engineer Microbial Traits.

    PubMed

    Freed, Emily F; Winkler, James D; Weiss, Sophie J; Garst, Andrew D; Mutalik, Vivek K; Arkin, Adam P; Knight, Rob; Gill, Ryan T

    2015-11-20

    The reliable engineering of biological systems requires quantitative mapping of predictable and context-independent expression over a broad range of protein expression levels. However, current techniques for modifying expression levels are cumbersome and are not amenable to high-throughput approaches. Here we present major improvements to current techniques through the design and construction of E. coli genome-wide libraries using synthetic DNA cassettes that can tune expression over a ∼10(4) range. The cassettes also contain molecular barcodes that are optimized for next-generation sequencing, enabling rapid and quantitative tracking of alleles that have the highest fitness advantage. We show these libraries can be used to determine which genes and expression levels confer greater fitness to E. coli under different growth conditions. PMID:26478262

  2. Serum Levels of Surfactant Proteins in Patients with Combined Pulmonary Fibrosis and Emphysema (CPFE)

    PubMed Central

    Papaioannou, Andriana I.; Kostikas, Konstantinos; Manali, Effrosyni D.; Papadaki, Georgia; Roussou, Aneza; Spathis, Aris; Mazioti, Argyro; Tomos, Ioannis; Papanikolaou, Ilias; Loukides, Stelios; Chainis, Kyriakos; Karakitsos, Petros; Griese, Matthias; Papiris, Spyros

    2016-01-01

    Introduction Emphysema and idiopathic pulmonary fibrosis (IPF) present either per se or coexist in combined pulmonary fibrosis and emphysema (CPFE). Serum surfactant proteins (SPs) A, B, C and D levels may reflect lung damage. We evaluated serum SP levels in healthy controls, emphysema, IPF, and CPFE patients and their associations to disease severity and survival. Methods 122 consecutive patients (31 emphysema, 62 IPF, and 29 CPFE) and 25 healthy controls underwent PFTs, ABG-measurements, 6MWT and chest HRCT. Serum levels of SPs were measured. Patients were followed-up for 1-year. Results SP-A and SP-D levels differed between groups (p = 0.006 and p<0.001 respectively). In post-hoc analysis, SP-A levels differed only between controls and CPFE (p<0.05) and CPFE and emphysema (p<0.05). SP-D differed between controls and IPF or CPFE (p<0.001 for both comparisons). In IPF SP-B correlated to pulmonary function while SP-A, correlated to the Composite Physiological Index (CPI). Controls current smokers had higher SP-A and SP-D levels compared to non-smokers (p = 0.026 and p = 0.023 respectively). SP-D levels were higher in CPFE patients with extended emphysema (p = 0.042). In patients with IPF, SP-B levels at the upper quartile of its range (≥26 ng/mL) presented a weak association with reduced survival (p = 0.05). Conclusion In conclusion, serum SP-A and SP-D levels were higher where fibrosis exists or coexists and related to disease severity, suggesting that serum SPs relate to alveolar damage in fibrotic lungs and may reflect either local overproduction or overleakage. The weak association between high levels of SP-B and survival needs further validation in clinical trials. PMID:27337142

  3. EFFECT OF DIETARY PROTEIN AND CARBOHYDRATE LEVELS ON WEIGHT GAIN AND GONAD PRODUCTION IN THE SEA URCHIN LYTECHINUS VARIEGATUS

    PubMed Central

    Heflin, Laura E.; Gibbs, Victoria K.; Powell, Mickie L; Makowsky, Robert; Lawrence, John M.; Lawrence, Addison L.; Watts, Stephen A.

    2014-01-01

    Adult Lytechinus variegatus were fed eight formulated diets with different protein (ranging from 12 to 36%) and carbohydrate (ranging from 21 to 39 %) levels. Each sea urchin (n = 8 per treatment) was fed a daily sub-satiation ration of 1.5% of average body weight for 9 weeks. Akaike information criterion analysis was used to compare six different hypothesized dietary composition models across eight growth measurements. Dietary protein level and protein: energy ratio were the best models for prediction of total weight gain. Diets with the highest (> 68.6 mg P kcal−-1) protein: energy ratios produced the most wet weight gain after 9 weeks. Dietary carbohydrate level was a poor predictor for most growth parameters examined in this study. However, the model containing a protein × carbohydrate interaction effect was the best model for protein efficiency ratio (PER). PER decreased with increasing dietary protein level, more so at higher carbohydrate levels. Food conversion ratio (FCR) was best modeled by total dietary energy levels: Higher energy diets produced lower FCRs. Dietary protein level was the best model of gonad wet weight gain. These data suggest that variations in dietary nutrients and energy differentially affect organismal growth and growth of body components. PMID:24994942

  4. Analysis of the Sperm Head Protein Profiles in Fertile Men: Consistency across Time in the Levels of Expression of Heat Shock Proteins and Peroxiredoxins

    PubMed Central

    Kichine, Elsa; Di Falco, Marcos; Hales, Barbara F.; Robaire, Bernard; Chan, Peter

    2013-01-01

    We investigated the identity and quantitative variations of proteins extracted from human sperm heads using a label-free Gel-MS approach. Sperm samples were obtained from three men with high sperm counts at three different time points. This design allowed us to analyse intra-individual and inter-individual variations of the human sperm head proteome. Each time point was analyzed in triplicate to minimize any background artifactual effects of the methodology on the variation analyses. Intra-individual analysis using the spectral counting method revealed that the expression levels of 90% of the common proteins identified in three samples collected at various time-points, separated by several months, had a coefficient of variation of less than 0.5 for each man. Across individuals, the expression level of more than 80% of the proteins had a CV under 0.7. Interestingly, 83 common proteins were found within the core proteome as defined by the intra- and inter-variation analyses set criteria (CV<0.7). Some of these uniformly expressed proteins were chaperones, peroxiredoxins, isomerases, and cytoskeletal proteins. Although there is a significant level of inter-individual variation in the protein profiles of human sperm heads even in a well-defined group of men with high sperm counts, the consistent expression levels of a wide range of proteins points to their essential role during spermatogenesis. PMID:24204839

  5. Effect of altered eating pattern on serum fructosamine: total protein ratio and plasma glucose level.

    PubMed

    Ch'ng, S L; Cheah, S H; Husain, R; Duncan, M T

    1989-05-01

    The effect of alteration of eating pattern during Ramadan on body mass index (BMI), serum fructosamine: total protein ratio (F/TP), and glucose level in 18 healthy male Asiatic Moslems were studied. The results showed a significant decrease (p less than 0.025) in F/TP at the second week of Ramadan in 11 subjects who experienced continuous decrease in BMI throughout Ramadan. The remaining 7 subjects showed no significant changes in BMI and F/TP. No evidence of hypoglycaemia was observed in the subjects during the study. Serum fructosamine: total protein ratio in subjects with altered eating pattern preferably should be interpreted along with the change in body mass index. PMID:2774480

  6. A Theoretical Lower Bound for Selection on the Expression Levels of Proteins.

    PubMed

    Price, Morgan N; Arkin, Adam P

    2016-01-01

    We use simple models of the costs and benefits of microbial gene expression to show that changing a protein's expression away from its optimum by 2-fold should reduce fitness by at least [Formula: see text], where P is the fraction the cell's protein that the gene accounts for. As microbial genes are usually expressed at above 5 parts per million, and effective population sizes are likely to be above 10(6), this implies that 2-fold changes to gene expression levels are under strong selection, as [Formula: see text], where Ne is the effective population size and s is the selection coefficient. Thus, most gene duplications should be selected against. On the other hand, we predict that for most genes, small changes in the expression will be effectively neutral. PMID:27289091

  7. Utility of C-Reactive Protein Levels for Early Prediction of Dengue Severity in Adults

    PubMed Central

    Chen, Chien-Chih; Lee, Ing-Kit; Liu, Jien-Wei; Huang, Shi-Yu; Wang, Lin

    2015-01-01

    Dengue has broad clinical presentation with unpredictable clinical evolution and outcome. We aimed to evaluate the utility of C-reactive protein (CRP) levels for distinguishing between mild and severe cases in the early phase of the dengue illness. We retrospectively evaluated adults with dengue from 2006 to 2014, according to 1997 and 2009 World Health Organization (WHO) criteria for severity. Of 191 included patients, 32.9% had nonshock dengue hemorrhagic fever (DHF), 3.1% dengue shock syndrome (DSS), and 7.9% severe dengue. The risk of DHF/DSS and severe dengue is significantly related to the increasing levels of CRP. Of 191 patients, 97 had CRP levels measured during the febrile (days 1–3); 85 during the critical (days 4–6); and 9 during the convalescent (days 7–10) illness phases. During the febrile phase, there was significant higher CRP level for DSS versus DF/nonshock DHF and severe dengue versus nonsevere dengue, with CRP cutoff level 30.1 mg/L (area under the receiver operating characteristic curve (AUC), 0.938; 100% sensitivity, 76.3% specificity) and 24.2 mg/L (AUC, 0.717; 70% sensitivity, 71.3% specificity), respectively. Our study highlights the utility of the CRP levels in early prediction of DSS and severe dengue in adult patients. PMID:26247033

  8. Mechanical Folding and Unfolding of Protein Barnase at the Single-Molecule Level.

    PubMed

    Alemany, Anna; Rey-Serra, Blanca; Frutos, Silvia; Cecconi, Ciro; Ritort, Felix

    2016-01-01

    The unfolding and folding of protein barnase has been extensively investigated in bulk conditions under the effect of denaturant and temperature. These experiments provided information about structural and kinetic features of both the native and the unfolded states of the protein, and debates about the possible existence of an intermediate state in the folding pathway have arisen. Here, we investigate the folding/unfolding reaction of protein barnase under the action of mechanical force at the single-molecule level using optical tweezers. We measure unfolding and folding force-dependent kinetic rates from pulling and passive experiments, respectively, and using Kramers-based theories (e.g., Bell-Evans and Dudko-Hummer-Szabo models), we extract the position of the transition state and the height of the kinetic barrier mediating unfolding and folding transitions, finding good agreement with previous bulk measurements. Measurements of the force-dependent kinetic barrier using the continuous effective barrier analysis show that protein barnase verifies the Leffler-Hammond postulate under applied force and allow us to extract its free energy of folding, ΔG0. The estimated value of ΔG0 is in agreement with our predictions obtained using fluctuation relations and previous bulk studies. To address the possible existence of an intermediate state on the folding pathway, we measure the power spectrum of force fluctuations at high temporal resolution (50 kHz) when the protein is either folded or unfolded and, additionally, we study the folding transition-path time at different forces. The finite bandwidth of our experimental setup sets the lifetime of potential intermediate states upon barnase folding/unfolding in the submillisecond timescale. PMID:26745410

  9. An alternative method of enhancing the expression level of heterologous protein in Escherichia coli.

    PubMed

    Yin, Jun; Tian, Hong; Bao, Lichen; Dai, Xin; Gao, Xiangdong; Yao, Wenbing

    2014-12-12

    Though numerous strategy options are available for achieving high expression levels of genes in Escherichia coli, not every gene can be efficiently expressed in this organism. By investigating the relationship between the mRNA secondary structure of translational initiation region (TIR) and gene expression in E.coli, we establish a simple method to design sequences of appropriate TIR (from -35 to +36) that meet a specific expression level as we need. Using this method, overexpression of native human humor necrosis factor α and extracellular domain of Her2/neu protein (aa 23-146) in E. coli were achieved. Differences in expression appeared was mainly related to the efficiency of translation initiation and the stability of mRNA secondary structure, because the intracellular mRNA levels analyzed by real-time RT-PCR were quite similar. Our approach can overcome the steric hindrance of translation startup, and therefore promote translation smoothly to acquire high expression of exogenous protein. PMID:25449272

  10. Physicochemical and sensory characteristics of whey protein hydrolysates generated at different total solids levels.

    PubMed

    Spellman, David; O'Cuinn, Gerard; FitzGerald, Richard J

    2005-05-01

    Whey protein hydrolysates were generated at different total solids (TS) levels (50-300 g/l) using the commercially available proteolytic preparation Debitrase HYW20, while enzyme to substrate ratio, pH and temperature were maintained constant. Hydrolysis proceeded at a faster rate at lower TS reaching a degree of hydrolysis (DH) of 16.6% at 300 g TS/l, compared with a DH of 22.7% at 50 g TS/l after 6 h hydrolysis. The slower breakdown of intact whey proteins at high TS was quantified by gel-permeation HPLC. Reversed-phase (RP) HPLC of hydrolysate samples of equivalent DH (approximately 15%) generated at different TS levels indicated that certain hydrophobic peptide peaks were present at higher levels in hydrolysates generated at low TS. Sensory evaluation showed that hydrolysates with equivalent DH values were significantly (P < 0.0005) less bitter when generated at 300 g TS/l (mean bitterness score = 25.4%) than hydrolysates generated at 50 g TS/l (mean bitterness score = 39.9%). A specific hydrophobic peptide peak present at higher concentrations in hydrolysates generated at low TS was isolated and identified as beta-lactoglobulin f(43-57), a fragment having the physical and chemical characteristics of a bitter peptide. PMID:15909678

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

    PubMed Central

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

    2015-01-01

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

  12. In vitro effect of dietary protein level and nondigestible oligosaccharides on feline fecal microbiota.

    PubMed

    Pinna, C; Stefanelli, C; Biagi, G

    2014-12-01

    The aim of the present study was to evaluate in vitro the effect of some prebiotic substances and 2 dietary protein levels on the composition and activity of feline fecal microbiota. Two in vitro studies were conducted. First, 6 nondigestible oligosaccharides were studied; treatments were control diet (CTRL), gluconic acid (GA), carrot fiber (CF), fructooligosaccharides (FOS), galactooligosaccharides (GOS), lactitol (LAC), and pectins from citrus fruit (PEC). Substrates were added to feline fecal cultures at 2 g/L for 24 h incubation. Compared with the CTRL, ammonia had been reduced (P<0.05) by GOS (-9%) after 6 h and by GA (-14%), LAC (-12%), and PEC (-10%) after 24 h. After 24 h, all treatments had resulted in a lower pH versus the CTRL. Putrescine concentrations at 24 h were greater (P<0.05) in cultures treated with FOS (+90%), GOS (+96%), and LAC (+87%). Compared with the CTRL, total VFA were higher (P<0.05) in bottles containing CF (+41%), whereas the acetic to propionic acid ratio was reduced by LAC (-51%; P<0.05). After 24 h, Enterobacteriaceae had been reduced (P<0.05) by LAC and PEC. In a second study, LAC and FOS were selected to be tested in the presence of 2 diets differing in their protein content. There were 6 treatments: low-protein (LP) CTRL with no addition of prebiotics (CTRL-LP), high-protein (HP) CTRL with no addition of prebiotics (CTRL-HP), LP diet plus FOS, CTRL-HP plus FOS, LP diet plus LAC, and CTRL-HP plus LAC. Both FOS and LAC were added to feline fecal cultures at 2 g/L for 24 h incubation. Ammonia at 24 h was affected (P<0.05) by the protein level (36.2 vs. 50.2 mmol/L for LP and HP, respectively). The CTRL-HPs resulted in a higher pH and increased concentrations of biogenic amines were found after 6 and 24 h of incubation (P<0.05); putrescine at 24 h showed an increase (P<0.05) in cultures treated with FOS. Total VFA were influenced (P<0.05) by the protein level (40.9 vs. 32.6 mmol/L for LP and HP, respectively). At 24 h, the CTRL

  13. The Nuclear Zinc Finger Protein Zfat Maintains FoxO1 Protein Levels in Peripheral T Cells by Regulating the Activities of Autophagy and the Akt Signaling Pathway.

    PubMed

    Ishikura, Shuhei; Iwaihara, Yuri; Tanaka, Yoko; Luo, Hao; Nishi, Kensuke; Doi, Keiko; Koyanagi, Midori; Okamura, Tadashi; Tsunoda, Toshiyuki; Shirasawa, Senji

    2016-07-15

    Forkhead box O1 (FoxO1) is a key molecule for the development and functions of peripheral T cells. However, the precise mechanisms regulating FoxO1 expression in peripheral T cells remain elusive. We previously reported that Zfat(f/f)-CD4Cre mice showed a marked decline in FoxO1 protein levels in peripheral T cells, partially through proteasomal degradation. Here we have identified the precise mechanisms, apart from proteasome-mediated degradation, of the decreased FoxO1 levels in Zfat-deficient T cells. First, we confirmed that tamoxifen-inducible deletion of Zfat in Zfat(f/f)-CreERT2 mice coincidently decreases FoxO1 protein levels in peripheral T cells, indicating that Zfat is essential for maintaining FoxO1 levels in these cells. Although the proteasome-specific inhibitors lactacystin and epoxomicin only moderately increase FoxO1 protein levels, the inhibitors of lysosomal proteolysis bafilomycin A1 and chloroquine restore the decreased FoxO1 levels in Zfat-deficient T cells to levels comparable with those in control cells. Furthermore, Zfat-deficient T cells show increased numbers of autophagosomes and decreased levels of p62 protein, together indicating that Zfat deficiency promotes lysosomal FoxO1 degradation through autophagy. In addition, Zfat deficiency increases the phosphorylation levels of Thr-308 and Ser-473 of Akt and the relative amounts of cytoplasmic to nuclear FoxO1 protein levels, indicating that Zfat deficiency causes Akt activation, leading to nuclear exclusion of FoxO1. Our findings have demonstrated a novel role of Zfat in maintaining FoxO1 protein levels in peripheral T cells by regulating the activities of autophagy and the Akt signaling pathway. PMID:27226588

  14. The Prognostic Value of C-Reactive Protein Serum Levels in Patients with Uterine Leiomyosarcoma

    PubMed Central

    Schwameis, Richard; Grimm, Christoph; Petru, Edgar; Natter, Camilla; Staudigl, Christine; Lamm, Wolfgang; Koelbl, Heinz; Krainer, Michael; Brodowicz, Thomas; Reinthaller, Alexander; Polterauer, Stephan

    2015-01-01

    Objective C-reactive protein (CRP) has previously been shown to serve as a prognostic parameter in women with gynecologic malignancies. Due to the lack of valid prognostic markers for uterine leiomyosarcoma (ULMS) this study set out to investigate the value of pre-treatment CRP serum levels as prognostic parameter. Methods Data of women with ULMS were extracted from databases of three Austrian centres for gynaecologic oncology. Pre-treatment CRP serum levels were measured and correlated with clinico-pathological parameters. Univariate and multivariable survival analyses were performed. Results In total, 53 patients with ULMS were included into the analysis. Mean (SD) CRP serum level was 3.46 mg/dL (3.96). Solely, an association between pre-treatment CRP serum levels and tumor size (p = 0.04) but no other clinic-pathologic parameter such as tumor stage (p = 0.16), or histological grade (p = 0.07), was observed. Univariate and multivariable survival analyses revealed that CRP serum levels (HR 2.7 [1.1–7.2], p = 0.037) and tumor stage (HR 6.1 [1.9–19.5], p = 0.002) were the only independent prognostic factors for overall survival (OS) in patients with ULMS. Patients with high pre-treatment CRP serum levels showed impaired OS compared to women with low levels (5-year-OS rates: 22.6% and 52.3%, p = 0.007). Conclusion High pre-treatment CRP serum levels were independently associated with impaired prognosis in women with ULMS and might serve as a prognostic parameter in these patients. PMID:26248232

  15. Generalized Anxiety and C-Reactive Protein Levels: A Prospective, Longitudinal Analysis

    PubMed Central

    Copeland, William E.; Shanahan, Lilly; Worthman, Carol; Angold, Adrian; Costello, E. Jane

    2012-01-01

    Background Generalized Anxiety Disorder (GAD) is highly comorbid with depression. Depression is associated with elevated levels of the inflammation marker C-reactive protein (CRP), cross-sectionally and over time. To date, no studies have looked at the association of CRP with GAD. Methods Ten waves of data from the prospective population-based Great Smoky Mountains Study (N = 1,420) were used, covering children in the community aged 9-16, 19, and 21 years old. Structured interviews were used at each assessment to assess GAD symptoms, diagnosis, and cumulative episodes. Bloodspots were collected and assayed for CRP levels. Results GAD was associated with increased levels of CRP in bivariate cross-sectional analyses. These bivariate associations, however, were attenuated after accounting for demographic, substance use, and health-related covariates. In longitudinal models, there was little evidence that CRP predicted later GAD. Associations from GAD to later CRP were attenuated in models adjusted for health-related coavariates and there was evidence that the GAD-CRP association was mediated by BMI and medication use. Conclusions Similar to depression, GAD was associated with elevated levels of CRP, but the effect of GAD on CRP levels was explained by the effect of GAD on health-related behaviors such as BMI and medication use. This study suggests differences in the association between inflammation and depression and GAD. PMID:22716910

  16. The impact of dietary protein levels on nutrient digestibility and water and nitrogen balances in eventing horses.

    PubMed

    Oliveira, C A A; Azevedo, J F; Martins, J A; Barreto, M P; Silva, V P; Julliand, V; Almeida, F Q

    2015-01-01

    This study was performed to evaluate the impact of dietary protein levels on nutrient digestibility and water and nitrogen balances in conditioning eventing horses. Twenty-four Brazilian Sport Horses, male and female (8.0 to 15.0 yr; 488 ± 32 kg BW), were used in a randomized design with 4 levels of CP diets: 7.5%, 9.0%, 11.0%, and 13.0%. A digestion assay was performed with partial feces collection over 4 d, followed by 1 d of total urine collection. Data were submitted to regression analysis and adjusted to linear and quadratic models (P < 0.05). No differences were observed in the intake of DM, OM, EE, ADF, and NDF as a function of dietary protein levels. Dry matter intake average was 1.7% of BW. CP and N intake showed a linear increase as a function of increasing protein level in diets. A quadratic response (P < 0.05) was observed on the CP and NDF digestibility coefficients, with the maximum estimated level of digestibility at 11.6% and 11.4% CP in the diet, respectively. There was a linear effect on ADF digestibility coefficients, digestible DM and protein intake, and CP/DE ratio according to dietary protein levels. There was no impact of dietary protein levels on daily water intake, total water intake, or fecal water excretion. Urinary excretion values showed a linear increase in response to increased dietary protein levels, but no impact was observed on water balance, with an average of 8.4 L/d. Nitrogen intake (NI), N absorption (NA), and urinary N increased linearly as a function of increasing dietary protein levels. There was no impact of dietary protein levels on N retention (NR), with an average of 7.5 g N/d. Nitrogen retention as a percentage of NI or NA showed no significant changes in the function of dietary protein levels. There was an impact of dietary protein levels on the digestibility coefficient of CP, NDF, ADF, and digestible protein intake on conditioning eventing horses. The 11.6% CP level in the diet provided an intake of 2.25 g CP/kg BW

  17. How Integrated Management Strategies Promote Protein Quality of Cotton Embryos: High Levels of Soil Available N, N Assimilation and Protein Accumulation Rate.

    PubMed

    Yang, HongKun; Meng, YaLi; Chen, BingLin; Zhang, XingYue; Wang, YouHua; Zhao, WenQing; Zhou, ZhiGuo

    2016-01-01

    Cottonseed is widely used as a source of ruminant feed and for industrial purposes. Therefore, there is a tremendous need to improve the nutritional value of cotton embryos. In this study, a conventional management (CM) and two integrated cotton management strategies (IMS1, IMS2) were performed at two soil fertility levels to study the relationships among soil N, N assimilation, embryonic protein accumulation and protein quality. The levels of proteins, essential amino acids, and semi-essential amino acids, especially those of glutamate, lysine, and methionine, were higher in IMS1 and IMS2 embryos than in CM embryos. These changes were significantly positively correlated with the soil-available N content, glutamine synthetase activity and peak value of protein accumulation rate and were negatively correlated with the free amino acid level. These results illustrated that integrated management strategies, especially the rates and timing of N application, raise the level of soil available N, which is beneficial for N assimilation in developing cotton embryos. The protein content was limited by the rate of protein accumulation rather than by the free amino acid content. The combination of target yield fertilization, a growth-driven N application schedule, a high plant density and the seedling raising with bio-organic fertilizer can substantially improve protein quality in cotton embryos, especially at a soil with low soil organic matter and total nitrogen. PMID:27532007

  18. How Integrated Management Strategies Promote Protein Quality of Cotton Embryos: High Levels of Soil Available N, N Assimilation and Protein Accumulation Rate

    PubMed Central

    Yang, HongKun; Meng, YaLi; Chen, BingLin; Zhang, XingYue; Wang, YouHua; Zhao, WenQing; Zhou, ZhiGuo

    2016-01-01

    Cottonseed is widely used as a source of ruminant feed and for industrial purposes. Therefore, there is a tremendous need to improve the nutritional value of cotton embryos. In this study, a conventional management (CM) and two integrated cotton management strategies (IMS1, IMS2) were performed at two soil fertility levels to study the relationships among soil N, N assimilation, embryonic protein accumulation and protein quality. The levels of proteins, essential amino acids, and semi-essential amino acids, especially those of glutamate, lysine, and methionine, were higher in IMS1 and IMS2 embryos than in CM embryos. These changes were significantly positively correlated with the soil-available N content, glutamine synthetase activity and peak value of protein accumulation rate and were negatively correlated with the free amino acid level. These results illustrated that integrated management strategies, especially the rates and timing of N application, raise the level of soil available N, which is beneficial for N assimilation in developing cotton embryos. The protein content was limited by the rate of protein accumulation rather than by the free amino acid content. The combination of target yield fertilization, a growth-driven N application schedule, a high plant density and the seedling raising with bio-organic fertilizer can substantially improve protein quality in cotton embryos, especially at a soil with low soil organic matter and total nitrogen. PMID:27532007

  19. BAG2 promotes tumorigenesis through enhancing mutant p53 protein levels and function

    PubMed Central

    Yue, Xuetian; Zhao, Yuhan; Liu, Juan; Zhang, Cen; Yu, Haiyang; Wang, Jiabei; Zheng, Tongsen; Liu, Lianxin; Li, Jun; Feng, Zhaohui; Hu, Wenwei

    2015-01-01

    Tumor suppressor p53 is the most frequently mutated gene in tumors. Many mutant p53 (mutp53) proteins promote tumorigenesis through the gain-of-function (GOF) mechanism. Mutp53 proteins often accumulate to high levels in tumors, which is critical for mutp53 GOF. Its underlying mechanism is poorly understood. Here, we found that BAG2, a protein of Bcl-2 associated athanogene (BAG) family, promotes mutp53 accumulation and GOF in tumors. Mechanistically, BAG2 binds to mutp53 and translocates to the nucleus to inhibit the MDM2-mutp53 interaction, and MDM2-mediated ubiquitination and degradation of mutp53. Thus, BAG2 promotes mutp53 accumulation and GOF in tumor growth, metastasis and chemoresistance. BAG2 is frequently overexpressed in tumors. BAG2 overexpression is associated with poor prognosis in patients and mutp53 accumulation in tumors. These findings revealed a novel and important mechanism for mutp53 accumulation and GOF in tumors, and also uncovered an important role of BAG2 in tumorigenesis through promoting mutp53 accumulation and GOF. DOI: http://dx.doi.org/10.7554/eLife.08401.001 PMID:26271008

  20. Suppression of cytochrome P450 3A protein levels by proteasome inhibitors.

    SciTech Connect

    Zangar, Richard C. ); Kocarek, Thomas A.; Shen, Shang; Bollinger, Nikki ); Dahn, Michael S.; Lee, Donna W.

    2003-06-01

    We have previously reported that CYP3A cross-links with polyubiquitinated proteins in microsomes from nicardipine-treated rats in a process that is distinct from classical polyubiquitination. To further examine the role of the proteasome in CYP3A degradation, we investigated the effects of proteasome inhibitors lactacystin, MG132, proteasome inhibitor 1, and hemin in primary cultures of rat and human hepatocytes. With the exception of hemin, these agents increased the total pool of ubiquitinated proteins in microsomes isolated from rat hepatocytes, indicating that lactacystin, MG132, and proteasome inhibitor 1 effectively inhibited the proteasome in these cells. All four agents caused a reduction in the amount of the major approximately 55-kDa CYP3A band, opposite to what would be expected if the ubiquitin-proteasome pathway degraded CYP3A. Only hemin treatment caused an increase in high molecular mass (HMM) CYP3A bands. Because hemin treatment did not alter levels of ubiquitin in CYP3 A immunoprecipitates, the HMM CYP3A bands formed in response to hemin treatment clearly were not due to proteasome inhibition. Rather, because hemin treatment also caused an increase in HMM CYP3A in the detergent-insoluble fraction of the 10,000g pellet, the HMM CYP3A seems to represent a large protein complex that is unlikely to primarily represent ubiquitination.

  1. The level of major urinary proteins is socially regulated in wild Mus musculus musculus.

    PubMed

    Janotova, Katerina; Stopka, Pavel

    2011-06-01

    Major urinary proteins (MUPs) are highly polymorphic proteins that have been shown to perform several important functions in the chemical communication of the house mouse, Mus musculus. Production of these proteins in C57Bl/6 females is cyclic, reaching the maximum just before the beginning of estrus. Social environment is an important factor that increases MUP production in both sexes. We examined responsiveness of MUP production to social stimuli in wild mice, Mus musculus musculus. The direction of change of MUP production in males depended on the sex of the stimulus animal. Males up-regulated MUP production when caged with a female, but down-regulated MUP production when caged with a male. Down-regulation was more pronounced in males that were defeated in a male-male encounter. Females responded to a male's presence with a decrease in MUP production. We conclude that social modulation of MUP production is specific and, in coordination with other mechanisms, facilitates adjustment of the animal's odor profile to different social contexts. Our results also suggest that in males, MUPs may play an important role in advertizing the male's quality to females. Furthermore, we highlight the importance of analyzing data corrected with creatinine, which show MUP production on the (post)translational level as well as raw data (non-corrected with creatinine), which represent actual concentrations of MUPs in the urine. PMID:21594616

  2. Fluctuations in Species-Level Protein Expression Occur during Element and Nutrient Cycling in the Subsurface

    PubMed Central

    Wilkins, Michael J.; Wrighton, Kelly C.; Nicora, Carrie D.; Williams, Kenneth H.; McCue, Lee Ann; Handley, Kim M.; Miller, Chris S.; Giloteaux, Ludovic; Montgomery, Alison P.; Lovley, Derek R.; Banfield, Jillian F.; Long, Philip E.; Lipton, Mary S.

    2013-01-01

    While microbial activities in environmental systems play a key role in the utilization and cycling of essential elements and compounds, microbial activity and growth frequently fluctuates in response to environmental stimuli and perturbations. To investigate these fluctuations within a saturated aquifer system, we monitored a carbon-stimulated in situ Geobacter population while iron reduction was occurring, using 16S rRNA abundances and high-resolution tandem mass spectrometry proteome measurements. Following carbon amendment, 16S rRNA analysis of temporally separated samples revealed the rapid enrichment of Geobacter-like environmental strains with strong similarity to G. bemidjiensis. Tandem mass spectrometry proteomics measurements suggest high carbon flux through Geobacter respiratory pathways, and the synthesis of anapleurotic four carbon compounds from acetyl-CoA via pyruvate ferredoxin oxidoreductase activity. Across a 40-day period where Fe(III) reduction was occurring, fluctuations in protein expression reflected changes in anabolic versus catabolic reactions, with increased levels of biosynthesis occurring soon after acetate arrival in the aquifer. In addition, localized shifts in nutrient limitation were inferred based on expression of nitrogenase enzymes and phosphate uptake proteins. These temporal data offer the first example of differing microbial protein expression associated with changing geochemical conditions in a subsurface environment. PMID:23472107

  3. Postnatal alterations in GABAB receptor tone produce sensorimotor gating deficits and protein level differences in adulthood.

    PubMed

    Bolton, Monica M; Heaney, Chelcie F; Murtishaw, Andrew S; Sabbagh, Jonathan J; Magcalas, Christy M; Kinney, Jefferson W

    2015-04-01

    The GABA transmitter system plays a vital role in modulating synaptic formation and activity during development. The GABAB receptor subtype in particular has been implicated in cell migration, promotion of neuronal differentiation, neurite outgrowth, and synapse formation but it's role in development is not well characterized. In order to investigate the effects of brief alterations in GABAB signaling in development, we administered to rats the GABAB agonist baclofen (2.0mg/kg) or antagonist phaclofen (0.3mg/kg) on postnatal days 7, 9, and 12, and evaluated sensorimotor gating in adulthood. We also examined tissue for changes in multiple proteins associated with GABAB receptor function and proteins associated with synapse formation. Our data indicate that early postnatal alterations to GABAB receptor-mediated signaling produced sex differences in sensorimotor gating in adulthood. Additionally, we found differences in GABAB receptor subunits and kalirin protein levels in the brain versus saline treated controls. Our data demonstrate that a subtle alteration in GABAB receptor function in early postnatal life induces changes that persist into adulthood. PMID:25314921

  4. Dependence of intestinal amino acid uptake on dietary protein or amino acid levels

    SciTech Connect

    Karasov, W.H.; Solberg, D.H.; Diamond, J.M.

    1987-05-01

    To understand how intestinal amino acid (AA) transport is regulated by dietary substrate levels, the authors measured uptake of seven radioactively-labelled AAs and glucose across the jejunal brush-border membrane of mice kept on one of three isocaloric rations differing in nitrogen content. In the high-protein ration, uptake increased by 77-81% for the nonessential, less toxic AAs, proline, and aspartate but only by 32-61% for the more toxic essential AAs tested. In the nitrogen-deficient ration, uptake decreased for the nonessential aspartate and proline but stayed constant or increased for essential AAs and for the nonessential alanine. These patterns imply independent regulation of the intestine's various AA transporters. With decreasing dietary AA (or protein), the imino acid and acidic AA private transporters are repressed, while activities of the basic AA transporter and the neutral AA public transporter decrease to an asymptote or else go through a minimum. These regulatory patterns can be understood as a compromise among conflicting constraints imposed by protein's multiple roles as a source of calories, nitrogen, and essential AAs and by the toxicity of essential AAs at high concentrations.

  5. Fluctuations in species-level protein expression occur during element and nutrient cycling in the subsurface.

    PubMed

    Wilkins, Michael J; Wrighton, Kelly C; Nicora, Carrie D; Williams, Kenneth H; McCue, Lee Ann; Handley, Kim M; Miller, Chris S; Giloteaux, Ludovic; Montgomery, Alison P; Lovley, Derek R; Banfield, Jillian F; Long, Philip E; Lipton, Mary S

    2013-01-01

    While microbial activities in environmental systems play a key role in the utilization and cycling of essential elements and compounds, microbial activity and growth frequently fluctuates in response to environmental stimuli and perturbations. To investigate these fluctuations within a saturated aquifer system, we monitored a carbon-stimulated in situ Geobacter population while iron reduction was occurring, using 16S rRNA abundances and high-resolution tandem mass spectrometry proteome measurements. Following carbon amendment, 16S rRNA analysis of temporally separated samples revealed the rapid enrichment of Geobacter-like environmental strains with strong similarity to G. bemidjiensis. Tandem mass spectrometry proteomics measurements suggest high carbon flux through Geobacter respiratory pathways, and the synthesis of anapleurotic four carbon compounds from acetyl-CoA via pyruvate ferredoxin oxidoreductase activity. Across a 40-day period where Fe(III) reduction was occurring, fluctuations in protein expression reflected changes in anabolic versus catabolic reactions, with increased levels of biosynthesis occurring soon after acetate arrival in the aquifer. In addition, localized shifts in nutrient limitation were inferred based on expression of nitrogenase enzymes and phosphate uptake proteins. These temporal data offer the first example of differing microbial protein expression associated with changing geochemical conditions in a subsurface environment. PMID:23472107

  6. Fluctuations in Species-Level Protein Expression Occur during Element and Nutrient Cycling in the Subsurface

    SciTech Connect

    Wilkins, Michael J.; Wrighton, Kelly C.; Nicora, Carrie D.; Williams, Kenneth H.; McCue, Lee Ann; Handley, Kim M.; Miller, C. S.; Giloteaux, L.; Montgomery, A. P.; Lovley, Derek R.; Banfield, Jillian F.; Long, Philip E.; Lipton, Mary S.

    2013-03-05

    While microbial activities in environmental systems play a key role in the utilization and cycling of essential elements and compounds, microbial activity and growth frequently fluctuates in response to environmental stimuli and perturbations. To investigate these fluctuations within a saturated aquifer system, we monitored a carbon-stimulated in situ Geobacter population while iron reduction was occurring, using 16S rRNA abundances and high-resolution tandem mass spectrometry proteome measurements. Following carbon amendment, 16S rRNA analysis of temporally separated samples revealed the rapid enrichment of Geobacter-like environmental strains with strong similarity to G. bemidjiensis. Tandem mass spectrometry proteomics measurements suggest high carbon flux through Geobacter respiratory pathways, and the synthesis of anapleurotic four carbon compounds from acetyl-CoA via pyruvate ferredoxin oxidoreductase activity. Across a 40-day period where Fe(III) reduction was occurring, fluctuations in protein expression reflected changes in anabolic versus catabolic reactions, with increased levels of biosynthesis occurring soon after acetate arrival in the aquifer. In addition, localized shifts in nutrient limitation were inferred based on expression of nitrogenase enzymes and phosphate uptake proteins. These temporal data offer the first example of differing microbial protein expression associated with changing geochemical conditions in a subsurface environment.

  7. Racial Differences in C-Reactive Protein Levels During Normal Pregnancy

    PubMed Central

    Picklesimer, Amy H; Jared, Heather L.; Moss, Kevin; Offenbacher, Steven; Beck, James D; Boggess, Kim A

    2008-01-01

    Objective Characterization of serum C-Reactive Protein (CRP) levels in a diverse population of healthy pregnant women using a high sensitivity assay. Study Design Cross-sectional analysis of a cohort of 775 pregnant women. CRP measured on serum specimens drawn <26 weeks gestation using highly sensitive ELISA kits. Results Median CRP was 4.8 mg/L (inter-quartile range 0.63 – 15.7). Black women had higher median CRP values than whites (7.68 mg/L vs 2.59 mg/L, p<.001). Black women demonstrated higher levels of CRP even after controlling for known confounding factors such as smoking and maternal weight. Conclusion Pregnancy is an inflammatory stressor. The etiology of racial differences is unclear, but may be important for understanding racial disparities in the incidence inflammatory disorders such as preterm labor and pre-eclampsia. PMID:18539258

  8. Cypermethrin alters Glial Fibrillary Acidic Protein levels in the rat brain.

    PubMed

    Malkiewicz, Katarzyna; Koteras, Marcin; Folkesson, Ronnie; Brzezinski, Jacek; Winblad, Bengt; Szutowski, Miroslaw; Benedikz, Eirikur

    2006-01-01

    Pyrethroids, widely used insecticides, are biologically active in neurons. Whether they act on the non-neuronal brain cells remains an open question. Thus, the aim of this study was to examine whether Cypermethrin intoxication affects astroglial cells in the rat brain. The levels of Glial Fibrillary Acidic Protein (GFAP) in different brain regions were measured by ELISA following oral treatment with 5 or 10% of LD(50) of Cypermethrin per day for 6 days. A significant decrease of GFAP was observed in different brain regions of treated animals. The cerebral cortex showed the most pronounced effect with GFAP levels reduced to 81% of the controls 2 days after treatment and 77% 21 days after treatment. Although we did not find profound changes in the morphology of astrocytes in Cypermethrin treated animals, the decrease in GFAP suggests that astrocytes were affected by low doses of pyrethroids. The possible consequences were discussed. PMID:21783638

  9. Increased CSF Levels of Phosphorylated Neurofilament Heavy Protein following Bout in Amateur Boxers

    PubMed Central

    Neselius, Sanna; Zetterberg, Henrik; Blennow, Kaj; Marcusson, Jan; Brisby, Helena

    2013-01-01

    Introduction Diagnosis of mild TBI is hampered by the lack of imaging or biochemical measurements for identifying or quantifying mild TBI in a clinical setting. We have previously shown increased biomarker levels of protein reflecting axonal (neurofilament light protein and tau) and glial (GFAP and S-100B) damage in cerebrospinal fluid (CSF) after a boxing bout. The aims of this study were to find other biomarkers of mild TBI, which may help clinicians diagnose and monitor mild TBI, and to calculate the role of APOE ε4 allele genotype which has been associated with poor outcome after TBI. Materials and Methods Thirty amateur boxers with a minimum of 45 bouts and 25 non-boxing matched controls were included in a prospective cohort study. CSF and blood were collected at one occasion between 1 and 6 days after a bout, and after a rest period for at least 14 days (follow up). The controls were tested once. CSF levels of neurofilament heavy (pNFH), amyloid precursor proteins (sAPPα and sAPPβ), ApoE and ApoA1 were analyzed. In blood, plasma levels of Aβ42 and ApoE genotype were analyzed. Results CSF levels of pNFH were significantly increased between 1 and 6 days after boxing as compared with controls (p<0.001). The concentrations decreased at follow up but were still significantly increased compared to controls (p = 0.018). CSF pNFH concentrations correlated with NFL (r =  0.57 after bout and 0.64 at follow up, p<0.001). No significant change was found in the other biomarkers, as compared to controls. Boxers carrying the APOE ε4 allele had similar biomarker concentrations as non-carriers. Conclusions Subconcussive repetitive trauma in amateur boxing causes a mild TBI that may be diagnosed by CSF analysis of pNFH, even without unconsciousness or concussion symptoms. Possession of the APOE ε4 allele was not found to influence biomarker levels after acute TBI. PMID:24260563

  10. Ck2-Dependent Phosphorylation Is Required to Maintain Pax7 Protein Levels in Proliferating Muscle Progenitors

    PubMed Central

    González, Natalia; Moresco, James J.; Bustos, Francisco; Yates, John R.; Olguín, Hugo C.

    2016-01-01

    Skeletal muscle regeneration and long term maintenance is directly link to the balance between self-renewal and differentiation of resident adult stem cells known as satellite cells. In turn, satellite cell fate is influenced by a functional interaction between the transcription factor Pax7 and members of the MyoD family of muscle regulatory factors. Thus, changes in the Pax7-to-MyoD protein ratio may act as a molecular rheostat fine-tuning acquisition of lineage identity while preventing precocious terminal differentiation. Pax7 is expressed in quiescent and proliferating satellite cells, while its levels decrease sharply in differentiating progenitors Pax7 is maintained in cells (re)acquiring quiescence. While the mechanisms regulating Pax7 levels based on differentiation status are not well understood, we have recently described that Pax7 levels are directly regulated by the ubiquitin-ligase Nedd4, thus promoting proteasome-dependent Pax7 degradation in differentiating satellite cells. Here we show that Pax7 levels are maintained in proliferating muscle progenitors by a mechanism involving casein kinase 2-dependent Pax7 phosphorylation at S201. Point mutations preventing S201 phosphorylation or casein kinase 2 inhibition result in decreased Pax7 protein in proliferating muscle progenitors. Accordingly, this correlates directly with increased Pax7 ubiquitination. Finally, Pax7 down regulation induced by casein kinase 2 inhibition results in precocious myogenic induction, indicating early commitment to terminal differentiation. These observations highlight the critical role of post translational regulation of Pax7 as a molecular switch controlling muscle progenitor fate. PMID:27144531

  11. Ck2-Dependent Phosphorylation Is Required to Maintain Pax7 Protein Levels in Proliferating Muscle Progenitors.

    PubMed

    González, Natalia; Moresco, James J; Cabezas, Felipe; de la Vega, Eduardo; Bustos, Francisco; Yates, John R; Olguín, Hugo C

    2016-01-01

    Skeletal muscle regeneration and long term maintenance is directly link to the balance between self-renewal and differentiation of resident adult stem cells known as satellite cells. In turn, satellite cell fate is influenced by a functional interaction between the transcription factor Pax7 and members of the MyoD family of muscle regulatory factors. Thus, changes in the Pax7-to-MyoD protein ratio may act as a molecular rheostat fine-tuning acquisition of lineage identity while preventing precocious terminal differentiation. Pax7 is expressed in quiescent and proliferating satellite cells, while its levels decrease sharply in differentiating progenitors Pax7 is maintained in cells (re)acquiring quiescence. While the mechanisms regulating Pax7 levels based on differentiation status are not well understood, we have recently described that Pax7 levels are directly regulated by the ubiquitin-ligase Nedd4, thus promoting proteasome-dependent Pax7 degradation in differentiating satellite cells. Here we show that Pax7 levels are maintained in proliferating muscle progenitors by a mechanism involving casein kinase 2-dependent Pax7 phosphorylation at S201. Point mutations preventing S201 phosphorylation or casein kinase 2 inhibition result in decreased Pax7 protein in proliferating muscle progenitors. Accordingly, this correlates directly with increased Pax7 ubiquitination. Finally, Pax7 down regulation induced by casein kinase 2 inhibition results in precocious myogenic induction, indicating early commitment to terminal differentiation. These observations highlight the critical role of post translational regulation of Pax7 as a molecular switch controlling muscle progenitor fate. PMID:27144531

  12. Effect of temperature on oxidative stress, antioxidant levels and uncoupling protein expression in striped hamsters.

    PubMed

    Zhou, Si-Si; Cao, Li-Li; Xu, Wei-Dong; Cao, Jing; Zhao, Zhi-Jun

    2015-11-01

    According to the rate of living-free radical hypothesis, higher metabolic rates should increase reactive oxygen species (ROS) production. However, the "uncoupling to survive" hypothesis postulates that uncoupling proteins (UCPs) can decrease ROS production by lowering the potential of the inner mitochondrial membrane, in which case the correlation between metabolic rate and ROS levels would be a negative rather than positive. In this study, we examined energy intake, oxidative stress levels, antioxidant activity and the expression of UCPs in brown adipose tissue (BAT), and in the liver, heart, skeletal muscle and brain, of striped hamsters (Cricetulus barabensis) acclimated to either 5 °C or 32.5 °C. The energy intake of hamsters acclimated to 5 °C increased by 70.7%, whereas the energy intake of hamsters acclimated to 32.5 °C decreased by 31.3%, relative to hamsters kept at room temperature (21 °C) (P<0.05). Malonadialdehyde (MDA) levels, total antioxidant capacity (T-AOC) and glutathione peroxidase (GSH-PX) activity in BAT significantly decreased in 5 °C group, but increased in 32.5 °C group, relative to the 21 °C group. Neither ROS levels (i.e. H2O2 levels), nor antioxidants in skeletal muscle, liver, heart or brain tissue, were affected by temperature. UCP1 expression in BAT was significantly up-regulated in 5 °C group, but down-regulated in 32.5 °C group, relative to the 21 °C group. UCP3 expression of skeletal muscle was also up-regulated significantly in hamsters acclimated to 5 °C. These results suggest that the relationship between ROS levels and metabolic rate was negative, rather than positive. UCP1 expression in BAT may have played a role in lowering ROS levels. PMID:26244518

  13. Infection and immunoglobulin levels in Sudanese children with severe protein-energy malnutrition*

    PubMed Central

    Suliman, Omer S. M.; Salih, Mustafa A. M.; Karrar, Zein A.; Mohammed, Abdelrahim O.; Helsing, Chrestover

    2011-01-01

    A hospital-based case control study was carried out to determine the pattern of infections and immunoblobulin levels in Sudanese children with severe protein energy malnutrition (PEM). The pre-dietary rehabilitation levels of the three major immunoglobulins (IgG, IgA and IgM) were compared with those of normal controls, and with the levels after dietary rehabilitation. Eighty one children were included in the study: 49 with severe PEM (23 with marasmus, 17 with marasmic – kwashiorkor and 9 with kwashiorkor), 13 with tuberculosis and 19 healthy children as controls. The study showed high incidence of infections, especially pneumonia and gastrointestinal infections in the malnourished children. Of special concern was the high incidence of urinary tract infection: 13 (26.5%) had significant pyuria and 9 of them had positive urine cultures, mainly Escherichia coli. Eight of the malnourished children also had pulmonary TB, and the ESR and Mantoux tests were not helpful in the diagnosis. The Mantoux test was negative in 88.8% of the malnourished group compared to 62.5% in those malnourished with TB. The malnourished groups had significantly higher plasma levels of the 3 immunoglobulins. While the maramic group attained significantly higher levels of IgG and IgA compared to the marasmic –kwashiorkor and kwashiorkor groups, the 3 groups of PEM showed a uniformly higher level of the IgM. After 2 weeks of rehabilitation, the levels of the 3 immunoglobulins showed no significant changes, except for the IgA which significantly decreased in all malnourished and the oedematous groups, and the IgM which increased significantly in the oedematous group.

  14. Effect of nutrient deprivation on lipid, carbohydrate, DNA, RNA, and protein levels in Vibrio cholerae.

    PubMed Central

    Hood, M A; Guckert, J B; White, D C; Deck, F

    1986-01-01

    The response of Vibrio cholerae to low nutrient levels was determined by measuring the concentrations of lipids, carbohydrates, DNA, RNA, and proteins over a 30-day starvation period. Ultrastructural integrity was observed by transmission electron microscopy. Total lipids and carbohydrates declined rapidly within the first 7 days, while DNA and protein exhibited a more constant decline over the 30 days of starvation. In contrast, RNA showed little decrease upon starvation. Although neutral lipids were lost, the percentage of neutral lipids did not decline as rapidly as the phospholipids. Detectable levels of poly-beta-hydroxybutyrate disappeared completely by 7 days. Carbohydrate profiles revealed the relative loss of the five-carbon sugar ribose and N-acetylglucosamine and a relative increase in the total six-carbon sugars, especially glucose. Morphologically, ribosomes appeared to exhibit no structural change, while inclusion bodies and mesosomelike structures disappeared completely, and cell wall and membrane integrity was lost. The data suggest that V. cholerae differs somewhat from other marine vibrios in its response to low nutrients but shares some characteristics in common with them. The data also suggest that certain lipids and carbohydrates may provide the endogenous energy sources needed for dormancy preparation and cell maintenance under nutrient starvation. Images PMID:2430523

  15. Myb-domain protein Teb1 controls histone levels and centromere assembly in fission yeast

    PubMed Central

    Valente, Luis P; Dehé, Pierre-Marie; Klutstein, Michael; Aligianni, Sofia; Watt, Stephen; Bähler, Jürg; Promisel Cooper, Julia

    2013-01-01

    The TTAGGG motif is common to two seemingly unrelated dimensions of chromatin function—the vertebrate telomere repeat and the promoter regions of many Schizosaccharomyces pombe genes, including all of those encoding canonical histones. The essential S. pombe protein Teb1 contains two Myb-like DNA binding domains related to those found in telomere proteins and binds the human telomere repeat sequence TTAGGG. Here, we analyse Teb1 binding throughout the genome and the consequences of reduced Teb1 function. Chromatin immunoprecipitation (ChIP)-on-chip analysis reveals robust Teb1 binding at many promoters, notably including all of those controlling canonical histone gene expression. A hypomorphic allele, teb1-1, confers reduced binding and reduced levels of histone transcripts. Prompted by previously suggested connections between histone expression and centromere identity, we examined localization of the centromeric histone H3 variant Cnp1 and found reduced centromeric binding along with reduced centromeric silencing. These data identify Teb1 as a master regulator of histone levels and centromere identity. PMID:23314747

  16. Myb-domain protein Teb1 controls histone levels and centromere assembly in fission yeast.

    PubMed

    Valente, Luis P; Dehé, Pierre-Marie; Klutstein, Michael; Aligianni, Sofia; Watt, Stephen; Bähler, Jürg; Cooper, Julia Promisel

    2013-02-01

    The TTAGGG motif is common to two seemingly unrelated dimensions of chromatin function-the vertebrate telomere repeat and the promoter regions of many Schizosaccharomyces pombe genes, including all of those encoding canonical histones. The essential S. pombe protein Teb1 contains two Myb-like DNA binding domains related to those found in telomere proteins and binds the human telomere repeat sequence TTAGGG. Here, we analyse Teb1 binding throughout the genome and the consequences of reduced Teb1 function. Chromatin immunoprecipitation (ChIP)-on-chip analysis reveals robust Teb1 binding at many promoters, notably including all of those controlling canonical histone gene expression. A hypomorphic allele, teb1-1, confers reduced binding and reduced levels of histone transcripts. Prompted by previously suggested connections between histone expression and centromere identity, we examined localization of the centromeric histone H3 variant Cnp1 and found reduced centromeric binding along with reduced centromeric silencing. These data identify Teb1 as a master regulator of histone levels and centromere identity. PMID:23314747

  17. Maternal High Fat Diet Affects Offspring’s Vitamin K-Dependent Proteins Expression Levels

    PubMed Central

    Lanham, Stuart; Cagampang, Felino R.; Oreffo, Richard O. C.

    2015-01-01

    Studies suggest bone growth & development and susceptibility to vascular disease in later life are influenced by maternal nutrition, during intrauterine and early postnatal life. There is evidence for a role of vitamin K-dependent proteins (VKDPs) including Osteocalcin, Matrix-gla protein, Periostin, and Gas6, in bone and vascular development. This study extends the analysis of VKDPs previously conducted in 6 week old offspring, into offspring of 30 weeks of age, to assess the longer term effects of a maternal and postnatal high fat (HF) diet on VKDP expression. Overall a HF maternal diet and offspring diet exacerbated the bone changes observed. Sex specific and tissue specific differences were observed in VKDP expression for both aorta and femoral tissues. In addition, significant correlations were observed between femoral OCN, Periostin Gas6, and Vkor expression levels and measures of femoral bone structure. Furthermore, MGP, OCN, Ggcx and Vkor expression levels correlated to mass and fat volume, in both sexes. In summary the current study has highlighted the importance of the long-term effects of maternal nutrition on offspring bone development and the correlation of VKDPs to bone structure. PMID:26381752

  18. Heat shock proteins in relation to heat stress tolerance of creeping bentgrass at different N levels.

    PubMed

    Wang, Kehua; Zhang, Xunzhong; Goatley, Mike; Ervin, Erik

    2014-01-01

    Heat stress is a primary factor causing summer bentgrass decline. Changes in gene expression at the transcriptional and/or translational level are thought to be a fundamental mechanism in plant response to environmental stresses. Heat stress redirects protein synthesis in higher plants and results in stress protein synthesis, particularly heat shock proteins (HSPs). The goal of this work was to analyze the expression pattern of major HSPs in creeping bentgrass (Agrostis stolonifera L.) during different heat stress periods and to study the influence of nitrogen (N) on the HSP expression patterns. A growth chamber study on 'Penn-A4' creeping bentgrass subjected to 38/28°C day/night for 50 days, was conducted with four nitrate rates (no N-0, low N-2.5, medium N-7.5, and high N-12.5 kg N ha-1) applied biweekly. Visual turfgrass quality (TQ), normalized difference vegetation index (NDVI), photochemical efficiency of photosystem II (Fv/Fm), shoot electrolyte leakage (ShEL), and root viability (RV) were monitored, along with the expression pattern of HSPs. There was no difference in measured parameters between treatments until week seven, except TQ at week five. At week seven, grass at medium N had better TQ, NDVI, and Fv/Fm accompanied by lower ShEL and higher RV, suggesting a major role in improved heat tolerance. All the investigated HSPs (HSP101, HSP90, HSP70, and sHSPs) were up-regulated by heat stress. Their expression patterns indicated cooperation between different HSPs and their roles in bentgrass thermotolerance. In addition, their production seems to be resource dependent. This study could further improve our understanding about how different N levels affect bentgrass thermotolerance. PMID:25050702

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

    PubMed

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

    2015-03-15

    Maize (Zea mays) root system architecture has a complex organization, with adventitious and lateral roots determining its overall absorptive capacity. To generate basic information about the earlier stages of root development, we compared the post-embryonic growth of maize seedlings germinated in water-embedded cotton beds with that of plants obtained from embryonic axes cultivated in liquid medium. In addition, the effect of four different auxins, namely indole-3-acetic acid (IAA), 1-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA) and 2,4-dichlorophenoxyacetic acid (2,4-D) on root architecture and levels of the heat shock protein HSP101 and the cell cycle proteins CKS1, CYCA1 and CDKA1 were analyzed. Our data show that during the first days after germination, maize seedlings develop several root types with a simultaneous and/or continuous growth. The post-embryonic root development started with the formation of the primary root (PR) and seminal scutellar roots (SSR) and then continued with the formation of adventitious crown roots (CR), brace roots (BR) and lateral roots (LR). Auxins affected root architecture in a dose-response fashion; whereas NAA and IBA mostly stimulated crown root formation, 2,4-D showed a strong repressing effect on growth. The levels of HSP101, CKS1, CYCA1 and CDKA in root and leaf tissues were differentially affected by auxins and interestingly, HSP101 registered an auxin-inducible and root specific expression pattern. Taken together, our results show the timing of early branching patterns of maize and indicate that auxins regulate root development likely through modulation of the HSP101 and cell cycle proteins. PMID:25615607

  20. Fish protein intake induces fast-muscle hypertrophy and reduces liver lipids and serum glucose levels in rats.

    PubMed

    Kawabata, Fuminori; Mizushige, Takafumi; Uozumi, Keisuke; Hayamizu, Kohsuke; Han, Li; Tsuji, Tomoko; Kishida, Taro

    2015-01-01

    In our previous study, fish protein was proven to reduce serum lipids and body fat accumulation by skeletal muscle hypertrophy and enhancing basal energy expenditure in rats. In the present study, we examined the precise effects of fish protein intake on different skeletal muscle fiber types and metabolic gene expression of the muscle. Fish protein increased fast-twitch muscle weight, reduced liver triglycerides and serum glucose levels, compared with the casein diet after 6 or 8 weeks of feeding. Furthermore, fish protein upregulated the gene expressions of a fast-twitch muscle-type marker and a glucose transporter in the muscle. These results suggest that fish protein induces fast-muscle hypertrophy, and the enhancement of basal energy expenditure by muscle hypertrophy and the increase in muscle glucose uptake reduced liver lipids and serum glucose levels. The present results also imply that fish protein intake causes a slow-to-fast shift in muscle fiber type. PMID:25198797

  1. Effects of Different Exercise Intensities with Isoenergetic Expenditures on C-Reactive Protein and Blood Lipid Levels

    ERIC Educational Resources Information Center

    Tsao, Te Hung; Yang, Chang Bin; Hsu, Chin Hsing

    2012-01-01

    We investigated the effects of different exercise intensities on C-reactive protein (CRP), and whether changes in CRP levels correlated with blood lipid levels. Ten men exercised at 25%, 65%, and 85% of their maximum oxygen consumption rates. Participants' blood was analyzed for CRP and blood lipid levels before and after the exercise sessions.…

  2. Low-level lasers affect uncoupling protein gene expression in skin and skeletal muscle tissues

    NASA Astrophysics Data System (ADS)

    Canuto, K. S.; Sergio, L. P. S.; Paoli, F.; Mencalha, A. L.; Fonseca, A. S.

    2016-03-01

    Wavelength, frequency, power, fluence, and emission mode determine the photophysical, photochemical, and photobiological responses of biological tissues to low-level lasers. Free radicals are involved in these responses acting as second messengers in intracellular signaling processes. Irradiated cells present defenses against these chemical species to avoid unwanted effects, such as uncoupling proteins (UCPs), which are part of protective mechanisms and minimize the effects of free radical generation in mitochondria. In this work UCP2 and UCP3 mRNA gene relative expression in the skin and skeletal muscle tissues of Wistar rats exposed to low-level red and infrared lasers was evaluated. Samples of the skin and skeletal muscle tissue of Wistar rats exposed to low-level red and infrared lasers were withdrawn for total RNA extraction, cDNA synthesis, and the evaluation of gene expression by quantitative polymerase chain reaction. UCP2 and UCP3 mRNA expression was differently altered in skin and skeletal muscle tissues exposed to lasers in a wavelength-dependent effect, with the UCP3 mRNA expression dose-dependent. Alteration on UCP gene expression could be part of the biostimulation effect and is necessary to make cells exposed to red and infrared low-level lasers more resistant or capable of adapting in damaged tissues or diseases.

  3. Increased Circulating Levels of Vitamin D Binding Protein in MS Patients

    PubMed Central

    Rinaldi, Arturo Ottavio; Sanseverino, Isabella; Purificato, Cristina; Cortese, Antonio; Mechelli, Rosella; Francisci, Silvia; Salvetti, Marco; Millefiorini, Enrico; Gessani, Sandra; Gauzzi, Maria Cristina

    2015-01-01

    Vitamin D (vitD) low status is currently considered a main environmental factor in multiple sclerosis (MS) etiology and pathogenesis. VitD and its metabolites are highly hydrophobic and circulate mostly bound to the vitamin D binding protein (DBP) and with lower affinity to albumin, while less than 1% are in a free form. The aim of this study was to investigate whether the circulating levels of either of the two vitD plasma carriers and/or their relationship are altered in MS. We measured DBP and albumin plasma levels in 28 MS patients and 24 healthy controls. MS patients were found to have higher DBP levels than healthy subjects. Concomitant interferon beta therapy did not influence DBP concentration, and the difference with the control group was significant in both females and males. No significant correlation between DBP and albumin levels was observed either in healthy controls or in patients. These observations suggest the involvement of DBP in the patho-physiology of MS. PMID:25590278

  4. Serum C-reactive protein level as a biomarker for differentiation of ischemic from hemorrhagic stroke.

    PubMed

    Roudbary, Seyed Ali; Saadat, Farshid; Forghanparast, Kambiz; Sohrabnejad, Reza

    2011-01-01

    Cerebrovascular accidents rank first in the frequency and importance among all neurological disease. Although a number of studies had shown increased level of the high sensitive C-reactive protein (hs-CRP) in patients with ischemic stroke, the association of increased hs-CRP with various type of stroke especially the assessment hs-CRP level in ischemic and hemorrhagic stroke have not been investigated. In the present study, we assessed the concentration of hs-CRP in patients with documented ischemic and hemorrhagic stroke in the first 24 hours of the onset of symptoms. Thirty-two patients with Ischemic and hemorrhagic stroke were evaluated at neurology department of Poursina Hospital. The presence of baseline vascular risk factors, including hypertension, diabetes mellitus, hypercholesterolemia, obesity, and smoking, was determined. The blood samples were then collected and routine hematology and biochemistry tests were done. hs-CRP levels were determined using a highly sensitive immunonephelometric method. In this cross sectional study, the age of patient varied from 45-85 years (Mean 70.9 ± 9.4). Serum level of hs-CRP in Ischemic patients were 18.92 ± 11.28 and in hemorrhagic group was 2.65 ± 1.7. This relationship was statistically significant (P<0.0001). It might be concluded that hs-CRP might be considered as a usefully adjunct method for the initial diagnosis of the type of stroke. PMID:21681701

  5. ELEVATED LEVELS OF SOLUBLE ST2 PROTEIN IN DENGUE VIRUS INFECTED PATIENTS

    PubMed Central

    Becerra, Aniuska; Warke, Rajas V.; de Bosch, Norma; Rothman, Alan L.; Bosch, Irene

    2008-01-01

    Levels of the soluble form of the interleukin-1 receptor like 1 protein (IL-1RL-1 / ST2) are elevated in the serum of patients with diseases characterized by an inflammatory response. The objective of this study was to determine the concentration of soluble ST2 (sST2) in dengue infected patients during the course of the disease. Twenty four patients with confirmed dengue infection, classified as dengue fever, and eleven patients with other febrile illness (OFI) were evaluated. Levels of sST2 in serum and laboratory variables usually altered during dengue infections were measured. Dengue infected patients had higher serum sST2 levels than OFI at the end of the febrile stage and at defervescence (p=0.0088 and p=0.0004 respectively). Patients with secondary dengue infections had higher serum sST2 levels compared with patients with primary dengue infections (p=0.047 at last day of fever and p=0.030 at defervescence). Furthermore, in dengue infected patients, we found a significant negative correlation of sST2 with platelet and WBC counts, and positive correlation with thrombin time and transaminases activity. We suggest that sST2 could be a potential marker of dengue infection, could be associated with severity or could play a role in the immune response in secondary dengue virus infection. PMID:18226917

  6. Comparative and evolutionary studies of vertebrate ALDH1A-like genes and proteins.

    PubMed

    Holmes, Roger S

    2015-06-01

    Vertebrate ALDH1A-like genes encode cytosolic enzymes capable of metabolizing all-trans-retinaldehyde to retinoic acid which is a molecular 'signal' guiding vertebrate development and adipogenesis. Bioinformatic analyses of vertebrate and invertebrate genomes were undertaken using known ALDH1A1, ALDH1A2 and ALDH1A3 amino acid sequences. Comparative analyses of the corresponding human genes provided evidence for distinct modes of gene regulation and expression with putative transcription factor binding sites (TFBS), CpG islands and micro-RNA binding sites identified for the human genes. ALDH1A-like sequences were identified for all mammalian, bird, lizard and frog genomes examined, whereas fish genomes displayed a more restricted distribution pattern for ALDH1A1 and ALDH1A3 genes. The ALDH1A1 gene was absent in many bony fish genomes examined, with the ALDH1A3 gene also absent in the medaka and tilapia genomes. Multiple ALDH1A1-like genes were identified in mouse, rat and marsupial genomes. Vertebrate ALDH1A1, ALDH1A2 and ALDH1A3 subunit sequences were highly conserved throughout vertebrate evolution. Comparative amino acid substitution rates showed that mammalian ALDH1A2 sequences were more highly conserved than for the ALDH1A1 and ALDH1A3 sequences. Phylogenetic studies supported an hypothesis for ALDH1A2 as a likely primordial gene originating in invertebrate genomes and undergoing sequential gene duplication to generate two additional genes, ALDH1A1 and ALDH1A3, in most vertebrate genomes. PMID:25446856

  7. Association of exome sequences with plasma C-reactive protein levels in >9000 participants

    PubMed Central

    Schick, Ursula M.; Auer, Paul L.; Bis, Joshua C.; Lin, Honghuang; Wei, Peng; Pankratz, Nathan; Lange, Leslie A.; Brody, Jennifer; Stitziel, Nathan O.; Kim, Daniel S.; Carlson, Christopher S.; Fornage, Myriam; Haessler, Jeffery; Hsu, Li; Jackson, Rebecca D.; Kooperberg, Charles; Leal, Suzanne M.; Psaty, Bruce M.; Boerwinkle, Eric; Tracy, Russell; Ardissino, Diego; Shah, Svati; Willer, Cristen; Loos, Ruth; Melander, Olle; Mcpherson, Ruth; Hovingh, Kees; Reilly, Muredach; Watkins, Hugh; Girelli, Domenico; Fontanillas, Pierre; Chasman, Daniel I.; Gabriel, Stacey B.; Gibbs, Richard; Nickerson, Deborah A.; Kathiresan, Sekar; Peters, Ulrike; Dupuis, Josée; Wilson, James G.; Rich, Stephen S.; Morrison, Alanna C.; Benjamin, Emelia J.; Gross, Myron D.; Reiner, Alex P.

    2015-01-01

    C-reactive protein (CRP) concentration is a heritable systemic marker of inflammation that is associated with cardiovascular disease risk. Genome-wide association studies have identified CRP-associated common variants associated in ∼25 genes. Our aims were to apply exome sequencing to (1) assess whether the candidate loci contain rare coding variants associated with CRP levels and (2) perform an exome-wide search for rare variants in novel genes associated with CRP levels. We exome-sequenced 6050 European-Americans (EAs) and 3109 African-Americans (AAs) from the NHLBI-ESP and the CHARGE consortia, and performed association tests of sequence data with measured CRP levels. In single-variant tests across candidate loci, a novel rare (minor allele frequency = 0.16%) CRP-coding variant (rs77832441-A; p.Thr59Met) was associated with 53% lower mean CRP levels (P = 2.9 × 10−6). We replicated the association of rs77832441 in an exome array analysis of 11 414 EAs (P = 3.0 × 10−15). Despite a strong effect on CRP levels, rs77832441 was not associated with inflammation-related phenotypes including coronary heart disease. We also found evidence for an AA-specific association of APOE-ε2 rs7214 with higher CRP levels. At the exome-wide significance level (P < 5.0 × 10−8), we confirmed associations for reported common variants of HNF1A, CRP, IL6R and TOMM40-APOE. In gene-based tests, a burden of rare/lower frequency variation in CRP in EAs (P ≤ 6.8 × 10−4) and in retinoic acid receptor-related orphan receptor α (RORA) in AAs (P = 1.7 × 10−3) were associated with CRP levels at the candidate gene level (P < 2.0 × 10−3). This inquiry did not elucidate novel genes, but instead demonstrated that variants distributed across the allele frequency spectrum within candidate genes contribute to CRP levels. PMID:25187575

  8. Association of exome sequences with plasma C-reactive protein levels in >9000 participants.

    PubMed

    Schick, Ursula M; Auer, Paul L; Bis, Joshua C; Lin, Honghuang; Wei, Peng; Pankratz, Nathan; Lange, Leslie A; Brody, Jennifer; Stitziel, Nathan O; Kim, Daniel S; Carlson, Christopher S; Fornage, Myriam; Haessler, Jeffery; Hsu, Li; Jackson, Rebecca D; Kooperberg, Charles; Leal, Suzanne M; Psaty, Bruce M; Boerwinkle, Eric; Tracy, Russell; Ardissino, Diego; Shah, Svati; Willer, Cristen; Loos, Ruth; Melander, Olle; Mcpherson, Ruth; Hovingh, Kees; Reilly, Muredach; Watkins, Hugh; Girelli, Domenico; Fontanillas, Pierre; Chasman, Daniel I; Gabriel, Stacey B; Gibbs, Richard; Nickerson, Deborah A; Kathiresan, Sekar; Peters, Ulrike; Dupuis, Josée; Wilson, James G; Rich, Stephen S; Morrison, Alanna C; Benjamin, Emelia J; Gross, Myron D; Reiner, Alex P

    2015-01-15

    C-reactive protein (CRP) concentration is a heritable systemic marker of inflammation that is associated with cardiovascular disease risk. Genome-wide association studies have identified CRP-associated common variants associated in ∼25 genes. Our aims were to apply exome sequencing to (1) assess whether the candidate loci contain rare coding variants associated with CRP levels and (2) perform an exome-wide search for rare variants in novel genes associated with CRP levels. We exome-sequenced 6050 European-Americans (EAs) and 3109 African-Americans (AAs) from the NHLBI-ESP and the CHARGE consortia, and performed association tests of sequence data with measured CRP levels. In single-variant tests across candidate loci, a novel rare (minor allele frequency = 0.16%) CRP-coding variant (rs77832441-A; p.Thr59Met) was associated with 53% lower mean CRP levels (P = 2.9 × 10(-6)). We replicated the association of rs77832441 in an exome array analysis of 11 414 EAs (P = 3.0 × 10(-15)). Despite a strong effect on CRP levels, rs77832441 was not associated with inflammation-related phenotypes including coronary heart disease. We also found evidence for an AA-specific association of APOE-ε2 rs7214 with higher CRP levels. At the exome-wide significance level (P < 5.0 × 10(-8)), we confirmed associations for reported common variants of HNF1A, CRP, IL6R and TOMM40-APOE. In gene-based tests, a burden of rare/lower frequency variation in CRP in EAs (P ≤ 6.8 × 10(-4)) and in retinoic acid receptor-related orphan receptor α (RORA) in AAs (P = 1.7 × 10(-3)) were associated with CRP levels at the candidate gene level (P < 2.0 × 10(-3)). This inquiry did not elucidate novel genes, but instead demonstrated that variants distributed across the allele frequency spectrum within candidate genes contribute to CRP levels. PMID:25187575

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  11. mTOR inhibition and levels of the DNA repair protein MGMT in T98G glioblastoma cells

    PubMed Central

    2014-01-01

    Background Glioblastoma multiforme (GBM), the most common and most aggressive type of primary adult brain tumour, responds poorly to conventional treatment. Temozolomide (TMZ) chemotherapy remains the most commonly used treatment, despite a large proportion of tumours displaying TMZ resistance. 60% of GBM tumours have unmethylated MGMT promoter regions, resulting in an overexpression of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT), which is responsible for tumour resistance to TMZ chemotherapy. Tumours also often exhibit hyperactive PI3-kinase/mTOR signalling, which enables them to resynthesise proteins quickly. Since MGMT is a suicide protein that is degraded upon binding to and repairing TMZ-induced O6-methylguanine adducts, it has been hypothesized that inhibition of translation via the mTOR signalling pathway could generate a tumour-specific reduction in MGMT protein and increase TMZ sensitivity. Methods MGMT was monitored at the post-transcriptional, translational and protein levels, to determine what effect mTOR inhibition was having on MGMT protein expression in vitro. Results We show that inhibiting mTOR signalling is indeed associated with acute inhibition of protein synthesis. Western blots show that despite this, relative to loading control proteins, steady state levels of MGMT protein increased and MGMT mRNA was retained in heavy polysomes. Whilst TMZ treatment resulted in maintained MGMT protein levels, concomitant treatment of T98G cells with TMZ and KU0063794 resulted in increased MGMT protein levels without changes in total mRNA levels. Conclusions These in vitro data suggest that, counterintuitively, mTOR inhibition may not be a useful adjunct to TMZ therapy and that more investigation is needed before applying mTOR inhibitors in a clinical setting. PMID:24909675

  12. Cumulative Depression Episodes Predicts Later C-Reactive Protein Levels: A Prospective Analysis

    PubMed Central

    Copeland, William E.; Shanahan, Lilly; Worthman, Carol; Angold, Adrian; Costello, E. Jane

    2013-01-01

    Background Depression is associated with elevated levels of the inflammation marker C -reactive protein (CRP), yet the direction of this association remains unclear. This study tested bi-directional longitudinal associations between CRP and depression in a sample of adolescent and young adults. The study compared the effects of current depression to the cumulative episodes of depression over time. Methods Nine waves of data from the prospective population-based Great Smoky Mountains Study (N = 1,420) were used, covering children in the community aged 9–16, 19, and 21 years old. Structured interviews were used to assess depressive symptoms, depression diagnosis, and cumulative depressive episodes. Bloodspots were collected at each observation and assayed for CRP levels. Results CRP levels were not associated with later depression status. In contrast, all depression-related variables displayed evidence of association with later CRP levels. The associations with depressive symptoms and diagnostic status were attenuated after controlling for covariates particularly body mass index, smoking, and medication use. The effect of cumulative depressive episodes, however, continued to be significant after accounting for a range of covariates. Body mass index, smoking behavior and recent infections may mediate a portion of the effect of cumulative episodes on later CRP, but cumulative depressive episodes continued to predict CRP levels independently. Conclusions The occurrence of multiple depressive episodes exerted the greatest effect on later CRP levels. This suggests that risk for the diseases of middle age - cardiovascular and metabolic disease – may begin in childhood and depend, in part, upon long-term emotional functioning. PMID:22047718

  13. Effect of dietary protein quality and feeding level on milk secretion and mammary protein synthesis in the rat

    SciTech Connect

    Sampson, D.A.; Jansen, G.R.

    1985-04-01

    Protein synthesis was studied in mammary tissue of rats fed diets deficient in protein quality and/or restricted in food intake throughout gestation and lactation. Diets containing 25% wheat gluten (WG), wheat gluten plus lysine and threonine (WGLT), or casein (C) were pair-fed from conception until day 15 of lactation at 100% or 85% of WG ad libitum consumption (PF100 and PF85, respectively). A seventh group was fed C ad libitum. Rates of protein synthesis were measured in vivo at day 15 of lactation from incorporation of (3-/sup 3/H)phenylalanine. At both PF100 and PF85, fractional and absolute rates of mammary gland protein synthesis were two- to three-fold higher in rats fed C than in those fed WG. Pup weights showed similar treatment effects. Both mammary protein synthesis rates and pup weights were significantly higher in rats fed C at PF85 than rats fed WG ad libitum. Food restriction from PF100 to PF85 depressed pup weights and mammary protein synthesis rates in rats fed WGLT, but had no effect in rats fed WG. These results demonstrate that when food intake is restricted, improvement of protein quality of the maternal diet increases milk output in the rat in association with increased rates of mammary protein synthesis.

  14. Levels of synthesis of primate-specific nuclear proteins differ between growth-arrested and proliferating cells

    SciTech Connect

    Celis, J.E.; Madsen, P.; Nielsen, S.; Ratz, G.P.; Lauridsen, J.B.; Celis, A.

    1987-02-01

    A monoclonal antibody that reacts specifically with the proliferation-sensitive nuclear proteins, isoelectric focusing (IEF) 8Z31 (molecular weight (MW), 76,000 charge variants, HeLa protein catalogue number) has been characterized. As determined by indirect immunofluorescence, the antibody stains the nucleolus and nucleoplasm of interphase-cultured cells of primate origin, but does not react with cells of other species. Proteins having similar MWs and isoelectric points as the human or monkey (primates) proteins were not observed in cultured cells of the following species: aves, bat, dog, dolphin, goat, hamster, mink, mouse, pisces, potoroo, rabbit and rat. Quantitative two-dimensional (2D) gel electrophoretic analysis of (/sup 35/S)methionine-labelled proteins synthesized by normal (quiescent, proliferating) and SV40-transformed human MRC-5 fibroblasts revealed significant differences in the levels of synthesis of both IEF 8Z30 and 8Z31. In quiescent cells the main labelled product corresponded to IEF 8Z31 (ratio IEF 8Z31/8Z30, 2.3), while in the transformed cells the major product was IEF 8Z30 (ratio, 0.62). Normal proliferating fibroblasts exhibited similar levels of both proteins (ratio, 1.21). Combined levels of synthesis of both proteins were 1.50 and 1.20 times as high in the transformed cells as in the quiescent and proliferating cells, respectively. Modulation of the levels of synthesis of these proteins may play a role in cell proliferation.

  15. Identification of a functional SNP in the 3'-UTR of caprine MTHFR gene that is associated with milk protein levels.

    PubMed

    An, Xiaopeng; Song, Yuxuan; Hou, Jinxing; Wang, Shan; Gao, Kexin; Cao, Binyun

    2016-08-01

    Xinong Saanen (n = 305) and Guanzhong (n = 317) dairy goats were used to detect SNPs in the caprine MTHFR 3'-UTR by DNA sequencing. One novel SNP (c.*2494G>A) was identified in the said region. Individuals with the AA genotype had greater milk protein levels than did those with the GG genotype at the c.*2494 G>A locus in both dairy goat breeds (P < 0.05). Functional assays indicated that the MTHFR:c.2494G>A substitution could increase the binding activity of bta-miR-370 with the MTHFR 3'-UTR. In addition, we observed a significant increase in the MTHFR protein level of AA carriers relative to that of GG carriers. These altered levels of MTHFR protein may account for the association of the SNP with milk protein level. PMID:27062401

  16. Chronic Low Level Complement Activation within the Eye Is Controlled by Intraocular Complement Regulatory Proteins

    PubMed Central

    Sohn, Jeong-Hyeon; Kaplan, Henry J.; Suk, Hye-Jung; Bora, Puran S.; Bora, Nalini S.

    2007-01-01

    -Crry mAb. Intracameral injection of anti-rat CD59 (6D1), anti-rat MHC class I antigen (OX-18), anti-rat Ig (G-16-510E3), or MOPC-21 caused no inflammatory reaction. Conclusions The results suggest that the complement system is continuously active at a low level in the normal eye and is tightly regulated by intraocular complement regulatory proteins. PMID:11006244

  17. C-reactive protein levels in stable COPD patients: a case-control study

    PubMed Central

    Silva, Denise Rossato; Gazzana, Marcelo Basso; Knorst, Marli Maria

    2015-01-01

    Background Previous studies have documented that C-reactive protein (CRP) levels are increased in stable COPD patients. However, most studies have also shown that higher CRP levels are observed in patients with comorbidities like diabetes mellitus and cardiovascular disease. We aimed to investigate if CRP levels are increased in stable COPD patients, and if there is an association between CRP levels and pulmonary function tests and clinical characteristics. Methods We conducted a case-control study in a tertiary care, university-affiliated hospital. COPD patients and controls were matched for sex and age in a 2:1 matching ratio. We included only those patients who had quit smoking. CRP levels were determined and pulmonary function tests were performed in both the groups. Results A total of 60 COPD patients and 30 controls were included in the analysis. The study subjects had a mean age of 64.8±8.5 years in COPD group and 64.3±9.2 years in control group (P=0.214). The median of CRP levels was 3.17 mg/L (interquartile range [IQR]: 1.73–5.99 mg/L) in COPD group and 2.13 mg/L (IQR: 1.18–7.69 mg/L) in control group (P=0.370). There were 34 (56.7%) patients in COPD group and 14 (46.7%) patients in control group with CRP levels greater than 3 mg/dL (P=0.382). Using bivariate correlations, we found significant positive correlations in COPD patients between body mass index (BMI) and CRP (r=0.3, P=0.045), and between CRP and forced vital capacity (FVC, % of predicted) (r=−0.3; P=0.023). In a multivariate model, female sex and FVC (% of predicted) were associated with a CRP value greater than 3 mg/dL in the COPD group. Conclusion The levels of CRP in the stable COPD patients were not significantly different when compared to those in the control subjects. Female sex and FVC (% predicted) were associated with CRP levels greater than 3 mg/dL in the COPD group. PMID:26357470

  18. A novel cryptochrome in the diatom Phaeodactylum tricornutum influences the regulation of light-harvesting protein levels.

    PubMed

    Juhas, Matthias; von Zadow, Andrea; Spexard, Meike; Schmidt, Matthias; Kottke, Tilman; Büchel, Claudia

    2014-05-01

    Diatoms possess several genes for proteins of the cryptochrome/photolyase family. A typical sequence for a plant cryptochrome was not found in our analysis of the Phaeodactylum tricornutum genome, but one protein grouped with higher plant and green algal cryptochromes. This protein, CryP, binds FAD and 5,10-methenyltetrahydrofolate, according to our spectroscopic studies on heterologously expressed protein. 5,10-Methenyltetrahydrofolate binding is a feature common to both cyclobutane pyrimidine dimer photolyases and DASH cryptochromes. In recombinant CryP, however, the FAD chromophore was present in its neutral radical state and had a red-shifted absorption maximum at 637 nm, which is more characteristic for a DASH cryptochrome than a cyclobutane pyrimidine dimer photolyase. Upon illumination with blue light, the fully reduced state of FAD was formed in the presence of reductant. Expression of CryP was silenced by antisense approaches, and the resulting cell lines showed increased levels of proteins of light-harvesting complexes, the Lhcf proteins, in vivo. In contrast, the levels of proteins active in light protection, the Lhcx proteins, were reduced. Thus, CryP cannot be directly grouped with known members of the cryptochrome/photolyase family. Of all P. tricornutum proteins, it is the most similar in sequence to a plant cryptochrome, and is involved in the regulation of light-harvesting protein expression, but shows spectroscopic features and a chromophore composition that are most typical of a DASH cryptochrome. PMID:24628952

  19. Prediction of the Grade of Acute Cholecystitis by Plasma Level of C-Reactive Protein

    PubMed Central

    Kabul Gurbulak, Esin; Gurbulak, Bunyamin; Akgun, Ismail Ethem; Duzkoylu, Yigit; Battal, Muharrem; Fevzi Celayir, Mustafa; Demir, Uygar

    2015-01-01

    Background: Acute cholecystitis is the most common complication of gallbladder stones. Today, Tokyo guidelines criteria are recommended for diagnosis, grading, and management of acute cholecystitis. Objectives: We aimed to evaluate the levels of C-reactive protein (CRP) at different cut-off values to predict the severity of the disease and its possible role in grading the disease with regard to the guideline. Patients and Methods: This is a retrospective study, analyzing 682 cases out of consecutive 892 patients with acute cholecystitis admitted to two different general surgery clinics in Istanbul, Turkey. Records of patients diagnosed with acute cholecystitis were screened retrospectively from the hospital computer database between January 2011 and July 2014. A total of 210 patients with concomitant diseases causing high CRP levels were excluded from the study. The criteria of Tokyo guidelines were used in grading the severity of acute cholecystitis, and patients were divided into 3 groups. CRP values at the time of admission were analyzed and compared among the groups. Results: Mean CRP levels of groups were found to be significantly different, 18.96 mg/L in Group I, 133.51 mg/L in Group II, and 237.23 mg/L in Group III (P < 0.001). Having examined CRP values among the groups, they were found to be highly and significantly correlated with the disease grade (P < 0.0001). After evaluating CRP levels according to the grade of the disease, group 2 was distinguished from group 1 with a cut-off CRP level of 70.65 mg/L, and from group 3 with a value of 198.95 mg/L. Those results were found to be statistically significant (P < 0.001). Conclusions: CRP, a well-known acute phase reactant that increases rapidly in various inflammatory processes, can be accepted as a strong predictor in classifying different grades of the disease, and treatment can be reliably planned according to this classification. PMID:26023353

  20. Retinol Binding Protein-4 and Adiponectin Levels in Thyroid Overt and Subclinical Dysfunction.

    PubMed

    Kokkinos, S; Papazoglou, D; Zisimopoulos, A; Papanas, N; Tiaka, E; Antonoglou, C; Maltezos, E

    2016-02-01

    Thyroid dysfunction is accompanied by numerous changes in intermediary metabolism. Retinol binding protein-4 (RBP-4) and adiponectin are 2 adipocytokines that have multiple metabolic functions. The aim of our study was to examine serum RBP4 and adiponectin levels in clinical (before and after therapy) and subclinical hyperthyroid and hypothyroid subjects as compared to controls.150 patients with thyroid dysfunction were recruited (65 hyperthyroid and 85 hypothyroid) while 28 euthyroid subjects served as a control group. We measured anthropometric, biochemical and hormonal (free T4, free T3, TSH, insulin) parameters in all participants. RBP-4 and adiponectin were measured using commercial ELISA kits.Mean baseline levels of RBP-4 were higher in patients with clinical hypothyroidism (29.0±10.2 ng/ml, 25.1±12.6 ng/ml, 38.8±16.5 ng/ml, 31.9±13.2 ng/ml, 20.4±8.2 ng/ml in patients with hyperthyroidism, subclinical hyperthryrodism, hypothyroidism, subclinical hypothyroidism and controls respectively, F=4.86, P<0.001) and decreased significantly in patients with clinical hyperthyroidism and hypothyroidism after normalization of thyroid hormones' levels (from 29.0±10.2 to 24.9±8.4 ng/ml, p=0.003 and from 38.8±16.5 to 29.0±10.8 ng/ml, p=0.001 respectively). We did not observe analogous changes in adiponectin levels in any of the studied groups.RBP-4 levels are higher in patients with clinical hypothyroidism and exhibit a marked decrease after normalization of thyroid function in both hyper and hypothyroid patients. We suggest that RBP-4 may play a role in the metabolic disturbances which accompany thyroid dysfunction. PMID:26575118

  1. A comparison of ghrelin, glucose, alpha-amylase and protein levels in saliva from diabetics.

    PubMed

    Aydin, Suleyman

    2007-01-31

    During the past decade, many salivary parameters have been used to characterize disease states. Ghrelin (GAH) is recently-discovered peptide hormone secreted mainly from the stomach but also produced in a number of other tissues including salivary glands. The aim of this work was to examine the relationship between active (aGAH) and inactive (dGAH) ghrelin in the saliva and other salivary parameters in type II diabetic patients and healthy controls. Salivary parameters were assessed in a single measurement of unstimulated whole saliva from 20 obese and 20 non-obese type II diabetes patients, and in 22 healthy controls. Total protein and alpha-amylase were determined by colorimetric methods, and glucose by the glucose-oxidase method. Saliva aGAH and dGAH levels were measured using a commercial radioimmunoassay (RIA) kit. Salivary concentrations of aGAH and dGAH ghrelin were more markedly decreased in obese diabetic subjects than in the two other groups. Glucose and alpha-amylase levels were higher in diabetic subjects than in controls. Furthermore, there were correlations between GAH levels and BMI, and between GAH and blood pressure. However, there was no marked variability in saliva flow rates among the groups. These results indicate that measurement of salivary GAH and its relationship to other salivary parameters might help to provide insight into the role of ghrelin in diabetes. PMID:17244479

  2. Dynamic regulation of basement membrane protein levels promotes egg chamber elongation in Drosophila.

    PubMed

    Isabella, Adam J; Horne-Badovinac, Sally

    2015-10-15

    Basement membranes (BMs) are sheet-like extracellular matrices that provide essential support to epithelial tissues. Recent evidence suggests that regulated changes in BM architecture can direct tissue morphogenesis, but the mechanisms by which cells remodel BMs are largely unknown. The Drosophila egg chamber is an organ-like structure that transforms from a spherical to an ellipsoidal shape as it matures. This elongation coincides with a stage-specific increase in Type IV Collagen (Col IV) levels in the BM surrounding the egg chamber; however, the mechanisms and morphogenetic relevance of this remodeling event have not been established. Here, we identify the Collagen-binding protein SPARC as a negative regulator of egg chamber elongation, and show that SPARC down-regulation is necessary for the increase in Col IV levels to occur. We find that SPARC interacts with Col IV prior to secretion and propose that, through this interaction, SPARC blocks the incorporation of newly synthesized Col IV into the BM. We additionally observe a decrease in Perlecan levels during elongation, and show that Perlecan is a negative regulator of this process. These data provide mechanistic insight into SPARC's conserved role in matrix dynamics and demonstrate that regulated changes in BM composition influence organ morphogenesis. PMID:26348027

  3. Measurement of pulmonary status and surfactant protein levels during dexamethasone treatment of neonatal respiratory distress syndrome.

    PubMed Central

    Wang, J. Y.; Yeh, T. F.; Lin, Y. C.; Miyamura, K.; Holmskov, U.; Reid, K. B.

    1996-01-01

    BACKGROUND: Early postnatal use of dexamethasone in infants with respiratory distress syndrome (RDS) has been shown effectively to improve pulmonary status and to allow early weaning off mechanical ventilation. However, the mechanisms to explain the beneficial effects of dexamethasone in ventilatory dependent preterm infants remain unclear. METHODS: A double blind, placebo controlled study was performed to determine the change in pulmonary ventilation of premature infants with RDS as a result of dexamethasone treatment, and to evaluate the effect of dexamethasone on the levels of surfactant-associated proteins A (SP-A) and D (SP-D) in the tracheal fluid from 34 premature infants with RDS and 29 control subjects. RESULTS: Dexamethasone treatment decreased fractional inspired oxygen concentration (FIO2), arterial carbon dioxide tension (PCO2), mean airway pressure (MAP), and facilitated successful weaning from mechanical ventilation. SP-A concentrations in the tracheal aspirates were increased at days 7 and 14, and SP-D concentrations were increased during the period from days 3 to 14 in the dexamethasone treated group compared with the control group. However, albumin levels in the tracheal aspirate samples were decreased after dexamethasone treatment over the period from days 3 to 14. There was an inverse correlation between PCO2 values and SP-A concentrations. CONCLUSIONS: These results suggest that early use of dexamethasone can improve pulmonary status and also increase SP-A and SP-D levels in the tracheal fluid in premature infants with RDS. PMID:8984701

  4. Reduced levels of protein recoding by A-to-I RNA editing in Alzheimer's disease

    PubMed Central

    Khermesh, Khen; D'Erchia, Anna Maria; Barak, Michal; Annese, Anita; Wachtel, Chaim; Levanon, Erez Y.; Picardi, Ernesto; Eisenberg, Eli

    2016-01-01

    Adenosine to inosine (A-to-I) RNA editing, catalyzed by the ADAR enzyme family, acts on dsRNA structures within pre-mRNA molecules. Editing of the coding part of the mRNA may lead to recoding, amino acid substitution in the resulting protein, possibly modifying its biochemical and biophysical properties. Altered RNA editing patterns have been observed in various neurological pathologies. Here, we present a comprehensive study of recoding by RNA editing in Alzheimer's disease (AD), the most common cause of irreversible dementia. We have used a targeted resequencing approach supplemented by a microfluidic-based high-throughput PCR coupled with next-generation sequencing to accurately quantify A-to-I RNA editing levels in a preselected set of target sites, mostly located within the coding sequence of synaptic genes. Overall, editing levels decreased in AD patients’ brain tissues, mainly in the hippocampus and to a lesser degree in the temporal and frontal lobes. Differential RNA editing levels were observed in 35 target sites within 22 genes. These results may shed light on a possible association between the neurodegenerative processes typical for AD and deficient RNA editing. PMID:26655226

  5. Ursolic acid regulates aging process through enhancing of metabolic sensor proteins level.

    PubMed

    Bahrami, Soroush Alaghehband; Bakhtiari, Nuredin

    2016-08-01

    We previously reported that Ursolic Acid (UA) ameliorates skeletal muscle performance through satellite cells proliferation and cellular energy status. In studying the potential role of the hypothalamus in aging, we developed a strategy to pursue UA effects on the hypothalamus anti-aging proteins such as; SIRT1, SIRT6, PGC-1β and α-Klotho. In this study, we used a model of aging animals (C57BL/6). UA dissolved in Corn oil (20mg/ml) and then administrated (200mg/Kg i.p injection) to mice, twice daily for 7days. After treatment times, the mice perfused and the hypothalamus isolated for preparing of tissue to Immunofluorescence microscopy. The data illustrated that UA significantly increased SIRT1 (∼3.5±0.3 folds) and SIRT-6 (∼1.5±0.2 folds) proteins overexpression (P<0.001). In addition, our results showed that UA enhanced α-Klotho (∼3.3±0.3) and PGC-1β (∼2.6±0.2 folds) proteins levels (P<0. 01). In this study, data were analyzed using SPSS 16 (ANOVA test). To the best of our knowledge, it seems that UA through enhancing of anti-aging biomarkers (SIRT1 and SIRT6) and PGC-1β in hypothalamus regulates aging-process and attenuates mitochondrial-related diseases. In regard to the key role of α-Klotho in aging, our data indicate that UA may be on the horizon to forestall diseases of aging. PMID:27470332

  6. Increased levels of myelin basic protein transcripts in virus-induced demyelination.

    PubMed

    Kristensson, K; Holmes, K V; Duchala, C S; Zeller, N K; Lazzarini, R A; Dubois-Dalcq, M

    In multiple sclerosis, a demyelinating disease of young adults, there is a paucity of myelin repair in the central nervous system (CNS) which is necessary for the restoration of fast saltatory conduction in axons. Consequently, this relapsing disease often causes marked disability. In similar diseases of small rodents, however, remyelination can be quite extensive, as in the demyelinating disease caused by the A59 strain of mouse hepatitis virus (MHV-A59), a coronavirus of mice. To investigate when and where oligodendrocytes are first triggered to repair CNS myelin in such disease, we have used a complementary DNA probe specific for one major myelin protein gene, myelin basic protein (MBP), which hybridizes with the four forms of MBP messenger RNA in rodents. Using Northern blot and in situ hybridization techniques, we previously found that MBP mRNA is first detected at about 5 days after birth, peaks at 18 days and progressively decreases to 25% of the peak levels in the adult. We now report that in spinal cord sections of adult animals with active demyelination and inflammatory cells, in situ hybridization reveals a dramatic increase in probe binding to MBP-specific mRNA at 2-3 weeks after virus inoculation and before remyelination can be detected by morphological methods. This increase of MBP-specific mRNA is found at the edge of the demyelinating area and extends into surrounding areas of normal-appearing white matter. Thus, in situ hybridization with myelin-specific probes appears to be a useful method for detecting the timing, intensity and location of myelin protein gene reactivation preceding remyelination. This method could be used to elucidate whether such a reactivation occurs in multiple sclerosis brain tissue. Our results suggest that in mice, glial cells react to a demyelinating process with widespread MBP mRNA synthesis which may be triggered by a diffusible factor released in the demyelinated areas. PMID:2426599

  7. The Influence of Tobacco Smoke on Protein and Metal Levels in the Serum of Women during Pregnancy

    PubMed Central

    Wrześniak, Marta; Kepinska, Marta; Królik, Małgorzata; Milnerowicz, Halina

    2016-01-01

    Background Tobacco smoking by pregnant women has a negative effect on fetal development and increases pregnancy risk by changing the oxidative balance and microelements level. Smoking affects the concentration, structure and function of proteins, potentially leading to various negative effects on pregnancy outcomes. Methodology/Principal Findings The influence of tobacco smoke on key protein fractions in smoking and non-smoking healthy pregnant women was determined by capillary electrophoresis (CE). Concentrations of the proteins α1-antitrypsin, α1-acid glycoprotein, α2-macroglobulin and transferrin were determined by ELISA tests. Total protein concentration was measured by the Biuret method. Smoking status was established by cotinine levels. Cadmium (Cd) and Zinc (Zn) concentrations were determined by flame atomic absorption spectrometry and the Zn/Cd ratio was calculated based on these numbers. Smoking women had a 3.7 times higher level of Cd than non-smoking women. Zn levels decreased during pregnancy for all women. The Zn/Cd ratio was three times lower in smoking women. The differences between the changes in the protein profile for smoking and non-smoking women were noted. Regarding proteins, α1-antitrypsin and α2-macroglobulin levels were lower in the non-smoking group than in the smoking group and correlated with Cd levels (r = -0.968, p = 0.032 for non-smokers; r = −0.835, p = 0.019 for smokers). Zn/Cd ratios correlated negatively with α1-, α2- and β-globulins. Conclusions/Significance Exposure to tobacco smoke increases the concentration of Cd in the blood of pregnant women and may lead to an elevated risk of pregnancy disorders. During pregnancy alter concentrations of some proteins. The correlation of Cd with proteins suggests that it is one of the causes of protein aberrations. PMID:27548057

  8. Endogenous salicylic acid levels correlate with accumulation of pathogenesis-related proteins and virus resistance in tobacco

    SciTech Connect

    Yalpani, N.; Shulaev, V.; Raskin, I. )

    1993-07-01

    Salicylic acid (SA) is hypothesized to be an endogenous regulator of local and systemic disease resistance and an inducer of pathogenesis-related (PR) proteins among plants. High levels of PR proteins have been observed in an uninoculated amphidiploid hybrid of Nicotiana glutinosa [times] N. debneyi, which is highly resistant to tobacco mosaic virus (TMV). Fluoresence, UV, and mass spectral analysis established that the levels of SA in healthy N. glutinosa [times] N. debneyi leaves were 30 times greater than in N. tabacum [open quotes]Xanthi-nc[close quotes] tobacco, which does not constitutively express PR proteins and is less resistant to TMV. Upon TMV-inoculation SA levels increased at least 70-fold leaves of Xanthi-nc but role only slightly in the hybrid. Phloem exudates of N. glutinosa [times] N. debneyi contained at least 500 times more SA than those of Xanthi-nc. SA treatment caused the appearance of PR-1 protein in Xanthi-nc but did not affect constitutively high levels of PR-1 protein in N. glutinosa [times] N. debneyi. In contrast to Xanthi-nc tobacco, TMV-inoculated N. glutinosa [times] N. debneyi kept at 32 C accumulated more than 0.5 [mu]g SA/g fresh weight, maintained high levels of PR proteins, and developed a hypersensitive response to TMV. PR proteins have previously been shown to accumulate in the lower leaves of healthy, flowering Xanthi-nc tobacco, which exhibited increased resistance to TMV. These developmentally induced increases in resistance and PR-1 proteins positively correlated with tissue levels of SA. These results affirm the regulatory role of SA in disease resistance and PR protein production. 31 refs., 9 figs., 1 tab.

  9. Influence of casein as a percentage of true protein and protein level on color and texture of milks containing 1 and 2% fat.

    PubMed

    Misawa, Noriko; Barbano, David M; Drake, MaryAnne

    2016-07-01

    Combinations of fresh liquid microfiltration retentate of skim milk, ultrafiltered retentate and permeate produced from microfiltration permeate, cream, and dried lactose monohydrate were used to produce a matrix of 20 milks. The milks contained 5 levels of casein as a percentage of true protein of about 5, 25, 50, 75, and 80% and 4 levels of true protein of 3.0, 3.76, 4.34, and 5.0% with constant lactose percentage of 5%. The experiment was replicated twice and repeated for both 1 and 2% fat content. Hunter color measurements, relative viscosity, and fat globule size distribution were measured, and a trained panel documented appearance and texture attributes on all milks. Overall, casein as a percentage of true protein had stronger effects than level of true protein on Hunter L, a, b values, relative viscosity, and fat globule size when using fresh liquid micellar casein concentrates and milk serum protein concentrates produced by a combination of microfiltration and ultrafiltration. As casein as a percentage of true protein increased, the milks became more white (higher L value), less green (lower negative a value), and less yellow (lower b value). Relative viscosity increased and d(0.9) generally decreased with increasing casein as a percentage of true protein. Panelists perceived milks with increasing casein as a percentage of true protein as more white, more opaque, and less yellow. Panelists were able to detect increased throat cling and mouthcoating with increased casein as a percentage of true protein in 2% milks, even when differences in appearance among milks were masked. PMID:27157580

  10. The Highwire ubiquitin ligase promotes axonal degeneration by tuning levels of Nmnat protein.

    PubMed

    Xiong, Xin; Hao, Yan; Sun, Kan; Li, Jiaxing; Li, Xia; Mishra, Bibhudatta; Soppina, Pushpanjali; Wu, Chunlai; Hume, Richard I; Collins, Catherine A

    2012-01-01

    Axonal degeneration is a hallmark of many neuropathies, neurodegenerative diseases, and injuries. Here, using a Drosophila injury model, we have identified a highly conserved E3 ubiquitin ligase, Highwire (Hiw), as an important regulator of axonal and synaptic degeneration. Mutations in hiw strongly inhibit Wallerian degeneration in multiple neuron types and developmental stages. This new phenotype is mediated by a new downstream target of Hiw: the NAD+ biosynthetic enzyme nicotinamide mononucleotide adenyltransferase (Nmnat), which acts in parallel to a previously known target of Hiw, the Wallenda dileucine zipper kinase (Wnd/DLK) MAPKKK. Hiw promotes a rapid disappearance of Nmnat protein in the distal stump after injury. An increased level of Nmnat protein in hiw mutants is both required and sufficient to inhibit degeneration. Ectopically expressed mouse Nmnat2 is also subject to regulation by Hiw in distal axons and synapses. These findings implicate an important role for endogenous Nmnat and its regulation, via a conserved mechanism, in the initiation of axonal degeneration. Through independent regulation of Wnd/DLK, whose function is required for proximal axons to regenerate, Hiw plays a central role in coordinating both regenerative and degenerative responses to axonal injury. PMID:23226106

  11. Protein carbonyl levels correlate with performance in elite field hockey players.

    PubMed

    Rosa-Lima, Frederico Luis; Lannes, Luiz; Viana-Gomes, Diego; Pierucci, Anna Paola T R; Salerno, Verônica P

    2015-07-01

    Excess and incorrectly selected exercise can degrade athletic performance from an imbalance in redox homeostasis and oxidative stress, but well-planned training and nutrition can improve antioxidant capacity. The aim of the study was to investigate how nutrient intake could influence oxidative stress and cell lesion biomarkers after 5 days of training followed by a game. Blood was collected from 10 athletes at the start of training (basal), after training (pre-game), and postgame. Their acceleration capacity also was measured pre- and postgame. Blood analysis showed an increase in lactate concentration postgame (13%) and total antioxidant capacity increased both pre-game (13.1%) and postgame (12.7%), all in comparison with basal levels. An oxidative stress marker, protein carbonyl (PC), increased 3-fold over the course of the game, which correlated with a decreased acceleration (r = 0.749). For biomarkers of tissue damage, creatine kinase and aspartate transaminase (AST) increased postgame by 150% and 75%, respectively. The AST variation had a high negative correlation with energy and carbohydrate consumption and a moderate correlation with lipid and vitamin C intake. Protein intake had a positive but moderate correlation with reduced glutathione. The observed correlations suggest that nutritional monitoring can improve exercise physiological homeostasis and that PC serves as a good biomarker for oxidative stress and performance loss. PMID:25962716

  12. Fused kinase is stabilized by Cdc37/Hsp90 and enhances Gli protein levels

    SciTech Connect

    Kise, Yoshiaki; Takenaka, Kei; Tezuka, Tohru; Yamamoto, Tadashi; Miki, Hiroaki . E-mail: miki@ims.u-tokyo.ac.jp

    2006-12-08

    Serine/threonine kinase Fused (Fu) is an essential component of Hedgehog (Hh) signaling in Drosophila, but the biochemical functions of Fu remain unclear. Here, we have investigated proteins co-precipitated with mammalian Fu and identified a kinase-specific chaperone complex, Cdc37/Hsp90, as a novel-binding partner of Fu. Inhibition of Hsp90 function by geldanamycin (GA) induces rapid degradation of Fu through a ubiquitin-proteasome pathway. We next show that co-expression of Fu with transcription factors Gli1 and Gli2 significantly increases their protein levels and luciferase reporter activities, which are blocked by GA. These increases can be ascribed to Fu-mediated stabilization of Gli because co-expression of Fu prolongs half-life of Gli1 and reduces polyubiquitination of Gli1. Finally, we show that GA inhibits proliferation of PC3, a Hh signaling-activated prostate cancer cell line. This growth inhibition is partially rescued by expression of ectopic Gli1, suggesting that Fu may contribute to enhance Hh signaling activity in cancer cells.

  13. Correlation between erythrocyte sedimentation rate and C-reactive protein level in patients with rheumatic diseases

    PubMed Central

    Kotulska, Anna; Kopeć-Mędrek, Magdalena; Grosicka, Anida; Kubicka, Monika

    2015-01-01

    Objectives Erythrocyte sedimentation rate (ESR) and serum level of C-reactive protein (CRP) are the acute phase reactants most commonly determined in patients with rheumatic diseases. The indices are affected by different factors, but both of them are applied for evaluation of the disease activity in patients with inflammatory disorders of the musculoskeletal system. Material and methods The authors compared the results of ESR and CRP, which were carried out during routine diagnosis in 200 patients admitted to the Department of Rheumatology. Results A significant correlation between ESR and CRP was found (ESR after 1 h/CRP: correlation coefficient 0.6944, ESR after 2 h/CRP: correlation coefficient 0.6126). There was no difference in ESR or CRP between male and female patients, and patients older than 40 years had higher ESR and CRP. Conclusions The obtained results support the usefulness of both indices in the clinical practice of rheumatologists.

  14. A Theoretical Lower Bound for Selection on the Expression Levels of Proteins

    PubMed Central

    Price, Morgan N.; Arkin, Adam P.

    2016-01-01

    We use simple models of the costs and benefits of microbial gene expression to show that changing a protein’s expression away from its optimum by 2-fold should reduce fitness by at least 0.2·P, where P is the fraction the cell’s protein that the gene accounts for. As microbial genes are usually expressed at above 5 parts per million, and effective population sizes are likely to be above 106, this implies that 2-fold changes to gene expression levels are under strong selection, as Ne·s≫1, where Ne is the effective population size and s is the selection coefficient. Thus, most gene duplications should be selected against. On the other hand, we predict that for most genes, small changes in the expression will be effectively neutral. PMID:27289091

  15. Effect of vitamins, protein level and probiotics on immune response of moulted male broiler breeders.

    PubMed

    Khan, R U; Rahman, Z U; Javed, I; Muhammad, F

    2014-08-01

    This study was planned to investigate the comparative effect of vitamins C (L-ascorbic acid), E (DL-α-tocopherol acetate), probiotics, lower than normal protein level (14%) and combination of these treatments on immune response of male broiler breeders after zinc-induced moulting. One hundred and eighty birds at the age of 65 weeks were induced to moult by mixing zinc oxide (ZnO) in feed at the rate 3000 IU/kg of feed. Upon completion of moulting, birds were divided into six groups (five replicates per group) in a completely randomized design and were fed vitamin C (500 IU/kg), vitamin E (100 IU/kg), lower protein level, probiotics (50 mg/l), and a combination of these components, while one group was kept as control. After completion of moulting phase (5 weeks), the treatment effects were tested as in vitro macrophages engulfment percentage, nitric oxide (NO) production, serum antibody titres against Newcastle disease (ND) and infectious bronchitis (IB). The results showed that in vitro macrophage engulfment percentage in unopsonized conditions was significantly higher in vitamin E-supplemented group. In addition, in opsonized condition, the macrophage engulfment percentage was significantly higher in both vitamin E- and C-supplemented groups. The NO (opsonized and unopsonized) production and antibody titre against ND and IB were significantly higher in vitamin E-supplemented group. It was concluded that vitamin E is a better option for enhanced immune response in broiler breeders after zinc-induced moulting. PMID:23782414

  16. Involvement of decreased neuroglobin protein level in cognitive dysfunction induced by 1-bromopropane in rats.

    PubMed

    Guo, Ying; Yuan, Hua; Jiang, Lulu; Yang, Junlin; Zeng, Tao; Xie, Keqin; Zhang, Cuili; Zhao, Xiulan

    2015-03-10

    1-Bromopropane (1-BP) is used as a substitute for ozone-depleting solvents (ODS) in industrial applications. 1-BP could display central nervous system (CNS) neurotoxicity manifested by cognitive dysfunction. Neuroglobin (Ngb) is an endogenous neuroprotectant and is predominantly expressed in the nervous system. The present study aimed to investigate Ngb involvement in CNS neurotoxicity induced by 1-BP in rats. Male Wistar rats were randomly divided into 5 groups (n=14) and treated with 0, 100, 200, 400 and 800 mg/kg bw 1-BP, respectively, by gavage for consecutive 12 days. Rats displayed cognitive dysfunction dose-dependently through Morris water maze (MWM) test. Significant neuron loss in layer 5 of the prelimbic cortex (PL) was observed. Moreover, 1-BP decreased Ngb protein level in cerebral cortex and Ngb decrease was significantly positively correlated with cognitive dysfunction. Glutathione (GSH) content, GSH/oxidized glutathione (GSSG) ratio and glutamate cysteine ligase (GCL) activity decreased in cerebral cortex, coupled with the increase in GSSG content. GSH and GSH/GSSG ratio decrease were significantly positively correlated with cortical Ngb decrease. Additionally, levels of N-epsilon-hexanoyl-lysine (HEL) and 4-hydroxy-2-nonenal (4-HNE) modified proteins in cerebral cortex of 1-BP-treated rats increased significantly. In conclusion, it was suggested that 1-BP resulted in decreased endogenous neuroprotectant Ngb in cerebral cortex, which might play an important role in CNS neurotoxicity induced by 1-BP and that 1-BP-induced oxidative stress in cerebral cortex might partly be responsible for Ngb decrease. PMID:25557405

  17. Dietary protein quality differentially regulates trypsin enzymes at the secretion and transcription level in Panulirus argus by distinct signaling pathways.

    PubMed

    Perera, Erick; Rodríguez-Viera, Leandro; Rodríguez-Casariego, Javier; Fraga, Iliana; Carrillo, Olimpia; Martínez-Rodríguez, Gonzalo; Mancera, Juan M

    2012-03-01

    The effects of pelleted diets with different protein composition (fish, squid or soybean meals as main protein sources) on trypsin secretion and expression were studied in the lobster Panulirus argus. Trypsin secretion was shown to be maximal 4 h after ingestion. At this time, fish- and squid-based diets induced trypsin secretion, as well as up-regulation of the major trypsin isoform at the transcription level. While fish- and squid-based diets elicited a prandial response, soybean-based diet failed to stimulate the digestive gland to secrete trypsin into the gastric fluid or induce trypsin expression above the levels observed in fasting lobsters. In vitro assays showed that intact proteins rather than protein hydrolysates stimulate trypsin secretion in the lobster. However, the signal for trypsin transcription appears to be different to that for secretion and is probably mediated by the appearance of free amino acids in the digestive gland, suggesting a stepwise regulation of trypsin enzymes during digestion. We conclude that trypsin enzymes in P. argus are regulated at the transcription and secretion level by the quality of dietary proteins through two distinct signaling pathways. Our results indicate that protein digestion efficiency in spiny lobsters can be improved by selecting appropriated protein sources. However, other factors like the poor solubility of dietary proteins in dry diets could hamper further enhancement of digestion efficiency. PMID:22323208

  18. Effects of dietary lysine/protein ratio and fat levels on growth performance and meat quality of finishing pigs.

    PubMed

    Maeda, Keisuke; Yamamoto, Fumika; Toyoshi, Masanari; Irie, Masakazu

    2014-04-01

    This study aimed to evaluate the effects of dietary lysine/protein ratio and fat levels on the growth, carcass characteristics and meat quality of finishing pigs fed feed made from food waste, including noodles and chocolate. Four dietary treatments, 2 levels of lysine/protein ratio (0.035 and 0.046) and 2 levels of fat (3.3% and 6.0%), were adapted to a 2 × 2 factorial arrangement. Each diet for the finishing pigs contained the same levels of adequate crude protein (16%) and lysine (0.58-0.75%), and similar levels of high total digestible nutrients (90.2-92.6%). In total, 32 LWD pigs with an average body weight of 57.2 kg were assigned to 4 dietary groups. The pigs were slaughtered at about 115 kg. Growth performance was not influenced by the dietary treatments. Carcass characteristics were slightly influenced by the dietary fat level. As the dietary lysine/protein ratio decreased, the marbling score of Longissimus dorsi muscle increased and the intramuscular fat (IMF) increased from 6.82% to 9.46%. Marbling score was not significantly influenced by the dietary fat level. These results indicate that IMF increased without adverse effects on growth, carcass characteristics and meat quality, when pigs were fed a diet with low lysine/protein ratio. PMID:24261827

  19. Elevated urinary level of vitamin D-binding protein as a novel biomarker for diabetic nephropathy

    PubMed Central

    TIAN, XIAO-QIN; ZHAO, LI-MIN; GE, JIA-PU; ZHANG, YAN; XU, YAN-CHENG

    2014-01-01

    Improving the early prediction and detection of diabetic nephropathy (DN) remains a great challenge in disease management. The aim of this study was to evaluate the early detection power of urinary vitamin D-binding protein (VDBP) for the diagnosis of DN. Urine samples were obtained from 45 healthy volunteers and 105 diabetic patients with normoalbuminuria (DM group), microalbuminuria (DN1 group) and macroalbuminuria (DN2 group) (n=35 per group). The VDBP expression patterns in urine from patients and controls were quantified by western blot analysis. The excretion levels of urinary VDBP were quantified with enzyme-linked immunosorbent assay. The quantification results were obtained by correcting for creatinine expression and showed that urinary VDBP levels were significantly elevated in the patients of the DN1 and DN2 groups compared with those of the DM group and normal controls (1,011.33±325.30 and 1,406.34±239.66 compared with 466.54±213.63 and 125.48±98.27 ng/mg, respectively) (P<0.001). Receiver operating characteristic analysis of urinary VDBP levels for the diagnosis of DN rendered an optimum cut-off value of 552.243 ng/mg corresponding to 92.86% sensitivity and 85.00% specificity, which also showed an area under the ROC curve of 0.966. In conclusion, the findings of the present study suggest that urinary VDBP may be a potential biomarker for the early detection and prevention of DN. Further studies are required to examine the pathogenic mechanisms of elevated VDBP levels and their role in the diagnosis of DN. PMID:24396416

  20. Diet quality is associated with circulating C-reactive protein but not irisin levels in humans

    PubMed Central

    Park, Kyung Hee; Zaichenko, Lesya; Peter, Patricia; Davis, Cynthia R.; Crowell, Judith A.; Mantzoros, Christos S.

    2015-01-01

    Objective Adherence to a healthy diet has been shown to decrease the incidence of obesity and associated comorbidities. C-reactive protein (CRP) is an established inflammatory marker and irisin was recently identified as a molecule which may play a role in energy regulation and obesity but whether diet alters irisin levels remains unknown. We aimed to investigate the association between circulating irisin, leptin, and CRP levels and dietary quantity and quality using the Alternate Healthy Eating Index (AHEI) and the Alternate Mediterranean Diet Score (aMED). Materials/Methods The study evaluated dietary data and biomarker levels of 151 participants between 2009 and 2011 (71 male vs. 80 female, over 35 years old, obese 43.7%). AHEI and aMED scores were calculated based on data derived from self-administered 110-item food-frequency questionnaires estimating usual nutrient intake over the past year. Cross-sectional associations between dietary quantity, quality, body composition by bioelectric impedance, and biomarker levels including irisin, leptin, and CRP after fasting were assessed. Results CRP, but not irisin, was negatively correlated with AHEI (r = −0.34) and aMED (r = −0.31). Irisin was positively correlated with BMI (r = 0.22), fat mass (r = 0.21), waist circumference (r = 0.24), waist-hip ratio (r = 0.20), leptin (r = 0.32), and CRP (r = 0.25). Participants with the highest AHEI scores tended to have 11.6% lower concentrations of irisin (P for trend =0.09), but they were not significant after adjustment for potential confounders. Better diet quality was associated with lower CRP concentrations (P for trend=0.02) in multivariate model. Percentage of energy from carbohydrate was inversely associated with CRP. Conclusions Unlike CRP, irisin is not associated with dietary quality or quantity. PMID:24315778

  1. YB-1 and MTA1 protein levels and not DNA or mRNA alterations predict for prostate cancer recurrence

    PubMed Central

    Sheridan, Christine Moore; Grogan, Tristan R.; Nguyen, Hao G.; Galet, Colette; Rettig, Matthew B.; Hsieh, Andrew C.; Ruggero, Davide

    2015-01-01

    Attempts to identify biomarkers to detect prostate tumorigenesis, and thus minimize prostate cancer progression and inform treatment decisions have primarily focused on alterations at the DNA and mRNA levels, ignoring alterations at the level of protein synthesis control. We have previously shown that the PI3K-AKT-mTOR pathway, frequently deregulated in prostate cancer, specifically induces the synthesis of proteins that contribute to metastasis, most notably YB-1 and MTA1, without altering mRNA levels thereby demonstrating the importance of translation control in driving the expression of these genes in cancer. Here, we analyze genomic sequencing and mRNA expression databases, as well as protein expression employing an annotated tissue microarray generated from 332 prostate cancer patients with 15 years of clinical follow-up to determine the combined prognostic capability of YB-1 and MTA1 alterations in forecasting prostate cancer outcomes. Remarkably, protein abundance, but not genomic or transcriptional alterations of YB-1 and MTA1, is predictive of disease recurrence, exhibiting a dose-dependent effect on time to PSA recurrence, an indicator of tumor relapse. Moreover, high protein levels of YB-1 and MTA1 are associated with a 3-fold increased risk for requiring future hormone therapy or radiation therapy. Importantly, YB-1 and MTA1 protein levels significantly increase the predictive capacity of a clinical model for prostate cancer recurrence. These findings demonstrate that protein abundance of YB-1 and MTA1, irrespective of DNA or mRNA status, can predict for prostate cancer relapse and uncover a vast underappreciated repository of biomarkers regulated at the level of protein expression. PMID:25797255

  2. Elevated C-reactive protein levels and metabolic syndrome in the elderly

    PubMed Central

    Zuliani, Giovanni; Volpato, Stefano; Galvani, Matteo; Blè, Alessandro; Bandinelli, Stefania; Corsi, Anna Maria; Lauretani, Fulvio; Maggio, Marcello; Guralnik, Jack M.; Fellin, Renato; Ferrucci, Luigi

    2009-01-01

    Metabolic syndrome (MS) and “low grade” systemic inflammation (LGSI) are very common findings in the older population. Although MS and LGSI have been associated in adults, it is not known what is the real contribution of MS, and its single components, to LGSI in older persons, due to the potential confounding effect of comorbidity and aging. We investigated the relationship between increased C-reactive protein (CRP) plasma levels, a marker of LGSI, and MS in 1044 older (≥65 years) community dwelling Italian individuals enrolled the InChianti study. Metabolic syndrome was defined by the NCEP-ATP III-AHA/NHLBI criteria. High sensitivity CRP (hs.CRP) levels were measured by enzyme-linked immunosorbent assay, and defined as high when >3 mg/L. The overall prevalence of MS was 31%. The prevalence of high hs.CRP was 54.5% in subjects with, and 41.3% in those without MS (p < 0.001). MS was associated with high hs.CRP levels after adjustment for age, gender, and comorbidity (OR: 1.93, 95% CI: 1.46-2.55). Compared to subjects with MS and no LGSI, individuals with MS and LGSI were characterized by higher waist circumference, BMI, and HOMA score. Multivariate logistic regression analysis confirmed the association between waist circumference and high hs.CRP levels in subjects with MS (waist circumference III vs. I tertile OR: 2.60, 95% CI: 1.79-3.77) independent of age, gender, and important confounding variables including comorbidity. Additional analyses, conducted with and without dichotomization of hs.CRP levels, confirmed the central role of waist circumference in the LGSI phenomenon, independent of gender and diagnosis of MS. We conclude that in older individuals, MS is associated with LGSI, but the association is mainly supported by a strong independent correlation between waist circumference and high hs.CRP levels. In the absence of this specific MS component, it seems that the contribution of MS to LGSI would be modest at best. PMID:18845301

  3. Protein pheromone expression levels predict and respond to the formation of social dominance networks

    PubMed Central

    Nelson, Adam C.; Cunningham, Christopher B.; Ruff, James S.; Potts, Wayne K.

    2015-01-01

    Communication signals are key regulators of social networks, and are thought to be under selective pressure to honestly reflect social status, including dominance status. The odors of dominants and nondominants differentially influence behavior, and identification of the specific pheromones associated with, and predictive of, dominance status is essential for understanding the mechanisms of network formation and maintenance. In mice, major urinary proteins (MUPs) are excreted in extraordinary large quantities and expression level has been hypothesized to provide an honest signal of dominance status. Here, we evaluate whether MUPs are associated with dominance in wild-derived mice by analyzing expression levels before, during, and after competition for reproductive resources over three days. During competition, dominant males have 24% greater urinary MUP expression than nondominants. The MUP darcin, a pheromone that stimulates female attraction, is predictive of dominance status: dominant males have higher darcin expression before competition. Dominants also have a higher ratio of darcin to other MUPs before and during competition. These differences appear transient, because there are no differences in MUPs or darcin after competition. We also find MUP expression is affected by sire dominance status: socially naive sons of dominant males have lower MUP expression, but this apparent repression is released during competition. A requisite condition for the evolution of communication signals is honesty, and we provide novel insight into pheromones and social networks by showing that MUP and darcin expression is a reliable signal of dominance status, a primary determinant of male fitness in many species. PMID:25867293

  4. Elevated C-reactive protein level predicts lower gastrointestinal tract bleeding

    PubMed Central

    TOMIZAWA, MINORU; SHINOZAKI, FUMINOBU; HASEGAWA, RUMIKO; SHIRAI, YOSHINORI; MOTOYOSHI, YASUFUMI; SUGIYAMA, TAKAO; YAMAMOTO, SHIGENORI; ISHIGE, NAOKI

    2016-01-01

    Lower gastrointestinal (GI) bleeding can be caused by colorectal polyps or cancer. The aim of the present study was to identify blood test variables and medications that can predict lower GI bleeding, which would allow for appropriate colonoscopy. The medical records of patients who underwent colonoscopy from September 2014 to September 2015 were retrospectively analyzed. The selected patients included 278 men (mean age, 67.0±11.5 years) and 249 women (mean age, 69.6±12.0 years). The diagnosis, medications, and blood test variables were compared between patients with and without bleeding. Logistic regression analysis was performed to determine the factors associated with lower GI bleeding. The presence of colorectal polyp and cancer was associated with lower GI bleeding (P=0.0044) with an odds ratio of 6.71 (P=0.0148). No lower GI bleeding was observed in patients taking non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, or anticoagulants. The C-reactive protein (CRP) levels were significantly higher in patients with lower GI bleeding (P=0.0227). The Hb levels were lower in patients with lower GI bleeding, however this finding was not statistically significant (P>0.05). No blood test variable was associated with lower GI bleeding. Elevated CRP was associated with lower GI bleeding, while there was no association between the medications and lower GI bleeding. PMID:27284411

  5. Neuron-Specific Enolase, S100 Calcium-Binding Protein B, and Heat Shock Protein 70 Levels in Patients With Intracranial Hemorrhage

    PubMed Central

    Alatas, Ömer Doğan; Gürger, Mehtap; Ateşçelik, Metin; Yildiz, Mustafa; Demir, Caner Feyzi; Kalayci, Mehmet; Ilhan, Nevin; Acar, Ethem

    2015-01-01

    Abstract The authors evaluated neuron-specific enolase (NSE), S100 calcium-binding protein B (S100B), and heat shock protein 70 (HSP 70) levels and their relationships with in-hospital mortality, Glasgow Coma Scale (GCS) scores, and National Institute of Health Stroke Scale (NIHSS) scores. In total, 35 patients older than 18 years were presented to our emergency department and were diagnosed with non-traumatic intracranial hemorrhage (ICH) and 32 healthy controls were included. Blood samples were drawn on days 0 and 5. S100 calcium-binding protein B and HSP levels were significantly higher in patients than in controls on days 0 and 5. Neuron-specific enolase levels were higher in patients than in controls on day 0, but there was no significant difference on day 5. S100 calcium-binding protein B was negatively correlated with GCS, whereas it was positively correlated with NIHSS and bleeding volume. There was also a negative correlation between NSE and GCS, but it was not statistically significant. In addition, no significant correlation was found in terms of bleeding volume or NIHSS. Heat shock protein 70 was negatively correlated with GCS and positively correlated with bleeding volume and NIHSS, but these results were not statistically significant. S100 calcium-binding protein B and HSP 70 levels were significantly higher in those who died compared with survivors. The areas under the curve of S100 B, NSE, and HSP 70 for mortality were 0.635, 0.477, and 0.770, respectively. Neuron-specific enolase, S100B, and HSP 70 levels are simple, inexpensive, and objective measures in cases of ICH. These tests can be used to support an assessment for screening ICH patients with clinical scoring systems, such as GCS and NIHSS. PMID:26559295

  6. Fluorescent labeling for clonal selection of Marc 145 cells secreting high levels of recombinant protein PBD-1.

    PubMed

    Huang, Hai-Jun; Peng, Xia; Deng, Bing; Huang, Cong; Li, Jie; Qian, Yun-Guo; Gao, Qi-Shuang; Xiang, Min; Lu, Shun; Chen, Zhi-Hua; Zhan, Cai-Yao; Zhou, Li; Tao, Bi-Fei; Liu, Jie; Tan, Ben-Zhong

    2016-03-01

    Despite the powerful impact gene expression markers like the green fluorescent protein (GFP) or enhanced GFP (EGFP) exert on linking the expression of recombinant protein for selection of high producers in recent years, there is still a strong incentive to develop more economical and efficient methods for isolating mammalian cell clones secreting high levels of recombinant proteins. Here we present a new method based on the co-expression of EGFP that allows clonal selection in standard 96-well cell culture plates. The genes encoding the EGFP protein and the related protein are linked by an internal ribosome entry site and thus are transcribed into the same mRNA in an independent translation process. Since both proteins arise from a common mRNA, the EGFP expression level correlates with the expression level of the therapeutic protein in each clone. By expressing recombinant porcine β-defensin 1 in Marc 145 cells, we demonstrate the robustness and performance of this technique. The method can be served as an alternative to identify high-producer clones with various cell sorting methods. PMID:25297006

  7. Baseline C-Reactive Protein Levels and Life Prognosis in Parkinson Disease

    PubMed Central

    Sawada, Hideyuki; Oeda, Tomoko; Umemura, Atsushi; Tomita, Satoshi; Kohsaka, Masayuki; Park, Kwiyoung; Yamamoto, Kenji; Sugiyama, Hiroshi

    2015-01-01

    Background C-reactive protein (CRP) is a biomarker of inflammation, and high levels of CRP correlate with vascular death. Chronic inflammation is considered to be involved in neurodegeneration, although there is no evidence linking it with the process of neurodegenerative diseases. Objective To determine the role of baseline CRP levels in the prognosis of patients with Parkinson disease (PD). Methods A cohort of 313 patients with a mean age of 69.1 and mean PD duration of 7.9 years was retrospectively followed for a mean observation time of 1,753 days. CRP was measured when patients were not diagnosed with any infections, and levels were repetitively measured to investigate a tendency of “regression to mean.” The primary outcome measure was a survival time from study enrollment to death. Results During the observation period 56 patients died. Baseline CRP was log-linearly associated with a risk of death in PD. Mean survival time was 3,149 (95% confidence interval; 3,009-3,289) days in patients with CRP ≤ 0.8mg/L (lower two thirds) and 2,620 (2,343-2,897) days in those with CRP > 0.8 mg/L (top third, p < 0.001, log-rank test). The adjusted hazard ratio (HR) per two-fold higher CRP concentration for all deaths was 1.29 (1.10-1.52), and after excluding PD-unrelated deaths, such as cancer or stroke, HR was 1.23 (1.01-1.49) (adjusted for age, sex, PD duration, modified Hohen-Yahr stages, MMSE scores, and serum albumin). Conclusions Baseline CRP concentrations were associated with the risk of death and predicted life prognosis of patients with PD. The associations were independent from PD duration, PD severity, cognitive function, ages, and nutritional conditions, suggesting the possibility that subclinical chronic inflammation is associated with a neurodegenerative process in PD. PMID:26218286

  8. Adult consumers of cranberry juice cocktail have lower C-reactive protein levels compared with nonconsumers.

    PubMed

    Duffey, Kiyah J; Sutherland, Lisa A

    2015-02-01

    Flavonoids are important bioactive plant constituents found in abundance in berries, including cranberries. Cranberry beverages have been shown to beneficially impact urinary and cardiovascular health in clinical and observational studies, but their association with anthropometric outcomes is unknown. We examined the association between cranberry juice cocktail (CJC) consumption with flavonoid intake, and cardiometabolic and anthropometric outcomes among adults in the US data for adults (≥19 years, n = 10334) were drawn from cross-sectional National Health and Nutrition Examination Survey combined 2005-2008 survey. We hypothesized that CJC consumers will have lower anthropometric measures and healthier cardiometabolic profiles, including lower cholesterol and C-reactive protein (CRP). A CJC consumer (n = 330) was defined as anyone consuming CJC for 2 nonconsecutive 24-hour dietary recalls. We used multivariate linear regression models to examine differences in anthropometric and cardiometabolic outcomes comparing CJC consumers to nonconsumers controlling for important confounders. Consumers drank an average 404 mL (14 fl oz) of CJC for 2 days and did not have higher total energy intakes compared with nonconsumers (mean [SD], 2259 [79] vs 2112 [24], respectively). In fully adjusted models, adult CJC consumers had significantly lower levels of CRP (mean [SD], -0.13 [0.05]; P = .015), results that were strengthened after further adjustment for body mass index (mean [SD], -0.98 [0.04]; P = .027). Trends toward lower weights and lower levels of cholesterol did not reach statistical significance. Intake of cranberry polyphenols may play a role in promoting anti-inflammatory markers among CJC consumers, specifically lowering CRP levels. PMID:25530012

  9. C-Reactive Protein Levels Among U.S. Adults Exposed to Parental Incarceration

    PubMed Central

    Boch, Samantha J.; Ford, Jodi L.

    2015-01-01

    Previous studies have linked childhood adversity to low-grade inflammation via C-reactive protein (CRP) levels. This study analyzed the association between low-grade inflammation and prior biological parental incarceration. Data from the National Longitudinal Study of Adolescent to Adult Health (1994–2008) were analyzed using multinomial logistic regression models. Measures included high-sensitivity (hs)-CRP (<3 mg/L = reference, 3–10 mg/L = low-grade inflammation, and >10 mg/L = acute inflammation), parent incarceration occurring in the child's lifetime, and frequency and timing of incarceration with respect to child's age (0–18 years or >18 years vs. never) of incarceration. Analyses were stratified by child's gender. Final sample sizes were n = 5,396 males and n = 6,447 females for maternal incarceration and n = 4,956 males and n = 5,860 females for paternal incarceration. In models with and without potential mediators, females whose fathers were ever incarcerated were more likely to have hs-CRP levels of 3–10 mg/L than females whose fathers were never incarcerated (adjusted odds ratio [AOR]: 1.44, 95% confidence interval [CI]: [1.09, 1.91]). Additionally, daughter's age (<18 years; AOR: 1.48, 95% CI: [1.11, 1.97]) and frequency of father's incarceration were significant (AOR: 1.24, 95% CI: [1.04, 1.49]). No mediating effects were observed. Males whose fathers were incarcerated when they were ≥ 18 years were less likely to have hs-CRP levels of 3–10 mg/L than those whose father was never incarcerated; the association was nonsignificant in the mediated model. Further investigation is needed on the physiological effects of exposure to parental incarceration and interventions to support children. PMID:25539769

  10. Determining synthesis rates of individual proteins in zebrafish (Danio rerio) with low levels of a stable isotope labelled amino acid.

    PubMed

    Geary, Bethany; Magee, Kieran; Cash, Phillip; Young, Iain S; Whitfield, Phillip D; Doherty, Mary K

    2016-05-01

    The zebrafish is a powerful model organism for the analysis of human cardiovascular development and disease. Understanding these processes at the protein level not only requires changes in protein concentration to be determined but also the rate at which these changes occur on a protein-by-protein basis. The ability to measure protein synthesis and degradation rates on a proteome-wide scale, using stable isotope labelling in conjunction with mass spectrometry is now a well-established experimental approach. With the advent of more selective and sensitive mass spectrometers, it is possible to accurately measure lower levels of stable isotope incorporation, even when sample is limited. In order to challenge the sensitivity of this approach, we successfully determined the synthesis rates of over 600 proteins from the cardiac muscle of the zebrafish using a diet where either 30% or 50% of the L-leucine was replaced with a stable isotope labelled analogue ([(2) H7 ]L-leucine]. It was possible to extract sufficient protein from individual zebrafish hearts to determine the incorporation rate of the label into hundreds of proteins simultaneously, with the two labelling regimens showing a good correlation of synthesis rates. PMID:26929125

  11. RESPONSE OF HEPATIC PROTEINS TO THE LOWERING OF HABITUAL DIETARY PROTEIN TO THE RECOMMENDED SAFE LEVEL OF INTAKE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The plasma concentrations of albumin, HDL apolipoprotein A1 (apoA1), retinol-binding protein (RBP), transthyretin (TTR), haptoglobulin, and fibrinogen were measured, and a stable isotope infusion protocol was used to determine the fractional and absolute synthesis rates of RBP, TTR, and fibrinogen i...

  12. Identification of novel candidate drivers connecting different dysfunctional levels for lung adenocarcinoma using protein-protein interactions and a shortest path approach

    PubMed Central

    Chen, Lei; Huang, Tao; Zhang, Yu-Hang; Jiang, Yang; Zheng, Mingyue; Cai, Yu-Dong

    2016-01-01

    Tumors are formed by the abnormal proliferation of somatic cells with disordered growth regulation under the influence of tumorigenic factors. Recently, the theory of “cancer drivers” connects tumor initiation with several specific mutations in the so-called cancer driver genes. According to the differentiation of four basic levels between tumor and adjacent normal tissues, the cancer drivers can be divided into the following: (1) Methylation level, (2) microRNA level, (3) mutation level, and (4) mRNA level. In this study, a computational method is proposed to identify novel lung adenocarcinoma drivers based on dysfunctional genes on the methylation, microRNA, mutation and mRNA levels. First, a large network was constructed using protein-protein interactions. Next, we searched all of the shortest paths connecting dysfunctional genes on different levels and extracted new candidate genes lying on these paths. Finally, the obtained candidate genes were filtered by a permutation test and an additional strict selection procedure involving a betweenness ratio and an interaction score. Several candidate genes remained, which are deemed to be related to two different levels of cancer. The analyses confirmed our assertions that some have the potential to contribute to the tumorigenesis process on multiple levels. PMID:27412431

  13. Identification of novel candidate drivers connecting different dysfunctional levels for lung adenocarcinoma using protein-protein interactions and a shortest path approach.

    PubMed

    Chen, Lei; Huang, Tao; Zhang, Yu-Hang; Jiang, Yang; Zheng, Mingyue; Cai, Yu-Dong

    2016-01-01

    Tumors are formed by the abnormal proliferation of somatic cells with disordered growth regulation under the influence of tumorigenic factors. Recently, the theory of "cancer drivers" connects tumor initiation with several specific mutations in the so-called cancer driver genes. According to the differentiation of four basic levels between tumor and adjacent normal tissues, the cancer drivers can be divided into the following: (1) Methylation level, (2) microRNA level, (3) mutation level, and (4) mRNA level. In this study, a computational method is proposed to identify novel lung adenocarcinoma drivers based on dysfunctional genes on the methylation, microRNA, mutation and mRNA levels. First, a large network was constructed using protein-protein interactions. Next, we searched all of the shortest paths connecting dysfunctional genes on different levels and extracted new candidate genes lying on these paths. Finally, the obtained candidate genes were filtered by a permutation test and an additional strict selection procedure involving a betweenness ratio and an interaction score. Several candidate genes remained, which are deemed to be related to two different levels of cancer. The analyses confirmed our assertions that some have the potential to contribute to the tumorigenesis process on multiple levels. PMID:27412431

  14. Lack of association of acute phase response proteins with hormone levels and antidepressant medication in perimenopausal depression

    PubMed Central

    2014-01-01

    Background Major depression is associated with higher plasma levels of positive acute-phase proteins, as well as with lower plasma levels of negative acute-phase proteins. The aim of this study is to examine the levels of acute-phase response proteins and whether these levels are influenced by reproductive hormones and antidepressant medication in the perimenopausal depression. Methods Sixty-five women (age range: 40–58 years old) participated in this study. All women were in the perimenopausal phase. The diagnosis of depression was made through a psychiatric interview and with the aid of Hamilton Depression Rating Scale 17 (HAM-D 17). The acute-phase response proteins, such as haptoglobin (HP), transferrine (TRf), α1-antitrypsin, complement protein 3 (C3), complement protein 4 (C4) and C-reactive protein (CRP) and the reproductive hormones, for example follicle-stimulating hormone (FSH), luteinizing hormone (LH) and estradiol (E2), were analyzed using standard laboratory methods. Pearson’s correlations were applied to evaluate the relationship between acute-phase proteins and hormones. Results Perimenopausal women were divided into three groups. The first group consisted of normal controls, the second one involved depressed perimenopausal women, who were taking selective serotonin reuptake inhibitors (SSRIs), and the third one included depressed women that were not treated with SSRIs. Depressed women in perimenopause, when being compared to non-depressed women, did not differ as to serum levels of acute-phase proteins. There was a positive correlation between HP and E2 in depressed perimenopausal women, who were not taking SSRIs. Conclusions The lack of association between acute-phase proteins and depressive mood mentioned in this study does not support previous findings in patients with major depression. This negative finding in perimenopausal depression indicates either the absence or a more complex nature of the interactions between acute-phase proteins

  15. Enhanced skeletal muscle protein synthesis rates in pigs treated with somatotropin requires fed amino acids levels

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  16. Effects of balanced dietary protein levels on egg production and egg quality parameters of individual commercial layers.

    PubMed

    Shim, M Y; Song, E; Billard, L; Aggrey, S E; Pesti, G M; Sodsee, P

    2013-10-01

    The effects of a series of balanced dietary protein levels on egg production and egg quality parameters of laying hens from 18 through 74 wk of age were investigated. One hundred forty-four pullets (Bovans) were randomly assigned to individual cages with separate feeders including 3 different protein level series of isocaloric diets. Diets were separated into 4 phases of 18-22, 23-32, 33-44, and 45-74 wk of age. The high protein (H) series contained 21.62, 19.05, 16.32, and 16.05% CP, respectively. Medium protein (M) and low protein (L) series were 2 and 4% lower in balanced dietary protein. The results clearly demonstrated that the balanced dietary protein level was a limiting factor for BW, ADFI, egg weight, hen day egg production (HDEP), and feed per kilogram of eggs. Feeding with the L series resulted in lower ADFI and HDEP (90.33% peak production) and more feed per kilogram of eggs compared with the H or M series (HDEP; 93.23 and 95.68% peak production, monthly basis). Egg weight responded in a linear manner to balanced dietary protein level (58.78, 55.94, and 52.73 g for H, M, and L, respectively). Feed intake of all hens, but especially those in the L series, increased considerably after wk 54 when the temperature of the house decreased due to winter conditions. Thus, hens fed the L series seemed particularly dependent on house temperature to maintain BW, ADFI, and HDEP. For egg quality parameters, percent yolk, Haugh units, and egg specific gravity were similar regardless of diets. Haugh units were found to be greatly affected by the variation of housing temperature (P = 0.025). Maximum performance cannot always be expected to lead to maximum profits. Contrary to the idea of a daily amino acid requirement for maximum performance, these results may be used to determine profit-maximizing levels of balanced dietary protein based on the cost of protein and returns from different possible protein levels that may be fed. PMID:24046416

  17. E3 ubiquitin ligase RFWD2 controls lung branching through protein-level regulation of ETV transcription factors.

    PubMed

    Zhang, Yan; Yokoyama, Shigetoshi; Herriges, John C; Zhang, Zhen; Young, Randee E; Verheyden, Jamie M; Sun, Xin

    2016-07-01

    The mammalian lung is an elaborate branching organ, and it forms following a highly stereotypical morphogenesis program. It is well established that precise control at the transcript level is a key genetic underpinning of lung branching. In comparison, little is known about how regulation at the protein level may play a role. Ring finger and WD domain 2 (RFWD2, also termed COP1) is an E3 ubiquitin ligase that modifies specific target proteins, priming their degradation via the ubiquitin proteasome system. RFWD2 is known to function in the adult in pathogenic processes such as tumorigenesis. Here, we show that prenatal inactivation of Rfwd2 gene in the lung epithelium led to a striking halt in branching morphogenesis shortly after secondary branch formation. This defect is accompanied by distalization of the lung epithelium while growth and cellular differentiation still occurred. In the mutant lung, two E26 transformation-specific (ETS) transcription factors essential for normal lung branching, ETS translocation variant 4 (ETV4) and ETV5, were up-regulated at the protein level, but not at the transcript level. Introduction of Etv loss-of-function alleles into the Rfwd2 mutant background attenuated the branching phenotype, suggesting that RFWD2 functions, at least in part, through degrading ETV proteins. Because a number of E3 ligases are known to target factors important for lung development, our findings provide a preview of protein-level regulatory network essential for lung branching morphogenesis. PMID:27335464

  18. Copper uptake is required for pyrrolidine dithiocarbamate-mediated oxidation and protein level increase of p53 in cells.

    PubMed Central

    Furuta, Saori; Ortiz, Fausto; Zhu Sun, Xiu; Wu, Hsiao-Huei; Mason, Andrew; Momand, Jamil

    2002-01-01

    The p53 tumour-suppressor protein is a transcription factor that activates the expression of genes involved in cell cycle arrest, apoptosis and DNA repair. The p53 protein is vulnerable to oxidation at cysteine thiol groups. The metal-chelating dithiocarbamates, pyrrolidine dithiocarbamate (PDTC), diethyldithiocarbamate, ethylene(bis)dithiocarbamate and H(2)O(2) were tested for their oxidative effects on p53 in cultured human breast cancer cells. Only PDTC oxidized p53, although all oxidants tested increased the p53 level. Inductively coupled plasma MS analysis indicated that the addition of 60 microM PDTC increased the cellular copper concentration by 4-fold, which was the highest level of copper accumulated amongst all the oxidants tested. Bathocuproinedisulphonic acid, a membrane-impermeable Cu(I) chelator inhibited the PDTC-mediated copper accumulation. Bathocuproinedisulphonic acid as well as the hydroxyl radical scavenger d-mannitol inhibited the PDTC-dependent increase in p53 protein and oxidation. Our results show that a low level of copper accumulation in the range of 25-40 microg/g of cellular protein increases the steady-state levels of p53. At copper accumulation levels higher than 60 microg/g of cellular protein, p53 is oxidized. These results suggest that p53 is vulnerable to free radical-mediated oxidation at cysteine residues. PMID:11964141

  19. Spaceflight has compartment- and gene-specific effects on mRNA levels for bone matrix proteins in rat femur

    NASA Technical Reports Server (NTRS)

    Evans, G. L.; Morey-Holton, E.; Turner, R. T.

    1998-01-01

    In the present study, we evaluated the possibility that the abnormal bone matrix produced during spaceflight may be associated with reduced expression of bone matrix protein genes. To test this possibility, we investigated the effects of a 14-day spaceflight (SLS-2 experiment) on steady-state mRNA levels for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), osteocalcin, osteonectin, and prepro-alpha(1) subunit of type I collagen in the major bone compartments of rat femur. There were pronounced site-specific differences in the steady-state levels of expression of the mRNAs for the three bone matrix proteins and GAPDH in normal weight-bearing rats, and these relationships were altered after spaceflight. Specifically, spaceflight resulted in decreases in mRNA levels for GAPDH (decreased in proximal metaphysis), osteocalcin (decreased in proximal metaphysis), osteonectin (decreased in proximal and distal metaphysis), and collagen (decreased in proximal and distal metaphysis) compared with ground controls. There were no changes in mRNA levels for matrix proteins or GAPDH in the shaft and distal epiphysis. These results demonstrate that spaceflight leads to site- and gene-specific decreases in mRNA levels for bone matrix proteins. These findings are consistent with the hypothesis that spaceflight-induced decreases in bone formation are caused by concomitant decreases in expression of genes for bone matrix proteins.

  20. High-level expression, purification, and characterization of bifunctional ScFv-9R fusion protein.

    PubMed

    Zhang, Xiguang; Xie, Jiasen; Sun, Yan; Xu, Huijing; Du, Tonghua; Liu, Zixuan; Chen, Jinhui; Zheng, Zhong; Liu, Keqiang; Zhang, Jizhou; Kan, Mujie; Li, Xiaokun; Xiao, Yechen

    2014-06-01

    Fibroblast growth factor receptor 3 (FGFR3) is a noted proto-oncogene involved in the pathogenesis of many tumors, so more and more studies focus on the potential use of receptor kinase inhibitor and therapeutic antibodies against FGFR3. In this study, we designed a novel fusion protein containing the single-chain Fv (ScFv) against FGFR3 and 9-arginine, denoted as ScFv-9R. To achieve the high-level production and soluble expression, ScFv and ScFv-9R were fused with small ubiquitin-related modifier (Sumo) by polymerase chain reaction and expressed in Escherichia coli BL21 (DE3). The recombinant bacteria was induced by 0.5 mM isopropyl-β-D-thiogalactopyranoside for 20 h at 20 °C; supernatants of Sumo-ScFv was harvested and purified by DEAE Sepharose FF and Ni-NTA orderly, and supernatants of Sumo-ScFv-9R was harvested and purified by Ni-NTA. After cleaved by the Sumo protease, the recombinant ScFv or ScFv-9R was released from the fusion protein, respectively. The purity of ScFv or ScFV-9R was shown to be higher than 90 %, and their yield reached 3-5 mg per liter of bacterial culture. In vitro data showed that ScFV-9R can attenuate the phosphorylation of FGFR3 and ERK in the absence or presence of FGF9. Gel retardation assay showed that 1 μg of ScFv-9R could efficiently bind to about 4 pmol siRNA. Fluorescent microscope analysis showed that ScFv-9R can efficiently bind and deliver siRNA into RT112 cells. In conclusion, we use Sumo fusion system to acquire high-level production, soluble expression, and bifunctional activity of ScFv-9R in E. coli. Our results also revealed that ScFv-9R, as a novel carrier, may have potential applications in antitumor studies and pharmaceutical development. PMID:24519456

  1. Extracellular matrix controls tubulin monomer levels in hepatocytes by regulating protein turnover

    NASA Technical Reports Server (NTRS)

    Mooney, D. J.; Hansen, L. K.; Langer, R.; Vacanti, J. P.; Ingber, D. E.

    1994-01-01

    Cells have evolved an autoregulatory mechanism to dampen variations in the concentration of tubulin monomer that is available to polymerize into microtubules (MTs), a process that is known as tubulin autoregulation. However, thermodynamic analysis of MT polymerization predicts that the concentration of free tubulin monomer must vary if MTs are to remain stable under different mechanical loads that result from changes in cell adhesion to the extracellular matrix (ECM). To determine how these seemingly contradictory regulatory mechanisms coexist in cells, we measured changes in the masses of tubulin monomer and polymer that resulted from altering cell-ECM contacts. Primary rat hepatocytes were cultured in chemically defined medium on bacteriological petri dishes that were precoated with different densities of laminin (LM). Increasing the LM density from low to high (1-1000 ng/cm2), promoted cell spreading (average projected cell area increased from 1200 to 6000 microns2) and resulted in formation of a greatly extended MT network. Nevertheless, the steady-state mass of tubulin polymer was similar at 48 h, regardless of cell shape or ECM density. In contrast, round hepatocytes on low LM contained a threefold higher mass of tubulin monomer when compared with spread cells on high LM. Furthermore, similar results were obtained whether LM, fibronectin, or type I collagen were used for cell attachment. Tubulin autoregulation appeared to function normally in these cells because tubulin mRNA levels and protein synthetic rates were greatly depressed in round cells that contained the highest level of free tubulin monomer. However, the rate of tubulin protein degradation slowed, causing the tubulin half-life to increase from approximately 24 to 55 h as the LM density was lowered from high to low and cell rounding was promoted. These results indicate that the set-point for the tubulin monomer mass in hepatocytes can be regulated by altering the density of ECM contacts and

  2. Influence of chlortetracycline and dietary protein level on visceral organ mass of growing beef steers.

    PubMed

    Baldwin, R L; McLeod, K R; Elsassert, T H; Kahl, S; Rumsey, T S; Streeter, M N

    2000-12-01

    Thirty-two beef steers (285 +/- 3 kg BW) were used to determine the effects of chlortetracycline and dietary protein level on visceral tissue mass, chemical composition, intestinal morphology, and proliferation rate indices. Steers were allotted randomly by weight to a factorial arrangement of dietary treatments consisting of either 10 or 13% CP diets top-dressed with a corn meal carrier (500 g/d) containing either 0 or 350 mg of chlortetracycline. After 84 d, steers were slaughtered and visceral organs removed and separated. Rinsed wet tissue mass was recorded; total RNA, total DNA, tissue DM, and tissue N content were determined; and tissue sections were prepared for immunohistochemical analysis. Thin tissue sections were evaluated to determine crypt depth and villus height as well as proliferation rate by immunohistochemical detection of the nuclear antigen Ki67. Rumen and abomasum weights and small intestinal length were greater (P < 0.04) in steers fed the 13% CP diet than in those fed the 10% CP diet on both an absolute weight basis and a percentage of empty BW. Chemical composition of the small intestinal and ruminal segments were largely unaffected by increased dietary protein. Increasing the dietary CP also increased the villus height in duodenal (P = 0.02) and the crypt depth of jejunal (P = 0.03) sections. Dietary administration of chlortetracycline decreased (P < 0.01) small intestinal weight both on absolute and empty BW bases. Nitrogen and RNA concentrations of the small intestinal segments were unaffected (P > 0.1) by dietary administration of subtherapeutic levels of chlortetracycline; however, because of increases (P < 0.05), or tendencies for an increase (P < 0.1), in the tissue content of DNA, the ratio of N to DNA was decreased (P < 0.05) or tended to be decreased (P < 0.1) in the small intestinal segments of the chlortetracycline-treated animals. The observed decrease in small intestinal epithelial mass does not appear to be due to alterations

  3. Identification of Leaf Proteins Differentially Accumulated between Wheat Cultivars Distinct in Their Levels of Drought Tolerance.

    PubMed

    Cheng, Zhiwei; Dong, Kun; Ge, Pei; Bian, Yanwei; Dong, Liwei; Deng, Xiong; Li, Xiaohui; Yan, Yueming

    2015-01-01

    The drought-tolerant 'Ningchun 47' (NC47) and drought-sensitive 'Chinese Spring' (CS) wheat (Triticum aestivum L.) cultivars were treated with different PEG6000 concentrations at the three-leaf stage. An analysis on the physiological and proteomic changes of wheat seedling in response to drought stress was performed. In total, 146 differentially accumulated protein (DAP) spots were separated and recognised using two-dimensional gel electrophoresis. In total, 101 DAP spots representing 77 unique proteins were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. These proteins were allocated to 10 groups according to putative functions, which were mainly involved in carbon metabolism (23.4%), photosynthesis/respiration (22.1%) and stress/defence/detoxification (18.2%). Some drought stress-related proteins in NC47, such as enolase, 6-phosphogluconate dehydrogenase, Oxygen-evolving enhancer protein 2, fibrillin-like protein, 2-Cys peroxiredoxin BAS1 and 70-kDa heat shock protein, were more upregulated than those in CS. Multivariate principal components analysis revealed obvious differences between the control and treatments in both NC47 and CS, while cluster analysis showed that the DAPs displayed five and six accumulation patterns in NC47 and CS, respectively. Protein-protein interaction network analysis showed that some key DAPs, such as 2-Cys peroxiredoxin BAS1, RuBisCO large subunit-binding protein, 50S ribosomal protein L1, 6-phosphogluconate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase isoenzyme and 70-kDa heat shock protein, with upregulated accumulation in NC47, had complex interactions with other proteins related to amino acid metabolism, carbon metabolism, energy pathway, signal transduction, stress/defence/detoxification, protein folding and nucleotide metabolism. These proteins could play important roles in drought-stress tolerance and contribute to the relatively stronger drought tolerance of NC47. PMID

  4. Cholesterol and steroid synthesizing smooth endoplasmic reticulum of adrenocortical cells contains high levels of translocation apparatus proteins.

    PubMed

    Black, V H; Sanjay, A; van Leyen, K; Möeller, I; Lauring, B; Kreibich, G

    2002-11-01

    Steroid-secreting cells possess abundant smooth endoplasmic reticulum whose membranes contain many enzymes involved in sterol and steroid synthesis. In this study we demonstrate that adrenal smooth microsomal subfractions enriched in these membranes also possess high levels of proteins belonging to the translocation apparatus, proteins previously assumed to be confined to morphologically identifiable rough endoplasmic reticulum (RER). We further demonstrate that these smooth microsomal subfractions are capable of effecting the functions of these protein complexes: co-translational translocation, signal peptide cleavage and N-glycosylation of newly synthesized polypeptides. We hypothesize that these elements participate in regulating the levels of ER-targeted membrane proteins involved in cholesterol and steroid metabolism in a sterol-dependent and hormonally-regulated manner. PMID:12530645

  5. The nuclear localization of SOCS6 requires the N-terminal region and negatively regulates Stat3 protein levels

    SciTech Connect

    Hwang, Mi-Na; Min, Chan-Hee; Kim, Hyung Sik; Lee, Ho; Yoon, Kyong-Ah; Park, Sung Yong; Lee, Eun Sook; Yoon, Sungpil . E-mail: yoons@ncc.re.kr

    2007-08-24

    We determined that endogenous- and overexpressed- SOCS6 was localized in both the nucleus and cytoplasm. The localization of SOCS6 depended on amino acids 1-210 in the N-terminal region of the protein, which contains an unidentified domain. GFP-tagged SOCS6 or the N-terminal region, was exclusively localized and widely distributed throughout the entire nucleus, whereas the C-terminal region displayed a nuclear omission pattern. We also demonstrated that the SOCS6 protein could decrease the levels of the Stat3 protein in the nucleus, and that its negative regulation of the Stat3 protein level was dependent on its C-terminal region. These observations suggest that SOCS6 is composed of at least two functional domains required for its biological role in localizing and degrading Stat3 in the nucleus.

  6. Is serum or sputum eosinophil cationic protein level adequate for diagnosis of mild asthma?

    PubMed

    Khakzad, Mohammad Reza; Mirsadraee, Majid; Sankian, Mojtaba; Varasteh, Abdolreza; Meshkat, Mojtaba

    2009-09-01

    Spirometry has been used as a common diagnostic test in asthma. Most of the patients with a mild asthma have a FEV1 within normal range. Hence, other diagnostic methods are usually used. The aim of this study was to evaluate whether eosinophil Cationic Protein (ECP) could be an accurate diagnostic marker of mild asthma. In this study diagnosis of asthma was made according to internationally accepted criteria. Asthma severity was evaluated according to frequency of symptoms and FEV1.Adequate sputum samples were obtained in 50 untreated subjects. A control group of 12 normal subjects that showed PC20 more than 8 mg/dl was also examined. Sputum was induced by inhalation of hypertonic saline. Inflammatory cells in sputum smears were assessed semi-quantitatively. ECP and IgE concentrations, eosinophil (EO) percentage and ECP/EO ratio in serum and sputum were also determined. The results revealed that Cough and dyspnea were the most frequent clinical findings. Dyspnea and wheezing were the symptoms that correlated with staging of asthma. FEV1 was within normal range (more than 80% of predicted) in 22 (44%) subjects.Asthmatic patients showed significantly higher numbers of blood eosinophils (4.5+/- 3.1% vs. 1.2+/-0.2%, P=0.009), and higher levels of serum ECP than control group (3.1+/- 2.6 % and 22.6+/- 15.8 ng/ml, respectively). Sputum ECP level in asthmatics was significantly higher than non- asthmatics (55.3+/-29.8ng/mL vs. 25.0+/-24.7ng/mL, P=0.045). Regression analysis showed no significant correlation between spirometric parameters and biomarkers, the only exception was significant correlation between FEF(25-75) and serum ECP (r= 0.28, P 0.041). Regarding clinical symptoms, wheezing was significantly correlated with elevation of most of biomarkers. Since, serum and sputum ECP levels are elevated in untreated asthmatics, the ECP level could be used for accurate diagnosis of mild form of asthma in which spirometry is unremarkable. PMID:20124607

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  9. In Situ Molecular Level Studies on Membrane Related Peptides and Proteins in Real Time Using Sum Frequency Generation Vibrational Spectroscopy

    PubMed Central

    Ye, Shuji; Nguyen, Khoi Tan; Le Clair, Stéphanie V.; Chen, Zhan

    2009-01-01

    Sum frequency generation (SFG) vibrational spectroscopy has been demonstrated to be a powerful technique to study the molecular structures of surfaces and interfaces in different chemical environments. This review summarizes recent SFG studies on hybrid bilayer membranes and substrate-supported lipid monolayers and bilayers, the interaction between peptides/proteins and lipid monolayers/bilayers, and bilayer perturbation induced by peptides/proteins. To demonstrate the ability of SFG to determine the orientations of various secondary structures, studies on the interaction between different peptides/proteins (melittin, G proteins, almethicin, and tachyplesin I) and lipid bilayers are discussed. Molecular level details revealed by SFG in these studies show that SFG can provide a unique understanding on the interactions between a lipid monolayer/bilayer and peptides/proteins in real time, in situ and without any exogenous labeling. PMID:19306928

  10. Effect of level of dietary protein on arginine-stimulated citrulline synthesis. Correlation with mitochondrial N-acetylglutamate concentrations.

    PubMed Central

    Morimoto, B H; Brady, J F; Atkinson, D E

    1990-01-01

    Increases in dietary protein have been reported to increase the rate of citrulline synthesis and the level of N-acetylglutamate in liver. We have confirmed this effect of diet on citrulline synthesis in rat liver mitochondria and show parallel increases in N-acetylglutamate concentration. The magnitude of the effect of arginine in the suspending medium on citrulline synthesis was also dependent on dietary protein content. Mitochondria from rats fed on a protein-free diet initially contained low levels of N-acetylglutamate, and addition of arginine increased the rate of its synthesis. Citrulline synthesis and acetylglutamate content in these mitochondria increased more than 5-fold when 1 mM-arginine was added. A diet high in protein results in mitochondria with increased N-acetylglutamate and a high rate of citrulline synthesis; 1 mM-arginine increased citrulline synthesis in such mitochondria by only 36%. The concentration of arginine in portal blood was 47 microM in rats fed on a diet lacking protein, and 182 microM in rats fed on a diet containing 60% protein, suggesting that arginine may be a regulatory signal to the liver concerning the dietary protein intake. The rates of citrulline synthesis were proportional to the mitochondrial content of acetylglutamate in mitochondria obtained from rats fed on diets containing 0, 24, or 60% protein, whether incubated in the absence or presence of arginine. Although the effector concentrations are higher than the Ka for the enzymes, these results support the view that concentrations of both arginine and acetylglutamate are important in the regulation of synthesis of citrulline and urea. Additionally, the effects of dietary protein level (and of arginine) are exerted in large part by way of modulation of the concentration of acetylglutamate. PMID:2268294

  11. Atomic-level description of protein-lipid interactions using an accelerated membrane model.

    PubMed

    Baylon, Javier L; Vermaas, Josh V; Muller, Melanie P; Arcario, Mark J; Pogorelov, Taras V; Tajkhorshid, Emad

    2016-07-01

    Peripheral membrane proteins are structurally diverse proteins that are involved in fundamental cellular processes. Their activity of these proteins is frequently modulated through their interaction with cellular membranes, and as a result techniques to study the interfacial interaction between peripheral proteins and the membrane are in high demand. Due to the fluid nature of the membrane and the reversibility of protein-membrane interactions, the experimental study of these systems remains a challenging task. Molecular dynamics simulations offer a suitable approach to study protein-lipid interactions; however, the slow dynamics of the lipids often prevents sufficient sampling of specific membrane-protein interactions in atomistic simulations. To increase lipid dynamics while preserving the atomistic detail of protein-lipid interactions, in the highly mobile membrane-mimetic (HMMM) model the membrane core is replaced by an organic solvent, while short-tailed lipids provide a nearly complete representation of natural lipids at the organic solvent/water interface. Here, we present a brief introduction and a summary of recent applications of the HMMM to study different membrane proteins, complementing the experimental characterization of the presented systems, and we offer a perspective of future applications of the HMMM to study other classes of membrane proteins. This article is part of a Special Issue entitled: Membrane proteins edited by J.C. Gumbart and Sergei Noskov. PMID:26940626

  12. SET overexpression in HEK293 cells regulates mitochondrial uncoupling proteins levels within a mitochondrial fission/reduced autophagic flux scenario

    SciTech Connect

    Almeida, Luciana O.; Goto, Renata N.; Neto, Marinaldo P.C.; Sousa, Lucas O.; Curti, Carlos; Leopoldino, Andréia M.

    2015-03-06

    We hypothesized that SET, a protein accumulated in some cancer types and Alzheimer disease, is involved in cell death through mitochondrial mechanisms. We addressed the mRNA and protein levels of the mitochondrial uncoupling proteins UCP1, UCP2 and UCP3 (S and L isoforms) by quantitative real-time PCR and immunofluorescence as well as other mitochondrial involvements, in HEK293 cells overexpressing the SET protein (HEK293/SET), either in the presence or absence of oxidative stress induced by the pro-oxidant t-butyl hydroperoxide (t-BHP). SET overexpression in HEK293 cells decreased UCP1 and increased UCP2 and UCP3 (S/L) mRNA and protein levels, whilst also preventing lipid peroxidation and decreasing the content of cellular ATP. SET overexpression also (i) decreased the area of mitochondria and increased the number of organelles and lysosomes, (ii) increased mitochondrial fission, as demonstrated by increased FIS1 mRNA and FIS-1 protein levels, an apparent accumulation of DRP-1 protein, and an increase in the VDAC protein level, and (iii) reduced autophagic flux, as demonstrated by a decrease in LC3B lipidation (LC3B-II) in the presence of chloroquine. Therefore, SET overexpression in HEK293 cells promotes mitochondrial fission and reduces autophagic flux in apparent association with up-regulation of UCP2 and UCP3; this implies a potential involvement in cellular processes that are deregulated such as in Alzheimer's disease and cancer. - Highlights: • SET, UCPs and autophagy prevention are correlated. • SET action has mitochondrial involvement. • UCP2/3 may reduce ROS and prevent autophagy. • SET protects cell from ROS via UCP2/3.

  13. Activation of TLR3 Promotes the Degeneration of Retinal Ganglion Cells by Upregulating the Protein Levels of JNK3

    PubMed Central

    Chintala, Shravan K.; Putris, Nahrain; Geno, Mason

    2015-01-01

    Purpose. To investigate whether activation of Toll-like receptor 3 (TLR3) promotes the degeneration of retinal ganglion cells (RGCs) by upregulating the protein levels of c-jun N-terminal kinase 3 (JNK3). Methods. Toll-like receptor 3-specific activator, Poly(I:C) (polyinosinic-polycytidylic acid), or PBS was injected into the vitreous humor of Thy1-YFP mice. At 24, 48, and 72 hours after treatments, degeneration of RGCs was assessed by using antibodies against brain-specific homeobox/POU domain protein 3a (Brn3a). A TLR3-specific inhibitor was injected into the vitreous humor with or without Poly(I:C). Western blot assays were performed to determine relative levels of TLR3, JNK3, pJNK3, and sterile alpha and HEAT/Armadillo motif-containing 1 (SARM1) proteins in retinal protein extracts, and immunohistochemistry assays were performed to determine their cellular localization in the retina. Mouse eyes were treated with Poly(I:C) or PBS along with MitoTracker Red, and colocalization of MitoTracker Red and JNK3 in the retinas was determined by using antibodies against JNK3. Results. Poly(I:C) activated TLR3 and upregulated its downstream target protein JNK3 but not SARM1 in the retina. Poly(I:C) activated TLR3 and upregulated JNK3 specifically in RGCs and promoted a significant degeneration of RGCs over a 72-hour time period. Toll-like receptor 3 upregulated the levels of JNK3 protein in the cytoplasm of RGCs, but not in the mitochondria. Toll-like receptor 3-specific inhibitor downregulated Poly(I:C)-mediated upregulation of JNK3 protein, and, in turn, significantly attenuated TLR3-induced degeneration of RGCs. Conclusions. Results presented in this study show that the activation of TLR3 alone promotes the degeneration of RGCs by upregulating the protein levels of JNK3. PMID:25564448

  14. Computational Design of Self-Assembling Protein Nanomaterials with Atomic Level Accuracy

    SciTech Connect

    King, Neil P.; Sheffler, William; Sawaya, Michael R.; Vollmar, Breanna S.; Sumida, John P.; André, Ingemar; Gonen, Tamir; Yeates, Todd O.; Baker, David

    2015-09-17

    We describe a general computational method for designing proteins that self-assemble to a desired symmetric architecture. Protein building blocks are docked together symmetrically to identify complementary packing arrangements, and low-energy protein-protein interfaces are then designed between the building blocks in order to drive self-assembly. We used trimeric protein building blocks to design a 24-subunit, 13-nm diameter complex with octahedral symmetry and a 12-subunit, 11-nm diameter complex with tetrahedral symmetry. The designed proteins assembled to the desired oligomeric states in solution, and the crystal structures of the complexes revealed that the resulting materials closely match the design models. The method can be used to design a wide variety of self-assembling protein nanomaterials.

  15. Protein-Level Fluctuation Correlation at the Microcolony Level and Its Application to the Vibrio harveyi Quorum-Sensing Circuit

    PubMed Central

    Wang, Yufang; Tu, Kimberly C.; Ong, N.P.; Bassler, Bonnie L.; Wingreen, Ned S.

    2011-01-01

    Gene expression is stochastic, and noise that arises from the stochastic nature of biochemical reactions propagates through active regulatory links. Thus, correlations in gene-expression noise can provide information about regulatory links. We present what to our knowledge is a new approach to measure and interpret such correlated fluctuations at the level of single microcolonies, which derive from single cells. We demonstrated this approach mathematically using stochastic modeling, and applied it to experimental time-lapse fluorescence microscopy data. Specifically, we investigated the relationships among LuxO, LuxR, and the small regulatory RNA qrr4 in the model quorum-sensing bacterium Vibrio harveyi. Our results show that LuxR positively regulates the qrr4 promoter. Under our conditions, we find that qrr regulation weakly depends on total LuxO levels and that LuxO autorepression is saturated. We also find evidence that the fluctuations in LuxO levels are dominated by intrinsic noise. We furthermore propose LuxO and LuxR interact at all autoinducer levels via an unknown mechanism. Of importance, our new method of evaluating correlations at the microcolony level is unaffected by partition noise at cell division. Moreover, the method is first-order accurate and requires less effort for data analysis than single-cell-based approaches. This new correlation approach can be applied to other systems to aid analysis of gene regulatory circuits. PMID:21689539

  16. [Interest of S100B protein blood level determination in severe or moderate head injury].

    PubMed

    Bouvier, Damien

    2013-01-01

    S100B, a suffering brain marker, exhibits a different interest in traumatic brain injury (TBI) as the trauma is severe (sTBI) or mild (mTBI). Our works presented for the attribution of the SFBC 2012 price talked about both aspects. Firstly, the extent of S100B elevation has been found to be useful in predicting clinical outcome after sTBI. However, few studies were realized with jugular venous blood samples. After comparing the interest between jugular venous and arterial blood concentrations evaluation of serum S100B protein in patients with sTBI, determination of S100B concentration in jugular blood samples appears to be better than in arterial ones to predict clinical outcome after brain injury. Secondly, it's difficult to determine the indication of cranial computed tomography (CCT) for patients with mTBI. Actually, 90% of patients with mTBI have unnecessary CCT or short hospitalization for observation. Serum concentrations of S100B were found to provide useful information. We have investigated in 2 studies (1 for adult, 1 for children) whether S100B concentrations in patients with mTBI could provide additional information to improve indication of the need for an initial CCT scan or for a short hospitalization. Patients with intracerebral lesions on the CCT scan (CCT+) or with bad clinical evolution were identified with a sensitivity level of 100% and a specificity level of 30%. Adding the measurement of S-100B serum concentration to the clinical decision rules for a CCT scan or hospitalization in patients with mTBI could allow a 30% reduction in scans and in hospitalization for clinical observation. PMID:23587577

  17. Identification of Leaf Proteins Differentially Accumulated between Wheat Cultivars Distinct in Their Levels of Drought Tolerance

    PubMed Central

    Bian, Yanwei; Dong, Liwei; Deng, Xiong; Li, Xiaohui; Yan, Yueming

    2015-01-01

    The drought-tolerant ‘Ningchun 47’ (NC47) and drought-sensitive ‘Chinese Spring’ (CS) wheat (Triticum aestivum L.) cultivars were treated with different PEG6000 concentrations at the three-leaf stage. An analysis on the physiological and proteomic changes of wheat seedling in response to drought stress was performed. In total, 146 differentially accumulated protein (DAP) spots were separated and recognised using two-dimensional gel electrophoresis. In total, 101 DAP spots representing 77 unique proteins were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. These proteins were allocated to 10 groups according to putative functions, which were mainly involved in carbon metabolism (23.4%), photosynthesis/respiration (22.1%) and stress/defence/detoxification (18.2%). Some drought stress-related proteins in NC47, such as enolase, 6-phosphogluconate dehydrogenase, Oxygen-evolving enhancer protein 2, fibrillin-like protein, 2-Cys peroxiredoxin BAS1 and 70-kDa heat shock protein, were more upregulated than those in CS. Multivariate principal components analysis revealed obvious differences between the control and treatments in both NC47 and CS, while cluster analysis showed that the DAPs displayed five and six accumulation patterns in NC47 and CS, respectively. Protein–protein interaction network analysis showed that some key DAPs, such as 2-Cys peroxiredoxin BAS1, RuBisCO large subunit-binding protein, 50S ribosomal protein L1, 6-phosphogluconate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase isoenzyme and 70-kDa heat shock protein, with upregulated accumulation in NC47, had complex interactions with other proteins related to amino acid metabolism, carbon metabolism, energy pathway, signal transduction, stress/defence/detoxification, protein folding and nucleotide metabolism. These proteins could play important roles in drought-stress tolerance and contribute to the relatively stronger drought tolerance of NC47

  18. Ezrin Binds to DEAD-Box RNA Helicase DDX3 and Regulates Its Function and Protein Level

    PubMed Central

    Çelik, Haydar; Sajwan, Kamal P.; Selvanathan, Saravana P.; Marsh, Benjamin J.; Pai, Amrita V.; Kont, Yasemin Saygideger; Han, Jenny; Minas, Tsion Z.; Rahim, Said; Erkizan, Hayriye Verda; Toretsky, Jeffrey A.

    2015-01-01

    Ezrin is a key regulator of cancer metastasis that links the extracellular matrix to the actin cytoskeleton and regulates cell morphology and motility. We discovered a small-molecule inhibitor, NSC305787, that directly binds to ezrin and inhibits its function. In this study, we used a nano-liquid chromatography-tandem mass spectrometry (nano-LC–MS-MS)-based proteomic approach to identify ezrin-interacting proteins that are competed away by NSC305787. A large number of the proteins that interact with ezrin were implicated in protein translation and stress granule dynamics. We validated direct interaction between ezrin and the RNA helicase DDX3, and NSC305787 blocked this interaction. Downregulation or long-term pharmacological inhibition of ezrin led to reduced DDX3 protein levels without changes in DDX3 mRNA. Ectopic overexpression of ezrin in low-ezrin-expressing osteosarcoma cells caused a notable increase in DDX3 protein levels. Ezrin inhibited the RNA helicase activity of DDX3 but increased its ATPase activity. Our data suggest that ezrin controls the translation of mRNAs preferentially with a structured 5′ untranslated region, at least in part, by sustaining the protein level of DDX3 and/or regulating its function. Therefore, our findings suggest a novel function for ezrin in regulation of gene translation that is distinct from its canonical role as a cytoskeletal scaffold at the cell membrane. PMID:26149384

  19. The Drosophila F-box protein Archipelago controls levels of the Trachealess transcription factor in the embryonic tracheal system

    PubMed Central

    Mortimer, Nathan T.; Moberg, Kenneth H.

    2007-01-01

    The archipelago gene (ago) encodes the F-box specificity subunit of an SCF(skp-cullin-f box) ubiquitin ligase that inhibits cell proliferation in Drosophila melanogaster and suppresses tumorigenesis in mammals. ago limits mitotic activity by targeting cell cycle and cell growth proteins for ubiquitin-dependent degradation, but the diverse developmental roles of other F-box proteins suggests that it is likely to have additional protein targets. Here we show that ago is required for the post-mitotic shaping of the Drosophila embryonic tracheal system, and that it acts in this tissue by targeting the Trachealess (Trh) protein, a conserved bHLH-PAS transcription factor. ago restricts Trh levels in vivo and antagonizes transcription of the breathless FGF receptor, a known target of Trh in the tracheal system. At a molecular level, the Ago protein binds Trh and is required for proteasome-dependent elimination of Trh in response to expression of the Dysfusion protein. ago mutations that elevate Trh levels in vivo are defective in binding forms of Trh found in Dysfusion-positive cells. These data identify a novel function for the ago ubiquitin-ligase in tracheal morphogenesis via Trh and its target breathless, and suggest that ago has distinct functions in mitotic and post-mitotic cells that influence its role in development and disease. PMID:17976568

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

    PubMed

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

    2015-10-21

    Hydrothermal treatment (heating in water) is a common method of general food processing and preparation. For red-meat-based foods, boiling is common; however, how the molecular level effects of this treatment correlate to the overall food properties is not yet well-understood. The effects of differing boiling times on lamb meat and the resultant cooking water were here examined through proteomic evaluation. The longer boiling time was found to result in increased protein aggregation involving particularly proteins such as glyceraldehyde-3-phosphate dehydrogenase, as well as truncation in proteins such as in α-actinin-2. Heat-induced protein backbone cleavage was observed adjacent to aspartic acid and asparagine residues. Side-chain modifications of amino acid residues resulting from the heating, including oxidation of phenylalanine and formation of carboxyethyllysine, were characterized in the cooked samples. Actin and myoglobin bands from the cooked meat per se remained visible on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, even after significant cooking time. These proteins were also found to be the major source of observed heat-induced modifications. This study provides new insights into molecular-level modifications occurring in lamb meat proteins during boiling and a protein chemistry basis for better understanding the effect of this common treatment on the nutritional and functional properties of red-meat-based foods. PMID:26381020

  1. JAK2 and MPL protein levels determine TPO-induced megakaryocyte proliferation vs differentiation

    PubMed Central

    Besancenot, Rodolphe; Roos-Weil, Damien; Tonetti, Carole; Abdelouahab, Hadjer; Lacout, Catherine; Pasquier, Florence; Willekens, Christophe; Rameau, Philippe; Lecluse, Yann; Micol, Jean-Baptiste; Constantinescu, Stefan N.; Vainchenker, William; Solary, Eric

    2014-01-01

    Megakaryopoiesis is a 2-step differentiation process, regulated by thrombopoietin (TPO), on binding to its cognate receptor myeloproliferative leukemia (MPL). This receptor associates with intracytoplasmic tyrosine kinases, essentially janus kinase 2 (JAK2), which regulates MPL stability and cell-surface expression, and mediates TPO-induced signal transduction. We demonstrate that JAK2 and MPL mediate TPO-induced proliferation arrest and megakaryocytic differentiation of the human megakaryoblastic leukemia cell line UT7-MPL. A decrease in JAK2 or MPL protein expression, and JAK2 chemical inhibition, suppress this antiproliferative action of TPO. The expression of JAK2 and MPL, which progressively increases along normal human megakaryopoiesis, is decreased in platelets of patients diagnosed with JAK2- or MPL-mutated essential thrombocytemia and primary myelofibrosis, 2 myeloproliferative neoplasms in which megakaryocytes (MKs) proliferate excessively. Finally, low doses of JAK2 chemical inhibitors are shown to induce a paradoxical increase in MK production, both in vitro and in vivo. We propose that JAK2 and MPL expression levels regulate megakaryocytic proliferation vs differentiation in both normal and pathological conditions, and that JAK2 chemical inhibitors could promote a paradoxical thrombocytosis when used at suboptimal doses. PMID:25143485

  2. Unravelling Protein-DNA Interactions at Molecular Level: A DFT and NCI Study.

    PubMed

    González, J; Baños, I; León, I; Contreras-García, J; Cocinero, E J; Lesarri, A; Fernández, J A; Millán, J

    2016-02-01

    Histone-DNA interactions were probed computationally at a molecular level, by characterizing the bimolecular clusters constituted by selected amino acid derivatives with polar (asparagine and glutamine), nonpolar (alanine, valine, and isoleucine), and charged (arginine) side chains and methylated pyrimidinic (1-methylcytosine and 1-methylthymine) and puric (9-methyladenine and 9-methylguanine) DNA bases. The computational approach combined different methodologies: a molecular mechanics (MMFFs forced field) conformational search and structural and vibrational density-functional calculations (M06-2X with double and triple-ζ Pople's basis sets). To dissect the interactions, intermolecular forces were analyzed with the Non-Covalent Interactions (NCI) analysis. The results for the 24 different clusters studied show a noticeable correlation between the calculated binding energies and the propensities for protein-DNA base interactions found in the literature. Such correlation holded even for the interaction of the selected amino acid derivatives with Watson and Crick pairs. Therefore, the balance between hydrogen bonds and van der Waals interactions (specially stacking) in the control of the final shape of the investigated amino acid-DNA base pairs seems to be well reproduced in dispersion-corrected DFT molecular models, reinforcing the idea that the specificity between the amino acids and the DNA bases play an important role in the regulation of DNA. PMID:26765058

  3. Inhibition of Fatty Acid Binding Proteins Elevates Brain Anandamide Levels and Produces Analgesia

    PubMed Central

    Kaczocha, Martin; Rebecchi, Mario J.; Ralph, Brian P.; Teng, Yu-Han Gary; Berger, William T.; Galbavy, William; Elmes, Matthew W.; Glaser, Sherrye T.; Wang, Liqun; Rizzo, Robert C.; Deutsch, Dale G.; Ojima, Iwao

    2014-01-01

    The endocannabinoid anandamide (AEA) is an antinociceptive lipid that is inactivated through cellular uptake and subsequent catabolism by fatty acid amide hydrolase (FAAH). Fatty acid binding proteins (FABPs) are intracellular carriers that deliver AEA and related N-acylethanolamines (NAEs) to FAAH for hydrolysis. The mammalian brain expresses three FABP subtypes: FABP3, FABP5, and FABP7. Recent work from our group has revealed that pharmacological inhibition of FABPs reduces inflammatory pain in mice. The goal of the current work was to explore the effects of FABP inhibition upon nociception in diverse models of pain. We developed inhibitors with differential affinities for FABPs to elucidate the subtype(s) that contributes to the antinociceptive effects of FABP inhibitors. Inhibition of FABPs reduced nociception associated with inflammatory, visceral, and neuropathic pain. The antinociceptive effects of FABP inhibitors mirrored their affinities for FABP5, while binding to FABP3 and FABP7 was not a predictor of in vivo efficacy. The antinociceptive effects of FABP inhibitors were mediated by cannabinoid receptor 1 (CB1) and peroxisome proliferator-activated receptor alpha (PPARα) and FABP inhibition elevated brain levels of AEA, providing the first direct evidence that FABPs regulate brain endocannabinoid tone. These results highlight FABPs as novel targets for the development of analgesic and anti-inflammatory therapeutics. PMID:24705380

  4. Ski protein levels increase during in vitro progression of HPV16-immortalized human keratinocytes and in cervical cancer

    SciTech Connect

    Chen, Yi; Pirisi, Lucia; Creek, Kim E.

    2013-09-15

    We compared the levels of the Ski oncoprotein, an inhibitor of transforming growth factor-beta (TGF-β) signaling, in normal human keratinocytes (HKc), HPV16 immortalized HKc (HKc/HPV16), and differentiation resistant HKc/HPV16 (HKc/DR) in the absence and presence of TGF-β. Steady-state Ski protein levels increased in HKc/HPV16 and even further in HKc/DR, compared to HKc. TGF-β treatment of HKc, HKc/HPV16, and HKc/DR dramatically decreased Ski. TGF-β-induced Ski degradation was delayed in HKc/DR. Ski and phospho-Ski protein levels are cell cycle dependent with maximal Ski expression and localization to centrosomes and mitotic spindles during G2/M. ShRNA knock down of Ski in HKc/DR inhibited cell proliferation. More intense nuclear and cytoplasmic Ski staining and altered Ski localization were found in cervical cancer samples compared to adjacent normal tissue in a cervical cancer tissue array. Overall, these studies demonstrate altered Ski protein levels, degradation and localization in HPV16-transformed human keratinocytes and in cervical cancer. - Highlights: • Ski oncoprotein levels increase during progression of HPV16-transformed cells. • Ski and phospho-Ski protein levels are cell cycle dependent. • Ski knock-down in HPV16-transformed keratinocytes inhibited cell proliferation. • Cervical cancer samples overexpress Ski.

  5. Genome-Wide Association Study of CSF Levels of 59 Alzheimer's Disease Candidate Proteins: Significant Associations with Proteins Involved in Amyloid Processing and Inflammation

    PubMed Central

    Kauwe, John S. K.; Bailey, Matthew H.; Ridge, Perry G.; Perry, Rachel; Wadsworth, Mark E.; Hoyt, Kaitlyn L.; Staley, Lyndsay A.; Karch, Celeste M.; Harari, Oscar; Cruchaga, Carlos; Ainscough, Benjamin J.; Bales, Kelly; Pickering, Eve H.; Bertelsen, Sarah; Fagan, Anne M.; Holtzman, David M.; Morris, John C.; Goate, Alison M.

    2014-01-01

    Cerebrospinal fluid (CSF) 42 amino acid species of amyloid beta (Aβ42) and tau levels are strongly correlated with the presence of Alzheimer's disease (AD) neuropathology including amyloid plaques and neurodegeneration and have been successfully used as endophenotypes for genetic studies of AD. Additional CSF analytes may also serve as useful endophenotypes that capture other aspects of AD pathophysiology. Here we have conducted a genome-wide association study of CSF levels of 59 AD-related analytes. All analytes were measured using the Rules Based Medicine Human DiscoveryMAP Panel, which includes analytes relevant to several disease-related processes. Data from two independently collected and measured datasets, the Knight Alzheimer's Disease Research Center (ADRC) and Alzheimer's Disease Neuroimaging Initiative (ADNI), were analyzed separately, and combined results were obtained using meta-analysis. We identified genetic associations with CSF levels of 5 proteins (Angiotensin-converting enzyme (ACE), Chemokine (C-C motif) ligand 2 (CCL2), Chemokine (C-C motif) ligand 4 (CCL4), Interleukin 6 receptor (IL6R) and Matrix metalloproteinase-3 (MMP3)) with study-wide significant p-values (p<1.46×10−10) and significant, consistent evidence for association in both the Knight ADRC and the ADNI samples. These proteins are involved in amyloid processing and pro-inflammatory signaling. SNPs associated with ACE, IL6R and MMP3 protein levels are located within the coding regions of the corresponding structural gene. The SNPs associated with CSF levels of CCL4 and CCL2 are located in known chemokine binding proteins. The genetic associations reported here are novel and suggest mechanisms for genetic control of CSF and plasma levels of these disease-related proteins. Significant SNPs in ACE and MMP3 also showed association with AD risk. Our findings suggest that these proteins/pathways may be valuable therapeutic targets for AD. Robust associations in cognitively normal

  6. Estrogen Affects Levels of Bcl-2 Protein and mRNA in Medial Amygdala of Ovariectomized Rats

    PubMed Central

    Fan, Lu; Pandey, Subhash C.; Cohen, Rochelle S.

    2013-01-01

    The survival factor Bcl-2 is a cyclic AMP response element-binding protein (CREB) gene product implicated in mediating some of estrogen’s effects on neuroprotection. Previously, we showed an effect of estradiol benzoate (E) on numbers of neuron-specific protein (NeuN)- and phosphorylated CREB (pCREB)-positive cells in medial (MeA), but not central (CeA), amygdala of ovariectomized rats. To determine whether these effects are accompanied by an E-induced increase in Bcl-2, we examined the effects of E on levels of Bcl-2 protein and mRNA in MeA and CeA of ovariectomized rats treated with E regimens resulting in moderate (2.5μg E for 4 or 14 days) or high (10μg E for 14 days) plasma estradiol levels. As a physiological control, we showed that all E treatments increased uterine wet weight relative to vehicle; 10μg E for 14 days also increased uterine weight compared to that seen with lower E levels. Western blot analysis revealed that all E groups displayed an increase in uterine Bcl-2 protein levels compared to vehicle. We found that 2.5μg and 10μg E for 14 days increased levels of Bcl-2 gold immunolabeling compared to vehicle and 2.5μg E for 4 days in MeA, but not CeA. We measured Bcl-2 mRNA levels in vehicle and 2.5μg E for 14 days groups. There was a significant increase in Bcl-2 mRNA levels in MeA, but not CeA, of E-treated ovariectomized rats compared with vehicle controls. The E-induced increase in protein and mRNA levels of Bcl-2 in MeA may be important for neuroprotection in this region. PMID:18655204

  7. Glutamine metabolism in uricotelic species: variation in skeletal muscle glutamine synthetase, glutaminase, glutamine levels and rates of protein synthesis.

    PubMed

    Watford, Malcolm; Wu, Guoyao

    2005-04-01

    High intracellular glutamine levels have been implicated in promoting net protein synthesis and accretion in mammalian skeletal muscle. Little is known regarding glutamine metabolism in uricotelic species but chicken breast muscle exhibits high rates of protein accretion and would be predicted to maintain high glutamine levels. However, chicken breast muscle expresses high glutaminase activity and here we report that chicken breast muscle also expresses low glutamine synthetase activity (0.07+/-0.01 U/g) when compared to leg muscle (0.50+/-0.04 U/g). Free glutamine levels were 1.38+/-0.09 and 9.69+/-0.12 nmol/mg wet weight in breast and leg muscles of fed chickens, respectively. Glutamine levels were also lower in dove breast muscle (4.82+/-0.35 nmol/mg wet weight) when compared to leg muscle (16.2+/-1.0 nmol/mg wet weight) and much lower (1.80+/-0.46 nmol/mg wet weight) in lizard leg muscle. In fed chickens, rates of fractional protein synthesis were higher in leg than in breast muscle, and starvation (48 h) resulted in a decrease in both glutamine content and rate of protein synthesis in leg muscle. Thus, although tissue-specific glutamine metabolism in uricotelic species differs markedly from that in ureotelic animals, differences in rates of skeletal muscle protein synthesis are associated with corresponding differences in intramuscular glutamine content. PMID:15763516

  8. Relationship between VEGF Gene Polymorphisms and Serum VEGF Protein Levels in Patients with Rheumatoid Arthritis

    PubMed Central

    Paradowska-Gorycka, Agnieszka; Pawlik, Andrzej; Romanowska-Prochnicka, Katarzyna; Haladyj, Ewa; Malinowski, Damian; Stypinska, Barbara; Manczak, Malgorzata; Olesinska, Marzena

    2016-01-01

    lower than the number of women with wild type allele -2578A (p = 0.006). Serum VEGF levels were significantly higher in RA patients than in control groups (both p = 0,0001). Conclusion Present findings indicated that VEGF genetic polymorphism as well as VEGF protein levels may be associated with the susceptibility to RA in the Polish population. PMID:27513931

  9. Cerebrospinal fluid neurofilament light chain protein levels in subtypes of frontotemporal dementia

    PubMed Central

    2013-01-01

    Background Frontotemporal dementia (FTD) is recognised as a clinically and morphologically heterogeneous group of interrelated neurodegenerative conditions. One of the subtypes within this disease spectrum is the behavioural variant FTD (bvFTD). This is known to be a varied disorder with a mixture of tau-positive and tau-negative underlying pathologies. The other subtypes include semantic dementia (SD), which generally exhibits tau-negative pathology, and progressive non-fluent aphasia (PNFA), which is usually tau-positive. As the clinical presentation of these subtypes may overlap, a specific diagnosis can be difficult to attain and today no specific biomarker can predict the underlying pathology. Neurofilament light chain protein (NFL), a cytoskeletal constituent of intermediate filaments, is thought to reflect neuronal and axonal death when appearing in the cerebrospinal fluid (CSF). NFL has been shown to be elevated in CSF in patients with FTD compared with AD and controls. Our hypothesis was that the levels of NFL also differ between the subtypes of FTD and may indicate the underlying pathological subtype. Methods We retrospectively analysed data from previous CSF analyses in 34 FTD cases (23 bvFTD, seven SD, four PNFA), 20 AD cases, and 26 healthy controls. A separate group of 10 neuropathologically verified and subtyped FTD cases (seven tau-negative, three tau-positive) were also analysed. Result NFL levels were significantly higher in FTD compared with both AD (p<0.001) and controls (p<0.001). The NFL levels of SD and bvFTD were significantly higher (p<0.001) compared with AD. The biomarker profiles of PNFA and AD were similar. In the neuropathologically verified FTD cases, NFL was higher in the tau-negative than in the tau-positive cases (exact p=0.017). Conclusions The marked NFL elevation in some but not all FTD cases is likely to reflect the different underlying pathologies. The highest NFL values found in the SD group as well as in the

  10. Detection and identification of sub-nanogram levels of protein in a nanoLC-trypsin-MS system.

    PubMed

    Slysz, Gordon W; Lewis, Darren F; Schriemer, David C

    2006-08-01

    Proteomic workflows involving liquid-based protein separations are an alternative to gel-based protein analysis, however the trypsin digestion procedure is usually difficult to implement, particularly when processing low abundance proteins from capillary column effluent. To convert the protein to peptides for the purpose of identification, current protocols require several sample handling steps, and sample losses become an issue. In this study, we present an improved system that conducts reversed-phase protein chromatography and rapid on-line tryptic digestion requiring sub-nanogram quantities of protein. This system employs a novel mirror-gradient concept that allows for dynamic titration of the column effluent to create optimal conditions for real-time tryptic digestion. The purpose behind this development was to improve the limits of detection of the online concept, to support flow-based alternatives to gel-based proteomics and to simplify the characterization of low abundance proteins. Using test mixtures of proteins, we show that peptide mass fingerprinting with high sequence representation can be easily achieved at the 20 fmol level, with detection limits down to 5 fmol (85 pg myoglobin). Limits of identification using standard data-dependent MS/MS experiments are as low as 10 fmol. These results suggest that the nanoLC-trypsin-MS/MS system could represent an alternative to the conventional "1D-gel to MS" proteomic strategy. PMID:16889418

  11. Associations Between Common and Rare Exonic Genetic Variants and Serum Levels of 20 Cardiovascular-Related Proteins

    PubMed Central

    Solomon, Terry; Smith, Erin N.; Matsui, Hiroko; Braekkan, Sigrid K.; Wilsgaard, Tom; Njølstad, Inger; Mathiesen, Ellisiv B.; Hansen, John-Bjarne

    2016-01-01

    Background— Genetic variation can be used to study causal relationships between biomarkers and diseases. Here, we identify new common and rare genetic variants associated with cardiovascular-related protein levels (protein quantitative trait loci [pQTLs]). We functionally annotate these pQTLs, predict and experimentally confirm a novel molecular interaction, and determine which pQTLs are associated with diseases and physiological phenotypes. Methods and Results— As part of a larger case–control study of venous thromboembolism, serum levels of 51 proteins implicated in cardiovascular diseases were measured in 330 individuals from the Tromsø Study. Exonic genetic variation near each protein’s respective gene (cis) was identified using sequencing and arrays. Using single site and gene-based tests, we identified 27 genetic associations between pQTLs and the serum levels of 20 proteins: 14 associated with common variation in cis, of which 6 are novel (ie, not previously reported); 7 associations with rare variants in cis, of which 4 are novel; and 6 associations in trans. Of the 20 proteins, 15 were associated with single sites and 7 with rare variants. cis-pQTLs for kallikrein and F12 also show trans associations for proteins (uPAR, kininogen) known to be cleaved by kallikrein and with NTproBNP. We experimentally demonstrate that kallikrein can cleave proBNP (NTproBNP precursor) in vitro. Nine of the pQTLs have previously identified associations with 17 disease and physiological phenotypes. Conclusions— We have identified cis and trans genetic variation associated with the serum levels of 20 proteins and utilized these pQTLs to study molecular mechanisms underlying disease and physiological phenotypes. PMID:27329291

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

    PubMed

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

    2011-01-01

    Cassava is the major source of calories for more than 250 million Sub-Saharan Africans, however, it has the lowest protein-to-energy ratio of any major staple food crop in the world. A cassava-based diet provides less than 30% of the minimum daily requirement for protein. Moreover, both leaves and roots contain potentially toxic levels of cyanogenic glucosides. The major cyanogen in cassava is linamarin which is stored in the vacuole. Upon tissue disruption linamarin is deglycosylated by the apolplastic enzyme, linamarase, producing acetone cyanohydrin. Acetone cyanohydrin can spontaneously decompose at pHs >5.0 or temperatures >35°C, or is enzymatically broken down by hydroxynitrile lyase (HNL) to produce acetone and free cyanide which is then volatilized. Unlike leaves, cassava roots have little HNL activity. The lack of HNL activity in roots is associated with the accumulation of potentially toxic levels of acetone cyanohydrin in poorly processed roots. We hypothesized that the over-expression of HNL in cassava roots under the control of a root-specific, patatin promoter would not only accelerate cyanogenesis during food processing, resulting in a safer food product, but lead to increased root protein levels since HNL is sequestered in the cell wall. Transgenic lines expressing a patatin-driven HNL gene construct exhibited a 2-20 fold increase in relative HNL mRNA levels in roots when compared with wild type resulting in a threefold increase in total root protein in 7 month old plants. After food processing, HNL overexpressing lines had substantially reduced acetone cyanohydrin and cyanide levels in roots relative to wild-type roots. Furthermore, steady state linamarin levels in intact tissues were reduced by 80% in transgenic cassava roots. These results suggest that enhanced linamarin metabolism contributed to the elevated root protein levels. PMID:21799761

  13. Residue-level global and local ensemble-ensemble comparisons of protein domains.

    PubMed

    Clark, Sarah A; Tronrud, Dale E; Karplus, P Andrew

    2015-09-01

    Many methods of protein structure generation such as NMR-based solution structure determination and template-based modeling do not produce a single model, but an ensemble of models consistent with the available information. Current strategies for comparing ensembles lose information because they use only a single representative structure. Here, we describe the ENSEMBLATOR and its novel strategy to directly compare two ensembles containing the same atoms to identify significant global and local backbone differences between them on per-atom and per-residue levels, respectively. The ENSEMBLATOR has four components: eePREP (ee for ensemble-ensemble), which selects atoms common to all models; eeCORE, which identifies atoms belonging to a cutoff-distance dependent common core; eeGLOBAL, which globally superimposes all models using the defined core atoms and calculates for each atom the two intraensemble variations, the interensemble variation, and the closest approach of members of the two ensembles; and eeLOCAL, which performs a local overlay of each dipeptide and, using a novel measure of local backbone similarity, reports the same four variations as eeGLOBAL. The combination of eeGLOBAL and eeLOCAL analyses identifies the most significant differences between ensembles. We illustrate the ENSEMBLATOR's capabilities by showing how using it to analyze NMR ensembles and to compare NMR ensembles with crystal structures provides novel insights compared to published studies. One of these studies leads us to suggest that a "consistency check" of NMR-derived ensembles may be a useful analysis step for NMR-based structure determinations in general. The ENSEMBLATOR 1.0 is available as a first generation tool to carry out ensemble-ensemble comparisons. PMID:26032515

  14. Alternations of 14-3-3 θ and β protein levels in brain during experimental sepsis.

    PubMed

    Memos, Nikolaos; Kataki, Agapi; Chatziganni, Emmy; Nikolopoulou, Marilena; Skoulakis, Euthimios; Consoulas, Christos; Zografos, George; Konstadoulakis, Manousos

    2011-09-01

    The 14-3-3 family members play a crucial role in the determination of cell fate, exerting their antiapoptotic activity through directly interfering with the critical function of the mitochondrial core proapoptotic machinery. Dimerization of 14-3-3 is vital for the interaction with many of its client proteins and is regulated by phosphorylation. In a previous study, we observed time-dependent neuronal apoptosis during sepsis. Therefore, in the present study, we sought to evaluate the expression of 14-3-3 θ and β isoforms in septic brain and their association with apoptosis. Sepsis was induced by a CLP model in Wistar rats that were sacrificed at predefined time points. Flow cytometric analysis showed a sepsis-induced, time-dependent alteration of 14-3-3 θ and β isoforms in both Neun(+) and GFAP(+) cells. 14-3-3 θ was linearly correlated with apoptosis, and stratified analysis for alive and apoptotic neuronal cells demonstrated a gradual down-regulation of θ isoform in alive neurons and astrocytes. The phospho-P38 (pP38) MAP kinase levels were altered in a time-dependent manner during sepsis, presenting a peak at 6 hr post-CLP. A significant correlation between the two isoforms of 14-3-3 was observed in septic rats, with the θ isoform predominant at all time points. The hippocampus, Purkinje cells, and glia-like cells showed intense immunohistochemical reactivity for 14-3-3 θ isoform, whereas the choroid plexus showed constantly increased β isoform expression. Our results showed that sepsis alters the expression of both 14-3-3 θ and β isoforms in a time-, cell-, and topography-dependent manner. PMID:21618583

  15. Comparison of Channel and Blue Catfish Fed Diets Containing Various Levels of Protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A comparative study was conducted on growth and protein requirements of channel catfish, Ictalurus punctatus, and blue catfish, Ictalurus furcatus. Four diets containing 24, 28, 32, or 36% protein were fed to both channel (initial weight 6.9 g/fish) and blue (6.6 g/fish) catfish for two growing seas...

  16. Epitopes expressed in different adenovirus capsid proteins induce different levels of epitope-specific immunity.

    PubMed

    Krause, Anja; Joh, Ju H; Hackett, Neil R; Roelvink, Peter W; Bruder, Joseph T; Wickham, Thomas J; Kovesdi, Imre; Crystal, Ronald G; Worgall, Stefan

    2006-06-01

    On the basis of the concept that the capsid proteins of adenovirus (Ad) gene transfer vectors can be genetically manipulated to enhance the immunogenicity of Ad-based vaccines, the present study compared the antiantigen immunogenicity of Ad vectors with a common epitope of the hemagglutinin (HA) protein of the influenza A virus incorporated into the outer Ad capsid protein hexon, penton base, fiber knob, or protein IX. Incorporation of the same epitope into the different capsid proteins provided insights into the correlation between epitope position and antiepitope immunity. Following immunization of three different strains of mice (C57BL/6, BALB/c, and CBA) with either an equal number of Ad particles (resulting in a different total HA copy number) or different Ad particle numbers (to achieve the same HA copy number), the highest primary (immunoglobulin M [IgM]) and secondary (IgG) anti-HA humoral and cellular CD4 gamma interferon and interleukin-4 responses against HA were always achieved with the Ad vector carrying the HA epitope in fiber knob. These observations suggest that the immune response against an epitope inserted into Ad capsid proteins is not necessarily dependent on the capsid protein number and imply that the choice of incorporation site in Ad capsid proteins in their use as vaccines needs to be compared in vivo. PMID:16699033

  17. EXPANSIN PROTEIN LEVELS DECLINE WITH THE DEVELOPMENT OF MEALINESS IN PEACHES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Expansin mRNA and protein expression in peaches was examined to investigate the possibility that expansins may be involved in the development of mealy flesh texture. Immunoblot analysis, using an expansin antibody, detected a 27-KDa protein corresponding to the predicted molecular mass of expansins...

  18. Evidence of normal functional levels of activated protein C inhibitor in combined Factor V/VIII deficiency disease.

    PubMed Central

    Canfield, W M; Kisiel, W

    1982-01-01

    Human activated protein C (APC) is a plasma serine protease that possesses amidolytic and anticoagulant activity. The rate at which the amidolytic and anticoagulant activity of APC was neutralized in normal plasma was essentially identical to that observed in plasma obtained from four individuals with combined Factor V/VIII deficiency disease. Incubation of radioiodinated APC with either normal human plasma or the combined Factor V/VIII-deficient plasmas resulted in the formation of a stable complex (Mr = 96,000) of the enzyme and a plasma protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Pretreatment of the radiolabeled APC with diisopropyl fluorophosphate prevented the formation of the enzyme-protein complex. On the basis of its ability to form a complex with radiolabeled APC, the APC-binding protein was purified to homogeneity from normal human plasma by ammonium sulfate fractionation, heparin-agarose chromatography, and QAE-Sephadex A-50 chromatography. The APC-binding protein (Mr = 54,000) is a glycoprotein, and possesses an amino-terminal sequence of Gly-Arg-Thr-Cys-Pro-Lys-Pro-Asp. The amino-terminal sequence of the APC-binding protein exhibited considerable homology with bovine colostrum inhibitor and pancreatic trypsin inhibitor, but no apparent sequence homology with the plasma serine protease inhibitors. Affinity-purified antibody against APC-binding protein immunoprecipitated a complex of radiolabeled APC and native APC-binding protein from normal human plasma. Complex formation was virtually eliminated in plasma immunodepleted of the APC-binding protein. Quantitative electroimmunoassay indicated essentially equal levels of APC-binding protein antigen in normal plasma compared with plasma from four patients with combined Factor V/VIII deficiency disease. Images PMID:6294139

  19. Outcomes for Single-Level Lumbar Fusion: The Role of Bone Morphogenetic Protein

    PubMed Central

    Cahill, Kevin S.; Chi, John H.; Groff, Michael W.; McGuire, Kevin; Afendulis, Christopher C.; Claus, Elizabeth B.

    2011-01-01

    Study Design Retrospective analysis of a population-based insurance claims dataset. Objective To determine the risk of repeat fusion and total costs associated with bone morphogenetic protein (BMP) use in single-level lumbar fusion for degenerative spinal disease. Summary of Background Data The use of BMP has been proposed to reduce overall costs of spinal fusion through prevention of repeat fusion procedures. Although radiographic fusion rates associated with BMP use have been examined in clinical trials, little data exists regarding outcomes associated with BMP use in the general population. Methods Using the MarketScan© claims dataset, 15,862 patients that underwent single-level lumbar fusion from 2003 to 2007 for degenerative disease were identified. Propensity scores were used to match 2,372 patients that underwent fusion with BMP to patients that underwent fusion without BMP. Logistic regression models, Kaplan-Meier estimates, and Cox proportional hazards models were used to examine risk of repeat fusion, length of stay, and 30-day readmission by BMP use. Cost comparisons were evaluated with linear regression models using logarithmic transformed data. Results At one year from surgery, BMP was associated with a 1.1% absolute decrease in the risk of repeat fusion (2.3% with BMP vs 3.4% without BMP, p=.03) and an odds ratio for repeat fusion of 0.66 (95% confidence interval 0.47-0.94) after multivariate adjustment. BMP was also associated with a decreased hazards for long-term repeat fusion (adjusted hazards ratio =0.74, 95% confidence interval 0.58-0.93). Cost analysis indicated that BMP was associated with initial increased costs for the surgical procedure (13.9% adjusted increase, 95% confidence interval 9.9%-17.9%) as well as total one year costs (10.1% adjusted increase, 95% confidence interval 6.2%-14.0%). Conclusions At one year, BMP use was associated with a decreased risk of repeat fusion but also increased healthcare costs. PMID:21311404

  20. Effect of selenium and vitamin e supplementation on plasma protein carbonyl levels in patients with arsenic-related skin lesions.

    PubMed

    Mahata, Julie; Argos, Maria; Verret, Wendy; Kibriya, Muhammad G; Santella, Regina M; Ahsan, Habibul

    2008-01-01

    An estimated 35 million people in Bangladesh have been chronically exposed to arsenic in drinking water and are at risk of an array of adverse health conditions. The mechanisms of arsenic toxicity have not been well established; however, oxidative stress has been one commonly proposed pathway. In this study, we evaluated the effect of antioxidant supplementation on plasma protein oxidation among patients with arsenical skin lesions participating in a randomized double-blinded placebo-controlled trial of vitamin E and selenium. Subjects were randomized to 1 of 4 treatments arms (vitamin E, selenium, combination, or placebo) and were treated for a 6-mo period. We observed a dose-dependent increase in adjusted protein carbonyl levels by arsenic exposure status in the pretreatment samples, although trends were not statistically significant. Following the 6-mo intervention, there was a decrease in protein carbonyl levels in each treatment group, although no resultant decrease was significantly different from that seen in the placebo group. Although we did not see a notable effect of selenium or vitamin E supplementation on changes in protein carbonyl levels, these preliminary data demonstrate a feasible methodological approach for the assessment of plasma protein carbonyls in relation to environmental toxicants in a human population and their potential use as endpoints in intervention trials. PMID:18444136

  1. Post-harvest light treatment increases expression levels of recombinant proteins in transformed plastids of potato tubers.

    PubMed

    Larraya, Luis M; Fernández-San Millán, Alicia; Ancín, María; Farran, Inmaculada; Veramendi, Jon

    2015-09-01

    Plastid genetic engineering represents an attractive system for the production of foreign proteins in plants. Although high expression levels can be achieved in leaf chloroplasts, the results for non-photosynthetic plastids are generally discouraging. Here, we report the expression of two thioredoxin genes (trx f and trx m) from the potato plastid genome to study transgene expression in amyloplasts. As expected, the highest transgene expression was detected in the leaf (up to 4.2% of TSP). The Trx protein content in the tuber was approximately two to three orders of magnitude lower than in the leaf. However, we demonstrate that a simple post-harvest light treatment of microtubers developed in vitro or soil-grown tubers induces up to 55 times higher accumulation of the recombinant protein in just seven to ten days. After the applied treatment, the Trx f levels in microtubers and soil-grown tubers increased to 0.14% and 0.11% of TSP, respectively. Moreover, tubers stored for eight months maintained the capacity of increasing the foreign protein levels after the light treatment. Post-harvest cold induction (up to five times) at 4°C was also detected in microtubers. We conclude that plastid transformation and post-harvest light treatment could be an interesting approach for the production of foreign proteins in potato. PMID:26121393

  2. Serum and tissue thiocyanate concentrations in growing pigs fed cassava peel or corn based diets containing graded protein levels.

    PubMed

    Tewe, O O

    1984-11-01

    Thiocyanate concentrations of serum, liver, kidney, spleen and longissimus dorsi were determined in 64 growing Large White x Landrace pigs offered 8 experimental isocaloric diets containing different levels of cassava peel and crude protein. Cassava peel increased serum thiocyanate on day 60 (P less than 0.01) and day 90 (P less than 0.01) of the trial, while the crude protein level increased it (P less than 0.05) on days 30 and 90, respectively. Interaction of the two factors was significant on day 30 (P less than 0.05) and day 90 (P less than 0.05). There was a correlation between cyanide intake and serum thiocyanate level. Coefficient of determination revealed that cyanide alone accounted for 28.5; 60.6 and 48.8% variation in serum thiocyanate on days 30, 60 and 90, respectively. Liver, spleen and longissimus dorsi thiocyanate were affected by dietary protein intake (P less than 0.05). Thiocyanate concentration was higher (P less than 0.05) on cassava peel diet. Generally, crude protein at 5% reduced organ and muscle thiocyanate concentrations. A diet containing 112.2-117.3 mg/kg hydrocyanic acid (HCN) affected serum but not organ and muscle thiocyanate in protein-sufficient diets. PMID:6506092

  3. Design of a novel globular protein fold with atomic-level accuracy.

    PubMed

    Kuhlman, Brian; Dantas, Gautam; Ireton, Gregory C; Varani, Gabriele; Stoddard, Barry L; Baker, David

    2003-11-21

    A major challenge of computational protein design is the creation of novel proteins with arbitrarily chosen three-dimensional structures. Here, we used a general computational strategy that iterates between sequence design and structure prediction to design a 93-residue alpha/beta protein called Top7 with a novel sequence and topology. Top7 was found experimentally to be folded and extremely stable, and the x-ray crystal structure of Top7 is similar (root mean square deviation equals 1.2 angstroms) to the design model. The ability to design a new protein fold makes possible the exploration of the large regions of the protein universe not yet observed in nature. PMID:14631033

  4. IFN-γ Directly Controls IL-33 Protein Level through a STAT1- and LMP2-dependent Mechanism*

    PubMed Central

    Kopach, Pavel; Lockatell, Virginia; Pickering, Edward M.; Haskell, Ronald E.; Anderson, Richard D.; Hasday, Jeffrey D.; Todd, Nevins W.; Luzina, Irina G.; Atamas, Sergei P.

    2014-01-01

    IL-33 contributes to disease processes in association with Th1 and Th2 phenotypes. IL-33 mRNA is rapidly regulated, but the fate of synthesized IL-33 protein is unknown. To understand the interplay among IL-33, IFN-γ, and IL-4 proteins, recombinant replication-deficient adenoviruses were produced and used for dual expression of IL-33 and IFN-γ or IL-33 and IL-4. The effects of such dual gene delivery were compared with the effects of similar expression of each of these cytokines alone. In lung fibroblast culture, co-expression of IL-33 and IFN-γ resulted in suppression of the levels of both proteins, whereas co-expression of IL-33 and IL-4 led to mutual elevation. In vivo, co-expression of IL-33 and IFN-γ in the lungs led to attenuation of IL-33 protein levels. Purified IFN-γ also attenuated IL-33 protein in fibroblast culture, suggesting that IFN-γ controls IL-33 protein degradation. Specific inhibition of caspase-1, -3, and -8 had minimal effect on IFN-γ-driven IL-33 protein down-regulation. Pharmacological inhibition, siRNA-mediated silencing, or gene deficiency of STAT1 potently up-regulated IL-33 protein expression levels and attenuated the down-regulating effect of IFN-γ on IL-33. Stimulation with IFN-γ strongly elevated the levels of the LMP2 proteasome subunit, known for its role in IFN-γ-regulated antigen processing. siRNA-mediated silencing of LMP2 expression abrogated the effect of IFN-γ on IL-33. Thus, IFN-γ, IL-4, and IL-33 are engaged in a complex interplay. The down-regulation of IL-33 protein levels by IFN-γ in pulmonary fibroblasts and in the lungs in vivo occurs through STAT1 and non-canonical use of the LMP2 proteasome subunit in a caspase-independent fashion. PMID:24619410

  5. Nerve Growth Factor Increases mRNA Levels for the Prion Protein and the β -amyloid Protein Precursor in Developing Hamster Brain

    NASA Astrophysics Data System (ADS)

    Mobley, William C.; Neve, Rachael L.; Prusiner, Stanley B.; McKinley, Michael P.

    1988-12-01

    Deposition of amyloid filaments serves as a pathologic hallmark for some neurodegenerative disorders. The prion protein (PrP) is found in amyloid of animals with scrapie and humans with Creutzfeldt-Jakob disease; the β protein is present in amyloid deposits in Alzheimer disease and Down syndrome patients. These two proteins are derived from precursors that in the brain are expressed primarily in neurons and are membrane bound. We found that gene expression for PrP and the β -protein precursor (β -PP) is regulated in developing hamster brain. Specific brain regions showed distinct patterns of ontogenesis for PrP and β -PP mRNAs. The increases in PrP and β -PP mRNAs in developing basal forebrain coincided with an increase in choline acetyltransferase activity, raising the possibility that these markers might be coordinately controlled in cholinergic neurons and regulated by nerve growth factor (NGF). Injections of NGF into the brains of neonatal hamsters increased both PrP and β -PP mRNA levels. Increased PrP and β -PP mRNA levels induced by NGF were confined to regions that contain NGF-responsive cholinergic neurons and were accompanied by elevations in choline acetyltransferase. It remains to be established whether or not exogenous NGF acts to increase PrP and β -PP gene expression selectively in forebrain cholinergic neurons in the developing hamster and endogenous NGF regulates expression of these genes.

  6. Low Levels of p53 Protein and Chromatin Silencing of p53 Target Genes Repress Apoptosis in Drosophila Endocycling Cells

    PubMed Central

    Zhang, Bingqing; Mehrotra, Sonam; Ng, Wei Lun; Calvi, Brian R.

    2014-01-01

    Apoptotic cell death is an important response to genotoxic stress that prevents oncogenesis. It is known that tissues can differ in their apoptotic response, but molecular mechanisms are little understood. Here, we show that Drosophila polyploid endocycling cells (G/S cycle) repress the apoptotic response to DNA damage through at least two mechanisms. First, the expression of all the Drosophila p53 protein isoforms is strongly repressed at a post-transcriptional step. Second, p53-regulated pro-apoptotic genes are epigenetically silenced in endocycling cells, preventing activation of a paused RNA Pol II by p53-dependent or p53-independent pathways. Over-expression of the p53A isoform did not activate this paused RNA Pol II complex in endocycling cells, but over-expression of the p53B isoform with a longer transactivation domain did, suggesting that dampened p53B protein levels are crucial for apoptotic repression. We also find that the p53A protein isoform is ubiquitinated and degraded by the proteasome in endocycling cells. In mitotic cycling cells, p53A was the only isoform expressed to detectable levels, and its mRNA and protein levels increased after irradiation, but there was no evidence for an increase in protein stability. However, our data suggest that p53A protein stability is regulated in unirradiated cells, which likely ensures that apoptosis does not occur in the absence of stress. Without irradiation, both p53A protein and a paused RNA pol II were pre-bound to the promoters of pro-apoptotic genes, preparing mitotic cycling cells for a rapid apoptotic response to genotoxic stress. Together, our results define molecular mechanisms by which different cells in development modulate their apoptotic response, with broader significance for the survival of normal and cancer polyploid cells in mammals. PMID:25211335

  7. Tbx Protein Level Critical for Clock-Mediated Somite Positioning Is Regulated through Interaction between Tbx and Ripply

    PubMed Central

    Wanglar, Chimwar; Takahashi, Jun; Yabe, Taijiro; Takada, Shinji

    2014-01-01

    Somitogenesis in vertebrates is a complex and dynamic process involving many sequences of events generated from the segmentation clock. Previous studies with mouse embryos revealed that the presumptive somite boundary is periodically created at the anterior border of the expression domain of Tbx6 protein. Ripply1 and Ripply2 are required for the determination of the Tbx6 protein border, but the mechanism by which this Tbx6 domain is regulated remains unclear. Furthermore, since zebrafish and frog Ripplys are known to be able to suppress Tbx6 function at the transcription level, it is also unclear whether Ripply-mediated mechanism of Tbx6 regulation is conserved among different species. Here, we tested the generality of Tbx6 protein-mediated process in somite segmentation by using zebrafish and further examined the mechanism of regulation of Tbx6 protein. By utilizing an antibody against zebrafish Tbx6/Fss, previously referred to as Tbx24, we found that the anterior border of Tbx6 domain coincided with the presumptive intersomitic boundary even in the zebrafish and it shifted dynamically during 1 cycle of segmentation. Consistent with the findings in mice, the tbx6 mRNA domain was located far anterior to its protein domain, indicating the possibility of posttranscriptional regulation. When both ripply1/2 were knockdown, the Tbx6 domain was anteriorly expanded. We further directly demonstrated that Ripply could reduce the expression level of Tbx6 protein depending on physical interaction between Ripply and Tbx6. Moreover, the onset of ripply1 and ripply2 expression occurred after reduction of FGF signaling at the anterior PSM, but this expression initiated much earlier on treatment with SU5402, a chemical inhibitor of FGF signaling. These results strongly suggest that Ripply is a direct regulator of the Tbx6 protein level for the establishment of intersomitic boundaries and mediates a reduction in FGF signaling for the positioning of the presumptive intersomitic

  8. Circulating Heat Shock Protein 60 Levels Are Elevated in HIV Patients and Are Reduced by Anti-Retroviral Therapy

    PubMed Central

    Anraku, Itaru; Rajasuriar, Reena; Dobbin, Caroline; Brown, Richard; Lewin, Sharon R.; Suhrbier, Andreas

    2012-01-01

    Circulating heat shock protein 60 (Hsp60) and heat shock protein 10 (Hsp10) have been associated with pro- and anti-inflammatory activity, respectively. To determine whether these heat shock proteins might be associated with the immune activation seen in HIV-infected patients, the plasma levels of Hsp60 and Hsp10 were determined in a cohort of 20 HIV-infected patients before and after effective combination anti-retroviral therapy (cART). We show for the first time that circulating Hsp60 levels are elevated in HIV-infected patients, with levels significantly reduced after cART, but still higher than those in HIV-negative individuals. Hsp60 levels correlated significantly with viral load, CD4 counts, and circulating soluble CD14 and lipopolysaccharide levels. No differences or correlations were seen for Hsp10 levels. Elevated circulating Hsp60 may contribute to the immune dysfunction and non-AIDS clinical events seen in HIV-infected patients. PMID:23028910

  9. Identification, characterization, and expression levels of putative adhesive proteins from the tube-dwelling polychaete Sabellaria alveolata.

    PubMed

    Becker, Pierre T; Lambert, Aurélie; Lejeune, Annabelle; Lanterbecq, Déborah; Flammang, Patrick

    2012-10-01

    The shelter of the tube-dwelling polychaete Sabellaria alveolata is composed of mineral particles assembled with spots of a proteinaceous cement. The adhesive proteins constituting the cement were identified on the basis of their sequence similarity with proteins of a phylogenetically related species, Phragmatopoma californica. Two positively charged proteins, Sa-1 and Sa-2, share common features: they both have a mass of 22 kDa; are rich in glycine, tyrosine and basic residues; and show repeated peptide motifs. The consensus repeat of Sa-1 is KGAYGAKGLGYGNKAGYGAYG (occurring 6-8 times), while Sa-2 displays the consensus heptapeptide VHKAAWG (5 times) and undecapeptide VHKAAGYGGYG (8 times). Two variants of a serine-rich protein, Sa-3A (22 kDa) and Sa-3B (21 kDa), were also identified. Their serine residues account for 75 mol% and are probably phosphorylated, meaning that Sa-3 is very acidic and negatively charged. Moreover, tyrosine residues of all adhesive proteins are presumably modified into DOPA. Although protein sequences are not well-conserved between S. alveolata and P. californica, their main characteristics (including amino acid composition, post-translational modifications, repeated patterns, isoelectric point, and mass) are shared by both species. This suggests that these features are more important for their function than the primary structure of the proteins. The mRNA abundance for each protein was estimated by quantitative real-time PCR, revealing relative expression levels of about 5, 11, 1.5, and 1 for Sa-1, -2, -3A, and -3B, respectively. These levels could be indicative of charge neutralization phenomena or could reflect their function (interface vs. bulk) in the cement. PMID:23111133

  10. Effect of dietary supplementation of omega-3 fatty acids and high levels of dietary protein on performance of sows

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A study was conducted to determine the effect of dietary supplementation of omega-3 fatty acids (O3FA), with or without high levels of protein, on the performance of sows during first and subsequent parity. Sixty-four pregnant gilts with BW of 195.0 ± 2.1 kg and backfat (BF) thickness of 12.9 ± 0.2 ...

  11. Rhinacanthus nasutus Improves the Levels of Liver Carbohydrate, Protein, Glycogen, and Liver Markers in Streptozotocin-Induced Diabetic Rats

    PubMed Central

    Visweswara Rao, Pasupuleti; Madhavi, K.; Dhananjaya Naidu, M.; Gan, Siew Hua

    2013-01-01

    The present study was designed to investigate the total carbohydrate, total protein, and glycogen levels in the liver and to measure functional liver markers such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in streptozotocin-(STZ-) induced diabetic rats after treatment with methanolic extract of Rhinacanthus nasutus (R. nasutus). The methanolic extract of R. nasutus was orally administered at 200 mg/kg/day while glibenclamide was administered at 50 mg/kg/day. All animals were treated for 30 days before being sacrificed. The amounts of carbohydrate, glycogen, proteins, and liver markers (AST and ALT) were measured in the liver tissue of the experimental animals. The levels of carbohydrate, glycogen, and proteins were significantly reduced in the diabetic rats but were augmented considerably after 30 days of R. nasutus treatment. The elevated AST and ALT levels in diabetic rats showed a significant decline after treatment with R. nasutus for 30 days. These results show that the administration of R. nasutus ameliorates the altered levels of carbohydrate, glycogen, proteins, and AST and ALT observed in diabetic rats and indicate that R. nasutus restores overall metabolism and liver function in experimental diabetic rats. In conclusion, the outcomes of the present study support the traditional belief that R. nasutus could ameliorate the diabetic state. PMID:24204387

  12. Effect of dietary supplementation of omega-3 fatty acids and high levels of dietary protein on performance of sows.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study was conducted to determine the effect of dietary supplementation of omega-3 fatty acids (O3FA), with or without high levels of dietary protein supplementation, on the performance of sows and their litters during first and subsequent parities. Sixty-four pregnant gilts with body weight (BW...

  13. Gli2 protein expression level is a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms.

    PubMed

    Sugiyama, Y; Sasajima, J; Mizukami, Y; Koizumi, K; Kawamoto, T; Ono, Y; Karasaki, H; Tanabe, H; Fujiya, M; Kohgo, Y

    2016-06-01

    The hedgehog pathway is known to promote proliferation of pancreatic ductal adenocarcinoma (PDA) and has been shown to restrain tumor progression. To understand how hedgehog causes these effects, we sought to carefully examine protein expression of hedgehog signaling components during different tumor stages. Genetically engineered mice, Pdx1-Cre;LSL-KrasG12D and Pdx1-Cre;LSL-KrasG12D;p53lox/+, were utilized to model distinct phases of tumorigenesis, pancreatic intraepithelial neoplasm (PanIN) and PDA. Human pancreatic specimens of intraductal papillary mucinous neoplasm (IPMN) and PDA were also employed. PanIN and IPMN lesions highly express Sonic Hedgehog, at a level that is slightly higher than that observed in PDA. GLI2 protein is also expressed in both PanIN/IPMN and PDA. Although there was no difference in the nuclear staining, the cytoplasmic GLI2 level in PDA was modest in comparison to that in PanIN/IPMN. Hedgehog interacting protein was strongly expressed in the precursors, whereas the level in PDA was significantly attenuated. There were no differences in expression of Patched1 at early and late stages. Finally, a strong correlation between Sonic Hedgehog and GLI2 staining was found in both human and murine pancreatic tumors. The results indicate that the GLI2 protein level could serve as a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms. PMID:27543868

  14. Aerobic fitness level does not modulate changes in whole-body protein turnover produced by unaccustomed increases in energy expenditure

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effects of a sudden increase in energy expenditure (EE) on whole-body protein turnover vary between studies, and the possibility that fitness level modulates those responses has not been fully investigated. We hypothesized that aerobically trained individuals may exhibit adaptations that protec...

  15. Association of common C-protein (CRP) gene polymorphism with baseline plasma CRP levels and fenofibrate response: The GOLDN Study

    Technology Transfer Automated Retrieval System (TEKTRAN)

    OBJECTIVE-C-reactive protein (CRP) is an inflammatory marker that contributes to the prediction of cardiovascular diseases (CVD). We investigated the influence of CRP polymorphisms on baseline CRP levels and fenofibrate-induced CRP changes in subjects with the metabolic syndrome (MetS). RESEARCH DES...

  16. Nrbf2 Protein Suppresses Autophagy by Modulating Atg14L Protein-containing Beclin 1-Vps34 Complex Architecture and Reducing Intracellular Phosphatidylinositol-3 Phosphate Levels*

    PubMed Central

    Zhong, Yu; Morris, Deanna H.; Jin, Lin; Patel, Mittul S.; Karunakaran, Senthil K.; Fu, You-Jun; Matuszak, Emily A.; Weiss, Heidi L.; Chait, Brian T.; Wang, Qing Jun

    2014-01-01

    Autophagy is a tightly regulated lysosomal degradation pathway for maintaining cellular homeostasis and responding to stresses. Beclin 1 and its interacting proteins, including the class III phosphatidylinositol-3 kinase Vps34, play crucial roles in autophagy regulation in mammals. We identified nuclear receptor binding factor 2 (Nrbf2) as a Beclin 1-interacting protein from Becn1−/−;Becn1-EGFP/+ mouse liver and brain. We also found that Nrbf2-Beclin 1 interaction required the N terminus of Nrbf2. We next used the human retinal pigment epithelial cell line RPE-1 as a model system and showed that transiently knocking down Nrbf2 by siRNA increased autophagic flux under both nutrient-rich and starvation conditions. To investigate the mechanism by which Nrbf2 regulates autophagy, we demonstrated that Nrbf2 interacted and colocalized with Atg14L, suggesting that Nrbf2 is a component of the Atg14L-containing Beclin 1-Vps34 complex. Moreover, ectopically expressed Nrbf2 formed cytosolic puncta that were positive for isolation membrane markers. These results suggest that Nrbf2 is involved in autophagosome biogenesis. Furthermore, we showed that Nrbf2 deficiency led to increased intracellular phosphatidylinositol-3 phosphate levels and diminished Atg14L-Vps34/Vps15 interactions, suggesting that Nrbf2-mediated Atg14L-Vps34/Vps15 interactions likely inhibit Vps34 activity. Therefore, we propose that Nrbf2 may interact with the Atg14L-containing Beclin 1-Vps34 protein complex to modulate protein-protein interactions within the complex, leading to suppression of Vps34 activity, autophagosome biogenesis, and autophagic flux. This work reveals a novel aspect of the intricate mechanism for the Beclin 1-Vps34 protein-protein interaction network to achieve precise control of autophagy. PMID:25086043

  17. Study on the fluorescent enhancement effect in terbium-gadolinium-protein-sodium dodecyl benzene sulfonate system and its application on sensitive detection of protein at nanogram level.

    PubMed

    Sun, Changxia; Yang, Jinghe; Wu, Xia; Liu, Shufang; Su, Benyu

    2004-08-01

    The co-luminescence effect in a terbium-gadolinium-protein-sodium dodecyl benzene sulfonate (SDBS) system is reported here. Based on it, the sensitive quantitative analysis of protein at nanogram levels is established. The co-luminescence mechanism is studied using fluorescence, resonance light scattering (RLS), absorption spectroscopy and NMR measurement. It is considered that protein could be unfolded by SDBS, then a efficacious intramolecular fluorescent energy transfer occurs from unfolded protein to rare earth ions through SDBS acting as a "transfer bridge" to enhance the emission fluorescence of Tb3+ in this ternary complex of Tb-SDBS-BSA, where energy transfer from protein to SDBS by aromatic ring stacking is the most important step. Cooperating with the intramolecular energy transfer above is the intermolecular energy transfer between the simultaneous existing complexes of both Tb3+ and Gd3+. The fluorescence quantum yield is increased by an energy-insulating sheath, which is considered to be another reason for the resulting enhancement of the fluorescence. Förster theory is used to calculate the distribution of enhancing factors and has led to a greater understanding of the mechanisms of energy transfer. PMID:15388234

  18. The Level of Protein in Milk Formula Modifies Ileal Sensitivity to LPS Later in Life in a Piglet Model

    PubMed Central

    Gras-Le Guen, Christèle; Lallès, Jean-Paul; Le Huërou-Luron, Isabelle; Boudry, Gaëlle

    2011-01-01

    Background Milk formulas have higher protein contents than human milk. This high protein level could modify the development of intestinal microbiota, epithelial barrier and immune functions and have long-term consequences. Methodology/Principal findings We investigated the effect of a high protein formula on ileal microbiota and physiology during the neonatal period and later in life. Piglets were fed from 2 to 28 days of age either a normoprotein (NP, equivalent to sow milk) or a high protein formula (HP, +40% protein). Then, they received the same solid diet until 160 days. During the formula feeding period ileal microbiota implantation was accelerated in HP piglets with greater concentrations of ileal bacteria at d7 in HP than NP piglets. Epithelial barrier function was altered with a higher permeability to small and large probes in Ussing chambers in HP compared to NP piglets without difference in bacterial translocation. Infiltration of T cells was increased in HP piglets at d28. IL-1β and NF-κB sub-units mRNA levels were reduced in HP piglets at d7 and d28 respectively; plasma haptoglobin also tended to be reduced at d7. Later in life, pro-inflammatory cytokines secretion in response to high doses of LPS in explants culture was reduced in HP compared to NP piglets. Levels of mRNA coding the NF-κB pathway sub-units were increased by the challenge with LPS in NP piglets, but not HP ones. Conclusions/Significance A high protein level in formula affects the postnatal development of ileal microbiota, epithelial barrier and immune function in piglets and alters ileal response to inflammatory mediators later in life. PMID:21573022

  19. Whole cell, label free protein quantitation with data independent acquisition: quantitation at the MS2 level.

    PubMed

    McQueen, Peter; Spicer, Vic; Schellenberg, John; Krokhin, Oleg; Sparling, Richard; Levin, David; Wilkins, John A

    2015-01-01

    Label free quantitation by measurement of peptide fragment signal intensity (MS2 quantitation) is a technique that has seen limited use due to the stochastic nature of data dependent acquisition (DDA). However, data independent acquisition has the potential to make large scale MS2 quantitation a more viable technique. In this study we used an implementation of data independent acquisition--SWATH--to perform label free protein quantitation in a model bacterium Clostridium stercorarium. Four tryptic digests analyzed by SWATH were probed by an ion library containing information on peptide mass and retention time obtained from DDA experiments. Application of this ion library to SWATH data quantified 1030 proteins with at least two peptides quantified (∼ 40% of predicted proteins in the C. stercorarium genome) in each replicate. Quantitative results obtained were very consistent between biological replicates (R(2) ∼ 0.960). Protein quantitation by summation of peptide fragment signal intensities was also highly consistent between biological replicates (R(2) ∼ 0.930), indicating that this approach may have increased viability compared to recent applications in label free protein quantitation. SWATH based quantitation was able to consistently detect differences in relative protein quantity and it provided coverage for a number of proteins that were missed in some samples by DDA analysis. PMID:25348682

  20. Revealing Atomic-Level Mechanisms of Protein Allostery with Molecular Dynamics Simulations

    PubMed Central

    Hertig, Samuel

    2016-01-01

    Molecular dynamics (MD) simulations have become a powerful and popular method for the study of protein allostery, the widespread phenomenon in which a stimulus at one site on a protein influences the properties of another site on the protein. By capturing the motions of a protein’s constituent atoms, simulations can enable the discovery of allosteric binding sites and the determination of the mechanistic basis for allostery. These results can provide a foundation for applications including rational drug design and protein engineering. Here, we provide an introduction to the investigation of protein allostery using molecular dynamics simulation. We emphasize the importance of designing simulations that include appropriate perturbations to the molecular system, such as the addition or removal of ligands or the application of mechanical force. We also demonstrate how the bidirectional nature of allostery—the fact that the two sites involved influence one another in a symmetrical manner—can facilitate such investigations. Through a series of case studies, we illustrate how these concepts have been used to reveal the structural basis for allostery in several proteins and protein complexes of biological and pharmaceutical interest. PMID:27285999

  1. Adaptor protein containing PH domain, PTB domain and leucine zipper (APPL1) regulates the protein level of EGFR by modulating its trafficking

    SciTech Connect

    Lee, Jae-Rin; Hahn, Hwa-Sun; Kim, Young-Hoon; Nguyen, Hong-Hoa; Yang, Jun-Mo; Kang, Jong-Sun; Hahn, Myong-Joon

    2011-11-11

    Highlights: Black-Right-Pointing-Pointer APPL1 regulates the protein level of EGFR in response to EGF stimulation. Black-Right-Pointing-Pointer Depletion of APPL1 accelerates the movement of EGF/EGFR from the cell surface to the perinuclear region in response to EGF. Black-Right-Pointing-Pointer Knockdown of APPL1 enhances the activity of Rab5. -- Abstract: The EGFR-mediated signaling pathway regulates multiple biological processes such as cell proliferation, survival and differentiation. Previously APPL1 (adaptor protein containing PH domain, PTB domain and leucine zipper 1) has been reported to function as a downstream effector of EGF-initiated signaling. Here we demonstrate that APPL1 regulates EGFR protein levels in response to EGF stimulation. Overexpression of APPL1 enhances EGFR stabilization while APPL1 depletion by siRNA reduces EGFR protein levels. APPL1 depletion accelerates EGFR internalization and movement of EGF/EGFR from cell surface to the perinuclear region in response to EGF treatment. Conversely, overexpression of APPL1 decelerates EGFR internalization and translocation of EGF/EGFR to the perinuclear region. Furthermore, APPL1 depletion enhances the activity of Rab5 which is involved in internalization and trafficking of EGFR and inhibition of Rab5 in APPL1-depleted cells restored EGFR levels. Consistently, APPL1 depletion reduced activation of Akt, the downstream signaling effector of EGFR and this is restored by inhibition of Rab5. These findings suggest that APPL1 is required for EGFR signaling by regulation of EGFR stabilities through inhibition of Rab5.

  2. The effect of varying protein levels on blood chemistry, food consumption, and behavior of captive seaducks

    USGS Publications Warehouse

    Wells-Berlin, A. M.; Perry, M.C.; Olsen, G.H.

    2005-01-01

    The Chesapeake Bay is a primary wintering area for scoters and the long-tailed ducks (Clangia hyemalis) that migrate along the Atlantic Flyway. Recently, the Chesapeake Bay had undergone an ecosystem shift and little is known about how this is affecting the seaduck populations. We are determining what are the preferred food sources of the seaducks wintering on the Bay and analyzing the factors influencing prey selection whether it is prey composition, energy assimilated, prey availability, or a combination of any or all of these factors. We have established a captive colony of surf (Melanitta perspicillata) and white-winged scoters (Melanitta fusca) as well as long-tailed ducks at Patuxent Wildlife Research Center to allow us to examine these factors in a more controlled environment. This project contains a multitude of experiments and the resultant data will be compiled into a compartmental model on the feeding ecology of seaducks wintering on the Bay. The first experiment entailed feeding groups of each species (four ducks per pen of equal sex ratio, if possible, and four pens per species) three diets varying in percent protein levels from November to February. Each diet was randomly assigned to each pen and the amount of food consumed was recorded each day. New feed was given when all existing food was consumed. Behavioral trials and blood profiles were completed on all study birds to determine the effects of the varying diets. There were no significant differences in food consumption, blood chemistry, and behavior detected at the 5% level among the diets for all three species of interest. There was a seasonal effect determined based on the food consumption data for white-winged scoters, but not for surf scoters or long-tailed ducks. The blood profiles of the surf scoters were compared to blood profiles of wild surf scoters and a there was no difference detected at the 5% level. As a health check of the ducks an aspergillosis test was run on the blood obtained

  3. Effects of pH and Repellent Tactic Stimuli on Protein Methylation Levels in Escherichia coli

    PubMed Central

    Slonczewski, Joan L.; Macnab, Robert M.; Alger, Jeffry R.; Castle, Anna M.

    1982-01-01

    Intracellular pH (pHint) and extracellular pH (pHext) of Escherichia coli were measured at 12-s time resolution by 31P-nuclear magnetic resonance: a sudden neutral-to-acid shift in pHext (e.g., from 7.0 to 5.6) caused a transient failure of homeostasis, with pHint decreasing by about 0.4 unit in ca. 30 s and then returning to its original value (ca. 7.5) over a period of several minutes. Membrane proton conductance was estimated to be 20 pmol s−1 cm−2 pH unit−1. Addition of the membrane-permeant weak acid benzoate at constant pHext also caused a lowering of pHint; at high concentrations it generated an inverted transmembrane pH gradient (ΔpH). The buffering capacity of the cells was estimated by such experiments to be ca. 50 mM per pH unit. Effects of pH-related stimuli on the methyl-accepting chemotaxis proteins (MCPs) were examined: the steady-state methylation of MCP I was found to decrease when pHint was lowered by weak acid addition or when pHext was lowered. The extent of demethylation in the latter case was too great to be explained by imperfect steady-state homeostasis; a small but reproducible undershoot in methylation level correlated with the observed short-term homeostatic failure. MCP II underwent smaller and more complex changes than MCP I, in response to pH-related stimuli. The methylation level of MCP I could not, by any condition tested, be driven below a limit of ca. 15% of the control level (unstimulated cells at pHext 7.0). The weak-acid concentration needed to reach that limit was dependent on pHext, as would be expected on the basis of ΔpH-driven concentrative effects. The potency ranking of weak acids was the same with respect to lowering pHint, demethylating MCP I, and causing repellent behavioral responses. The data are consistent with a model whereby MCP I and hence tactic behavior are sensitive to both pHint and pHext. Evidence is presented that pHint may also have a direct (non-MCP-related) effect on motor function. Comparison of

  4. Relative expression levels of the HLA class-I proteins in normal and HIV-infected cells 1

    PubMed Central

    Apps, Richard; Meng, Zhaojing; Del Prete, Gregory Q.; Lifson, Jeffrey D.; Zhou, Ming; Carrington, Mary

    2015-01-01

    The expression level of human leukocyte antigens (HLA) is known to influence pathological outcomes: pathogens downregulate HLA to evade host immune responses, host inflammatory reactions upregulate HLA, and differences between people in steady-state expression levels of HLA associate with disease susceptibility. Yet precise quantification of relative expression levels of the various HLA loci is difficult due to the tremendous polymorphism of HLA. We report relative expression levels of HLA-A, HLA-B, HLA-C and HLA-E proteins for the specific haplotype A*02:01, B*44:02, C*05:01, characterized using two independent methods based on flow cytometry and mass spectrometry. Peripheral blood lymphocytes from normal donors showed that HLA-A and HLA-B proteins are expressed at similar levels, which are 13-18 times higher than HLA-C by flow cytometry and 4-5 times higher than HLA-C by mass spectrometry, differences that may reflect variation in the conformation or location of proteins detected. HLA-E was detected at a level 25 times lower than that of HLA-C by mass spectrometry. Primary CD4+ T cells infected with HIV in vitro were also studied since HIV downregulates selective HLA types. HLA-A and -B were reduced on HIV-infected cells by a magnitude that varied between cells in an infected culture. Averaging all infected cells from an individual showed HLA-A to be 1-3 and HLA-B to be 2-5 times higher than HLA-C for different individuals by flow cytometry. These results quantify substantial differences in expression levels of the proteins from different HLA loci, which are very likely physiologically significant on both uninfected and HIV-infected cells. PMID:25754738

  5. Deletions or duplications in the BtuB protein affect its level in the outer membrane of Escherichia coli.

    PubMed Central

    Köster, W; Gudmundsdottir, A; Lundrigan, M D; Seiffert, A; Kadner, R J

    1991-01-01

    The Escherichia coli btuB product is an outer membrane protein that mediates the TonB-coupled active transport of cobalamins and the uptake of the E colicins and bacteriophage BF23. The roles of various segments of the BtuB protein in its function or cellular localization were investigated by analysis of several genetic constructs. Hybrid proteins in which various lengths from the amino terminus of BtuB were linked to alkaline phosphatase (btuB::phoA genes) were all secreted across the cytoplasmic membrane. The BtuB-PhoA proteins that carried up to 327 amino acids of BtuB appeared to reside in the periplasmic space, whereas hybrid proteins containing at least 399 amino acids of BtuB were associated with the outer membrane. Eleven in-frame internal deletion mutations that spanned more than half of the mature sequence were prepared by combining appropriate restriction fragments from btuB variants with 6-bp linker insertions. None of the deleted proteins was able to complement any BtuB functions, and only three of them were detectable in the outer membrane, suggesting that most of the deletions affected sequences needed for stable association with the outer membrane. Duplications covering the same portions of BtuB were prepared in the same manner. All of these partial duplication variants complemented all BtuB functions, although some gave substantially reduced levels of activity. These proteins were found in the outer membrane, although some were subject to proteolytic cleavage within or near the duplicated segment. These results indicate that the insertion of BtuB into the outer membrane requires the presence of several regions of teh BtuB protein and that the presence of extra or redundant segments of the protein can be tolerated during its insertion and function. Images PMID:1885541

  6. Investigation into the Effects of Boron on Liver Tissue Protein Carbonyl, MDA, and Glutathione Levels in Endotoxemia.

    PubMed

    Balabanlı, Barbaros; Balaban, Tuba

    2015-10-01

    Endotoxin has been known to cause the formation and damage of free radical. The importance of boron for human life is increasing each passing day, and its consuming fields are continuing to expand due to the advances in science and technology. Therefore, in our study, we intended to investigate into the effects of boron on liver tissue oxidative events. Eighteen male Wistar albino rats were randomly separated into three equal groups in the experiments; control group, boron + endotoxin group, and endotoxin group. Dissolved in distilled water, boric acid (100 mg/kg) was administered to boron + endotoxin group via gavage procedure for 28 days. Only distilled water was administered to control and endotoxin groups via gavage procedure for 28 days. Then 4 mg/kg endotoxin (LPS; Escherichia coli 0111:B4) was intraperitoneally (ip) administered to boron + endotoxin and endotoxin groups on the 28th day. Sterile saline was injected into control group on the 28th day (ip). Malondialdehyde (MDA), which is the end product of lipid peroxidation in liver tissues, protein carbonyl compounds (PC), which are protein oxidization markers, and glutathione (GSH) levels were measured spectrophotometrically. The results were compared with Mann-Whitney U test. When boron + endotoxin group is compared with endotoxin group, PC levels of endotoxin group showed a significant increase. When GSH levels are compared, GSH level in boron + endotoxin group decreased according to endotoxin group. Variations among all groups in MDA levels were found to be statistically insignificant. We are of the opinion that endotoxin affects the proteins by forming free radicals, and boron may also cause the structural and/or functional changes in proteins in order to protect proteins from oxidization. PMID:25787825

  7. Canavanine Alters ROS/RNS Level and Leads to Post-translational Modification of Proteins in Roots of Tomato Seedlings.

    PubMed

    Krasuska, Urszula; Andrzejczak, Olga; Staszek, Paweł; Bogatek, Renata; Gniazdowska, Agnieszka

    2016-01-01

    Canavanine (CAN), a structural analog of arginine (Arg), is used as a selective inhibitor of inducible NOS in mammals. CAN is incorporated into proteins' structure in the place of Arg, leading to the formation of aberrant compounds. This non-protein amino acid is found in legumes, e.g., Canavalia ensiformis (L.) DC. or Sutherlandia frutescens (L.) R.Br. and acts as a strong toxin against herbivores or plants. Tomato (Solanum lycopersicum L.) seedlings were treated for 24-72 h with CAN (10 or 50 μM) inhibiting root growth by 50 or 100%, without lethal effect. We determined ROS level/production in root extracts, fluorescence of DAF-FM and APF derivatives corresponding to RNS level in roots of tomato seedlings and linked CAN-induced restriction of root growth to the post-translational modifications (PTMs) of proteins: carbonylation and nitration. Both PTMs are stable markers of nitro-oxidative stress, regarded as the plant's secondary response to phytotoxins. CAN enhanced H2O2 content and superoxide radicals generation in extracts of tomato roots and stimulated formation of protein carbonyl groups. An elevated level of carbonylated proteins was characteristic for the plants after 72 h of the culture, mainly for the roots exposed to 10 μM CAN. The proteolytic activity was stimulated by tested non-protein amino acid. CAN treatment led to decline of fluorescence of DAF-FM derivatives, and transiently stimulated fluorescence of APF derivatives. Short-term exposure of tomato seedlings to CAN lowered the protein nitration level. Activity of peroxidase, polyamine oxidase and NADPH oxidase, enzymes acting as modulators of H2O2 concentration and governing root architecture and growth were determined. Activities of all enzymes were stimulated by CAN, but no strict CAN concentration dependence was observed. We conclude, that although CAN treatment led to a decline in the nitric oxide level, PTMs observed in roots of plants exposed to CAN are linked rather to the formation of

  8. Regulation of mRNA and Protein Levels of β1 Integrin Variants in Human Prostate Carcinoma

    PubMed Central

    Perlino, Elda; Lovecchio, Mariarosaria; Vacca, Rosa A.; Fornaro, Mara; Moro, Loredana; Ditonno, Pasquale; Battaglia, Michele; Selvaggi, Francesco P.; Mastropasqua, Mauro G.; Bufo, Pantaleo; Languino, Lucia R.

    2000-01-01

    Alterations of integrin expression levels in cancer cells correlate with changes in invasiveness, tumor progression, and metastatic potential. The β1C integrin, an alternatively spliced form of the human β1 integrin, has been shown to inhibit prostate cell proliferation. Furthermore, β1C protein levels were found to be abundant in normal prostate glandular epithelium and down-regulated in prostatic adenocarcinoma. To gain further insights into the molecular mechanisms underlying abnormal cancer cell proliferation, we have studied β1C and β1 integrin expression at both mRNA and protein levels by Northern and immunoblotting analysis using freshly isolated neoplastic and normal human prostate tissue specimens. Steady-state mRNA levels were evaluated in 38 specimens: 33 prostatic adenocarcinomas exhibiting different Gleason’s grade and five normal tissue specimens that did not show any histological manifestation of benign prostatic hypertrophy. Our results demonstrate that β1C mRNA is expressed in normal prostate and is significantly down-regulated in neoplastic prostate specimens. In addition, using a probe that hybridizes with all β1 variants, mRNA levels of β1 are found reduced in neoplastic versus normal prostate tissues. We demonstrate that β1C mRNA down-regulation does not correlate with either tumor grade or differentiation according to Gleason’s grade and TNM system evaluation, and that β1C mRNA levels are not affected by hormonal therapy. In parallel, β1C protein levels were analyzed. As expected, β1C is found to be expressed in normal prostate and dramatically reduced in neoplastic prostate tissues; in contrast, using an antibody to β1 that recognizes all β1 variants, the levels of β1 are comparable in normal and neoplastic prostate, thus indicating a selective down-regulation of the β1C protein in prostate carcinoma. These results demonstrate for the first time that β1C and β1 mRNA expression is down-regulated in prostate carcinoma

  9. Prebiotics affect nutrient digestibility but not faecal ammonia in dogs fed increased dietary protein levels.

    PubMed

    Hesta, M; Roosen, W; Janssens, G P J; Millet, S; De Wilde, R

    2003-12-01

    An increased protein content and less digestible protein sources in the diet can induce bad faecal odour. The present study investigated the effect of adding prebiotics to dog diets enriched with animal-derived protein sources on apparent digestibilities and faecal ammonia concentration. In three subsequent periods eight healthy beagle dogs were fed a commercial dog diet that was gradually supplemented by up to 50 % with meat and bone meal (MBM), greaves meal (GM) or poultry meal (PM) respectively. Afterwards, 3 % fructo-oligosaccharides or 3 % isomalto-oligosaccharides were substituted for 3 % of the total diet. Supplementation with animal-derived protein sources did not decrease the apparent N digestibility significantly but oligosaccharides did. On the other hand the bacterial N content (% DM) in the faeces was highest in the oligosaccharide groups followed by the protein-supplemented groups and lowest in the control groups. When the apparent N digestibility was corrected for bacterial N no significant differences were noted anymore except for the GM group where the corrected N digestibility was still lower after oligosaccharide supplementation. The amount of faecal ammonia was significantly increased by supplementing with protein or oligosaccharides in the MBM and GM groups but not in the PM group. When apparent N digestibility is interpreted, a correction for bacterial N should be taken into account, especially when prebiotics are added to the diet. Oligosaccharides did not reduce the faecal ammonia concentrations as expected. PMID:14641959

  10. Visualizing Protein Movement on DNA at the Single-molecule Level using DNA Curtains

    PubMed Central

    Silverstein, Timothy D.; Gibb, Bryan; Greene, Eric C.

    2014-01-01

    A fundamental feature of many nucleic-acid binding proteins is their ability to move along DNA either by diffusion-based mechanisms or by ATP-hydrolysis driven translocation. For example, most site-specific DNA-binding proteins must diffuse to some extent along DNA to either find their target sites, or to otherwise fulfill their biological roles. Similarly, nucleic-acid translocases such as helicases and polymerases must move along DNA to fulfill their functions. In both instances, the proteins must also be capable of moving in crowded environments while navigating through DNA-bound obstacles. These types of behaviors can be challenging to analyze by bulk biochemical methods because of the transient nature of the interactions, and/or heterogeneity of the reaction intermediates. The advent of single-molecule methodologies has overcome some of these problems, and has led to many new insights into the mechanisms that contribute to protein motion along DNA. We have developed DNA curtains as a tool to facilitate single molecule observations of protein-nucleic acid interactions, and we have applied these new research tools to systems involving both diffusive-based motion as well as ATP directed translocation. Here we highlight these studies by first discussing how diffusion contributes to target searches by proteins involved in post-replicative mismatch repair. We then discuss DNA curtain assays of two different DNA translocases, RecBCD and FtsK, which participate in homologous DNA recombination and site-specific DNA recombination, respectively. PMID:24598576

  11. Enhanced expression of glucose-regulated protein 78 correlates with malondialdehyde levels during the formation of liver cirrhosis in rats

    PubMed Central

    ZHANG, YUN; ZHANG, HUIYING; ZHAO, ZHONGFU; LV, MINLI; JIA, JIANTAO; ZHANG, LILI; TIAN, XIAOXIA; CHEN, YUNXIA; LI, BAOHONG; LIU, MINGSHE; HAN, DEWU; JI, CHENG

    2015-01-01

    The aim of the present study was to explore the role of glucose-regulated protein 78 (GRP78) in the development of liver cirrhosis promoted by intestinal endotoxemia in rats. Fifty-one male Wistar rats were randomly divided into the liver cirrhosis 4-week, 6-week and 8-week groups and the normal control group at each time point. Liver cirrhosis was induced by employing multiple pathogenic factors in the rats. Blood and liver tissues were collected. The levels of alanine aminotransferase (ALT), homocysteine, endotoxin and tumor necrosis factor-α (TNF-α) in the plasma, and TNF-α, malondialdehyde (MDA) and procollagen type III peptide (PIIIP) in the liver tissues were determined. The mRNA and protein expression levels of GRP78 in the liver were detected using reverse transcription-quantitative polymerase chain reaction and immunohistochemistry. Morphological changes were observed through hematoxylin and eosin and van Gieson staining of the liver. Liver cirrhosis caused marked histopathological changes to the livers of the rats. Following significant increases in the levels of ALT, homocysteine, endotoxin and TNF-α in the plasma, and TNF-α, MDA and PIIIP in the liver tissues of all experimental groups with the progression of liver cirrhosis, the mRNA and protein expression levels of GRP78 also gradually increased. In addition, correlation analysis indicated that the enhanced expression of GRP78 correlated with the MDA levels of the rats during the formation of liver cirrhosis. PMID:26668603

  12. In utero exposure to benzene increases embryonic c-Myb and Pim-1 protein levels in CD-1 mice

    SciTech Connect

    Wan, Joanne; Winn, Louise M.

    2008-05-01

    Benzene is a known human leukemogen, but its role as an in utero leukemogen remains controversial. Epidemiological studies have correlated parental exposure to benzene with an increased incidence of childhood leukemias. We hypothesize that in utero exposure to benzene may cause leukemogenesis by affecting the embryonic c-Myb/Pim-1 signaling pathway and that this is mediated by oxidative stress. To investigate this hypothesis, pregnant CD-1 mice were treated with either 800 mg/kg of benzene or corn oil (i.p.) on days 10 and 11 of gestation and in some cases pretreated with 25 kU/kg of PEG-catalase. Phosphorylated and total embryonic c-Myb and Pim-1 protein levels were assessed using Western blotting and maternal and embryonic oxidative stress were assessed by measuring reduced to oxidized glutathione ratios. Our results show increased oxidative stress at 4 and 24 h after exposure, increased phosphorylated Pim-1 protein levels 4 h after benzene exposure, and increased Pim-1 levels at 24 and 48 h after benzene exposure. Embryonic c-Myb levels were elevated at 24 h after exposure. PEG-catalase pretreatment prevented benzene-mediated increases in embryonic c-Myb and Pim-1 protein levels, and benzene-induced oxidative stress. These results support a role for ROS in c-Myb and Pim-1 alterations after in utero benzene exposure.

  13. Combined Inflammatory and Metabolic Defects Reflected by Reduced Serum Protein Levels in Patients with Buruli Ulcer Disease

    PubMed Central

    Landier, Jordi; Oldenburg, Reid; Frimpong, Michael; Wansbrough-Jones, Mark; Abass, Kabiru; Thompson, William; Forson, Mark; Fontanet, Arnaud; Niang, Fatoumata; Demangel, Caroline

    2014-01-01

    Buruli ulcer is a skin disease caused by Mycobacterium ulcerans that is spreading in tropical countries, with major public health and economic implications in West Africa. Multi-analyte profiling of serum proteins in patients and endemic controls revealed that Buruli ulcer disease down-regulates the circulating levels of a large array of inflammatory mediators, without impacting on the leukocyte composition of peripheral blood. Notably, several proteins contributing to acute phase reaction, lipid metabolism, coagulation and tissue remodelling were also impacted. Their down-regulation was selective and persisted after the elimination of bacteria with antibiotic therapy. It involved proteins with various functions and origins, suggesting that M. ulcerans infection causes global and chronic defects in the host's protein metabolism. Accordingly, patients had reduced levels of total serum proteins and blood urea, in the absence of signs of malnutrition, or functional failure of liver or kidney. Interestingly, slow healers had deeper metabolic and coagulation defects at the start of antibiotic therapy. In addition to providing novel insight into Buruli ulcer pathogenesis, our study therefore identifies a unique proteomic signature for this disease. PMID:24722524

  14. Hipk2 and PP1c cooperate to maintain Dvl protein levels required for Wnt signal transduction.

    PubMed

    Shimizu, Nobuyuki; Ishitani, Shizuka; Sato, Atsushi; Shibuya, Hiroshi; Ishitani, Tohru

    2014-09-11

    The phosphoprotein Dishevelled (Dvl) is a common essential component of Wnt/β-catenin and Wnt/planar cell polarity (PCP) signaling pathways. However, the regulation and significance of Dvl phosphorylation are not fully understood. Here, we show that homeodomain-interacting protein kinase 2 (Hipk2) facilitates protein phosphatase 1 catalytic subunit (PP1c)-mediated dephosphorylation of Dvl via its C-terminal domain and that this dephosphorylation blocks ubiquitination and consequent degradation mediated by the E3 ubiquitin ligase Itch, which targets the phosphorylated form of Dvl proteins. Inhibition of Hipk2 or PP1c function reduces Dvl protein levels and suppresses Wnt/β-catenin and Wnt/PCP pathway-dependent events in mammalian cells and zebrafish embryos, suggesting that Hipk2 and PP1c are essential for maintaining Dvl protein levels that are sufficient to activate Wnt signaling. We also show that Wnt-3a, a Wnt/β-catenin ligand, induces dissociation of the Dvl-Hipk2-PP1c complex and Dvl degradation under high-cell-density conditions. This regulation may be a negative feedback mechanism that fine-tunes Wnt/β-catenin signaling. PMID:25159144

  15. Engineered mutations in fibrillin-1 leading to Marfan syndrome act at the protein, cellular and organismal levels.

    PubMed

    Zeyer, Karina A; Reinhardt, Dieter P

    2015-01-01

    Fibrillins are the major components of microfibrils in the extracellular matrix of elastic and non-elastic tissues. They are multi-domain proteins, containing primarily calcium binding epidermal growth factor-like (cbEGF) domains and 8-cysteine/transforming growth factor-beta binding protein-like (TB) domains. Mutations in the fibrillin-1 gene give rise to Marfan syndrome, a connective tissue disorder with clinical complications in the cardiovascular, skeletal, ocular and other organ systems. Here, we review the consequences of engineered Marfan syndrome mutations in fibrillin-1 at the protein, cellular and organismal levels. Representative point mutations associated with Marfan syndrome in affected individuals have been introduced and analyzed in recombinant fibrillin-1 fragments. Those mutations affect fibrillin-1 on a structural and functional level. Mutations which impair folding of cbEGF domains can affect protein trafficking. Protein folding disrupted by some mutations can lead to defective secretion in mutant fibrillin-1 fragments, whereas fragments with other Marfan mutations are secreted normally. Many Marfan mutations render fibrillin-1 more susceptible to proteolysis. There is also evidence that some mutations affect heparin binding. Few mutations have been further analyzed in mouse models. An extensively studied mouse model of Marfan syndrome expresses mouse fibrillin-1 with a missense mutation (p.C1039G). The mice display similar characteristics to human patients with Marfan syndrome. Overall, the analyses of engineered mutations leading to Marfan syndrome provide important insights into the pathogenic molecular mechanisms exerted by mutated fibrillin-1. PMID:26281765

  16. Genes adopt non-optimal codon usage to generate cell cycle-dependent oscillations in protein levels

    PubMed Central

    Frenkel-Morgenstern, Milana; Danon, Tamar; Christian, Thomas; Igarashi, Takao; Cohen, Lydia; Hou, Ya-Ming; Jensen, Lars Juhl

    2012-01-01

    The cell cycle is a temporal program that regulates DNA synthesis and cell division. When we compared the codon usage of cell cycle-regulated genes with that of other genes, we discovered that there is a significant preference for non-optimal codons. Moreover, genes encoding proteins that cycle at the protein level exhibit non-optimal codon preferences. Remarkably, cell cycle-regulated genes expressed in different phases display different codon preferences. Here, we show empirically that transfer RNA (tRNA) expression is indeed highest in the G2 phase of the cell cycle, consistent with the non-optimal codon usage of genes expressed at this time, and lowest toward the end of G1, reflecting the optimal codon usage of G1 genes. Accordingly, protein levels of human glycyl-, threonyl-, and glutamyl-prolyl tRNA synthetases were found to oscillate, peaking in G2/M phase. In light of our findings, we propose that non-optimal (wobbly) matching codons influence protein synthesis during the cell cycle. We describe a new mathematical model that shows how codon usage can give rise to cell-cycle regulation. In summary, our data indicate that cells exploit wobbling to generate cell cycle-dependent dynamics of proteins. PMID:22373820

  17. Steady state protein levels in Geobacter metallireducens grown with Iron (III) citrate or nitrate as terminal electron acceptor.

    SciTech Connect

    Ahrendt, A. J.; Tollaksen, S. L.; Lindberg, C.; Zhu, W.; Yates, J. R., III; Nevin, K. P.; Lovley, D.; Giometti, C. S.; Biosciences Division; The Scripps Research Inst.; Univ. of Massachusetts

    2007-01-01

    Geobacter species predominate in aquatic sediments and submerged soils where organic carbon sources are oxidized with the reduction of Fe(III). The natural occurrence of Geobacter in some waste sites suggests this microorganism could be useful for bioremediation if growth and metabolic activity can be regulated. 2-DE was used to monitor the steady state protein levels of Geobacter metallireducens grown with either Fe(III) citrate or nitrate to elucidate metabolic differences in response to different terminal electron acceptors present in natural environments populated by Geobacter. Forty-six protein spots varied significantly in abundance (p<0.05) between the two growth conditions; proteins were identified by tryptic peptide mass and peptide sequence determined by MS/MS. Enzymes involved in pyruvate metabolism and the tricarboxylic acid (TCA) cycle were more abundant in cells grown with Fe(III) citrate, while proteins associated with nitrate metabolism and sensing cellular redox status along with several proteins of unknown function were more abundant in cells grown with nitrate. These results indicate a higher level of flux through the TCA cycle in the presence of Fe(III) compared to nitrate. The oxidative stress response observed in previous studies of Geobacter sulfurreducens grown with Fe(III) citrate was not seen in G. metallireducens.

  18. Effect of Buddhist meditation on serum cortisol and total protein levels, blood pressure, pulse rate, lung volume and reaction time.

    PubMed

    Sudsuang, R; Chentanez, V; Veluvan, K

    1991-09-01

    Serum cortisol and total protein levels, blood pressure, heart rate, lung volume, and reaction time were studied in 52 males 20-25 years of age practicing Dhammakaya Buddhist meditation, and in 30 males of the same age group not practicing meditation. It was found that after meditation, serum cortisol levels were significantly reduced, serum total protein level significantly increased, and systolic pressure, diastolic pressure and pulse rate significantly reduced. Vital capacity, tidal volume and maximal voluntary ventilation were significantly lower after meditation than before. There were also significant decreases in reaction time after mediation practice. The percentage decrease in reaction time during meditation was 22%, while in subjects untrained in meditation, the percentage decrease was only 7%. Results from these studies indicate that practising Dhammakaya Buddhist meditation produces biochemical and physiological changes and reduces the reaction time. PMID:1801007

  19. Moderate hypothermia induces marked increase in levels and nuclear accumulation of SUMO2/3-conjugated proteins in neurons

    PubMed Central

    Wang, Liangli; Ma, Qing; Yang, Wei; Mackensen, G. Burkhard; Paschen, Wulf

    2012-01-01

    Deep hypothermia protects the brain from ischemic damage and is therefore used during major cardiovascular surgeries requiring cardiopulmonary bypass and a period of circulatory arrest. Here, we demonstrated that small ubiquitin-like modifier (SUMO1-3) conjugation is markedly activated in the brain during deep to moderate hypothermia. Animals were subjected to normothermic (37°C) or deep to moderate (18°C, 24°C, 30°C) hypothermic cardiopulmonary bypass, and the effects of hypothermia on SUMO conjugation were evaluated by Western blot and immunohistochemistry. Exposure to moderate 30°C hypothermia was sufficient to markedly increased levels and nuclear accumulation of SUMO2/3-conjugated proteins in these cells. Deep hypothermia induced nuclear translocation of the SUMO conjugating enzyme Ubc9, suggesting that the increase in nuclear levels of SUMO2/3-conjugated proteins observed in brains of hypothermic animals is an active process. Exposure of primary neuronal cultures to deep hypothermia induced only a moderate rise in levels of SUMO2/3-conjugated proteins. This suggests that neurons in vivo have a higher capacity than neurons in vitro to activate this endogenous potentially neuroprotective pathway upon exposure to hypothermia. Identifying proteins that are SUMO2/3 conjugated during hypothermia could help to design new strategies for preventive and therapeutic interventions to make neurons more resistant to a transient interruption of blood supply. PMID:22891650

  20. Serine/threonine-protein phosphatase 1 α levels are paralleling olfactory memory formation in the CD1 mouse.

    PubMed

    Winding, Christiana; Sun, Yanwei; Höger, Harald; Bubna-Littitz, Hermann; Pollak, Arnold; Schmidt, Peter; Lubec, Gert

    2011-06-01

    Although olfactory discrimination has already been studied in several mouse strains, data on protein levels linked to olfactory memory are limited. Wild mouse strains Mus musculus musculus, Mus musculus domesticus and CD1 laboratory outbred mice were tested in a conditioned odor preference task and trained to discriminate between two odors, Rose and Lemon, by pairing one odor with a sugar reward. Six hours following the final test, mice were sacrificed and olfactory bulbs (OB) were taken for gel-based proteomics analyses and immunoblotting. OB proteins were extracted, separated by 2-DE and quantified using specific software (Proteomweaver). Odor-trained mice showed a preference for the previously rewarded odor suggesting that conditioned odor preference occurred. In CD1 mice levels, one out of 482 protein spots was significantly increased in odor-trained mice as compared with the control group; it was in-gel digested by trypsin and chymotrypsin and analyzed by tandem mass spectrometry (nano-ESI-LC-MS/MS). The spot was unambiguously identified as serine/threonine-protein phosphatase PP1-α catalytic subunit (PP-1A) and differential levels observed in gel-based proteomic studies were verified by immunoblotting. PP-1A is a key signalling element in synaptic plasticity and memory processes and is herein shown to be paralleling olfactory discrimination representing olfactory memory. PMID:21647921

  1. Heat Shock Partially Dissociates the Overlapping Modules of the Yeast Protein-Protein Interaction Network: A Systems Level Model of Adaptation

    PubMed Central

    Mihalik, Ágoston; Csermely, Peter

    2011-01-01

    Network analysis became a powerful tool giving new insights to the understanding of cellular behavior. Heat shock, the archetype of stress responses, is a well-characterized and simple model of cellular dynamics. S. cerevisiae is an appropriate model organism, since both its protein-protein interaction network (interactome) and stress response at the gene expression level have been well characterized. However, the analysis of the reorganization of the yeast interactome during stress has not been investigated yet. We calculated the changes of the interaction-weights of the yeast interactome from the changes of mRNA expression levels upon heat shock. The major finding of our study is that heat shock induced a significant decrease in both the overlaps and connections of yeast interactome modules. In agreement with this the weighted diameter of the yeast interactome had a 4.9-fold increase in heat shock. Several key proteins of the heat shock response became centers of heat shock-induced local communities, as well as bridges providing a residual connection of modules after heat shock. The observed changes resemble to a ‘stratus-cumulus’ type transition of the interactome structure, since the unstressed yeast interactome had a globally connected organization, similar to that of stratus clouds, whereas the heat shocked interactome had a multifocal organization, similar to that of cumulus clouds. Our results showed that heat shock induces a partial disintegration of the global organization of the yeast interactome. This change may be rather general occurring in many types of stresses. Moreover, other complex systems, such as single proteins, social networks and ecosystems may also decrease their inter-modular links, thus develop more compact modules, and display a partial disintegration of their global structure in the initial phase of crisis. Thus, our work may provide a model of a general, system-level adaptation mechanism to environmental changes. PMID:22022244

  2. Dietary protein level affects iridescent coloration in Anna's hummingbirds, Calypte anna

    PubMed Central

    Meadows, Melissa G.; Roudybush, Thomas E.; McGraw, Kevin J.

    2012-01-01

    SUMMARY Many animal displays involve colorful ornamental traits that signal an individual's quality as a mate or rival. Brilliant iridescent ornaments are common, but little is currently known about their production cost and signaling value. One potential cost of colorful ornaments is the acquisition of limited dietary resources that may be involved, directly or indirectly, in their production. Protein, the primary component of bird feathers and of many nanostructural components of iridescent traits, is naturally restricted in hummingbird diets (comprised mostly of sugars), suggesting that iridescent coloration may be especially challenging to produce in these animals. In this study, we experimentally investigated the effect of dietary protein availability during molt on iridescent color expression in male Anna's hummingbirds (Calypte anna). We fed captive birds either a 6% (high) or a 3% (low) protein diet and stimulated molt by plucking half the gorget and crown ornaments on each bird as well as the non-ornamental iridescent green tail feathers. We found that birds receiving more protein grew significantly more colorful crown feathers (higher red chroma and redder hue) than those fed the low-protein diet. Diet did not affect gorget coloration, but regrowth of feathers in captivity affected both gorget and crown coloration. Additionally, birds on the high-protein diet grew yellower (higher hue) green tail feathers than birds on the low-protein diet. These results indicate that iridescent ornamental feathers are sensitive to diet quality and may serve as honest signals of nutrition to mates or rivals. Further, because both ornamental and non-ornamental iridescent coloration were affected by conditions during their growth, iridescent color in these birds appears to be generally condition dependent. PMID:22837446

  3. Dietary protein level affects iridescent coloration in Anna's hummingbirds, Calypte anna.

    PubMed

    Meadows, Melissa G; Roudybush, Thomas E; McGraw, Kevin J

    2012-08-15

    Many animal displays involve colorful ornamental traits that signal an individual's quality as a mate or rival. Brilliant iridescent ornaments are common, but little is currently known about their production cost and signaling value. One potential cost of colorful ornaments is the acquisition of limited dietary resources that may be involved, directly or indirectly, in their production. Protein, the primary component of bird feathers and of many nanostructural components of iridescent traits, is naturally restricted in hummingbird diets (comprised mostly of sugars), suggesting that iridescent coloration may be especially challenging to produce in these animals. In this study, we experimentally investigated the effect of dietary protein availability during molt on iridescent color expression in male Anna's hummingbirds (Calypte anna). We fed captive birds either a 6% (high) or a 3% (low) protein diet and stimulated molt by plucking half the gorget and crown ornaments on each bird as well as the non-ornamental iridescent green tail feathers. We found that birds receiving more protein grew significantly more colorful crown feathers (higher red chroma and redder hue) than those fed the low-protein diet. Diet did not affect gorget coloration, but regrowth of feathers in captivity affected both gorget and crown coloration. Additionally, birds on the high-protein diet grew yellower (higher hue) green tail feathers than birds on the low-protein diet. These results indicate that iridescent ornamental feathers are sensitive to diet quality and may serve as honest signals of nutrition to mates or rivals. Further, because both ornamental and non-ornamental iridescent coloration were affected by conditions during their growth, iridescent color in these birds appears to be generally condition dependent. PMID:22837446

  4. Quantitation of protein 3 content of circulating erythrocytes at the single-cell level

    SciTech Connect

    Jennings, L.K.; Brown, L.K.; Dockter, M.E.

    1985-05-01

    The density and size of human erythrocytes has been roughly correlated with cell age, with the denser and smaller cells being older. Observations of this type have led to a hypothesis that the membranes of circulating erythrocytes are dynamic with respect to composition and that material is lost from the membrane during cell maturation and circulation. In this study, flow cytofluorimetry was used to investigate the distribution of the human erythrocyte anion transport protein (protein 3) in heterogeneous samples of circulating red cells. We verified that protein 3 can be specifically and quantitatively labeled in intact human erythrocytes with eosin-5-maleimide, a luminescent probe. Individual cells were accordingly analyzed for size by forward light scattering and for protein 3 content by quantitation of eosin fluorescence. Initial results indicated that the smallest erythrocytes had a protein 3 content equal to that of the largest circulating erythrocytes. This result was independently verified by light scatter-activated cell sorting; direct measurement of cell diameters by microscopy verified that the cell sizes of erythrocytes showing the 10% greatest and 10% smallest light-scattering signal were indeed distinct. Independent analysis of the size-sorted erythrocytes for protein 3 content was accomplished by gel electrophoresis of stroma from 150,000 large and small erythrocytes. Quantitative scanning densitometry of silver-stained gels of prepared stroma showed that protein 3 content of each set of fractionated cells was equal and did not vary as a function of cell size. Taken in combination with the reported correlation between increasing red blood cell age and decreasing cell size, these results indicate that any loss of membranous material during the cell aging process is not random.

  5. A Phospholipid-Protein Complex from Antarctic Krill Reduced Plasma Homocysteine Levels and Increased Plasma Trimethylamine-N-Oxide (TMAO) and Carnitine Levels in Male Wistar Rats

    PubMed Central

    Bjørndal, Bodil; Ramsvik, Marie S.; Lindquist, Carine; Nordrehaug, Jan E.; Bruheim, Inge; Svardal, Asbjørn; Nygård, Ottar; Berge, Rolf K.

    2015-01-01

    Seafood is assumed to be beneficial for cardiovascular health, mainly based on plasma lipid lowering and anti-inflammatory effects of n-3 polyunsaturated fatty acids. However, other plasma risk factors linked to cardiovascular disease are less studied. This study aimed to penetrate the effect of a phospholipid-protein complex (PPC) from Antarctic krill on one-carbon metabolism and production of trimethylamine-N-oxide (TMAO) in rats. Male Wistar rats were fed isoenergetic control, 6%, or 11% PPC diets for four weeks. Rats fed PPC had reduced total homocysteine plasma level and increased levels of choline, dimethylglycine and cysteine, whereas the plasma level of methionine was unchanged compared to control. PPC feeding increased the plasma level of TMAO, carnitine, its precursors trimethyllysine and γ-butyrobetaine. There was a close correlation between plasma TMAO and carnitine, trimethyllysine, and γ-butyrobetaine, but not between TMAO and choline. The present data suggest that PPC has a homocysteine lowering effect and is associated with altered plasma concentrations of metabolites related to one-carbon metabolism and B-vitamin status in rats. Moreover, the present study reveals a non-obligatory role of gut microbiota in the increased plasma TMAO level as it can be explained by the PPC’s content of TMAO. The increased level of carnitine and carnitine precursors is interpreted to reflect increased carnitine biosynthesis. PMID:26371012

  6. A gradient-free method for the purification of infective dengue virus for protein-level investigations.

    PubMed

    Jensen, Stephanie M; Nguyen, Celina T; Jewett, John C

    2016-09-01

    Dengue virus (DENV) is a mosquito-transmitted flavivirus that infects approximately 100 million people annually. Multi-day protocols for purification of DENV reduce the infective titer due to viral sensitivity to both temperature and pH. Herein we describe a 5-h protocol for the purification of all DENV serotypes, utilizing traditional gradient-free ultracentrifugation followed by selective virion precipitation. This protocol allows for the separation of DENV from contaminating proteins - including intact C6/36 densovirus, for the production of infective virus at high concentration for protein-level analysis. PMID:27265428

  7. Effects of Ramadan Fasting on Serum Amyloid A and Protein Carbonyl Group Levels in Patients With Cardiovascular Diseases

    PubMed Central

    Asadi, Hami; Abolfathi, Ali Akbar; Badalzadeh, Reza; Majidinia, Maryam; Yaghoubi, Alireza; Asadi, Maryam; Yousefi, Bahman

    2015-01-01

    Introduction: Serum amyloid-A (SAA) and protein carbonyl group are rigorously related with cardiovascular diseases (CVDs) as a sensitive marker of an acute inflammatory state and as an important index of oxidative stress, respectively. Moreover, diet is one of the main factors that can modify cardiovascular risks. Therefore, this study aimed to investigate the effects of Ramadan fasting on SAA and protein carbonyl group levels in patients with CVDs. Methods: Twenty-one patients (21 male; mean age 52±9 years old) with CVDs (coronary artery disease, cerebrovascular, or peripheral arterial diseases) were participated in this study. Biochemical parameters were measured in patients 2 days before and 2 days after Ramadan fasting. SAA levels were assessed using enzyme-linked immunosorbent assay and Cayman’s protein carbonyl colorimetric assay was provided for measuring protein carbonyl groups. Results: According to the findings of the study, post-Ramadan levels of inflammatory biomarker, SAA was decreased significantly in patients with CVDs in comparison with the baseline before-fasting values (16.84±8.20 vs. 24.40±6.72 μg/ml, P = 0.021). In addition, Ramadan fasting significantly reduced the levels of protein carbonyl group in patients as compared with those of baseline values (33.08±15.31 vs. 43.65±16.88 nmol/ml, P = 0.039). Conclusion: Ramadan fasting has impressive effects on modulating CVDs by decreasing inflammation and oxidative stress markers. However, to get a clear conclusion with more results, further investigation is warranted. PMID:26191392

  8. Canavanine Alters ROS/RNS Level and Leads to Post-translational Modification of Proteins in Roots of Tomato Seedlings

    PubMed Central

    Krasuska, Urszula; Andrzejczak, Olga; Staszek, Paweł; Bogatek, Renata; Gniazdowska, Agnieszka

    2016-01-01

    Canavanine (CAN), a structural analog of arginine (Arg), is used as a selective inhibitor of inducible NOS in mammals. CAN is incorporated into proteins’ structure in the place of Arg, leading to the formation of aberrant compounds. This non-protein amino acid is found in legumes, e.g., Canavalia ensiformis (L.) DC. or Sutherlandia frutescens (L.) R.Br. and acts as a strong toxin against herbivores or plants. Tomato (Solanum lycopersicum L.) seedlings were treated for 24–72 h with CAN (10 or 50 μM) inhibiting root growth by 50 or 100%, without lethal effect. We determined ROS level/production in root extracts, fluorescence of DAF-FM and APF derivatives corresponding to RNS level in roots of tomato seedlings and linked CAN-induced restriction of root growth to the post-translational modifications (PTMs) of proteins: carbonylation and nitration. Both PTMs are stable markers of nitro-oxidative stress, regarded as the plant’s secondary response to phytotoxins. CAN enhanced H2O2 content and superoxide radicals generation in extracts of tomato roots and stimulated formation of protein carbonyl groups. An elevated level of carbonylated proteins was characteristic for the plants after 72 h of the culture, mainly for the roots exposed to 10 μM CAN. The proteolytic activity was stimulated by tested non-protein amino acid. CAN treatment led to decline of fluorescence of DAF-FM derivatives, and transiently stimulated fluorescence of APF derivatives. Short-term exposure of tomato seedlings to CAN lowered the protein nitration level. Activity of peroxidase, polyamine oxidase and NADPH oxidase, enzymes acting as modulators of H2O2 concentration and governing root architecture and growth were determined. Activities of all enzymes were stimulated by CAN, but no strict CAN concentration dependence was observed. We conclude, that although CAN treatment led to a decline in the nitric oxide level, PTMs observed in roots of plants exposed to CAN are linked rather to the

  9. Improving protein identification from peptide mass fingerprinting through a parameterized multi-level scoring algorithm and an optimized peak detection.

    PubMed

    Gras, R; Müller, M; Gasteiger, E; Gay, S; Binz, P A; Bienvenut, W; Hoogland, C; Sanchez, J C; Bairoch, A; Hochstrasser, D F; Appel, R D

    1999-12-01

    We have developed a new algorithm to identify proteins by means of peptide mass fingerprinting. Starting from the matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) spectra and environmental data such as species, isoelectric point and molecular weight, as well as chemical modifications or number of missed cleavages of a protein, the program performs a fully automated identification of the protein. The first step is a peak detection algorithm, which allows precise and fast determination of peptide masses, even if the peaks are of low intensity or they overlap. In the second step the masses and environmental data are used by the identification algorithm to search in protein sequence databases (SWISS-PROT and/or TrEMBL) for protein entries that match the input data. Consequently, a list of candidate proteins is selected from the database, and a score calculation provides a ranking according to the quality of the match. To define the most discriminating scoring calculation we analyzed the respective role of each parameter in two directions. The first one is based on filtering and exploratory effects, while the second direction focuses on the levels where the parameters intervene in the identification process. Thus, according to our analysis, all input parameters contribute to the score, however with different weights. Since it is difficult to estimate the weights in advance, they have been computed with a generic algorithm, using a training set of 91 protein spectra with their environmental data. We tested the resulting scoring calculation on a test set of ten proteins and compared the identification results with those of other peptide mass fingerprinting programs. PMID:10612280

  10. Electroacupuncture Improves Insulin Resistance by Reducing Neuroprotein Y/Agouti-Related Protein Levels and Inhibiting Expression of Protein Tyrosine Phosphatase 1B in Diet-induced Obese Rats.

    PubMed

    Liu, Xia; He, Jun-Feng; Qu, Ya-Ting; Liu, Zhi-Jun; Pu, Qing-Yang; Guo, Sheng-Tong; Du, Jia; Jiang, Peng-Fei

    2016-04-01

    Electroacupuncture (EA) has been shown to exert beneficial effects on obesity, but the mechanism is unclear. This study investigated the effects of EA on diet-induced obese (DIO) rats. Fifty male Sprague-Dawley rats were randomly divided into low-fat diet (LFD, 10 rats) and high-fat diet (HFD, 40 rats) groups. After the DIO models had been established, successful model rats were randomly divided into HFD, EA, and orlistat (OLST) groups. The EA group received EA at Zusanli (ST36) and Quchi (LI11) for 20 minutes once per day for 28 days. The OLST group was treated with orlistat by gavage. The body weight, homeostasis model assessment-insulin resistance index, adipocyte diameters, and neuroprotein Y/agouti-related protein and protein tyrosine phosphatase 1B levels were significantly lower in the EA group than in the HFD group. The rats of the OLST group showed watery stools and yellow hairs whereas those of the EA group had regular stools and sleek coats. The effect of EA on weight loss may be related to improved insulin resistance caused by changes in the adipocyte size and by reductions in the expressions of neuroprotein Y/agouti-related protein and protein tyrosine phosphatase 1B. This study indicates that EA may be a better method of alternative therapy for treating obesity and other metabolic diseases. PMID:27079226

  11. STAP-2 Protein Expression in B16F10 Melanoma Cells Positively Regulates Protein Levels of Tyrosinase, Which Determines Organs to Infiltrate in the Body*

    PubMed Central

    Sekine, Yuichi; Togi, Sumihito; Muromoto, Ryuta; Kon, Shigeyuki; Kitai, Yuichi; Yoshimura, Akihiko; Oritani, Kenji; Matsuda, Tadashi

    2015-01-01

    Melanoma is the most serious type of skin cancer, with a highly metastatic phenotype. In this report, we show that signal transducing adaptor protein 2 (STAP-2) is involved in cell migration, proliferation, and melanogenesis as well as chemokine receptor expression and tumorigenesis in B16F10 melanoma cells. This was evident in mice injected with STAP-2 shRNA (shSTAP-2)-expressing B16F10 cells, which infiltrated organs in a completely different pattern from the original cells, showing massive colonization in the liver, kidney, and neck but not in the lung. The most important finding was that STAP-2 expression determined tyrosinase protein content. STAP-2 colocalized with tyrosinase in lysosomes and protected tyrosinase from protein degradation. It is noteworthy that B16F10 cells with knocked down tyrosinase showed similar cell characteristics as shSTAP-2 cells. These results indicated that tyrosinase contributed to some cellular events beyond melanogenesis. Taken together, one possibility is that STAP-2 positively regulates the protein levels of tyrosinase, which determines tumor invasion via controlling chemokine receptor expression. PMID:26023234

  12. Src Homology 2 Domain Containing Protein 5 (SH2D5) Binds the Breakpoint Cluster Region Protein, BCR, and Regulates Levels of Rac1-GTP*

    PubMed Central

    Gray, Elizabeth J.; Petsalaki, Evangelia; James, D. Andrew; Bagshaw, Richard D.; Stacey, Melissa M.; Rocks, Oliver; Gingras, Anne-Claude; Pawson, Tony

    2014-01-01

    SH2D5 is a mammalian-specific, uncharacterized adaptor-like protein that contains an N-terminal phosphotyrosine-binding domain and a C-terminal Src homology 2 (SH2) domain. We show that SH2D5 is highly enriched in adult mouse brain, particularly in Purkinjie cells in the cerebellum and the cornu ammonis of the hippocampus. Despite harboring two potential phosphotyrosine (Tyr(P)) recognition domains, SH2D5 binds minimally to Tyr(P) ligands, consistent with the absence of a conserved Tyr(P)-binding arginine residue in the SH2 domain. Immunoprecipitation coupled to mass spectrometry (IP-MS) from cultured cells revealed a prominent association of SH2D5 with breakpoint cluster region protein, a RacGAP that is also highly expressed in brain. This interaction occurred between the phosphotyrosine-binding domain of SH2D5 and an NxxF motif located within the N-terminal region of the breakpoint cluster region. siRNA-mediated depletion of SH2D5 in a neuroblastoma cell line, B35, induced a cell rounding phenotype correlated with low levels of activated Rac1-GTP, suggesting that SH2D5 affects Rac1-GTP levels. Taken together, our data provide the first characterization of the SH2D5 signaling protein. PMID:25331951

  13. Effect of trichostatin A on gelsolin levels, proteolysis of amyloid precursor protein, and amyloid beta-protein load in the brain of transgenic mouse model of Alzheimer's disease.

    PubMed

    Yang, Wenzhong; Chauhan, Abha; Wegiel, Jerzy; Kuchna, Izabela; Gu, Feng; Chauhan, Ved

    2014-01-01

    In vivo and in vitro studies have shown that gelsolin is an anti-amyloidogenic protein. Trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, promotes the expression of gelsolin. Fibrillized amyoid beta-protein (Aβ) is a key constituent of amyloid plaques in the brains of patients with Alzheimer's disease (AD). We studied the effects of TSA on the levels of gelsolin; amyloid precursor protein (APP); proteolytic enzymes (γ-secretase and β-secretase) responsible for the production of Aβ; Aβ-cleaving enzymes, i.e., neprilysin (NEP) and insulin-degrading enzyme (IDE); and amyloid load in the double transgenic (Tg) APPswe/PS1(δE9) mouse model of AD. Intraperitoneal injection of TSA for two months (9-11 months of age) resulted in decreased activity of HDAC, and increased levels of gelsolin in the hippocampus and cortex of the brain in AD Tg mice as compared to vehicle-treated mice. TSA also increased the levels of γ-secretase and β-secretase activity in the brain. However, TSA did not show any effect on the activities or the expression levels of NEP and IDE in the brain. Furthermore, TSA treatment of AD Tg mice showed no change in the amyloid load (percent of examined area occupied by amyloid plaques) in the hippocampus and cortex, suggesting that TSA treatment did not result in the reduction of amyloid load. Interestingly, TSA prevented the formation of new amyloid deposits but increased the size of existing plaques. TSA treatment did not cause any apoptosis in the brain. These results suggest that TSA increases gelsolin expression in the brain, but the pleiotropic effects of TSA negate the anti-amyloidogenic effect of gelsolin in AD Tg mice. PMID:25387339

  14. Effect of the oral contraceptive pill on protein S and antithrombin-III levels in Malaysian women.

    PubMed

    Wong, K K; Ng, S C; Koong, P L

    Studies focusing on the relationship between oral contraceptive (OC) usage and occurrence of thromboembolism have been conducted for over 3 decades. Those studies centered on the effects OC use has on blood proteins and on measurable physiological changes that occurred in women with venous thrombosis. This article reports the findings of a study that investigated the effects of OC use on the levels of the anticoagulants antithrombin-III (AT-III), protein C (PC), and protein S (PS) in a group of Asian women. Previous studies had mostly been based on Caucasian women. Of the 21 women studied, 16 were Malaysian, 3 were Chinese, and 2 were Indian. Low-dose OCs containing 30 mcg of ethinyl estradiol and 150 mcg of either desogestrel or levonorgestrel were used. Blood was tested before OC use and 3 and 6 months after starting OC use. Levels of AT-III and PS were measured using the Laurell rocket immunoelectrophoresis technique. Statistical analysis was performed using the paired Student's t-test and an analysis of variance test. No statistically significant differences were found for the mean levels of AT-III and total PS when comparing the pre-OC with the 3- and 6-month post-OC values. Earlier studies based mostly on Caucasian women have reported lower levels of both total PS and free PS in OC users. PMID:12288974

  15. Viral adaptation to an antiviral protein enhances the fitness level to above that of the uninhibited wild type.

    PubMed

    Cherwa, James E; Sanchez-Soria, Pablo; Wichman, Holly A; Fane, Bentley A

    2009-11-01

    Viruses often evolve resistance to antiviral agents. While resistant strains are able to replicate in the presence of the agent, they generally exhibit lower fitness than the wild-type strain in the absence of the inhibitor. In some cases, resistant strains become dependent on the antiviral agent. However, the agent rarely, if ever, elevates dependent strain fitness above the uninhibited wild-type level. This would require an adaptive mechanism to convert the antiviral agent into a beneficial growth factor. Using an inhibitory scaffolding protein that specifically blocks phiX174 capsid assembly, we demonstrate that such mechanisms are possible. To obtain the quintuple-mutant resistant strain, the wild-type virus was propagated for approximately 150 viral life cycles in the presence of increasing concentrations of the inhibitory protein. The expression of the inhibitory protein elevated the strain's fitness significantly above the uninhibited wild-type level. Thus, selecting for resistance coselected for dependency, which was characterized and found to operate on the level of capsid nucleation. To the best of our knowledge, this is the first report of a virus evolving a mechanism to productively utilize an antiviral agent to stimulate its fitness above the uninhibited wild-type level. The results of this study may be predictive of the types of resistant phenotypes that could be selected by antiviral agents that specifically target capsid assembly. PMID:19726521

  16. Atomic level description of the protecting effect of osmolytes against thermal denaturation of proteins

    NASA Astrophysics Data System (ADS)

    Pieraccini, Stefano; Burgi, Luigi; Genoni, Alessandro; Benedusi, Anna; Sironi, Maurizio

    2007-04-01

    The protecting effect of the osmolyte molecule taurine against thermal denaturation of the protein Chimotripsin Inhibitor 2 was modelled using Molecular Dynamics simulations. The protein was simulated in denaturing conditions at different taurine concentrations. Analysis of the molecular details of its behaviour shows that the protective effect of the osmolyte is concentration dependent. Moreover, the influence of taurine on the solvent structure was studied. A concentration dependent ordering effect of taurine on water molecules emerges from solvent structure analysis and is well correlated to the protecting effect observed. Based on these observations an interpretation of the osmoprotective effect is proposed.

  17. Systemic and lung protein changes in sarcoidosis. Lymphocyte counts, gallium uptake values, and serum angiotensin-converting enzyme levels may reflect different aspects of disease activity

    SciTech Connect

    Check, I.J.; Kidd, M.R.; Staton, G.W. Jr.

    1986-01-01

    BAL lymphocyte percentages, quantitated gallium-67 lung uptake, and SACE levels have all been proposed as measures of disease activity in sarcoidosis. We analyzed 32 paired sera and BAL fluids from sarcoidosis patients by high-resolution agarose electrophoresis to look for protein changes characteristic of systemic or local inflammation and compared the results with those from the above tests. Nine patients (group 1) had serum inflammatory protein changes and increased total protein, albumin, beta 1-globulin (transferrin), and gamma-globulin levels in fluid recovered by BAL. Thirteen patients (group 2) had normal protein levels in sera but abnormal protein levels in BAL specimens. Ten patients (group 3) had normal protein levels in sera and in BAL specimens. Patients in groups 1 and 2 had a disproportionate increase in beta 1-globulin (transferrin) and gamma-globulin levels in their BAL specimens. The BAL lymphocyte percentage changes paralleled the BAL protein level changes, suggesting relationships among the immunoregulatory role of these cells, increased local immunoglobulin synthesis, and the pathogenesis of altered alveolar permeability. Gallium-67 uptake was highest in patients with serum inflammatory protein changes. Thus, systemic inflammation may facilitate pulmonary gallium-67 uptake, possibly by changes in BAL fluid or serum transferrin saturation and/or kinetics. SACE levels showed no relationship to changes in the levels of serum or BAL proteins. These data suggest that the various proposed measures of disease activity reflect different aspects of inflammation in sarcoidosis.

  18. Toxicity of TDCPP and TCEP on PC12 cell: changes in CAMKII, GAP43, tubulin and NF-H gene and protein levels.

    PubMed

    Ta, Na; Li, Chaonan; Fang, Yanjun; Liu, Huanliang; Lin, Bencheng; Jin, Hong; Tian, Lei; Zhang, Huashan; Zhang, Wei; Xi, Zhuge

    2014-06-16

    TDCPP and TCEP are two major types of organophosphorus flame retardants (OPFRs) that are bioaccumulative and persistent in the environment. The toxicity effects of TDCPP and TCEP on PC12 cell are not well understood. In the present study, we investigated morphology, viability and apoptosis in cultured PC12 cells in response to TDCPP and TCEP. The mRNA and protein expression levels of CAMKII, GAP43, tubulin and NF-H were quantified in PC12 cells treated with varying concentrations of the two agents. Results indicate that, upon treatment with the two OPFRs, cell growth decreased, apoptosis increased, morphology was altered and significant changes were found in the gene and protein levels. Treatment with TDCPP caused a reduction in the levels of each of the six proteins studied and in the gene levels of GAP43, NF-H and the two tubulins, but it resulted in an increase in CAMKII gene levels. Treatment with TCEP resulted in similar changes in gene levels to TDCPP and led to decreases in the protein levels of GAP43 and the tubulins while increasing the CAMKII and NF-H protein levels. These results suggest that changes in the gene and protein levels of the regulatory proteins (CAMKII, GAP43) and the structural proteins (tubulin, NF-H) are due to different mechanisms of the toxins, and these proteins may be useful biomarkers for the cytotoxicity and neurotoxicity. PMID:24717766

  19. Interactions of DNA binding proteins with G-Quadruplex structures at the single molecule level

    NASA Astrophysics Data System (ADS)

    Ray, Sujay

    Guanine-rich nucleic acid (DNA/RNA) sequences can form non-canonical secondary structures, known as G-quadruplex (GQ). Numerous in vivo and in vitro studies have demonstrated formation of these structures in telomeric and non-telomeric regions of the genome. Telomeric GQs protect the chromosome ends whereas non-telomeric GQs either act as road blocks or recognition sites for DNA metabolic machinery. These observations suggest the significance of these structures in regulation of different metabolic processes, such as replication and repair. GQs are typically thermodynamically more stable than the corresponding Watson-Crick base pairing formed by G-rich and C-rich strands, making protein activity a crucial factor for their destabilization. Inside the cell, GQs interact with different proteins and their enzymatic activity is the determining factor for their stability. We studied interactions of several proteins with GQs to understand the underlying principles of protein-GQ interactions using single-molecule FRET and other biophysical techniques. Replication Protein-A (RPA), a single stranded DNA (ssDNA) binding protein, is known to posses GQ unfolding activity. First, we compared the thermal stability of three potentially GQ-forming DNA sequences (PQS) to their stability against RPA-mediated unfolding. One of these sequences is the human telomeric repeat and the other two, located in the promoter region of tyrosine hydroxylase gene, are highly heterogeneous sequences that better represent PQS in the genome. The thermal stability of these structures do not necessarily correlate with their stability against protein-mediated unfolding. We conclude that thermal stability is not necessarily an adequate criterion for predicting the physiological viability of GQ structures. To determine the critical structural factors that influence protein-GQ interactions we studied two groups of GQ structures that have systematically varying loop lengths and number of G-tetrad layers. We

  20. Identification of endogenously S-nitrosylated proteins in Arabidopsis plantlets: effect of cold stress on cysteine nitrosylation level.

    PubMed

    Puyaubert, Juliette; Fares, Abasse; Rézé, Nathalie; Peltier, Jean-Benoît; Baudouin, Emmanuel

    2014-02-01

    S-nitrosylation is a nitric oxide (NO)-based post-translational modification regulating protein function and signalling. We used a combination between the biotin switch method and labelling with isotope-coded affinity tag to identify endogenously S-nitrosylated peptides in Arabidopsis thaliana proteins extracted from plantlets. The relative level of S-nitrosylation in the identified peptides was compared between unstressed and cold-stress seedlings. We thereby detected 62 endogenously nitrosylated peptides out of which 20 are over-nitrosylated following cold exposure. Taken together these data provide a new repertoire of endogenously S-nitrosylated proteins in Arabidopsis with cysteine S-nitrosylation site. Furthermore they highlight the quantitative modification of the S-nitrosylation status of specific cysteine following cold stress. PMID:24388526

  1. From transcriptomic to protein level changes in TDP-43 and FUS loss-of-function cell models.

    PubMed

    Colombrita, Claudia; Onesto, Elisa; Buratti, Emanuele; de la Grange, Pierre; Gumina, Valentina; Baralle, Francisco E; Silani, Vincenzo; Ratti, Antonia

    2015-12-01

    The full definition of the physiological RNA targets regulated by TDP-43 and FUS RNA-binding proteins (RBPs) represents an important issue in understanding the pathogenic mechanisms associated to these two proteins in amyotrophic lateral sclerosis and frontotemporal dementia. In the last few years several high-throughput screenings have generated a plethora of data, which are difficult to compare due to the different experimental designs and models explored. In this study by using the Affymetrix Exon Arrays, we were able to assess and compare the effects of both TDP-43 and FUS loss-of-function on the whole transcriptome using the same human neuronal SK-N-BE cell model. We showed that TDP-43 and FUS depletion induces splicing and gene expression changes mainly distinct for the two RBPs, although they may regulate common pathways, including neuron differentiation and cytoskeleton organization as evidenced by functional annotation analysis. In particular, TDP-43 and FUS were found to regulate splicing and expression of genes related to neuronal (SEPT6, SULT4A1, TNIK) and RNA metabolism (DICER, ELAVL3/HuC, POLDIP3). Our extended analysis at protein level revealed that these changes have also impact on the protein isoform ratio and content, not always in a direct correlation with transcriptomic data. Contrarily to a loss-of-function mechanism, we showed that mutant TDP-43 proteins maintained their splicing activity in human ALS fibroblasts and experimental cell lines. Our findings further contribute to define the biological functions of these two RBPs in physiological and disease state, strongly encouraging the evaluation of the identified transcriptomic changes at protein level in neuronal experimental models. PMID:26514432

  2. Methylation potential associated with diet, genotype, protein, and metabolite levels in the Delta Obesity Vitamin Study

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Micronutrient research typically focuses on analyzing the effects of single or a few nutrients on health by analyzing a limited number of biomarkers. The observational study described here analyzed micronutrients, plasma proteins, dietary intakes, and genotype using a systems approach. Participants ...

  3. Protein expression levels in the medullary visceral zone of rats with subarachnoid hemorrhage.

    PubMed

    Sun, L H; Xing, L F; Zhang, G H; Pan, S Y

    2015-01-01

    We investigated protein expression in the medullary visceral zone (MVZ) of rats with multiple-organ dysfunction syndrome (MODS) caused by subarachnoid hemorrhage (SAH) to discuss the possible regulatory mechanism of the MVZ in the course of SAH-induced MODS. A SAH-induced MODS model was established in rats by injecting arterial blood into the Willis' circle. Protein expression in the MVZ was analyzed by immunohistochemistry assay. Protein expression in the MVZ peaked 24-36 h after SAH, and was significantly higher than in the control and sham operation groups. Organs at each time point exhibited inflammatory injuries to varying degrees after SAH, which reached a maximum at 24-36 h. Incidences of systemic inflammatory response syndrome and MODS were 100 and 71.67%, respectively, after SAH. There is a consistency between MVZ protein expression and inflammatory changes in each organ after SAH. This prompts the suggestion that the MVZ may be one of the direct regulative centers in SAH-induced MODS, and may be involved in the functional regulation of the surrounding organs after SAH. PMID:26345815

  4. Xylella fastidiosa infection of grapevines affects xylem levels of phenolic compounds and pathogenesis-related proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pierce’s disease (PD), caused by the xylem-dwelling pathogen Xylella fastidiosa (X.f.), is a serious threat to grape production. The effects of X.f. infection six months post-inoculation on defense-associated proteins and phenolic compounds found in xylem sap and tissue were evaluated. Defense-assoc...

  5. Phytoestrogens regulate mRNA and protein levels of guanine nucleotide-binding protein, beta-1 subunit (GNB1) in MCF-7 cells.

    PubMed

    Naragoni, Srivatcha; Sankella, Shireesha; Harris, Kinesha; Gray, Wesley G

    2009-06-01

    Phytoestrogens (PEs) are non-steroidal ligands, which regulate the expression of number of estrogen receptor-dependent genes responsible for a variety of biological processes. Deciphering the molecular mechanism of action of these compounds is of great importance because it would increase our understanding of the role(s) these bioactive chemicals play in prevention and treatment of estrogen-based diseases. In this study, we applied suppression subtractive hybridization (SSH) to identify genes that are regulated by PEs through either the classic nuclear-based estrogen receptor or membrane-based estrogen receptor pathways. SSH, using mRNA from genistein (GE) treated MCF-7 cells as testers, resulted in a significant increase in GNB1 mRNA expression levels as compared with 10 nM 17beta estradiol or the no treatment control. GNB1 mRNA expression was up regulated two- to fivefold following exposure to 100.0 nM GE. Similarly, GNB1 protein expression was up regulated 12- to 14-fold. GE regulation of GNB1 was estrogen receptor-dependent, in the presence of the anti-estrogen ICI-182,780, both GNB1 mRNA and protein expression were inhibited. Analysis of the GNB1 promoter using ChIP assay showed a PE-dependent association of estrogen receptor alpha (ERalpha) and beta (ERbeta) to the GNB1 promoter. This association was specific for ERalpha since association was not observed when the cells were co-incubated with GE and the ERalpha antagonist, ICI. Our data demonstrate that the levels of G-protein, beta-1 subunit are regulated by PEs through an estrogen receptor pathway and further suggest that PEs may control the ratio of alpha-subunit to beta/gamma-subunits of the G-protein complex in cells. J. Cell. Physiol. 219: 584-594, 2009. (c) 2009 Wiley-Liss, Inc. PMID:19170076

  6. Role of Human Sec63 in Modulating the Steady-State Levels of Multi-Spanning Membrane Proteins

    PubMed Central

    Awe, Karin; Stieler, Jens; Döring, Tatjana; Füser, Sabine; Prange, Reinhild

    2012-01-01

    The Sec61 translocon of the endoplasmic reticulum (ER) membrane forms an aqueous pore, allowing polypeptides to be transferred across or integrated into membranes. Protein translocation into the ER can occur co- and posttranslationally. In yeast, posttranslational translocation involves the heptameric translocase complex including its Sec62p and Sec63p subunits. The mammalian ER membrane contains orthologs of yeast Sec62p and Sec63p, but their function is poorly understood. Here, we analyzed the effects of excess and deficit Sec63 on various ER cargoes using human cell culture systems. The overexpression of Sec63 reduces the steady-state levels of viral and cellular multi-spanning membrane proteins in a cotranslational mode, while soluble and single-spanning ER reporters are not affected. Consistent with this, the knock-down of Sec63 increases the steady-state pools of polytopic ER proteins, suggesting a substrate-specific and regulatory function of Sec63 in ER import. Overexpressed Sec63 exerts its down-regulating activity on polytopic protein levels independent of its Sec62-interacting motif, indicating that it may not act in conjunction with Sec62 in human cells. The specific action of Sec63 is further sustained by our observations that the up-regulation of either Sec62 or two other ER proteins with lumenal J domains, like ERdj1 and ERdj4, does not compromise the steady-state level of a multi-spanning membrane reporter. A J domain-specific mutation of Sec63, proposed to weaken its interaction with the ER resident BiP chaperone, reduces the down-regulating capacity of excess Sec63, suggesting an involvement of BiP in this process. Together, these results suggest that Sec63 may perform a substrate-selective quantity control function during cotranslational ER import. PMID:23166619

  7. Reduction of Cellular Expression Levels Is a Common Feature of Functionally Affected Pendrin (SLC26A4) Protein Variants

    PubMed Central

    de Moraes, Vanessa C S; Bernardinelli, Emanuele; Zocal, Nathalia; Fernandez, Jhonathan A; Nofziger, Charity; Castilho, Arthur M; Sartorato, Edi L; Paulmichl, Markus; Dossena, Silvia

    2016-01-01

    Sequence alterations in the pendrin gene (SLC26A4) leading to functionally affected protein variants are frequently involved in the pathogenesis of syndromic and nonsyndromic deafness. Considering the high number of SLC26A4 sequence alterations reported to date, discriminating between functionally affected and unaffected pendrin protein variants is essential in contributing to determine the genetic cause of deafness in a given patient. In addition, identifying molecular features common to the functionally affected protein variants can be extremely useful to design future molecule-directed therapeutic approaches. Here we show the functional and molecular characterization of six previously uncharacterized pendrin protein variants found in a cohort of 58 Brazilian deaf patients. Two variants (p.T193I and p.L445W) were undetectable in the plasma membrane, completely retained in the endoplasmic reticulum and showed no transport function; four (p.P142L, p.G149R, p.C282Y and p.Q413R) showed reduced function and significant, although heterogeneous, expression levels in the plasma membrane. Importantly, total expression levels of all of the functionally affected protein variants were significantly reduced with respect to the wild-type and a fully functional variant (p.R776C), regardless of their subcellular localization. Interestingly, reduction of expression may also reduce the transport activity of variants with an intrinsic gain of function (p.Q413R). As reduction of overall cellular abundance was identified as a common molecular feature of pendrin variants with affected function, the identification of strategies to prevent reduction in expression levels may represent a crucial step of potential future therapeutic interventions aimed at restoring the transport activity of dysfunctional pendrin variants. PMID:26752218

  8. The regulation of GRP78 and messenger RNA levels by hypoxia is modulated by protein kinase C activators and inhibitors

    SciTech Connect

    Koong, A.C.; Auger, E.A.; Chen, E.Y.; Giaccia, A.J.

    1994-04-01

    In this study, we have shown that steady-state levels of glucose-regulated 78 kDa (GRP78) protein and messenger RNA increase during a 5-h exposure to 0.02% oxygen. This increase in GRP78 protein and mRNA induced by hypoxia can be abolished by a 1-h pretreatment of cells before hypoxia with the protein kinase C (PKC) inhibitors staurosporine and H7 at concentrations at which the drugs themselves do not cause cytotoxicity. Although all studies using protein kinase inhibitors must be interpreted with caution, staurosporine and H7 have been shown to be potent inhibitors of PKC activity, suggesting a role for PKC in mediating the transcriptional regulation of GRP78 by hypoxia. Further support for PKC in regulating GRP78 gene expression by hypoxia stems from gel-mobility shift studies in mixtures of nuclear extracts from aerobic or hypoxic cells with a 36 bp region of the GRP78 promoter (-170 to -135). Binding of this factor could be inhibited by pretreating cells with the PKC inhibitor staurosporine before hypoxia or activated by treating cells with the PKC-activating phorbol ester TPA. These data suggest that activation of this hypoxia-responsive factor is sensitive to oxygen levels and seems to be mediated through a PKC signal transduction pathway. 13 refs., 4 figs.

  9. Enhancement of SMN protein levels in a mouse model of spinal muscular atrophy using novel drug-like compounds

    PubMed Central

    Cherry, Jonathan J; Osman, Erkan Y; Evans, Matthew C; Choi, Sungwoon; Xing, Xuechao; Cuny, Gregory D; Glicksman, Marcie A; Lorson, Christian L; Androphy, Elliot J

    2013-01-01

    Spinal muscular atrophy (SMA) is a neurodegenerative disease that causes progressive muscle weakness, which primarily targets proximal muscles. About 95% of SMA cases are caused by the loss of both copies of the SMN1 gene. SMN2 is a nearly identical copy of SMN1, which expresses much less functional SMN protein. SMN2 is unable to fully compensate for the loss of SMN1 in motor neurons but does provide an excellent target for therapeutic intervention. Increased expression of functional full-length SMN protein from the endogenous SMN2 gene should lessen disease severity. We have developed and implemented a new high-throughput screening assay to identify small molecules that increase the expression of full-length SMN from a SMN2 reporter gene. Here, we characterize two novel compounds that increased SMN protein levels in both reporter cells and SMA fibroblasts and show that one increases lifespan, motor function, and SMN protein levels in a severe mouse model of SMA. PMID:23740718

  10. Codon-level information improves predictions of inter-residue contacts in proteins by correlated mutation analysis

    PubMed Central

    Jacob, Etai; Unger, Ron; Horovitz, Amnon

    2015-01-01

    Methods for analysing correlated mutations in proteins are becoming an increasingly powerful tool for predicting contacts within and between proteins. Nevertheless, limitations remain due to the requirement for large multiple sequence alignments (MSA) and the fact that, in general, only the relatively small number of top-ranking predictions are reliable. To date, methods for analysing correlated mutations have relied exclusively on amino acid MSAs as inputs. Here, we describe a new approach for analysing correlated mutations that is based on combined analysis of amino acid and codon MSAs. We show that a direct contact is more likely to be present when the correlation between the positions is strong at the amino acid level but weak at the codon level. The performance of different methods for analysing correlated mutations in predicting contacts is shown to be enhanced significantly when amino acid and codon data are combined. DOI: http://dx.doi.org/10.7554/eLife.08932.001 PMID:26371555

  11. Effects of aerobic exercise intervention on serum cartilage oligomeric matrix protein levels and lymphocyte dna damage in obese elderly females

    PubMed Central

    Cho, Su Youn; Roh, Hee Tae

    2016-01-01

    [Purpose] The aim of the reported research was to investigate the effects of regular aerobic exercise on cartilage oligomeric matrix protein and oxidative DNA damage in obese, elderly females. [Subjects and Methods] Sixteen class I obese, elderly females, according to World Health Organization criteria, were randomly and equally assigned to a control group (n=8) or an exercise group (n=8). The exercise group participated in exercise sessions of 60 minutes per day, 3 days per week, for a period of 8 weeks. [Results] After aerobic exercise intervention, weight, body mass index, body fat, waist circumference, and DNA damage (Tail moment) were significantly decreased, compared with baseline values. In contrast, serum cartilage oligomeric matrix protein levels were not significantly different among any groups or time-points. [Conclusion] Regular aerobic exercise may be effective for reducing obesity-induced high DNA damage levels in obese females, without causing the deformation or degradation of lower extremity articular cartilage. PMID:27390441

  12. Lipopolysaccharide-binding protein plasma levels as a biomarker of obesity-related insulin resistance in adolescents

    PubMed Central

    Kim, Ki Eun; Cho, Young Sun; Baek, Kyung Suk; Li, Lan; Baek, Kwang-Hyun; Kim, Jung Hyun; Kim, Ho-Seong

    2016-01-01

    Purpose Lipopolysaccharide-binding protein (LBP) is a 65-kDa acute phase protein, derived from the liver, which is present in high concentrations in plasma. Data regarding the association between circulating plasma LBP levels and obesity-related biomarkers in the pediatric population are scarce. We aimed to determine whether there was a difference in plasma LBP levels between overweight/obese and normal-weight adolescents and to assess the correlation of circulating LBP levels with anthropometric measures and obesity-related biomarkers, including insulin resistance, liver enzyme levels, and lipid profiles. Methods The study included 87 adolescents aged 12–13 years; 44 were overweight/obese and 43 were of normal-weight. We assessed anthropometric and laboratory measures, including body mass index (BMI), blood pressure, insulin resistance, liver enzyme levels, and lipid profiles. Plasma LBP levels were measured using an enzyme-linked immunosorbent assay. Results The mean age of the participants was 12.9±0.3 years. Circulating plasma LBP levels were significantly increased in overweight/obese participants compared with those in normal-weight participants (7.8±1.9 µg/mL vs. 6.0±1.6 µg/mL, P<0.001). LBP levels were significantly and positively associated with BMI, systolic blood pressure, aspartate aminotransferase, alanine aminotransferase, total cholesterol, low density lipoprotein-cholesterol, fasting glucose and insulin, and insulin resistance as indicated by the homeostatic model assessment of insulin resistance (HOMA-IR) (all P<0.05). In multivariate linear regression analysis, BMI and HOMA-IR were independently and positively associated with plasma LBP levels. Conclusion LBP is an inflammatory biomarker associated with BMI and obesity-related insulin resistance in adolescents. The positive correlation between these parameters suggests a potentially relevant pathophysiological mechanism linking LBP to obesity-related insulin resistance in adolescents. PMID

  13. 3Drefine: Consistent Protein Structure Refinement by Optimizing Hydrogen Bonding Network and Atomic-Level Energy Minimization

    PubMed Central

    Bhattacharya, Debswapna; Cheng, Jianlin

    2013-01-01

    One of the major limitations of computational protein structure prediction is the deviation of predicted models from their experimentally derived true, native structures. The limitations often hinder the possibility of applying computational protein structure prediction methods in biochemical assignment and drug design that are very sensitive to structural details. Refinement of these low-resolution predicted models to high-resolution structures close to the native state, however, has proven to be extremely challenging. Thus, protein structure refinement remains a largely unsolved problem. Critical assessment of techniques for protein structure prediction (CASP) specifically indicated that most predictors participating in the refinement category still did not consistently improve model quality. Here, we propose a two-step refinement protocol, called 3Drefine, to consistently bring the initial model closer to the native structure. The first step is based on optimization of hydrogen bonding (HB) network and the second step applies atomic-level energy minimization on the optimized model using a composite physics and knowledge-based force fields. The approach has been evaluated on the CASP benchmark data and it exhibits consistent improvement over the initial structure in both global and local structural quality measures. 3Drefine method is also computationally inexpensive, consuming only few minutes of CPU time to refine a protein of typical length (300 residues). PMID:22927229

  14. Regulation of intracellular beta-catenin levels by the adenomatous polyposis coli (APC) tumor-suppressor protein.

    PubMed Central

    Munemitsu, S; Albert, I; Souza, B; Rubinfeld, B; Polakis, P

    1995-01-01

    The APC tumor-suppressor protein associates with beta-catenin, a cell adhesion protein that is upregulated by the WNT1 oncogene. We examined the effects of exogenous APC expression on the distribution and amount of beta-catenin in a colorectal cancer cell containing only mutant APC. Expression of wild-type APC caused a pronounced reduction in total beta-catenin levels by eliminating an excessive supply of cytoplasmic beta-catenin indigenous to the SW480 colorectal cancer cell line. This reduction was due to an enhanced rate of beta-catenin protein degradation. Truncated mutant APC proteins, characteristic of those associated with cancer, lacked this activity. Mutational analysis revealed that the central region of the APC protein, which is typically deleted or severely truncated in tumors, was responsible for the down-regulation of beta-catenin. These results suggest that the tumor-suppressor activity of mutant APC may be compromised due to a defect in its ability to regulate beta-catenin. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7708772

  15. A significant causal association between C-reactive protein levels and schizophrenia

    PubMed Central

    Inoshita, Masatoshi; Numata, Shusuke; Tajima, Atsushi; Kinoshita, Makoto; Umehara, Hidehiro; Nakataki, Masahito; Ikeda, Masashi; Maruyama, Souichiro; Yamamori, Hidenaga; Kanazawa, Tetsufumi; Shimodera, Shinji; Hashimoto, Ryota; Imoto, Issei; Yoneda, Hiroshi; Iwata, Nakao; Ohmori, Tetsuro

    2016-01-01

    Many observational studies have shown elevated blood CRP levels in schizophrenia compared with controls, and one population-based prospective study has reported that elevated plasma CRP levels were associated with late- and very-late-onset schizophrenia. Furthermore, several clinical studies have reported the efficacy of anti-inflammatory drugs on the symptoms in patients with schizophrenia. However, whether elevated CRP levels are causally related to schizophrenia is not still established because of confounding factors and reverse causality. In the present study, we demonstrated that serum CRP levels were significantly higher in patients with schizophrenia than in the controls by conducting a case-control study and a meta-analysis of case-control studies between schizophrenia and serum CRP levels. Furthermore, we provided evidence for a causal association between elevated CRP levels and increased schizophrenia risk by conducting a Mendelian randomization analysis. Our findings suggest that elevated CRP itself may be a causal risk factor for schizophrenia. PMID:27193331

  16. A significant causal association between C-reactive protein levels and schizophrenia.

    PubMed

    Inoshita, Masatoshi; Numata, Shusuke; Tajima, Atsushi; Kinoshita, Makoto; Umehara, Hidehiro; Nakataki, Masahito; Ikeda, Masashi; Maruyama, Souichiro; Yamamori, Hidenaga; Kanazawa, Tetsufumi; Shimodera, Shinji; Hashimoto, Ryota; Imoto, Issei; Yoneda, Hiroshi; Iwata, Nakao; Ohmori, Tetsuro

    2016-01-01

    Many observational studies have shown elevated blood CRP levels in schizophrenia compared with controls, and one population-based prospective study has reported that elevated plasma CRP levels were associated with late- and very-late-onset schizophrenia. Furthermore, several clinical studies have reported the efficacy of anti-inflammatory drugs on the symptoms in patients with schizophrenia. However, whether elevated CRP levels are causally related to schizophrenia is not still established because of confounding factors and reverse causality. In the present study, we demonstrated that serum CRP levels were significantly higher in patients with schizophrenia than in the controls by conducting a case-control study and a meta-analysis of case-control studies between schizophrenia and serum CRP levels. Furthermore, we provided evidence for a causal association between elevated CRP levels and increased schizophrenia risk by conducting a Mendelian randomization analysis. Our findings suggest that elevated CRP itself may be a causal risk factor for schizophrenia. PMID:27193331

  17. Serum levels of nitric oxide and protein oxidation in goats seropositive for Toxoplasma gondii and Neospora caninum.

    PubMed

    Tonin, A A; Weber, A; Ribeiro, A; Camillo, G; Vogel, F F; Moura, A B; Bochi, G V; Moresco, R N; Da Silva, A S

    2015-08-01

    The aim of this study was to assess and analyze the levels of nitric oxide (NO) and advanced oxidation protein products (AOPP) in serum of goats naturally infected by Toxoplasma gondii, Neospora caninum, or concomitantly infected by these two parasites. Thus, it was measured NOx and AOPP levels in twenty (n=20) sera samples of goats seronegative for T. gondii and N. caninum [negative control group (A)]; while the positive groups were composed by sera of infected animals, twelve (n=12) seropositive for N. caninum [group B]; eighteen (n=18) positive for T. gondii [group C]; and thirteen (n=13) seropositive for N. caninum and T. gondii [group D]. As results, it was observed that animals seropositive for N. caninum and T. gondii (Groups B to D) showed higher serum levels of NOx (P<0.001; F=9.5), when compared with seronegative animals. Additionally, it was observed a positive correlation between NOx levels and antibodies titrations for N. caninum (P<0.01; r=0.68) and T. gondii (P<0.05; r=0.56). AOPP levels were increase in groups C and D (P>0.05). Interestingly, group B did not show increase in AOPP, what led us to hypothesize that the major protein damage is linked to T. gondii infection. Therefore, our results showed an increased in NOx levels, which was probably related to the immune response, since it is an important inflammatory mediator; and AOPP were increased in groups where there was seropositivity for T. gondii, but not for the group composed only by animals seropositive for N. caninum, allowing us to suggest higher protein damage in toxoplasmosis. PMID:26031474

  18. Increased astrocyte expression of IL-6 or CCL2 in transgenic mice alters levels of hippocampal and cerebellar proteins

    PubMed Central

    Gruol, Donna L.; Vo, Khanh; Bray, Jennifer G.

    2014-01-01

    Emerging research has identified that neuroimmune factors are produced by cells of the central nervous system (CNS) and play critical roles as regulators of CNS function, directors of neurodevelopment and responders to pathological processes. A wide range of neuroimmune factors are produced by CNS cells, primarily the glial cells, but the role of specific neuroimmune factors and their glial cell sources in CNS biology and pathology have yet to be fully elucidated. We have used transgenic mice that express elevated levels of a specific neuroimmune factor, the cytokine IL-6 or the chemokine CCL2, through genetic modification of astrocyte expression to identify targets of astrocyte produced IL-6 or CCL2 at the protein level. We found that in non-transgenic mice constitutive expression of IL-6 and CCL2 occurs in the two CNS regions studied, the hippocampus and cerebellum, as measured by ELISA. In the CCL2 transgenic mice elevated levels of CCL2 were evident in the hippocampus and cerebellum, whereas in the IL-6 transgenic mice, elevated levels of IL-6 were only evident in the cerebellum. Western blot analysis of the cellular and synaptic proteins in the hippocampus and cerebellum of the transgenic mice showed that the elevated levels of CCL2 or IL-6 resulted in alterations in the levels of specific proteins and that these actions differed for the two neuroimmune factors and for the two brain regions. These results are consistent with cell specific profiles of action for IL-6 and CCL2, actions that may be an important aspect of their respective roles in CNS physiology and pathophysiology. PMID:25177271

  19. Association of levels of antibodies from patients with inflammatory bowel disease with extracellular proteins of food and probiotic bacteria.

    PubMed

    Hevia, Arancha; López, Patricia; Suárez, Ana; Jacquot, Claudine; Urdaci, María C; Margolles, Abelardo; Sánchez, Borja

    2014-01-01

    Inflammatory bowel disease (IBD) is an autoimmune disease characterized by a chronic inflammation of the gastrointestinal tract mucosa and is related to an abnormal immune response to commensal bacteria. Our aim of the present work has been to explore the levels of antibodies (IgG and IgA) raised against extracellular proteins produced by LAB and its association with IBD. We analyzed, by Western-blot and ELISA, the presence of serum antibodies (IgA and IgG) developed against extracellular protein fractions produced by different food bacteria from the genera Bifidobacterium and Lactobacillus. We used a sera collection consisting of healthy individuals (HC, n = 50), Crohn's disease patients (CD, n = 37), and ulcerative colitis patients (UC, n = 15). Levels of IgA antibodies developed against a cell-wall hydrolase from Lactobacillus casei subsp. rhamnosus GG (CWH) were significantly higher in the IBD group (P < 0.002; n = 52). The specificity of our measurements was confirmed by measuring IgA antibodies developed against the CWH peptide 365-VNTSNQTAAVSAS-377. IBD patients appeared to have different immune response to food bacteria. This paper sets the basis for developing systems for early detection of IBD, based on the association of high levels of antibodies developed against extracellular proteins from food and probiotic bacteria. PMID:24991549

  20. Alterations in skeletal protein, distribution of PKCalpha, and level of phospholipids in erythrocyte membranes of women with primary breast cancer.

    PubMed

    Kaczmarek, Jolanta; Thieleman, Anna; Kopczyński, Zygmunt; Goslar, Janina; Hoffmann, Stanisław Kazimierz; Rybczyńska, Maria

    2002-01-01

    The aim of our work was to study the influence of primary breast cancer on mature erythrocyte membranes. Blood was sampled from 29 women with primary breast cancer, aged 35-86 years, in different stages of clinical progression of the disease. In red blood cell membranes an increase of phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-diphosphate levels was observed. These changes were accompanied by a decrease in phospholipase C activity. Simultaneously, a significant decrease in concentration of phosphatidylserine, sphingomyelin, and phosphatidylinositol was found. Quantitative protein evaluation showed an increase in band 4.1 protein content with no changes in the level of constitutive PKCalpha responsible for the phosphorylation of this protein and its affinity to glycophorine C. In parallel a greater increase of PKCalpha translocation after PMA treatment compared to controls was observed. Possible oxidative damage of erythrocyte membranes indicated by an increase in malonyldialdehyde level and decrease in SH-group content as well as by an increase in the w/ ratio was documented. From the results it is concluded that primary breast cancer seems to affect the membranes of mature erythrocytes. PMID:12490289

  1. Fusion protein-based biofilm fabrication composed of recombinant azurin-myoglobin for dual-level biomemory application

    NASA Astrophysics Data System (ADS)

    Lee, Taek; Chung, Yong-Ho; Yoon, Jinho; Min, Junhong; Choi, Jeong-Woo

    2014-11-01

    In the present study, a fusion protein-based biofilm composed of a recombinant azurin-myoglobin (Azu-Myo) has been developed and confirmed its original electrochemical property for dual-level biomemory device application. For this purpose, the azurin was modified with cysteine residues for direct immobilization and conjugation. Then, the recombinant azurin was conjugated with the myoglobin via a sulfo-SMCC bifunctional linker using the chemical ligation method (CLM). The SDS-PAGE and UV-vis spectroscopy were performed to examine the fusion protein conjugates. The prepared Azu-Myo fusion protein was self-assembled onto Au substrate for the biofilm fabrication. Then, the atomic force microscopy (AFM) was used to confirm the immobilization and the surface-enhanced Raman spectroscopy (SERS) was carried out to the surface analysis. Also, the cyclic voltammetry (CV) was carried out to observe an electrochemical property of fabricated biofilm. As a result, the two pair of redox potential values was obtained for dual-level biomemory device application. Then, the dual-level biomemory function was verified by the multi-potential chronoamperometry (MPCA). The results indicate a new fabrication method and material combination for advances in bioelectronic device development.

  2. Association of Levels of Antibodies from Patients with Inflammatory Bowel Disease with Extracellular Proteins of Food and Probiotic Bacteria

    PubMed Central

    Hevia, Arancha; López, Patricia; Suárez, Ana; Jacquot, Claudine; Urdaci, María C.; Margolles, Abelardo; Sánchez, Borja

    2014-01-01

    Inflammatory bowel disease (IBD) is an autoimmune disease characterized by a chronic inflammation of the gastrointestinal tract mucosa and is related to an abnormal immune response to commensal bacteria. Our aim of the present work has been to explore the levels of antibodies (IgG and IgA) raised against extracellular proteins produced by LAB and its association with IBD. We analyzed, by Western-blot and ELISA, the presence of serum antibodies (IgA and IgG) developed against extracellular protein fractions produced by different food bacteria from the genera Bifidobacterium and Lactobacillus. We used a sera collection consisting of healthy individuals (HC, n = 50), Crohn's disease patients (CD, n = 37), and ulcerative colitis patients (UC, n = 15). Levels of IgA antibodies developed against a cell-wall hydrolase from Lactobacillus casei subsp. rhamnosus GG (CWH) were significantly higher in the IBD group (P < 0.002; n = 52). The specificity of our measurements was confirmed by measuring IgA antibodies developed against the CWH peptide 365-VNTSNQTAAVSAS-377. IBD patients appeared to have different immune response to food bacteria. This paper sets the basis for developing systems for early detection of IBD, based on the association of high levels of antibodies developed against extracellular proteins from food and probiotic bacteria. PMID:24991549

  3. CREB-binding protein (CBP) levels in the rat hippocampus fail to predict chronological or cognitive aging

    PubMed Central

    Pereira, Inês Tomás; Coletta, Christopher E.; Perez, Evelyn V.; Kim, David H.; Gallagher, Michela; Goldberg, Ilya G.; Rapp, Peter R.

    2012-01-01

    Normal cognitive aging is associated with deficits in memory processes dependent on the hippocampus, along with large-scale changes in the hippocampal expression of many genes. Histone acetylation can broadly influence gene expression and has been recently linked to learning and memory. We hypothesized that cAMP response element binding (CREB)-binding protein (CBP), a key histone acetyltransferase, may contribute to memory decline in normal aging. Here, we quantified CBP protein levels in the hippocampus of young, aged unimpaired and aged impaired rats, classified on the basis of spatial memory capacity documented in the Morris water maze. First, CBP-immunofluorescence was quantified across the principal cell layers of the hippocampus using both low and high resolution laser scanning imaging approaches. Second, digital images of CBP immunostaining were analyzed by a multi-purpose classifier algorithm (WND-CHARM) with validated sensitivity across many types of input materials. Finally, CBP protein levels in the principal subfields of the hippocampus were quantified by quantitative western blotting. CBP levels were equivalent as a function of age and cognitive status in all analyses. The sensitivity of the techniques used was substantial, sufficient to reveal differences across the principal cell fields of the hippocampus, and to correctly classify images from young and aged animals independent of CBP-immunoreactivity. The results are discussed in the context of recent evidence suggesting that CBP decreases may be most relevant in conditions of aging that, unlike normal cognitive aging, involve significant neuron loss. PMID:22884549

  4. Expression of the hereditary hemochromatosis protein HFE increases ferritin levels by inhibiting iron export in HT29 cells.

    PubMed

    Davies, Paige S; Enns, Caroline A

    2004-06-11

    Iron is essential for life in almost all organisms and, in mammals, is absorbed through the villus cells of the duodenum. Using a human colonic carcinoma cell line that has many duodenal characteristics, HT29, we show that genes involved in intestinal iron transport are endogenously expressed. When stably transfected to express the hereditary hemochromatosis protein HFE these cells have increased ferritin levels. We demonstrate that this is not due to an effect on the transferrin (TF)-mediated iron uptake pathway but rather due to inhibition of iron efflux from the cell. The effect of HFE was independent of its interaction with TF receptor 1 as indicated by similar results using both the wild type HFE and the W81A mutant that binds TF receptor 1 with greatly reduced affinity. HFE expression did not affect the mRNA levels of most of the genes involved in iron absorption that were tested; however, it did correspond to a decrease in hephaestin message levels. These results point to a role for HFE in inhibition of iron efflux in HT29 cells. This is a distinct role from that in HeLa and human embryonic kidney 293 cells where HFE has been shown to inhibit TF-mediated iron uptake resulting in decreased ferritin levels. Such a distinction suggests a multifunctional role for HFE that is dependent upon expression levels of proteins involved in iron transport. PMID:15044462

  5. TMEM106B p.T185S regulates TMEM106B protein levels: implications for frontotemporal dementia.

    PubMed

    Nicholson, Alexandra M; Finch, Nicole A; Wojtas, Aleksandra; Baker, Matt C; Perkerson, Ralph B; Castanedes-Casey, Monica; Rousseau, Linda; Benussi, Luisa; Binetti, Giuliano; Ghidoni, Roberta; Hsiung, Ging-Yuek R; Mackenzie, Ian R; Finger, Elizabeth; Boeve, Bradley F; Ertekin-Taner, Nilüfer; Graff-Radford, Neill R; Dickson, Dennis W; Rademakers, Rosa

    2013-09-01

    Frontotemporal lobar degeneration (FTLD) is the second leading cause of dementia in individuals under age 65. In many patients, the predominant pathology includes neuronal cytoplasmic or intranuclear inclusions of ubiquitinated TAR DNA binding protein 43 (FTLD-TDP). Recently, a genome-wide association study identified the first FTLD-TDP genetic risk factor, in which variants in and around the TMEM106B gene (top SNP rs1990622) were significantly associated with FTLD-TDP risk. Intriguingly, the most significant association was in FTLD-TDP patients carrying progranulin (GRN) mutations. Here, we investigated to what extent the coding variant, rs3173615 (p.T185S) in linkage disequilibrium with rs1990622, affects progranulin protein (PGRN) biology and transmembrane protein 106 B (TMEM106B) regulation. First, we confirmed the association of TMEM106B variants with FTLD-TDP in a new cohort of GRN mutation carriers. We next generated and characterized a TMEM106B-specific antibody for investigation of this protein. Enzyme-linked immunoassay analysis of progranulin protein levels showed similar effects upon T185 and S185 TMEM106B over-expression. However, over-expression of T185 consistently led to higher TMEM106B protein levels than S185. Cycloheximide treatment experiments revealed that S185 degrades faster than T185 TMEM106B, potentially due to differences in N-glycosylation at residue N183. Together, our results provide a potential mechanism by which TMEM106B variants lead to differences in FTLD-TDP risk. We studied the p.T185S TMEM106B genetic variant previously implicated in frontotemporal dementia with TAR DNA binding protein 43 pathology caused by progranulin mutations. Our cell culture studies provide evidence that the protective S185 isoform is degraded more rapidly than T185 TMEM106B, potentially due to differences in glycosylation. These findings suggest that low TMEM106B levels might protect against FTLD-TDP in these patients. PMID:23742080

  6. High-level expression and characterization of a glycosylated human cementum protein 1 with lectin activity.

    PubMed

    Romo-Arévalo, Enrique; Arzate, Higinio; Montoya-Ayala, Gonzalo; Rodríguez-Romero, Adela

    2016-01-01

    This work aims to contribute to the knowledge of human cementum protein 1 (CEMP1), its conformational characteristics and influence during the biomineralization process. The results revealed that hrCEMP1 expressed in Pichia pastoris is a 2.4% glycosylated, thermostable protein which possesses a molecular mass of 28 770 Da. The circular dichroism spectrum indicated a secondary structure content of 28.6% of alpha-helix, 9.9% of beta-sheet and 61.5% of random-coil forms. Biological activity assays demonstrated that hrCEMP1 nucleates and regulates hydroxyapatite crystal growth. Hereby, it is demonstrated for the first time that CEMP1 has a (C-type) lectin-like activity and specifically recognizes mannopyranoside. The information produced by this biochemical and structural characterization may contribute to understand more fully the biological functions of CEMP1. PMID:26763148

  7. Mitochondrial localization of the low level p53 protein in proliferative cells

    SciTech Connect

    Ferecatu, Ioana; Bergeaud, Marie; Rodriguez-Enfedaque, Aida; Le Floch, Nathalie; Oliver, Lisa; Rincheval, Vincent; Renaud, Flore; Vallette, Francois M.; Mignotte, Bernard; Vayssiere, Jean-Luc

    2009-10-02

    p53 protein plays a central role in suppressing tumorigenesis by inducing cell cycle arrest or apoptosis through transcription-dependent and -independent mechanisms. Emerging publications suggest that following stress, a fraction of p53 translocates to mitochondria to induce cytochrome c release and apoptosis. However, the localization of p53 under unstressed conditions remains largely unexplored. Here we show that p53 is localized at mitochondria in absence of apoptotic stimuli, when cells are proliferating, localization observed in various cell types (rodent and human). This is also supported by acellular assays in which p53 bind strongly to mitochondria isolated from rat liver. Furthermore, the mitochondria subfractionation study and the alkaline treatment of the mitochondrial p53 revealed that the majority of mitochondrial p53 is present in the membranous compartments. Finally, we identified VDAC, a protein of the mitochondrial outer-membrane, as a putative partner of p53 in unstressed/proliferative cells.

  8. An integrated superhydrophobic-plasmonic biosensor for mid-infrared protein detection at the femtomole level.

    PubMed

    De Ninno, Adele; Ciasca, Gabriele; Gerardino, Annamaria; Calandrini, Eugenio; Papi, Massimiliano; De Spirito, Marco; Nucara, Alessandro; Ortolani, Michele; Businaro, Luca; Baldassarre, Leonetta

    2015-09-01

    In this work we present an integrated biosensor that enables FTIR (Fourier Transform-Infrared) detection of analytes contained in diluted solutions. The fabricated nanosensor allows for the detection of proteins through the identification of the fine structure of their amide I and II bands, up to the nanomolar concentration range. We exploited two distinct effects to enhance the sensitivity: (i) the concentration effect due to the presence of the superhydrophobic surface that conveys molecules dispersed in solution directly inside the focus of a FTIR spectromicroscope; (ii) the plasmonic resonance of the nanoantenna array that provides electromagnetic field enhancement in the amide I and II spectral region (1500-1700 cm(-1)). We demonstrate the detection of ferritin in the nanomolar concentration range, a blood protein that is usually available in small amounts in typical blood samples. PMID:25712032

  9. Protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins are the major structural and functional components of all cells in the body. They are macromolecules that comprise 1 or more chains of amino acids that vary in their sequence and length and are folded into specific 3-dimensional structures. The sizes and conformations of proteins, therefor...

  10. Proteins.

    ERIC Educational Resources Information Center

    Doolittle, Russell F.

    1985-01-01

    Examines proteins which give rise to structure and, by virtue of selective binding to other molecules, make genes. Binding sites, amino acids, protein evolution, and molecular paleontology are discussed. Work with encoding segments of deoxyribonucleic acid (exons) and noncoding stretches (introns) provides new information for hypotheses. (DH)

  11. Penicillin-Binding Protein 5 Sequence Alteration and Levels of plp5 mRNA Expression in Clinical Isolates of Enterococcus faecium with Different Levels of Ampicillin Resistance.

    PubMed

    Belhaj, Mondher; Boutiba-Ben Boubaker, Ilhem; Slim, Amin

    2016-04-01

    Eighty-two nonduplicated ampicillin-resistant Enterococcus faecium (AREF) isolates from clinical infections at the Charles Nicolle Hospital of Tunisia were investigated. They were collected from January 2001 to December 2009. Genetic relationship between them was studied using pulsed-field gel electrophoresis. The amino acid sequence difference variations of the C-terminal part of penicillin-binding protein 5 (PBP5) versus levels of expressed mRNA were investigated by polymerase chain reaction (PCR), sequencing, and real-time PCR quantification of (PBP5), respectively. No β-lactamase activity was detected and none of our strains showed resistance to glycopeptides, which retain their therapeutic efficiency against enterococcal infections in our hospital. Pattern analysis of the strains revealed six main clones disseminating in different wards. Sequence data revealed the existence of 19 different plp5 alleles with a difference in 16 amino acid positions spanning from residue 414 to 632. Each allele presented at least five amino acid substitutions (His-470→Gln, Asn-496→Lys, Ala-499→Thr, Glu-525→Asp, and Glu-629→Val). No correlation between amino acid sequence polymorphism of PBP5 and levels of ampicillin resistance was detected. The levels of plp5 mRNA expression varied between strains and did not always correlate with levels of ampicillin resistance in clinical AREF. PMID:26618475

  12. Evaluation of three industrial Escherichia coli strains in fed-batch cultivations during high-level SOD protein production

    PubMed Central

    2013-01-01

    Background In the biopharmaceutical industry, Escherichia coli (E. coli) strains are among the most frequently used bacterial hosts for producing recombinant proteins because they allow a simple process set-up and they are Food and Drug Administration (FDA)-approved for human applications. Widespread use of E. coli in biotechnology has led to the development of many different strains, and selecting an ideal host to produce a specific protein of interest is an important step in developing a production process. E. coli B and K–12 strains are frequently employed in large-scale production processes, and therefore are of particular interest. We previously evaluated the individual cultivation characteristics of E. coli BL21 and the K–12 hosts RV308 and HMS174. To our knowledge, there has not yet been a detailed comparison of the individual performances of these production strains in terms of recombinant protein production and system stability. The present study directly compared the T7-based expression hosts E. coli BL21(DE3), RV308(DE3), and HMS174(DE3), focusing on evaluating the specific attributes of these strains in relation to high-level protein production of the model protein recombinant human superoxide dismutase (SOD). The experimental setup was an exponential carbon-limited fed-batch cultivation with minimal media and single-pulse induction. Results The host strain BL21(DE3) produced the highest amounts of specific protein, followed by HMS174(DE3) and RV308(DE3). The expression system HMS174(DE3) exhibited system stability by retaining the expression vector over the entire process time; however, it entirely stopped growing shortly after induction. In contrast, BL21(DE3) and RV308(DE3) encountered plasmid loss but maintained growth. RV308(DE3) exhibited the lowest ppGpp concentration, which is correlated with the metabolic stress level and lowest degradation of soluble protein fraction compared to both other strains. Conclusions Overall, this study provides

  13. Vascular endothelial growth factor A protein level and gene expression in intracranial meningiomas with brain edema.

    PubMed

    Nassehi, Damoun; Dyrbye, Henrik; Andresen, Morten; Thomsen, Carsten; Juhler, Marianne; Laursen, Henning; Broholm, Helle

    2011-12-01

    Meningiomas are the second most common primary intracranial tumors in adults. Although meningiomas are mostly benign, more than 50% of patients with meningioma develop peritumoral brain edema (PTBE), which may be fatal because of increased intracranial pressure. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen and angiogen. VEGF-A protein, which is identical to vascular permeability factor, is a regulator of angiogenesis. In this study, 101 patients with meningiomas, and possible co-factors to PTBE, such as meningioma subtypes and tumor location, were examined. Forty-three patients had primary, solitary, supratentorial meningiomas with PTBE. In these, correlations in PTBE, edema index, VEGF-A protein, VEGF gene expression, capillary length, and tumor water content were investigated. DNA-branched hybridization was used for measuring VEGF gene expression in tissue homogenates prepared from frozen tissue samples. The method for VEGF-A analysis resembled an ELISA assay, but was based on chemiluminescence. The edema index was positively correlated to VEGF-A protein (p = 0.014) and VEGF gene expression (p < 0.05). The capillary length in the meningiomas was positively correlated to the PTBE (p = 0.038). If VEGF is responsible for the formation of PTBE, the edema may be treated with the anti-VEGF drug Bevacizumab (Avastin), which has been shown to reduce PTBE in patients with glioblastoma multiforme. PMID:22085359

  14. Serum sex hormone and growth arrest-specific protein 6 levels in male patients with coronary heart disease

    PubMed Central

    Zhao, Rui; Li, Yan; Dai, Wen

    2016-01-01

    Epidemiological studies have shown a high prevalence of low serum testosterone levels in men with cardiovascular disease. Moreover, the tyrosine kinase receptor Axl, the ligand of which is growth arrest-specific protein 6 (GAS6), is expressed in the vasculature, and serum GAS6 levels are associated with endothelial dysfunction and cardiovascular events. Testosterone regulates GAS6 gene transcription directly, which inhibits calcification of vascular smooth muscle cells and provides a mechanistic insight into the cardioprotective action of androgens. This study was designed to determine the correlation between serum GAS6 and testosterone levels in male patients with coronary heart disease (CHD). We recruited 225 patients with CHD and 102 apparently healthy controls. Serum concentrations of GAS6 and soluble Axl were quantified by an enzyme-linked immunosorbent assay. Levels of high-sensitivity C-reactive protein, testosterone, estradiol, and other routine biochemical markers were also measured. Testosterone decreased from 432.69 ± 14.40 to 300.76 ± 6.23 ng dl−1 (P < 0.001) and GAS6 decreased from 16.20 ± 0.31 to 12.51 ± 0.19 ng ml−1 (P < 0.001) in patients with CHD, compared with control subjects. Multiple linear regression analysis showed that serum testosterone and GAS6 levels were positively associated in male patients with CHD. Alterations in GAS6 levels may influence the development of CHD. Downregulation of GAS6/Axl signaling in the presence of low sex hormone levels during disease progression is a potential mechanism by which GAS6 affects CHD. This study provides novel results regarding the influence of sex hormones on serum GAS6 levels in patients with CHD. PMID:26924277

  15. Elevated Serum Levels of the Antiapoptotic Protein Decoy-Receptor 3 Are Associated with Advanced Liver Disease

    PubMed Central

    Gizis, Michalis; Delladetsima, Ioanna; Laoudi, Eyfrosyni; Siakavellas, Spyros I.; Kaltsa, Garyfallia; Vlachogiannakos, John; Vafiadis-Zouboulis, Irene; Daikos, George L.; Papatheodoridis, George V.

    2016-01-01

    Background. Decoy-receptor 3 (DcR3) exerts antiapoptotic and immunomodulatory function and is overexpressed in neoplastic and inflammatory conditions. Serum DcR3 (sDcR3) levels during the chronic hepatitis/cirrhosis/hepatocellular carcinoma (HCC) sequence have not been explored. Objective. To assess the levels and significance of sDcR3 protein in various stages of chronic liver disease. Methods. We compared sDcR3 levels between healthy controls and patients with chronic viral hepatitis (CVH), decompensated cirrhosis (DC), and HCC. Correlations between sDcR3 levels and various patient- and disease-related factors were analyzed. Results. sDcR3 levels were significantly higher in patients with CVH than in controls (P < 0.01). sDcR3 levels were elevated in DC and HCC, being significantly higher compared not only to controls (P < 0.001 for both) but to CVH patients as well (P < 0.001 for both). In addition, DcR3 protein was detected in large quantities in the ascitic fluid of cirrhotics. In patients with CVH, sDcR3 significantly correlated to fibrosis severity, as estimated by Ishak score (P = 0.019) or by liver stiffness measured with elastography (Spearman r = 0.698, P < 0.001). In cirrhotic patients, significant positive correlations were observed between sDcR3 levels and markers of severity of hepatic impairment, including MELD score (r = 0.653, P < 0.001). Conclusions. Circulating levels of DcR3 are elevated during chronic liver disease and correlate with severity of liver damage. sDcR3 may serve as marker for liver fibrosis severity and progression to end-stage liver disease. PMID:27595094

  16. Serum sex hormone and growth arrest-specific protein 6 levels in male patients with coronary heart disease.

    PubMed

    Zhao, Rui; Li, Yan; Dai, Wen

    2016-01-01

    Epidemiological studies have shown a high prevalence of low serum testosterone levels in men with cardiovascular disease. Moreover, the tyrosine kinase receptor Axl, the ligand of which is growth arrest-specific protein 6 (GAS6), is expressed in the vasculature, and serum GAS6 levels are associated with endothelial dysfunction and cardiovascular events. Testosterone regulates GAS6 gene transcription directly, which inhibits calcification of vascular smooth muscle cells and provides a mechanistic insight into the cardioprotective action of androgens. This study was designed to determine the correlation between serum GAS6 and testosterone levels in male patients with coronary heart disease (CHD). We recruited 225 patients with CHD and 102 apparently healthy controls. Serum concentrations of GAS6 and soluble Axl were quantified by an enzyme-linked immunosorbent assay. Levels of high-sensitivity C-reactive protein, testosterone, estradiol, and other routine biochemical markers were also measured. Testosterone decreased from 432.69 ± 14.40 to 300.76 ± 6.23 ng dl-1 (P < 0.001) and GAS6 decreased from 16.20 ± 0.31 to 12.51 ± 0.19 ng ml-1 (P < 0.001) in patients with CHD, compared with control subjects. Multiple linear regression analysis showed that serum testosterone and GAS6 levels were positively associated in male patients with CHD. Alterations in GAS6 levels may influence the development of CHD. Downregulation of GAS6/Axl signaling in the presence of low sex hormone levels during disease progression is a potential mechanism by which GAS6 affects CHD. This study provides novel results regarding the influence of sex hormones on serum GAS6 levels in patients with CHD. PMID:26924277

  17. Elevated Serum Levels of the Antiapoptotic Protein Decoy-Receptor 3 Are Associated with Advanced Liver Disease.

    PubMed

    Bamias, Giorgos; Gizis, Michalis; Delladetsima, Ioanna; Laoudi, Eyfrosyni; Siakavellas, Spyros I; Koutsounas, Ioannis; Kaltsa, Garyfallia; Vlachogiannakos, John; Vafiadis-Zouboulis, Irene; Daikos, George L; Papatheodoridis, George V; Ladas, Spiros D

    2016-01-01

    Background. Decoy-receptor 3 (DcR3) exerts antiapoptotic and immunomodulatory function and is overexpressed in neoplastic and inflammatory conditions. Serum DcR3 (sDcR3) levels during the chronic hepatitis/cirrhosis/hepatocellular carcinoma (HCC) sequence have not been explored. Objective. To assess the levels and significance of sDcR3 protein in various stages of chronic liver disease. Methods. We compared sDcR3 levels between healthy controls and patients with chronic viral hepatitis (CVH), decompensated cirrhosis (DC), and HCC. Correlations between sDcR3 levels and various patient- and disease-related factors were analyzed. Results. sDcR3 levels were significantly higher in patients with CVH than in controls (P < 0.01). sDcR3 levels were elevated in DC and HCC, being significantly higher compared not only to controls (P < 0.001 for both) but to CVH patients as well (P < 0.001 for both). In addition, DcR3 protein was detected in large quantities in the ascitic fluid of cirrhotics. In patients with CVH, sDcR3 significantly correlated to fibrosis severity, as estimated by Ishak score (P = 0.019) or by liver stiffness measured with elastography (Spearman r = 0.698, P < 0.001). In cirrhotic patients, significant positive correlations were observed between sDcR3 levels and markers of severity of hepatic impairment, including MELD score (r = 0.653, P < 0.001). Conclusions. Circulating levels of DcR3 are elevated during chronic liver disease and correlate with severity of liver damage. sDcR3 may serve as marker for liver fibrosis severity and progression to end-stage liver disease. PMID:27595094

  18. Hepatic heat shock protein 70 and plasma cortisol levels in rainbow trout after tagging with a passive integrated transponder

    USGS Publications Warehouse

    Feldhaus, J.W.; Heppell, S.A.; Mesa, M.G.; Li, H.

    2008-01-01

    This study examined the potentially stressful effects of tagging juvenile rainbow trout Oncorhynchus mykiss with passive integrated transponder (PIT) tags by measuring short-term (<120-h) changes in plasma concentrations of cortisol and hepatic heat shock protein 70 (hsp70). In a laboratory experiment, plasma cortisol levels were measured in fish before they were tagged (0 h) and at 2, 6, 24, and 120 h after being tagged. Hepatic hsp70 levels were measured at 0, 24, and 120 h. All results were compared with those for fish that were handled but not tagged. Plasma cortisol levels were significantly higher in both treatment groups (tagged and handled but not tagged) at 2 h than in the pretreatment groups (0 h). Plasma cortisol levels in the treatment groups returned to near pretreatment levels by 6 h. However, there was a significant difference in plasma cortisol levels between treatment groups at 6 h. There were no significant differences in hepatic hsp70 levels among the two treatment groups, and hepatic hsp70 levels did not change through time. Our results suggest that PIT tagging is a low-impact tagging procedure for juvenile salmonids. ?? Copyright by the American Fisheries Society 2008.

  19. Effect of dietary level of rumen-degraded protein on production and nitrogen metabolism in lactating dairy cows.

    PubMed

    Reynal, S M; Broderick, G A

    2005-11-01

    Twenty-eight (8 with ruminal cannulas) lactating Holstein cows were assigned to 4 x 4 Latin squares and fed diets with different levels of rumen-degraded protein (RDP) to study the effect of RDP on production and N metabolism. Diets contained [dry matter (DM) basis] 37% corn silage, 13% alfalfa silage, and 50% concentrate. The concentrate contained solvent and lignosulfonate-treated soybean meal and urea, and was adjusted to provide RDP at: 13.2, 12.3, 11.7, and 10.6% of DM in diets A to D, respectively. Intake of DM and yield of milk, fat-corrected milk, and fat were not affected by treatments. Dietary RDP had positive linear effects on milk true protein content and microbial non-ammonia N (NAN) flow at the omasal canal, and a quadratic effect on true protein yield, with maximal protein production at 12.3% RDP. However, dietary RDP had a positive linear effect on total N excretion, with urinary N accounting for most of the increase, and a negative linear effect on environmental N efficiency (kg of milk produced per kg of N excreted). Therefore, a compromise between profitability and environmental quality was achieved at a dietary RDP level of 11.7% of DM. Observed microbial NAN flow and RDP supply were higher and RUP flow was lower than those predicted by the NRC (2001) model. The NRC (2001) model overpredicted production responses to RUP compared with the results in this study. Replacing default NRC degradation rates for protein supplements with rates measured in vivo resulted in similar observed and predicted values, suggesting that in situ degradation rates used by the NRC are slower than apparent rates in this study. PMID:16230710

  20. Development of a suspension array assay in multiplex for simultaneous measurement of serum levels of four eosinophil granule proteins

    PubMed Central

    Makiya, Michelle A.; Herrick, Jesica A.; Khoury, Paneez; Prussin, Calman P.; Nutman, Thomas B.; Klion, Amy D.

    2014-01-01

    The concentrations of major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil derived neurotoxin (EDN) and eosinophil peroxidase (EPO) have been associated with eosinophilic disease severity. Whereas a variety of techniques have been used to measure individual eosinophil granule protein concentration, none of these methods efficiently measures MBP, ECP, EDN and EPO simultaneously. A multiplex suspension array system was developed to simultaneously measure the concentration of MBP, ECP, EDN and EPO in serum. The assay showed excellent inter- and intra-assay reliability, and serum levels of MBP, ECP and EDN from eosinophilic subjects analyzed by ELISA and multiplex were highly correlated (r = 0.8579; P < 0.0001, r = 0.6356; P = 0.0006 and r = 0.8600; P < 0.0001, respectively, Spearman rank correlation). Moreover, the multiplex assay required 500-fold less serum than a single ELISA to achieve comparable sensitivity. Absolute eosinophil count and eosinophil surface expression of the activation marker, CD69, were significantly correlated with concentrations of MBP, EDN and EPO, but not ECP, in serum from eosinophilic subjects. Furthermore, subjects with eosinophilic gastrointestinal disorder and normal peripheral absolute eosinophil counts (< 0.5 × 109/L) had significantly increased concentrations of MBP (P < 0.0001), ECP (P < 0.0001), EDN (P = 0.0001) and EPO (P < 0.0001) compared to normal donors. In summary, the eosinophil granule protein multiplex assay provides a rapid and reliable way to measure eosinophil granule protein levels and should prove useful in assessing patterns of degranulation in patients with eosinophilic disorders. PMID:24914990

  1. Identification and Characterization of a Non-retinoid Ligand for Retinol-binding Protein 4 Which Lowers Serum Retinol-binding Protein 4 Levels in Vivo*

    PubMed Central

    Motani, Alykhan; Wang, Zhulun; Conn, Marion; Siegler, Karen; Zhang, Ying; Liu, Qingxiang; Johnstone, Sheree; Xu, Haoda; Thibault, Steve; Wang, Yingcai; Fan, Pingchen; Connors, Richard; Le, Hoa; Xu, Guifen; Walker, Nigel; Shan, Bei; Coward, Peter

    2009-01-01

    Retinol-binding protein 4 (RBP4) transports retinol from the liver to extrahepatic tissues, and RBP4 lowering is reported to improve insulin sensitivity in mice. We have identified A1120, a high affinity (Ki = 8.3 nm) non-retinoid ligand for RBP4, which disrupts the interaction between RBP4 and its binding partner transthyretin. Analysis of the RBP4-A1120 co-crystal structure reveals that A1120 induces critical conformational changes at the RBP4-transthyretin interface. Administration of A1120 to mice lowers serum RBP4 and retinol levels but, unexpectedly, does not improve insulin sensitivity. In addition, we show that Rpb4-/- mice display normal insulin sensitivity and are not protected from high fat diet-induced insulin resistance. We conclude that lowering RBP4 levels does not improve insulin sensitivity in mice. Therefore, RBP4 lowering may not be an effective strategy for treating diabetes. PMID:19147488

  2. Sequence Variations and Protein Expression Levels of the Two Immune Evasion Proteins Gpm1 and Pra1 Influence Virulence of Clinical Candida albicans Isolates

    PubMed Central

    Luo, Shanshan; Hipler, Uta-Christina; Münzberg, Christin; Skerka, Christine; Zipfel, Peter F.

    2015-01-01

    Candida albicans, the important human fungal pathogen uses multiple evasion strategies to control, modulate and inhibit host complement and innate immune attack. Clinical C. albicans strains vary in pathogenicity and in serum resistance, in this work we analyzed sequence polymorphisms and variations in the expression levels of two central fungal complement evasion proteins, Gpm1 (phosphoglycerate mutase 1) and Pra1 (pH-regulated antigen 1) in thirteen clinical C. albicans isolates. Four nucleotide (nt) exchanges, all representing synonymous exchanges, were identified within the 747-nt long GPM1 gene. For the 900-nt long PRA1 gene, sixteen nucleotide exchanges were identified, which represented synonymous, as well as non-synonymous exchanges. All thirteen clinical isolates had a homozygous exchange (A to G) at position 73 of the PRA1 gene. Surface levels of Gpm1 varied by 8.2, and Pra1 levels by 3.3 fold in thirteen tested isolates and these differences influenced fungal immune fitness. The high Gpm1/Pra1 expressing candida strains bound the three human immune regulators more efficiently, than the low expression strains. The difference was 44% for Factor H binding, 51% for C4BP binding and 23% for plasminogen binding. This higher Gpm1/Pra1 expressing strains result in enhanced survival upon challenge with complement active, Factor H depleted human serum (difference 40%). In addition adhesion to and infection of human endothelial cells was increased (difference 60%), and C3b surface deposition was less effective (difference 27%). Thus, variable expression levels of central immune evasion protein influences immune fitness of the human fungal pathogen C. albicans and thus contribute to fungal virulence. PMID:25692293

  3. Enhancement of the basal-level activity of HIV-1 long terminal repeat by HIV-1 nucleocapsid protein.

    PubMed

    Zhang, J L; Sharma, P L; Crumpacker, C S

    2000-03-15

    Two HIV-1 proteins, Tat and NCp7 (NC), have zinc finger-like structures. NC is a virion protein and has been shown to accumulate in the nucleus 8 h postinfection. Since transcription factors with zinc fingers assist the transcriptional activity of both RNA polymerases II and III, we examined the effect of NC on HIV-1 LTR-directed gene expression. The HIV-1 NC binds to the HIV-1 LTR and results in a mobility shift in polyacrylamide gel electrophoresis. Competition assays with cold probes revealed that the binding of NC and formation of a DNA-protein complex could be prevented by the addition of excess unlabeled LTR self-probe, but not the HIV-1 V3 envelope gene. The DNase I footprint analysis showed that NC binds to six regions within HIV-1 LTR, four of which are near the transcription start site. The NC alone enhances LTR basal-level activity in RNA runoff experiments. When the general transcription factors (GTFs) were added in the assay, NC enhances NF-kappaB, Sp1, and TFIIB-induced HIV-1 LTR-directed RNA transcription. RNA transcription directed by the adenovirus major late promoter, however, is not significantly affected by NC in the cell-free system. Transient transfection of human T lymphocytes with the plasmids containing HIV-1 nc or gag showed enhancement of LTR-CAT activity. Moreover, transfection of HIV-1 provirus containing mutations in NC zinc-finger domains dramatically decreases the enhancement activity in human T cells, in which HIV-1 LTR is stably integrated into the cellular genome. These observations show that NC binds to HIV-1 LTR and cooperatively enhances GTFs and NF-kappaB induced HIV-1 LTR basal-level activity. NC may play the role of a nucleation protein, which binds to LTR and enhances basal-level transcription by recruiting cellular transcription factors to the HIV-1 promoter in competition with cellular promoters. PMID:10704334

  4. Mitochondrial thiol modification by a targeted electrophile inhibits metabolism in breast adenocarcinoma cells by inhibiting enzyme activity and protein levels

    PubMed Central

    Smith, M. Ryan; Vayalil, Praveen K.; Zhou, Fen; Benavides, Gloria A.; Beggs, Reena R.; Golzarian, Hafez; Nijampatnam, Bhavitavya; Oliver, Patsy G.; Smith, Robin A.J.; Murphy, Michael P.; Velu, Sadanandan E.; Landar, Aimee

    2016-01-01

    Many cancer cells follow an aberrant metabolic program to maintain energy for rapid cell proliferation. Metabolic reprogramming often involves the upregulation of glutaminolysis to generate reducing equivalents for the electron transport chain and amino acids for protein synthesis. Critical enzymes involved in metabolism possess a reactive thiolate group, which can be modified by certain oxidants. In the current study, we show that modification of mitochondrial protein thiols by a model compound, iodobutyl triphenylphosphonium (IBTP), decreased mitochondrial metabolism and ATP in MDA-MB 231 (MB231) breast adenocarcinoma cells up to 6 days after an initial 24 h treatment. Mitochondrial thiol modification also depressed oxygen consumption rates (OCR) in a dose-dependent manner to a greater extent than a non-thiol modifying analog, suggesting that thiol reactivity is an important factor in the inhibition of cancer cell metabolism. In non-tumorigenic MCF-10A cells, IBTP also decreased OCR; however the extracellular acidification rate was significantly increased at all but the highest concentration (10 µM) of IBTP indicating that thiol modification can have significantly different effects on bioenergetics in tumorigenic versus non-tumorigenic cells. ATP and other adenonucleotide levels were also decreased by thiol modification up to 6 days post-treatment, indicating a decreased overall energetic state in MB231 cells. Cellular proliferation of MB231 cells was also inhibited up to 6 days post-treatment with little change to cell viability. Targeted metabolomic analyses revealed that thiol modification caused depletion of both Krebs cycle and glutaminolysis intermediates. Further experiments revealed that the activity of the Krebs cycle enzyme, aconitase, was attenuated in response to thiol modification. Additionally, the inhibition of glutaminolysis corresponded to decreased glutaminase C (GAC) protein levels, although other protein levels were unaffected. This study

  5. Mitochondrial thiol modification by a targeted electrophile inhibits metabolism in breast adenocarcinoma cells by inhibiting enzyme activity and protein levels.

    PubMed

    Smith, M Ryan; Vayalil, Praveen K; Zhou, Fen; Benavides, Gloria A; Beggs, Reena R; Golzarian, Hafez; Nijampatnam, Bhavitavya; Oliver, Patsy G; Smith, Robin A J; Murphy, Michael P; Velu, Sadanandan E; Landar, Aimee

    2016-08-01

    Many cancer cells follow an aberrant metabolic program to maintain energy for rapid cell proliferation. Metabolic reprogramming often involves the upregulation of glutaminolysis to generate reducing equivalents for the electron transport chain and amino acids for protein synthesis. Critical enzymes involved in metabolism possess a reactive thiolate group, which can be modified by certain oxidants. In the current study, we show that modification of mitochondrial protein thiols by a model compound, iodobutyl triphenylphosphonium (IBTP), decreased mitochondrial metabolism and ATP in MDA-MB 231 (MB231) breast adenocarcinoma cells up to 6 days after an initial 24h treatment. Mitochondrial thiol modification also depressed oxygen consumption rates (OCR) in a dose-dependent manner to a greater extent than a non-thiol modifying analog, suggesting that thiol reactivity is an important factor in the inhibition of cancer cell metabolism. In non-tumorigenic MCF-10A cells, IBTP also decreased OCR; however the extracellular acidification rate was significantly increased at all but the highest concentration (10µM) of IBTP indicating that thiol modification can have significantly different effects on bioenergetics in tumorigenic versus non-tumorigenic cells. ATP and other adenonucleotide levels were also decreased by thiol modification up to 6 days post-treatment, indicating a decreased overall energetic state in MB231 cells. Cellular proliferation of MB231 cells was also inhibited up to 6 days post-treatment with little change to cell viability. Targeted metabolomic analyses revealed that thiol modification caused depletion of both Krebs cycle and glutaminolysis intermediates. Further experiments revealed that the activity of the Krebs cycle enzyme, aconitase, was attenuated in response to thiol modification. Additionally, the inhibition of glutaminolysis corresponded to decreased glutaminase C (GAC) protein levels, although other protein levels were unaffected. This study

  6. Differential levels of specific cytochrome c biogenesis proteins in response to oxygen: analysis of the ccl operon in Rhodobacter capsulatus.

    PubMed Central

    Gabbert, K K; Goldman, B S; Kranz, R G

    1997-01-01

    The photosynthetic bacterium Rhodobacter capsulatus synthesizes c-type cytochromes under a variety of growth conditions. For example, under aerobic growth, c-type cytochromes are synthesized as part of an electron transport pathway, using oxygen as the terminal electron acceptor. Anaerobically in the light, R. capsulatus requires cytochrome bc1 and other c-type cytochromes for the photosynthetic electron transport pathway. It is shown here that the ccl1 and ccl2 genes of R. capsulatus are required for the synthesis of all c-type cytochromes, including the cytochrome c' protein of unknown function but of structural similarity to cytochrome b562. Polar and nonpolar mutations constructed in each gene demonstrated that the ccl12 genes form an operon. Expression of the ccl12 genes was examined by using lacZ and phoA fusions as translational reporters. Primer extension analysis was used to determine transcriptional control and the start site of the ccl12 promoter. Finally, antiserum to the Ccl2 protein was used to quantitate levels of Ccl2 under six different growth conditions. The Ccl2 protein is present at 20-fold-higher levels under conditions where oxygen is present. In contrast, other cytochromes c biogenesis proteins, HelA and HelX, previously shown to be part of an helABCDX operon, are at relatively similar levels under these six growth conditions. This discovery is discussed in terms of the physiology and evolution of cytochromes c biogenesis, with particular attention to oxidative environments. PMID:9286996

  7. High levels of p53 protein expression do not correlate with p53 gene mutations in anaplastic large cell lymphoma.

    PubMed Central

    Cesarman, E.; Inghirami, G.; Chadburn, A.; Knowles, D. M.

    1993-01-01

    Strong immunohistochemical reactivity for p53 tumor suppressor gene product has been reported in a variety of different human malignancies including CD30- (Ki-1) positive anaplastic large cell lymphoma (ALCL). Although high levels of p53 protein have been interpreted as abnormal, rapidly proliferating benign and neoplastic lymphoid cells may have increased p53 expression in the absence of structural alterations. On the other hand, mutations in the p53 gene can lead to a lack of p53 protein production. Structural alterations of the p53 gene have not been documented in cases of ALCL and the mechanism for an abnormal pattern of p53 expression in these lymphomas has not been elucidated. Therefore, to determine whether an altered pattern of p53 expression correlates with mutations in the p53 locus in ALCL, we analyzed the expression of p53 protein immunohistochemically, compared it with the proliferation index using monoclonal antibody Ki-67, and assessed the presence of mutations in exons 5 though 9 of the p53 gene using a single-strand conformation polymorphism assay in a panel of 17 ALCLs. Furthermore, we studied the presence of allelic deletions of chromosome 17p by restriction fragment length polymorphism analysis. We found significant levels of p53 protein expression in 12 of the 15 cases studied, but identified mutations in only one of 17 cases. An allelic deletion in chromosome 17p was identified only in the one case containing a mutated p53 gene. Whereas the case containing structural alterations in the p53 gene did have strong p53 immunoreactivity, 11 cases that lacked p53 mutations in the regions examined also had significant levels of p53. Thus, our studies indicate that strong immunohistochemical reactivity for p53 is not a reliable indicator of the presence of structural alterations of p53 gene exons 5 through 9 in ALCL. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8103295

  8. Effects of the dietary protein level on the microbial composition and metabolomic profile in the hindgut of the pig.

    PubMed

    Zhou, Liping; Fang, Lingdong; Sun, Yue; Su, Yong; Zhu, Weiyun

    2016-04-01

    The aim of this study was to investigate the effects of a long-term low protein diet on the microbial composition and metabolomic profile in the hindgut of the pig. Thirty-six Duroc × Landrace × Large White growing barrows (70 days of age, 23.57 ± 1.77 kg) were randomly allocated to normal protein diet (NP) and low protein diet (LP) groups using a randomized block design. At the age of 170 days, the digesta in the hindguts of the pigs were collected for microbial and metabolomic analysis. The results showed that there were no significant differences in the average daily gain, average daily feed intake, or feed:gain ratio between the NP and LP groups. The concentrations of isobutyrate, isovalerate, and branched-chain fatty acids (BCFAs)/short-chain fatty acids (SCFAs) in the cecum decreased with the reduction of dietary protein. Pyrosequencing of the V1-V3 region of the 16S rRNA genes showed that LP diet significantly decreased the relative abundance of Lactobacillus in the cecum, and Streptococcus in the colon; however, the relative abundance of Prevotella and Coprococcus in the LP group was significantly higher than in the NP group in the cecum, and Sarcina, Peptostreptococcaceae incertae sedis, Mogibacterium, Subdoligranulum, and Coprococcus was higher in the colon. The gas chromatography-mass spectrometry (GC-MS) analysis showed that the dietary protein level mainly affected phenylalanine metabolism; glycine, serine, and threonine metabolism; the citrate cycle; pyruvate metabolism; and the alanine, aspartate, and glutamate metabolism. Moreover, the correlation analysis of the combined datasets revealed some potential relationships between the colonic metabolites and certain microbial species. These results suggest that a low protein diet may modulate the microbial composition and metabolites of the hindgut, without affecting the growth performance of pigs; however, potential roles of this modulation to the health of pigs remains unknown. PMID:26723572

  9. Subunit composition and in vivo substrate-binding characteristics of Escherichia coli Tat protein complexes expressed at native levels.

    PubMed

    McDevitt, Christopher A; Buchanan, Grant; Sargent, Frank; Palmer, Tracy; Berks, Ben C

    2006-12-01

    The Tat system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. Substrates are targeted to the Tat pathway by signal peptides containing a pair of consecutive arginine residues. The membrane proteins TatA, TatB and TatC are the essential components of this pathway in Escherichia coli. The complexes that these proteins form at native levels of expression have been investigated by the use of affinity tag-coding sequences fused to chromosomal tat genes. Distinct TatA and TatBC complexes were identified using size-exclusion chromatography and shown to have apparent molecular masses of approximately 700 and 500 kDa, respectively. Following in vivo expression, the Tat substrate protein SufI was found to copurify with the TatBC, but not the TatA, complex. This binding required the SufI signal peptide. Substitution of the twin-arginine residues in the SufI signal peptide by either twin lysine or twin alanine residues abolished export. However, both variant SufI proteins still copurified with the TatBC complex. These data show that the twin-arginine residues of the Tat consensus motif are not essential for binding of precursor to the TatBC complex but are required for the successful entry of the precursor into the transport cycle. The effect on substrate binding of single amino acid substitutions in TatC that affect Tat transport were studied using TatC variants Phe94Ala, Glu103Ala, Glu103Arg and Asp211Ala. Only variant Glu103Arg showed reduced copurification of SufI with TatBC. The transport defects associated with the other TatC variants do not, therefore, arise from an inability to bind substrate proteins. PMID:17212781

  10. Pharmacological modulation of Alzheimer's beta-amyloid precursor protein levels in the CSF of rats with forebrain cholinergic system lesions.

    PubMed

    Haroutunian, V; Greig, N; Pei, X F; Utsuki, T; Gluck, R; Acevedo, L D; Davis, K L; Wallace, W C

    1997-06-01

    Abnormal deposition and accumulation of Alzheimer's amyloid beta-protein (A beta) and degeneration of forebrain cholinergic neurons are among the principal features of Alzheimer's disease. Studies in rat model systems have shown that forebrain cholinergic deficits are accompanied by induction of cortical beta-amyloid precursor protein (beta-APP) mRNAs and increased levels of secreted beta-APP in the CSF. The studies reported here determined whether the CSF levels of secreted beta-APP could be altered pharmacologically. In different experiments, rats with lesions of the forebrain cholinergic system received injections of vehicle, a muscarinic receptor antagonist scopolamine, or one of two cholinesterase inhibitors - diisopropyl phosphorofluoridate (DFP) or phenserine. Scopolamine was administered to determine whether the levels of beta-APP in the CSF could be increased by anticholinergic agents. The cholinesterase inhibitors were administered to determine whether the forebrain cholinergic system lesion-induced increases in CSF beta-APP could be reduced by cholinergic augmentation. Scopolamine administration led to a significant increase in the CSF levels of secreted beta-APP in sham-lesioned rats. Phenserine, a novel, reversible acetyl-selective cholinesterase inhibitor, significantly decreased the levels of secreted beta-APP in the CSF of forebrain cholinergic system-lesioned rats whereas DFP, a relatively non-specific cholinesterase inhibitor, failed to affect CSF levels of secreted beta-APP. These results suggest that the levels of secreted beta-APP in the CSF can be pharmacologically modulated but that this modulation is dependent upon the status of the forebrain cholinergic system and the pharmacological properties of the drugs used to influence it. PMID:9191090

  11. Protein level for alfalfa and corn silage-based diets: II. Nitrogen balance and manure characteristics.

    PubMed

    Wattiaux, M A; Karg, K L

    2004-10-01

    This N balance study was completed with 48 multiparous Holstein cows (body weight [BW] = 653 kg; days in milk = 89) blocked by calving date and assigned to a 2 x 2 factorial arrangement of dietary treatments. The total mixed ration included alfalfa silage (AS) or corn silage (CS) as the primary forage source (41 and 14% vs. 14 and 41% of diet dry matter (DM), respectively) and were formulated for recommended (RP) or excessive (HP) amounts of rumen degradable protein (RDP) and rumen undegradable protein (RUP) according to the guidelines of the National Research Council (NRC). Crude protein (CP) averaged 16.5, 18.0, 16.4, and 17.3% for the AS-RP; AS-HP; CS-RP; and CS-HP diet, respectively (DM basis). Regardless of primary forage source, the reduction in dietary CP to the NRC guidelines tended to improve milk yield (43.4 vs. 41.0 kg/d) but did not alter 3.5% fat-corrected milk (37.0 kg/d) or milk true protein yield (1167 g/d). In this trial, cows fed the CS-based diets consumed less DM than those fed the AS-based diets in part because of rumen acidosis. The adverse effect of low rumen pH was accompanied by an increase in urinary N (UN) as a percentage of N intake, but did not alter milk yield. Notwithstanding partial confounding, fecal N (FN) was 49 g/d lower (213 vs. 164 g/d), UN was unchanged (229 g/d), but milk N tended to be higher (194 vs. 206 g/d) when cows were fed the CS-based diets compared with AS-based diets. Compared with the HP diets, cows fed the RP diets had similar FN (189 g/d) and milk N (200 g/d), but UN and urine urea N were reduced by 41 g/d (249 vs. 208 g/d) and 40 g/d (210 vs. 171 g/d), respectively. Fecal N concentration was higher for CS-based diets, but urinary N concentration was higher for AS-based diets. The reduction in dietary CP did not influence these concentrations but lowered urine volume. The metabolic relationships between energy and protein in determining the fate of excess dietary N (primarily in the form of excess RUP in this

  12. Bladder cancer biomarker array to detect aberrant levels of proteins in urine.

    PubMed

    Gogalic, S; Sauer, U; Doppler, S; Preininger, C

    2015-02-01

    Bladder cancer (BCa) is a serious malignancy of the urinary tract worldwide and also prominent for its high rate of recurrence incorporating 50% of all treated patients. To reduce relapse of BCa, lifelong surveillance of patients is essential leading to high treatment costs. The gold standard for the diagnosis of bladder cancer is cystoscopy. It is very sensitive, but due to high costs and its invasive nature this method for routine diagnosis of bladder cancer remains questionable. Because of this and the required surveillance of patients suffering from bladder cancer, urine based markers represent a new potential field of investigation. Literature at the National Center of Biological Information (NCBI) was retrieved for a potential marker panel offering specific protein signatures and used to develop a sensitive and accurate chip assay to monitor BCa. Discovery of possible bladder cancer protein markers is compiled by extensive literature search including 1077 recently (15.01.2008-20.03.2014) published research articles. Validation of this literature is done by selection based on prior defined inclusion and exclusion criteria. A set of six putative biomarkers (VEGF, IL-8, MMP-9, MMP-7, survivin and Cyfra 21.1) was identified and a non-invasive microarray developed to be used for further clinical validation. Investigation regarding optimized urine preparation and assay development, to enhance assay sensitivity for the marker panel, was carried out. This protein based BCa chip enables the fast (within 5 h), simultaneous, easy to operate, cheap, early and non-invasive determination of BCa and is ready for clinical evaluation. PMID:25427191

  13. Intercellular chaperone transmission via exosomes contributes to maintenance of protein homeostasis at the organismal level

    PubMed Central

    Takeuchi, Toshihide; Suzuki, Mari; Fujikake, Nobuhiro; Popiel, H. Akiko; Kikuchi, Hisae; Futaki, Shiroh; Wada, Keiji; Nagai, Yoshitaka

    2015-01-01

    The heat shock response (HSR), a transcriptional response that up-regulates molecular chaperones upon heat shock, is necessary for cell survival in a stressful environment to maintain protein homeostasis (proteostasis). However, there is accumulating evidence that the HSR does not ubiquitously occur under stress conditions, but largely depends on the cell types. Despite such imbalanced HSR among different cells and tissues, molecular mechanisms by which multicellular organisms maintain their global proteostasis have remained poorly understood. Here, we report that proteostasis can be maintained by molecular chaperones not only in a cell-autonomous manner but also in a non–cell-autonomous manner. We found that elevated expression of molecular chaperones, such as Hsp40 and Hsp70, in a group of cells improves proteostasis in other groups of cells, both in cultured cells and in Drosophila expressing aggregation-prone polyglutamine proteins. We also found that Hsp40, as well as Hsp70 and Hsp90, is physiologically secreted from cells via exosomes, and that the J domain at the N terminus is responsible for its exosome-mediated secretion. Addition of Hsp40/Hsp70-containing exosomes to the culture medium of the polyglutamine-expressing cells results in efficient suppression of inclusion body formation, indicating that molecular chaperones non-cell autonomously improve the protein-folding environment via exosome-mediated transmission. Our study reveals that intercellular chaperone transmission mediated by exosomes is a novel molecular mechanism for non–cell-autonomous maintenance of organismal proteostasis that could functionally compensate for the imbalanced state of the HSR among different cells, and also provides a novel physiological role of exosomes that contributes to maintenance of organismal proteostasis. PMID:25918398

  14. Levels of heat shock protein transcripts in normal follicles and ovarian follicular cysts.

    PubMed

    Velázquez, Melisa M L; Alfaro, Natalia S; Salvetti, Natalia R; Stangaferro, Matías L; Rey, Florencia; Panzani, Carolina G; Ortega, Hugo H

    2011-11-01

    In the study, the gene expression of several heat shock proteins (HSPs) was determined in normal follicles and cystic follicles from cattle. A lower expression of HSP10 and HSP40 was observed in granulosa and theca cells of cysts compared to normal follicles. HSP27 was significantly less expressed in granulosa cells in cystic and large antral follicles than in other follicular categories. HSP60 and HSP90a expressions were highest in theca cells of cysts. However, HSP70 and HSP90b exhibited a lower expression in cysts than in healthy follicles. PMID:22139340

  15. Specific responses in rat small intestinal epithelial mRNA expression and protein levels during chemotherapeutic damage and regeneration.

    PubMed

    Verburg, Melissa; Renes, Ingrid B; Van Nispen, Danielle J P M; Ferdinandusse, Sacha; Jorritsma, Marieke; Büller, Hans A; Einerhand, Alexandra W C; Dekker, Jan

    2002-11-01

    The rapidly dividing small intestinal epithelium is very sensitive to the cytostatic drug methotrexate. We investigated the regulation of epithelial gene expression in rat jejunum during methotrexate-induced damage and regeneration. Ten differentiation markers were localized on tissue sections and quantified at mRNA and protein levels relative to control levels. We analyzed correlations in temporal expression patterns between markers. mRNA expression of enterocyte and goblet cell markers decreased significantly during damage for a specific period. Of these, sucrase-isomaltase (-62%) and CPS (-82%) were correlated. Correlations were also found between lactase (-76%) and SGLT1 (-77%) and between I-FABP (-52%) and L-FABP (-45%). Decreases in GLUT5 (-53%), MUC2 (-43%), and TFF3 (-54%) mRNAs occurred independently of any of the other markers. In contrast, lysozyme mRNA present in Paneth cells increased (+76%). At the protein level, qualitative and quantitative changes were in agreement with mRNA expression, except for Muc2 (+115%) and TFF3 (+81%), which increased significantly during damage, following independent patterns. During regeneration, expression of each marker returned to control levels. The enhanced expression of cytoprotective molecules (Muc2, TFF3, lysozyme) during damage represents maintenance of goblet cell and Paneth cell functions, most likely to protect the epithelium. Decreased expression of enterocyte-specific markers represents decreased enterocyte function, of which fatty acid transporters were least affected. PMID:12417619

  16. Cyclic Changes in the Level of the Innate Immune Molecule, Surfactant Protein-A, and Cytokines in Vaginal Fluid

    PubMed Central

    MacNeill, Colin; de Guzman, Glendell; Sousa, Grace E.; Umstead, Todd M.; Phelps, David S.; Floros, Joanna; Ahn, Kwangmi; Weisz, Judith

    2013-01-01

    PROBLEM Our knowledge of the innate host-defenses in the vagina, a site where these defenses are essential to protecting the host upper reproductive tract from invasion by pathogens, is as yet rudimentary. Specifically, little is known about the pattern-recognition component of vaginal innate immunity, the relationship of pattern-recognition molecules to known cytokine levels, and the role of gonadal hormones in their regulation. METHOD OF STUDY We measured levels of Surfactant Protein-A (SP-A), a prototypic innate pattern-recognition protein, in vaginal fluid (VF) and correlated them with levels of IL-1β and IL-8, two cytokines known to be present in VF. Assays were carried out on VF collected over three consecutive cycles from ten healthy naturally cycling women who were sampled at three specific time points in the menstrual cycle. The three time points were chosen to enable correlation with distinct hormonal states. RESULTS Both SP-A and cytokines levels were highest 5–6 days after menses (p < 0.05) and were significantly lower at ovulation and mid-luteal phase. CONCLUSION SP-A, like other host-defense molecules in the reproductive tract, appears to be regulated by gonadal hormones. PMID:22672628

  17. Lipid Loading of Human Vascular Smooth Muscle Cells Induces Changes in Tropoelastin Protein Levels and Physical Structure

    PubMed Central

    Samouillan, Valerie; Dandurand, Jany; Nasarre, Laura; Badimon, Lina; Lacabanne, Colette; Llorente-Cortés, Vicenta

    2012-01-01

    Aggregated low-density lipoprotein (agLDL), one of the main LDL modifications in the arterial intima, contributes to massive intracellular cholesteryl ester (CE) accumulation in human vascular smooth muscle cells (VSMC), which are major producers of elastin in the vascular wall. Our aim was to analyze the levels, physical structure, and molecular mobility of tropoelastin produced by agLDL-loaded human VSMC (agLDL-VSMC) versus that produced by control VSMC. Western blot analysis demonstrated that agLDL reduced VSMC-tropoelastin protein levels by increasing its degradation rate. Moreover, our results demonstrated increased levels of precursor and mature forms of cathepsin S in agLDL-VSMC. Fourier transform infrared analysis revealed modifications in the secondary structures of tropoelastin produced by lipid-loaded VSMCs. Thermal and dielectric analyses showed that agLDL-VSMC tropoelastin has decreased glass transition temperatures and distinct chain dynamics that, in addition to a loss of thermal stability, lead to strong changes in its mechanical properties. In conclusion, agLDL lipid loading of human vascular cells leads to an increase in cathepsin S production concomitantly with a decrease in cellular tropoelastin protein levels and dramatic changes in secreted tropoelastin physical structure. Therefore, VSMC-lipid loading likely determines alterations in the mechanical properties of the vascular wall and plays a crucial role in elastin loss during atherosclerosis. PMID:22947869

  18. Chronic obstructive pulmonary disease and inhaled steroids alter surfactant protein D (SP-D) levels: a cross-sectional study

    PubMed Central

    Sims, Michael W; Tal-Singer, Ruth M; Kierstein, Sonja; Musani, Ali I; Beers, Michael F; Panettieri, Reynold A; Haczku, Angela

    2008-01-01

    Background Surfactant protein D (SP-D), an innate immune molecule, plays an important protective role during airway inflammation. Deficiency of this molecule induces emphysematous changes in murine lungs, but its significance in human COPD remains unclear. Methods We collected bronchoalveolar lavage fluid from 20 subjects with varying degrees of COPD (8 former smokers and 12 current smokers) and 15 asymptomatic healthy control subjects (5 never smokers, 3 remote former smokers, and 7 current smokers). All subjects underwent a complete medical history and pulmonary function testing. SP-D was measured by Enzyme-Linked ImmunoSorbent Assay. Statistical analysis was performed using nonparametric methods and multivariable linear regression for control of confounding. The effect of corticosteroid treatment on SP-D synthesis was studied in vitro using an established model of isolated type II alveolar epithelial cell culture. Results Among former smokers, those with COPD had significantly lower SP-D levels than healthy subjects (median 502 and 1067 ng/mL, respectively, p = 0.01). In a multivariable linear regression model controlling for age, sex, race, and pack-years of tobacco, COPD was independently associated with lower SP-D levels (model coefficient -539, p = 0.04) and inhaled corticosteroid use was independently associated with higher SP-D levels (398, p = 0.046). To support the hypothesis that corticosteroids increase SP-D production we used type II alveolar epithelial cells isolated from adult rat lungs. These cells responded to dexamethasone treatment by a significant increase of SP-D mRNA (p = 0.041) and protein (p = 0.037) production after 4 days of culture. Conclusion Among former smokers, COPD is associated with lower levels of SP-D and inhaled corticosteroid use is associated with higher levels of SP-D in the lung. Dexamethasone induced SP-D mRNA and protein expression in isolated epithelial cells in vitro. Given the importance of this molecule as a modulator

  19. Poststroke Depression as a Factor Adversely Affecting the Level of Oxidative Damage to Plasma Proteins during a Brain Stroke

    PubMed Central

    Cichoń, Natalia; Bijak, Michał; Miller, Elżbieta; Niwald, Marta; Saluk, Joanna

    2015-01-01

    Poststroke depression, the second most serious psychosomatic complication after brain stroke, leads to delay of the rehabilitation process and is associated with an increased disability and cognitive impairment along with increase in term mortality. Research into the biochemical changes in depression is still insufficiently described. The aim of our study was therefore to evaluate the possible association between plasma protein oxidative/nitrative damages and the development of poststroke depression. We evaluated oxidative/nitrative modifications of specific proteins by measurement of 3-nitrotyrosine and carbonyl groups levels using ELISA test. Additionally, we checked differences in proteins thiol groups by spectrophotometric assay based on reaction between DTNB and thiols. We also evaluated catalase activity in erythrocytes measured as ability to decompose H2O2. Correlation analysis was performed using Spearman's rank. We observed significant (P < 0.001) differences in all oxidative/nitrative stress parameters in brain stroke patients compared to healthy group. Our research shows that oxidative damage of proteins is correlated with the degree of poststroke depression, while nitrative changes do not show any relationship. We demonstrate a positive correlation between the concentration of carbonyl groups and the Geriatric Depression Scale and a negative correlation between the degree of depression and the concentration of -SH groups or catalase activity. PMID:25838867

  20. Effects of macromolecular crowding on a small lipid binding protein probed at the single-amino acid level.

    PubMed

    Pérez Santero, Silvia; Favretto, Filippo; Zanzoni, Serena; Chignola, Roberto; Assfalg, Michael; D'Onofrio, Mariapina

    2016-09-15

    Macromolecular crowding is a distinctive feature of the cellular interior, influencing the behaviour of biomacromolecules. Despite significant advancements in the description of the effects of crowding on global protein properties, the influence of cellular components on local protein attributes has received limited attention. Here, we describe a residue-level systematic interrogation of the structural, dynamic, and binding properties of the liver fatty acid binding protein (LFABP) in crowded solutions. Two-dimensional NMR spectral fingerprints and relaxation data were collected on LFABP in the presence of polymeric and biomolecular crowders. Non-interacting crowders produced minimal site-specific spectral perturbations on ligand-free and lipid-bound LFABP. Conformational adaptations upon ligand binding reproduced those observed in dilute solution, but a perturbation of the free oleate state resulted in less favorable uptake. When LFABP engaged in direct interactions with background molecules, changes in local chemical environments were detected for residues of the internal binding pocket and of the external surface. Enhanced complexity was introduced by investigating LFABP in cell lysates, and in membrane-bounded compartments. LFABP was able to capture ligands from prokaryotic and eukaryotic cell lysates, and from artificial cells (water-in-oil emulsion droplets). The data suggest that promiscuous interactions are a major factor influencing protein function in the cell. PMID:27457417