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Sample records for core metabolic proteins

  1. Adaptive Evolution and Functional Redesign of Core Metabolic Proteins in Snakes

    OpenAIRE

    Castoe, Todd A.; Jiang, Zhi J.; Wanjun Gu; Wang, Zhengyuan O.; Pollock, David D.

    2008-01-01

    BACKGROUND: Adaptive evolutionary episodes in core metabolic proteins are uncommon, and are even more rarely linked to major macroevolutionary shifts. METHODOLOGY/PRINCIPAL FINDINGS: We conducted extensive molecular evolutionary analyses on snake mitochondrial proteins and discovered multiple lines of evidence suggesting that the proteins at the core of aerobic metabolism in snakes have undergone remarkably large episodic bursts of adaptive change. We show that snake mitochondrial proteins ex...

  2. Adaptive evolution and functional redesign of core metabolic proteins in snakes.

    Science.gov (United States)

    Castoe, Todd A; Jiang, Zhi J; Gu, Wanjun; Wang, Zhengyuan O; Pollock, David D

    2008-05-21

    Adaptive evolutionary episodes in core metabolic proteins are uncommon, and are even more rarely linked to major macroevolutionary shifts. We conducted extensive molecular evolutionary analyses on snake mitochondrial proteins and discovered multiple lines of evidence suggesting that the proteins at the core of aerobic metabolism in snakes have undergone remarkably large episodic bursts of adaptive change. We show that snake mitochondrial proteins experienced unprecedented levels of positive selection, coevolution, convergence, and reversion at functionally critical residues. We examined Cytochrome C oxidase subunit I (COI) in detail, and show that it experienced extensive modification of normally conserved residues involved in proton transport and delivery of electrons and oxygen. Thus, adaptive changes likely altered the flow of protons and other aspects of function in CO, thereby influencing fundamental characteristics of aerobic metabolism. We refer to these processes as "evolutionary redesign" because of the magnitude of the episodic bursts and the degree to which they affected core functional residues. The evolutionary redesign of snake COI coincided with adaptive bursts in other mitochondrial proteins and substantial changes in mitochondrial genome structure. It also generally coincided with or preceded major shifts in ecological niche and the evolution of extensive physiological adaptations related to lung reduction, large prey consumption, and venom evolution. The parallel timing of these major evolutionary events suggests that evolutionary redesign of metabolic and mitochondrial function may be related to, or underlie, the extreme changes in physiological and metabolic efficiency, flexibility, and innovation observed in snake evolution.

  3. Adaptive evolution and functional redesign of core metabolic proteins in snakes.

    Directory of Open Access Journals (Sweden)

    Todd A Castoe

    Full Text Available BACKGROUND: Adaptive evolutionary episodes in core metabolic proteins are uncommon, and are even more rarely linked to major macroevolutionary shifts. METHODOLOGY/PRINCIPAL FINDINGS: We conducted extensive molecular evolutionary analyses on snake mitochondrial proteins and discovered multiple lines of evidence suggesting that the proteins at the core of aerobic metabolism in snakes have undergone remarkably large episodic bursts of adaptive change. We show that snake mitochondrial proteins experienced unprecedented levels of positive selection, coevolution, convergence, and reversion at functionally critical residues. We examined Cytochrome C oxidase subunit I (COI in detail, and show that it experienced extensive modification of normally conserved residues involved in proton transport and delivery of electrons and oxygen. Thus, adaptive changes likely altered the flow of protons and other aspects of function in CO, thereby influencing fundamental characteristics of aerobic metabolism. We refer to these processes as "evolutionary redesign" because of the magnitude of the episodic bursts and the degree to which they affected core functional residues. CONCLUSIONS/SIGNIFICANCE: The evolutionary redesign of snake COI coincided with adaptive bursts in other mitochondrial proteins and substantial changes in mitochondrial genome structure. It also generally coincided with or preceded major shifts in ecological niche and the evolution of extensive physiological adaptations related to lung reduction, large prey consumption, and venom evolution. The parallel timing of these major evolutionary events suggests that evolutionary redesign of metabolic and mitochondrial function may be related to, or underlie, the extreme changes in physiological and metabolic efficiency, flexibility, and innovation observed in snake evolution.

  4. Adaptive Evolution and Functional Redesign of Core Metabolic Proteins in Snakes

    Science.gov (United States)

    Gu, Wanjun; Wang, Zhengyuan O.; Pollock, David D.

    2008-01-01

    Background Adaptive evolutionary episodes in core metabolic proteins are uncommon, and are even more rarely linked to major macroevolutionary shifts. Methodology/Principal Findings We conducted extensive molecular evolutionary analyses on snake mitochondrial proteins and discovered multiple lines of evidence suggesting that the proteins at the core of aerobic metabolism in snakes have undergone remarkably large episodic bursts of adaptive change. We show that snake mitochondrial proteins experienced unprecedented levels of positive selection, coevolution, convergence, and reversion at functionally critical residues. We examined Cytochrome C oxidase subunit I (COI) in detail, and show that it experienced extensive modification of normally conserved residues involved in proton transport and delivery of electrons and oxygen. Thus, adaptive changes likely altered the flow of protons and other aspects of function in CO, thereby influencing fundamental characteristics of aerobic metabolism. We refer to these processes as “evolutionary redesign” because of the magnitude of the episodic bursts and the degree to which they affected core functional residues. Conclusions/Significance The evolutionary redesign of snake COI coincided with adaptive bursts in other mitochondrial proteins and substantial changes in mitochondrial genome structure. It also generally coincided with or preceded major shifts in ecological niche and the evolution of extensive physiological adaptations related to lung reduction, large prey consumption, and venom evolution. The parallel timing of these major evolutionary events suggests that evolutionary redesign of metabolic and mitochondrial function may be related to, or underlie, the extreme changes in physiological and metabolic efficiency, flexibility, and innovation observed in snake evolution. PMID:18493604

  5. Fluorescence lifetime imaging of alterations to cellular metabolism by domain 2 of the hepatitis C virus core protein.

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    Nirmal Mazumder

    Full Text Available Hepatitis C virus (HCV co-opts hepatic lipid pathways to facilitate its pathogenesis. The virus alters cellular lipid biosynthesis and trafficking, and causes an accumulation of lipid droplets (LDs that gives rise to hepatic steatosis. Little is known about how these changes are controlled at the molecular level, and how they are related to the underlying metabolic states of the infected cell. The HCV core protein has previously been shown to independently induce alterations in hepatic lipid homeostasis. Herein, we demonstrate, using coherent anti-Stokes Raman scattering (CARS microscopy, that expression of domain 2 of the HCV core protein (D2 fused to GFP is sufficient to induce an accumulation of larger lipid droplets (LDs in the perinuclear region. Additionally, we performed fluorescence lifetime imaging of endogenous reduced nicotinamide adenine dinucleotides [NAD(PH], a key coenzyme in cellular metabolic processes, to monitor changes in the cofactor's abundance and conformational state in D2-GFP transfected cells. When expressed in Huh-7 human hepatoma cells, we observed that the D2-GFP induced accumulation of LDs correlated with an increase in total NAD(PH fluorescence and an increase in the ratio of free to bound NAD(PH. This is consistent with an approximate 10 fold increase in cellular NAD(PH levels. Furthermore, the lifetimes of bound and free NAD(PH were both significantly reduced--indicating viral protein-induced alterations in the cofactors' binding and microenvironment. Interestingly, the D2-expressing cells showed a more diffuse localization of NAD(PH fluorescence signal, consistent with an accumulation of the co-factor outside the mitochondria. These observations suggest that HCV causes a shift of metabolic control away from the use of the coenzyme in mitochondrial electron transport and towards glycolysis, lipid biosynthesis, and building of new biomass. Overall, our findings demonstrate that HCV induced alterations in hepatic

  6. Packing in protein cores

    Science.gov (United States)

    Gaines, J. C.; Clark, A. H.; Regan, L.; O'Hern, C. S.

    2017-07-01

    Proteins are biological polymers that underlie all cellular functions. The first high-resolution protein structures were determined by x-ray crystallography in the 1960s. Since then, there has been continued interest in understanding and predicting protein structure and stability. It is well-established that a large contribution to protein stability originates from the sequestration from solvent of hydrophobic residues in the protein core. How are such hydrophobic residues arranged in the core; how can one best model the packing of these residues, and are residues loosely packed with multiple allowed side chain conformations or densely packed with a single allowed side chain conformation? Here we show that to properly model the packing of residues in protein cores it is essential that amino acids are represented by appropriately calibrated atom sizes, and that hydrogen atoms are explicitly included. We show that protein cores possess a packing fraction of φ ≈ 0.56 , which is significantly less than the typically quoted value of 0.74 obtained using the extended atom representation. We also compare the results for the packing of amino acids in protein cores to results obtained for jammed packings from discrete element simulations of spheres, elongated particles, and composite particles with bumpy surfaces. We show that amino acids in protein cores pack as densely as disordered jammed packings of particles with similar values for the aspect ratio and bumpiness as found for amino acids. Knowing the structural properties of protein cores is of both fundamental and practical importance. Practically, it enables the assessment of changes in the structure and stability of proteins arising from amino acid mutations (such as those identified as a result of the massive human genome sequencing efforts) and the design of new folded, stable proteins and protein-protein interactions with tunable specificity and affinity.

  7. The core and unique proteins of haloarchaea

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    Capes Melinda D

    2012-01-01

    Full Text Available Abstract Background Since the first genome of a halophilic archaeon was sequenced in 2000, biologists have been advancing the understanding of genomic characteristics that allow for survival in the harsh natural environments of these organisms. An increase in protein acidity and GC-bias in the genome have been implicated as factors in tolerance to extreme salinity, desiccation, and high solar radiation. However, few previous attempts have been made to identify novel genes that would permit survival in such extreme conditions. Results With the recent release of several new complete haloarchaeal genome sequences, we have conducted a comprehensive comparative genomic analysis focusing on the identification of unique haloarchaeal conserved proteins that likely play key roles in environmental adaptation. Using bioinformatic methods, we have clustered 31,312 predicted proteins from nine haloarchaeal genomes into 4,455 haloarchaeal orthologous groups (HOGs. We assigned likely functions by association with established COG and KOG databases in NCBI. After identifying homologs in four additional haloarchaeal genomes, we determined that there were 784 core haloarchaeal protein clusters (cHOGs, of which 83 clusters were found primarily in haloarchaea. Further analysis found that 55 clusters were truly unique (tucHOGs to haloarchaea and qualify as signature proteins while 28 were nearly unique (nucHOGs, the vast majority of which were coded for on the haloarchaeal chromosomes. Of the signature proteins, only one example with any predicted function, Ral, involved in desiccation/radiation tolerance in Halobacterium sp. NRC-1, was identified. Among the core clusters, 33% was predicted to function in metabolism, 25% in information transfer and storage, 10% in cell processes and signaling, and 22% belong to poorly characterized or general function groups. Conclusion Our studies have established conserved groups of nearly 800 protein clusters present in all

  8. Inferring ancient metabolism using ancestral core metabolic models of enterobacteria.

    Science.gov (United States)

    Baumler, David J; Ma, Bing; Reed, Jennifer L; Perna, Nicole T

    2013-06-11

    Enterobacteriaceae diversified from an ancestral lineage ~300-500 million years ago (mya) into a wide variety of free-living and host-associated lifestyles. Nutrient availability varies across niches, and evolution of metabolic networks likely played a key role in adaptation. Here we use a paleo systems biology approach to reconstruct and model metabolic networks of ancestral nodes of the enterobacteria phylogeny to investigate metabolism of ancient microorganisms and evolution of the networks. Specifically, we identified orthologous genes across genomes of 72 free-living enterobacteria (16 genera), and constructed core metabolic networks capturing conserved components for ancestral lineages leading to E. coli/Shigella (~10 mya), E. coli/Shigella/Salmonella (~100 mya), and all enterobacteria (~300-500 mya). Using these models we analyzed the capacity for carbon, nitrogen, phosphorous, sulfur, and iron utilization in aerobic and anaerobic conditions, identified conserved and differentiating catabolic phenotypes, and validated predictions by comparison to experimental data from extant organisms. This is a novel approach using quantitative ancestral models to study metabolic network evolution and may be useful for identification of new targets to control infectious diseases caused by enterobacteria.

  9. HBV Core Protein Enhances Cytokine Production

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    Tatsuo Kanda

    2015-09-01

    Full Text Available Hepatitis B virus (HBV infection, a cause of hepatocellular carcinoma (HCC, remains a serious global health concern. HCC development and human hepatocarcinogenesis are associated with hepatic inflammation caused by host interferons and cytokines. This article focused on the association between the HBV core protein, which is one of the HBV-encoding proteins, and cytokine production. The HBV core protein induced the production of interferons and cytokines in human hepatoma cells and in a mouse model. These factors may be responsible for persistent HBV infection and hepatocarcinogenesis. Inhibitors of programmed death (PD-1 and HBV core and therapeutic vaccines including HBV core might be useful for the treatment of patients with chronic HBV infection. Inhibitors of HBV core, which is important for hepatic inflammation, could be helpful in preventing the progression of liver diseases in HBV-infected patients.

  10. Metabolism of biologics: biotherapeutic proteins.

    Science.gov (United States)

    Hamuro, Lora L; Kishnani, Narendra S

    2012-01-01

    Recombinant therapeutic protein drugs have now been in clinical use for nearly three decades and have advanced considerably in complexity over this time period. Regulatory approvals of some early pioneering protein drugs did not require characterization of metabolism, but more recently regulatory expectations and guidance have appropriately evolved. Sponsors may now be expected to investigate metabolism of newer biologics as the structural complexity of proteins has increased markedly, particularly with the introduction of conjugated and modified proteins. This review discusses the value and need for metabolite characterization of some therapeutic proteins by presenting select examples. Regulatory expectations will undoubtedly evolve further with the development of other novel macromolecular biologic therapeutics based on modified nucleic acids, novel conjugated lipids and polysaccharides.

  11. Core-shell microparticles for protein sequestration and controlled release of a protein-laden core.

    Science.gov (United States)

    Rinker, Torri E; Philbrick, Brandon D; Temenoff, Johnna S

    2017-07-01

    Development of multifunctional biomaterials that sequester, isolate, and redeliver cell-secreted proteins at a specific timepoint may be required to achieve the level of temporal control needed to more fully regulate tissue regeneration and repair. In response, we fabricated core-shell heparin-poly(ethylene-glycol) (PEG) microparticles (MPs) with a degradable PEG-based shell that can temporally control delivery of protein-laden heparin MPs. Core-shell MPs were fabricated via a re-emulsification technique and the number of heparin MPs per PEG-based shell could be tuned by varying the mass of heparin MPs in the precursor PEG phase. When heparin MPs were loaded with bone morphogenetic protein-2 (BMP-2) and then encapsulated into core-shell MPs, degradable core-shell MPs initiated similar C2C12 cell alkaline phosphatase (ALP) activity as the soluble control, while non-degradable core-shell MPs initiated a significantly lower response (85+19% vs. 9.0+4.8% of the soluble control, respectively). Similarly, when degradable core-shell MPs were formed and then loaded with BMP-2, they induced a ∼7-fold higher C2C12 ALP activity than the soluble control. As C2C12 ALP activity was enhanced by BMP-2, these studies indicated that degradable core-shell MPs were able to deliver a bioactive, BMP-2-laden heparin MP core. Overall, these dynamic core-shell MPs have the potential to sequester, isolate, and then redeliver proteins attached to a heparin core to initiate a cell response, which could be of great benefit to tissue regeneration applications requiring tight temporal control over protein presentation. Tissue repair requires temporally controlled presentation of potent proteins. Recently, biomaterial-mediated binding (sequestration) of cell-secreted proteins has emerged as a strategy to harness the regenerative potential of naturally produced proteins, but this strategy currently only allows immediate amplification and re-delivery of these signals. The multifunctional, dynamic

  12. The Activity Reaction Core and Plasticity of Metabolic Networks.

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    2005-12-01

    Full Text Available Understanding the system-level adaptive changes taking place in an organism in response to variations in the environment is a key issue of contemporary biology. Current modeling approaches, such as constraint-based flux-balance analysis, have proved highly successful in analyzing the capabilities of cellular metabolism, including its capacity to predict deletion phenotypes, the ability to calculate the relative flux values of metabolic reactions, and the capability to identify properties of optimal growth states. Here, we use flux-balance analysis to thoroughly assess the activity of Escherichia coli, Helicobacter pylori, and Saccharomyces cerevisiae metabolism in 30,000 diverse simulated environments. We identify a set of metabolic reactions forming a connected metabolic core that carry non-zero fluxes under all growth conditions, and whose flux variations are highly correlated. Furthermore, we find that the enzymes catalyzing the core reactions display a considerably higher fraction of phenotypic essentiality and evolutionary conservation than those catalyzing noncore reactions. Cellular metabolism is characterized by a large number of species-specific conditionally active reactions organized around an evolutionary conserved, but always active, metabolic core. Finally, we find that most current antibiotics interfering with bacterial metabolism target the core enzymes, indicating that our findings may have important implications for antimicrobial drug-target discovery.

  13. Differential Stoichiometry among Core Ribosomal Proteins

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    Nikolai Slavov

    2015-11-01

    Full Text Available Understanding the regulation and structure of ribosomes is essential to understanding protein synthesis and its dysregulation in disease. While ribosomes are believed to have a fixed stoichiometry among their core ribosomal proteins (RPs, some experiments suggest a more variable composition. Testing such variability requires direct and precise quantification of RPs. We used mass spectrometry to directly quantify RPs across monosomes and polysomes of mouse embryonic stem cells (ESC and budding yeast. Our data show that the stoichiometry among core RPs in wild-type yeast cells and ESC depends both on the growth conditions and on the number of ribosomes bound per mRNA. Furthermore, we find that the fitness of cells with a deleted RP-gene is inversely proportional to the enrichment of the corresponding RP in polysomes. Together, our findings support the existence of ribosomes with distinct protein composition and physiological function.

  14. Differential Stoichiometry among Core Ribosomal Proteins

    Science.gov (United States)

    Slavov, Nikolai; Semrau, Stefan; Airoldi, Edoardo; Budnik, Bogdan; van Oudenaarden, Alexander

    2015-01-01

    Summary Understanding the regulation and structure of ribosomes is essential to understanding protein synthesis and its dysregulation in disease. While ribosomes are believed to have a fixed stoichiometry among their core ribosomal proteins (RPs), some experiments suggest a more variable composition. Testing such variability requires direct and precise quantification of RPs. We used mass spectrometry to directly quantify RPs across monosomes and polysomes of mouse embryonic stem cells (ESC) and budding yeast. Our data show that the stoichiometry among core RPs in wild-type yeast cells and ESC depends both on the growth conditions and on the number of ribosomes bound per mRNA. Furthermore, we find that the fitness of cells with a deleted RP-gene is inversely proportional to the enrichment of the corresponding RP in polysomes. Together, our findings support the existence of ribosomes with distinct protein composition and physiological function. PMID:26565899

  15. Exploration of the core metabolism of symbiotic bacteria

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    Klein Cecilia Coimbra

    2012-08-01

    Full Text Available Abstract Background A large number of genome-scale metabolic networks is now available for many organisms, mostly bacteria. Previous works on minimal gene sets, when analysing host-dependent bacteria, found small common sets of metabolic genes. When such analyses are restricted to bacteria with similar lifestyles, larger portions of metabolism are expected to be shared and their composition is worth investigating. Here we report a comparative analysis of the small molecule metabolism of symbiotic bacteria, exploring common and variable portions as well as the contribution of different lifestyle groups to the reduction of a common set of metabolic capabilities. Results We found no reaction shared by all the bacteria analysed. Disregarding those with the smallest genomes, we still do not find a reaction core, however we did find a core of biochemical capabilities. While obligate intracellular symbionts have no core of reactions within their group, extracellular and cell-associated symbionts do have a small core composed of disconnected fragments. In agreement with previous findings in Escherichia coli, their cores are enriched in biosynthetic processes whereas the variable metabolisms have similar ratios of biosynthetic and degradation reactions. Conversely, the variable metabolism of obligate intracellular symbionts is enriched in anabolism. Conclusion Even when removing the symbionts with the most reduced genomes, there is no core of reactions common to the analysed symbiotic bacteria. The main reason is the very high specialisation of obligate intracellular symbionts, however, host-dependence alone is not an explanation for such absence. The composition of the metabolism of cell-associated and extracellular bacteria shows that while they have similar needs in terms of the building blocks of their cells, they have to adapt to very distinct environments. On the other hand, in obligate intracellular bacteria, catabolism has largely disappeared

  16. Protein coalitions in a core mammalian biochemical network linked by rapidly evolving proteins

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    Tsoka Sophia

    2011-05-01

    Full Text Available Abstract Background Cellular ATP levels are generated by glucose-stimulated mitochondrial metabolism and determine metabolic responses, such as glucose-stimulated insulin secretion (GSIS from the β-cells of pancreatic islets. We describe an analysis of the evolutionary processes affecting the core enzymes involved in glucose-stimulated insulin secretion in mammals. The proteins involved in this system belong to ancient enzymatic pathways: glycolysis, the TCA cycle and oxidative phosphorylation. Results We identify two sets of proteins, or protein coalitions, in this group of 77 enzymes with distinct evolutionary patterns. Members of the glycolysis, TCA cycle, metabolite transport, pyruvate and NADH shuttles have low rates of protein sequence evolution, as inferred from a human-mouse comparison, and relatively high rates of evolutionary gene duplication. Respiratory chain and glutathione pathway proteins evolve faster, exhibiting lower rates of gene duplication. A small number of proteins in the system evolve significantly faster than co-pathway members and may serve as rapidly evolving adapters, linking groups of co-evolving genes. Conclusions Our results provide insights into the evolution of the involved proteins. We find evidence for two coalitions of proteins and the role of co-adaptation in protein evolution is identified and could be used in future research within a functional context.

  17. redGEM: Systematic reduction and analysis of genome-scale metabolic reconstructions for development of consistent core metabolic models.

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    Meric Ataman

    2017-07-01

    Full Text Available Genome-scale metabolic reconstructions have proven to be valuable resources in enhancing our understanding of metabolic networks as they encapsulate all known metabolic capabilities of the organisms from genes to proteins to their functions. However the complexity of these large metabolic networks often hinders their utility in various practical applications. Although reduced models are commonly used for modeling and in integrating experimental data, they are often inconsistent across different studies and laboratories due to different criteria and detail, which can compromise transferability of the findings and also integration of experimental data from different groups. In this study, we have developed a systematic semi-automatic approach to reduce genome-scale models into core models in a consistent and logical manner focusing on the central metabolism or subsystems of interest. The method minimizes the loss of information using an approach that combines graph-based search and optimization methods. The resulting core models are shown to be able to capture key properties of the genome-scale models and preserve consistency in terms of biomass and by-product yields, flux and concentration variability and gene essentiality. The development of these "consistently-reduced" models will help to clarify and facilitate integration of different experimental data to draw new understanding that can be directly extendable to genome-scale models.

  18. Whole body protein metabolism in chronic hemodialysis

    NARCIS (Netherlands)

    Veeneman, Jorden Marcus

    2004-01-01

    To summarise, evidence suggests that protein-energy malnutrition is associated with mor-bidity and mortality in chronic hemodialysis patients. Urea kinetics are used as a clinical marker for protein intake and oxidation. Chapter 2 focuses on the relation between pro-tein and urea metabolism, which

  19. Articulation of three core metabolic processes in Arabidopsis: Fatty acid biosynthesis, leucine catabolism and starch metabolism

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    Nikolau Basil J

    2008-07-01

    Full Text Available Abstract Background Elucidating metabolic network structures and functions in multicellular organisms is an emerging goal of functional genomics. We describe the co-expression network of three core metabolic processes in the genetic model plant Arabidopsis thaliana: fatty acid biosynthesis, starch metabolism and amino acid (leucine catabolism. Results These co-expression networks form modules populated by genes coding for enzymes that represent the reactions generally considered to define each pathway. However, the modules also incorporate a wider set of genes that encode transporters, cofactor biosynthetic enzymes, precursor-producing enzymes, and regulatory molecules. We tested experimentally the hypothesis that one of the genes tightly co-expressed with starch metabolism module, a putative kinase AtPERK10, will have a role in this process. Indeed, knockout lines of AtPERK10 have an altered starch accumulation. In addition, the co-expression data define a novel hierarchical transcript-level structure associated with catabolism, in which genes performing smaller, more specific tasks appear to be recruited into higher-order modules with a broader catabolic function. Conclusion Each of these core metabolic pathways is structured as a module of co-expressed transcripts that co-accumulate over a wide range of environmental and genetic perturbations and developmental stages, and represent an expanded set of macromolecules associated with the common task of supporting the functionality of each metabolic pathway. As experimentally demonstrated, co-expression analysis can provide a rich approach towards understanding gene function.

  20. Cytokines: muscle protein and amino acid metabolism

    DEFF Research Database (Denmark)

    van Hall, Gerrit

    2012-01-01

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

  1. Proteasome Activation by Hepatitis C Core Protein is Reversed by Ethanol-Induced Oxidative Stress

    Science.gov (United States)

    Osna, Natalia A.; White, Ronda L.; Krutik, Viatcheslav M.; Wang, Ting; Weinman, Steven A.; Donohue, Terrence M.

    2008-01-01

    Background and Aims The proteasome is a major cellular proteinase. Its activity is modulated by cellular oxidants. Hepatitis C core protein and ethanol exposure both cause enhanced oxidant generation. The aim was to investigate whether core protein, by its ability to generate oxidants, alters proteasome activity and whether these alterations are further affected byethanol exposure. Methods These interactions were examined in Huh-7 cell lines that expressed inducible HCV core protein and/or constitutive cytochrome P450 2E1 (CYP2E1) and as purified components in a cell free system. Chymotrypsin-like proteasome activity was measured fluorometrically. Results Proteasome activity in core-positive 191-20 cells was 20% higher than that in core-negative cells and was enhanced three-fold in CYP2E1-expressing L14 cells. Exposure of core-positive cells to glutathione ethyl ester, catalase, or the CYP2E1 inhibitor, DAS, partially reversed the elevation of proteasome activity in core-positive cells, while ethanol exposure suppressed proteasome activity. The results indicate that proteasome activity was up-regulated by low levels of core-induced oxidative stress and down-regulated by high levels of ethanol-elicited stress. These findings were partially mimicked in cell free system. Addition of core protein enhanced the peptidase activity of purified 20S proteasome containing the proteasome activator, PA28 and was further potentiated by addition of liver mitochondrial and/or microsome fractions. However, proteasome activation was significantly attenuated when fractions were obtained from ethanol-fed animals. Conclusions HCV core protein interacts with PA28, mitochondrial and ER proteins to cause low levels of oxidant stress and proteasome activation, which is dampened during ethanol metabolism when oxidant generation is higher. PMID:18549882

  2. Chimeric hepatitis B virus core particles with parts or copies of the hepatitis C virus core protein.

    OpenAIRE

    Yoshikawa, A.; Tanaka, T.; Hoshi, Y.; Kato, N; Tachibana, K; Iizuka, H; Machida, A; Okamoto, H.; Yamasaki, M; Miyakawa, Y

    1993-01-01

    Either parts or multiple copies of the core gene of hepatitis C virus (HCV) were fused to the 3' terminus of the hepatitis B virus (HBV) core gene with 34 codons removed. As many as four copies of HCV core protein (720 amino acids) were fused to the carboxy terminus of truncated HBV core protein (149 amino acids) without preventing the assembly of HBV core particles. Chimeric core particles were sandwiched between monoclonal antibody to HBV core and that to HCV core, thereby indicating that a...

  3. Modulation of mitogen-activated protein kinase-activated protein kinase 3 by hepatitis C virus core protein

    DEFF Research Database (Denmark)

    Ngo, HT; Pham, Long; Kim, JW

    2013-01-01

    Hepatitis C virus (HCV) is highly dependent on cellular proteins for its own propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assays using the HCV core protein as a probe. Of ~9,000 host proteins immobilized in a microarray...... inducers. Binding of HCV core to MAPKAPK3 was confirmed by in vitro pulldown assay and further verified by coimmunoprecipitation assay. HCV core protein interacted with MAPKAPK3 through amino acid residues 41 to 75 of core and the N-terminal half of kinase domain of MAPKAPK3. In addition, both RNA...... increased HCV IRES-mediated translation and MAPKAPK3-dependent HCV IRES activity was further increased by core protein. These data suggest that HCV core may modulate MAPKAPK3 to facilitate its own propagation....

  4. An analysis of core deformations in protein superfamilies.

    Science.gov (United States)

    Leo-Macias, Alejandra; Lopez-Romero, Pedro; Lupyan, Dmitry; Zerbino, Daniel; Ortiz, Angel R

    2005-02-01

    An analysis is presented on how structural cores modify their shape across homologous proteins, and whether or not a relationship exists between these structural changes and the vibrational normal modes that proteins experience as a result of the topological constraints imposed by the fold. A set of 35 representative, well-populated protein families is studied. The evolutionary directions of deformation are obtained by using multiple structural alignments to superimpose the structures and extract a conserved core, together with principal components analysis to extract the main deformation modes from the three-dimensional superimposition. In parallel, a low-resolution normal mode analysis technique is employed to study the properties of the mechanical core plasticity of these same families. We show that the evolutionary deformations span a low dimensional space of 4-5 dimensions on average. A statistically significant correspondence exists between these principal deformations and the approximately 20 slowest vibrational modes accessible to a particular topology. We conclude that, to a significant extent, the structural response of a protein topology to sequence changes takes place by means of collective deformations along combinations of a small number of low-frequency modes. The findings have implications in structure prediction by homology modeling.

  5. Relationship between asparagine metabolism and protein concentration in soybean seed

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    The relationship between asparagine metabolism and protein concentration was investigated in soybean seed. Phenotyping of a population of recombinant inbred lines adapted to Illinois confirmed a positive correlation between free asparagine levels in developing seeds and protein concentration at matu...

  6. Homogeneous protein analysis by magnetic core-shell nanorod probes

    KAUST Repository

    Schrittwieser, Stefan

    2016-03-29

    Studying protein interactions is of vital importance both to fundamental biology research and to medical applications. Here, we report on the experimental proof of a universally applicable label-free homogeneous platform for rapid protein analysis. It is based on optically detecting changes in the rotational dynamics of magnetically agitated core-shell nanorods upon their specific interaction with proteins. By adjusting the excitation frequency, we are able to optimize the measurement signal for each analyte protein size. In addition, due to the locking of the optical signal to the magnetic excitation frequency, background signals are suppressed, thus allowing exclusive studies of processes at the nanoprobe surface only. We study target proteins (soluble domain of the human epidermal growth factor receptor 2 - sHER2) specifically binding to antibodies (trastuzumab) immobilized on the surface of our nanoprobes and demonstrate direct deduction of their respective sizes. Additionally, we examine the dependence of our measurement signal on the concentration of the analyte protein, and deduce a minimally detectable sHER2 concentration of 440 pM. For our homogeneous measurement platform, good dispersion stability of the applied nanoprobes under physiological conditions is of vital importance. To that end, we support our measurement data by theoretical modeling of the total particle-particle interaction energies. The successful implementation of our platform offers scope for applications in biomarker-based diagnostics as well as for answering basic biology questions.

  7. Chemical reporter for visualizing metabolic cross-talk between carbohydrate metabolism and protein modification.

    Science.gov (United States)

    Zaro, Balyn W; Chuh, Kelly N; Pratt, Matthew R

    2014-09-19

    Metabolic chemical reporters have been largely used to study posttranslational modifications. Generally, it was assumed that these reporters entered one biosynthetic pathway, resulting in labeling of one type of modification. However, because they are metabolized by cells before their addition onto proteins, metabolic chemical reporters potentially provide a unique opportunity to read-out on both modifications of interest and cellular metabolism. We report here the development of a metabolic chemical reporter 1-deoxy-N-pentynyl glucosamine (1-deoxy-GlcNAlk). This small-molecule cannot be incorporated into glycans; however, treatment of mammalian cells results in labeling of a variety proteins and enables their visualization and identification. Competition of this labeling with sodium acetate and an acetyltransferase inhibitor suggests that 1-deoxy-GlcNAlk can enter the protein acetylation pathway. These results demonstrate that metabolic chemical reporters have the potential to isolate and potentially discover cross-talk between metabolic pathways in living cells.

  8. Protein design in systems metabolic engineering for industrial strain development.

    Science.gov (United States)

    Chen, Zhen; Zeng, An-Ping

    2013-05-01

    Accelerating the process of industrial bacterial host strain development, aimed at increasing productivity, generating new bio-products or utilizing alternative feedstocks, requires the integration of complementary approaches to manipulate cellular metabolism and regulatory networks. Systems metabolic engineering extends the concept of classical metabolic engineering to the systems level by incorporating the techniques used in systems biology and synthetic biology, and offers a framework for the development of the next generation of industrial strains. As one of the most useful tools of systems metabolic engineering, protein design allows us to design and optimize cellular metabolism at a molecular level. Here, we review the current strategies of protein design for engineering cellular synthetic pathways, metabolic control systems and signaling pathways, and highlight the challenges of this subfield within the context of systems metabolic engineering. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Ethanol metabolism modifies hepatic protein acylation in mice.

    Directory of Open Access Journals (Sweden)

    Kristofer S Fritz

    Full Text Available Mitochondrial protein acetylation increases in response to chronic ethanol ingestion in mice, and is thought to reduce mitochondrial function and contribute to the pathogenesis of alcoholic liver disease. The mitochondrial deacetylase SIRT3 regulates the acetylation status of several mitochondrial proteins, including those involved in ethanol metabolism. The newly discovered desuccinylase activity of the mitochondrial sirtuin SIRT5 suggests that protein succinylation could be an important post-translational modification regulating mitochondrial metabolism. To assess the possible role of protein succinylation in ethanol metabolism, we surveyed hepatic sub-cellular protein fractions from mice fed a control or ethanol-supplemented diet for succinyl-lysine, as well as acetyl-, propionyl-, and butyryl-lysine post-translational modifications. We found mitochondrial protein propionylation increases, similar to mitochondrial protein acetylation. In contrast, mitochondrial protein succinylation is reduced. These mitochondrial protein modifications appear to be primarily driven by ethanol metabolism, and not by changes in mitochondrial sirtuin levels. Similar trends in acyl modifications were observed in the nucleus. However, comparatively fewer acyl modifications were observed in the cytoplasmic or the microsomal compartments, and were generally unchanged by ethanol metabolism. Using a mass spectrometry proteomics approach, we identified several candidate acetylated, propionylated, and succinylated proteins, which were enriched using antibodies against each modification. Additionally, we identified several acetyl and propionyl lysine residues on the same sites for a number of proteins and supports the idea of the overlapping nature of lysine-specific acylation. Thus, we show that novel post-translational modifications are present in hepatic mitochondrial, nuclear, cytoplasmic, and microsomal compartments and ethanol ingestion, and its associated

  10. Quantitative Protein And Fat Metabolism In West African Dwarf ...

    African Journals Online (AJOL)

    Quantitative Protein And Fat Metabolism In West African Dwarf Sheep Fed Margaritaria Discoidea As Supplement. ... Animal Research International ... Protein and energy utilization and quantitative retention of protein, fat and energy was investigated with twelve castrated Djallonke sheep averaging (20.0 ± 2.2kg BW) in ...

  11. BCL-2 family proteins as regulators of mitochondria metabolism.

    Science.gov (United States)

    Gross, Atan

    2016-08-01

    The BCL-2 family proteins are major regulators of apoptosis, and one of their major sites of action are the mitochondria. Mitochondria are the cellular hubs for metabolism and indeed selected BCL-2 family proteins also possess roles related to mitochondria metabolism and dynamics. Here we discuss the link between mitochondrial metabolism/dynamics and the fate of stem cells, with an emphasis on the role of the BID-MTCH2 pair in regulating this link. We also discuss the possibility that BCL-2 family proteins act as metabolic sensors/messengers coming on and off of mitochondria to "sample" the cytosol and provide the mitochondria with up-to-date metabolic information. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Proteomic analysis of pear (Pyrus pyrifolia) ripening process provides new evidence for the sugar/acid metabolism difference between core and mesocarp.

    Science.gov (United States)

    Gao, Zhen; Zhang, Chengjun; Luo, Meng; Wu, Yusen; Duan, Shuyan; Li, Jiefa; Wang, Lei; Song, Shiren; Xu, Wenping; Wang, Shiping; Zhang, Caixi; Ma, Chao

    2016-12-01

    Pears are one of the most popular nutrient-rich fruits in the world. The pear core and mesocarp have significantly different metabolism, although they display similar profiles. Most strikingly, the core is more acidic in taste. Our results showed that there is more titrated acid but lower total soluble solids in the core compared to the mesocarp, and the content of citric acid was more than 17-fold higher in the core compared to the mesocarp at the ripening stage. Proteomics was used to investigate the difference between core and mesocarp tissues during "Cuiguan" pear ripening. Fifty-four different protein expression patterns were identified in the core and mesocarp. In general, common variably expressed proteins between the core and mesocarp were associated with important physiological processes, such as glycolysis, pyruvate metabolic processes, and oxidative stress. Further, protein level associated qRT-PCR verification revealed a higher abundance of fructose-bisphosphate aldolase and NADP-dependent malic enzymes, which may play a role in the low acid content in the mesocarp, whereas a higher abundance of disulfide isomerase-like 2-2 and calcium-dependent lipid-binding in the core may explain why it is less prone to accumulate sugar. The different levels of a few typical ROS scavenger enzymes suggested that oxidative stress is higher in the core than in the mesocarp. This study provides the first characterization of the pear core proteome and a description of its variation compared to the mesocarp during ripening. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. MANET: tracing evolution of protein architecture in metabolic networks

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    Caetano-Anollés Gustavo

    2006-07-01

    Full Text Available Abstract Background Cellular metabolism can be characterized by networks of enzymatic reactions and transport processes capable of supporting cellular life. Our aim is to find evolutionary patterns and processes embedded in the architecture and function of modern metabolism, using information derived from structural genomics. Description The Molecular Ancestry Network (MANET project traces evolution of protein architecture in biomolecular networks. We describe metabolic MANET, a database that links information in the Structural Classification of Proteins (SCOP, the Kyoto Encyclopedia of Genes and Genomes (KEGG, and phylogenetic reconstructions depicting the evolution of protein fold architecture. Metabolic MANET literally 'paints' the ancestries of enzymes derived from rooted phylogenomic trees directly onto over one hundred metabolic subnetworks, enabling the study of evolutionary patterns at global and local levels. An initial analysis of painted subnetworks reveals widespread enzymatic recruitment and an early origin of amino acid metabolism. Conclusion MANET maps evolutionary relationships directly and globally onto biological networks, and can generate and test hypotheses related to evolution of metabolism. We anticipate its use in the study of other networks, such as signaling and other protein-protein interaction networks.

  14. The Metabolic Core and Catalytic Switches Are Fundamental Elements in the Self-Regulation of the Systemic Metabolic Structure of Cells

    Science.gov (United States)

    De la Fuente, Ildefonso M.; Cortes, Jesus M.; Perez-Pinilla, Martin B.; Ruiz-Rodriguez, Vicente; Veguillas, Juan

    2011-01-01

    Background Experimental observations and numerical studies with dissipative metabolic networks have shown that cellular enzymatic activity self-organizes spontaneously leading to the emergence of a metabolic core formed by a set of enzymatic reactions which are always active under all environmental conditions, while the rest of catalytic processes are only intermittently active. The reactions of the metabolic core are essential for biomass formation and to assure optimal metabolic performance. The on-off catalytic reactions and the metabolic core are essential elements of a Systemic Metabolic Structure which seems to be a key feature common to all cellular organisms. Methodology/Principal Findings In order to investigate the functional importance of the metabolic core we have studied different catalytic patterns of a dissipative metabolic network under different external conditions. The emerging biochemical data have been analysed using information-based dynamic tools, such as Pearson's correlation and Transfer Entropy (which measures effective functionality). Our results show that a functional structure of effective connectivity emerges which is dynamical and characterized by significant variations of bio-molecular information flows. Conclusions/Significance We have quantified essential aspects of the metabolic core functionality. The always active enzymatic reactions form a hub –with a high degree of effective connectivity- exhibiting a wide range of functional information values being able to act either as a source or as a sink of bio-molecular causal interactions. Likewise, we have found that the metabolic core is an essential part of an emergent functional structure characterized by catalytic modules and metabolic switches which allow critical transitions in enzymatic activity. Both, the metabolic core and the catalytic switches in which also intermittently-active enzymes are involved seem to be fundamental elements in the self-regulation of the Systemic

  15. The metabolic core and catalytic switches are fundamental elements in the self-regulation of the systemic metabolic structure of cells.

    Directory of Open Access Journals (Sweden)

    Ildefonso M De la Fuente

    Full Text Available BACKGROUND: Experimental observations and numerical studies with dissipative metabolic networks have shown that cellular enzymatic activity self-organizes spontaneously leading to the emergence of a metabolic core formed by a set of enzymatic reactions which are always active under all environmental conditions, while the rest of catalytic processes are only intermittently active. The reactions of the metabolic core are essential for biomass formation and to assure optimal metabolic performance. The on-off catalytic reactions and the metabolic core are essential elements of a Systemic Metabolic Structure which seems to be a key feature common to all cellular organisms. METHODOLOGY/PRINCIPAL FINDINGS: In order to investigate the functional importance of the metabolic core we have studied different catalytic patterns of a dissipative metabolic network under different external conditions. The emerging biochemical data have been analysed using information-based dynamic tools, such as Pearson's correlation and Transfer Entropy (which measures effective functionality. Our results show that a functional structure of effective connectivity emerges which is dynamical and characterized by significant variations of bio-molecular information flows. CONCLUSIONS/SIGNIFICANCE: We have quantified essential aspects of the metabolic core functionality. The always active enzymatic reactions form a hub--with a high degree of effective connectivity--exhibiting a wide range of functional information values being able to act either as a source or as a sink of bio-molecular causal interactions. Likewise, we have found that the metabolic core is an essential part of an emergent functional structure characterized by catalytic modules and metabolic switches which allow critical transitions in enzymatic activity. Both, the metabolic core and the catalytic switches in which also intermittently-active enzymes are involved seem to be fundamental elements in the self

  16. Protein Metabolism in Senescing Wheat Leaves 1

    Science.gov (United States)

    Lamattina, Lorenzo; Lezica, Rafael Pont; Conde, Rubén D.

    1985-01-01

    Wheat leaves (Triticum aestivum L.) at the moment of their maximum expansion were detached and put in darkness. Their protein, RNA and DNA contents, as well as their rates of protein synthesis and degradation, were measured at different times from 0 to 5 days after detachment. Rates of protein synthesis were measured by incorporation into proteins of large amounts of [3H]leucine. Fractional rates of protein degradation were estimated either from the difference between the rates of synthesis and the net protein change or by the disappearance of radioactivity from proteins previously labeled with [3H]leucine or [14C]proline. Protein loss reached a value of 20% during the first 48 hours of the process. RNA loss paralleled that of protein, whereas DNA content proved to be almost constant during the first 3 days and decreased dramatically thereafter. Measurements of protein synthesis and degradation indicate that, in spite of a slowdown in rate of protein synthesis, an increased rate of protein breakdown is mainly responsible for the observed rapid protein loss. PMID:16664103

  17. Altered expression patterns of lipid metabolism genes in an animal model of HCV core-related, nonobese, modest hepatic steatosis

    Directory of Open Access Journals (Sweden)

    Chang Ming-Ling

    2008-02-01

    Full Text Available Abstract Background Because the gene expression patterns of nonobese hepatic steatosis in affected patients remain unclear, we sought to explore these patterns using an animal model of nonobese hepatic steatosis. Methods We developed mice that conditionally express the hepatitis C virus (HCV core protein regulated by the tetracycline transactivator (tTA. Microarray analyses and reverse-transcription polymerase chain reaction were performed using liver samples of both the double transgenic mice (DTM, which express both the HCV core and tTA, and single transgenic mice (STM, which express tTA alone, at 2 months of age. Functional categories of genes with altered expression were classified using gene ontology programs. Serum glucose, lipid levels, and systemic blood pressure were also measured. Results Approximately 20–30% of hepatocytes from the DTM were steatotic. No significant differences were observed in the serum glucose, lipid content, or blood pressure levels between the DTM and STM. Gene expression analyses revealed Sterol-regulatory element-binding protein (SREBP pathway activation and dysregulation of the following genes involved in lipid metabolism: 3-hydroxy-3-methylglutaryl-coenzyme A synthase 1, Apolipoprotein AII, Apolipoprotein CI, acyl-CoA thioesterase I, and fatty acid binding protein 1; in mitochondrial function: solute carrier family 25 member 25 and cytochrome c oxidase subunit II; in immune reaction: complement component 3, lymphocyte antigen 6 complex, locus A, lymphocyte antigen 6 complex, locus C, lymphocyte antigen 6 complex, locus D, and lymphocyte antigen 6 complex, locus E. Conclusion Some genes of lipid metabolism, mitochondrial function, and immune reaction and the SREBP pathway are involved in HCV core-related, nonobese, modest hepatic steatosis.

  18. The Expanded FindCore Method for Identification of a Core Atom Set for Assessment of Protein Structure Prediction

    Science.gov (United States)

    Snyder, David A.; Grullon, Jennifer; Huang, Yuanpeng J.; Tejero, Roberto; Montelione, Gaetano T.

    2014-01-01

    Maximizing the scientific impact of NMR-based structure determination requires robust and statistically sound methods for assessing the precision of NMR-derived structures. In particular, a method to define a core atom set for calculating superimpositions and validating structure predictions is critical to the use of NMR-derived structures as targets in the CASP competition. FindCore (D.A. Snyder and G.T. Montelione PROTEINS 2005;59:673–686) is a superimposition independent method for identifying a core atom set, and partitioning that set into domains. However, as FindCore optimizes superimposition by sensitively excluding not-well-defined atoms, the FindCore core may not comprise all atoms suitable for use in certain applications of NMR structures, including the CASP assessment process. Adapting the FindCore approach to assess predicted models against experimental NMR structures in CASP10 required modification of the FindCore method. This paper describes conventions and a standard protocol to calculate an “Expanded FindCore” atom set suitable for validation and application in biological and biophysical contexts. A key application of the Expanded FindCore method is to identify a core set of atoms in the experimental NMR structure for which it makes sense to validate predicted protein structure models. We demonstrate the application of this Expanded FindCore method in characterizing well-defined regions of 18 NMR-derived CASP10 target structures. The Expanded FindCore protocol defines “expanded core atom sets” that match an expert’s intuition of which parts of the structure are sufficiently well-defined to use in assessing CASP model predictions. We also illustrate the impact of this analysis on the CASP GDT assessment scores. PMID:24327305

  19. Both core and F proteins of hepatitis C virus could enhance cell proliferation in transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Wen-Ta [Graduate Institute of Medical Biotechnology, Tzu Chi University, Hualien, Taiwan (China); Li, Hui-Chun [Department of Biochemistry, Tzu Chi University, Hualien, Taiwan (China); Lee, Shen-Kao; Ma, Hsin-Chieh; Yang, Chee-Hing; Chen, Hung-Ling [Graduate Institute of Medical Biotechnology, Tzu Chi University, Hualien, Taiwan (China); Lo, Shih-Yen, E-mail: losylo@mail.tcu.edu.tw [Graduate Institute of Medical Biotechnology, Tzu Chi University, Hualien, Taiwan (China); Department of Laboratory Medicine, Buddhist Tzu Chi General Hospital, Hualien, Taiwan (China)

    2013-05-24

    Highlights: •HCV core and F proteins could induce hepatocyte proliferation in the transgenic mice. •β-Catenin signaling pathway was activated by core protein in the transgenic mice. •β-Catenin signaling pathway was activated by myc-F protein in the transgenic mice. •Expression of SMA protein was enhanced by core but not myc-F protein. -- Abstract: The role of the protein encoded by the alternative open reading frame (ARF/F/core+1) of the Hepatitis C virus (HCV) genome in viral pathogenesis remains unknown. The different forms of ARF/F/core+1 protein were labile in cultured cells, a myc-tag fused at the N-terminus of the F protein made it more stable. To determine the role of core and F proteins in HCV pathogenesis, transgenic mice with either protein expression under the control of Albumin promoter were generated. Expression of core protein and F protein with myc tag (myc-F) could be detected by Western blotting analysis in the livers of these mice. The ratio of liver to body weight is increased for both core and myc-F transgenic mice compared to that of wild type mice. Indeed, the proliferating cell nuclear antigen protein, a proliferation marker, was up-regulated in the transgenic mice with core or myc-F protein. Further analyses by microarray and Western blotting suggested that β-catenin signaling pathway was activated by either core or myc-F protein in the transgenic mice. These transgenic mice were further treated with either Diethynitrosamine (a tumor initiator) or Phenobarbital (a tumor promoter). Phenobarbital but not Diethynitrosamine treatment could increase the liver/body weight ratio of these mice. However, no tumor formation was observed in these mice. In conclusion, HCV core and myc-F proteins could induce hepatocyte proliferation in the transgenic mice possibly through β-catenin signaling pathway.

  20. The Membrane M Protein Carboxy Terminus Binds to Transmissible Gastroenteritis Coronavirus Core and Contributes to Core Stability

    Science.gov (United States)

    Escors, David; Ortego, Javier; Laude, Hubert; Enjuanes, Luis

    2001-01-01

    The architecture of transmissible gastroenteritis coronavirus includes three different structural levels, the envelope, an internal core, and the nucleocapsid that is released when the core is disrupted. Starting from purified virions, core structures have been reproducibly isolated as independent entities. The cores were stabilized at basic pH and by the presence of divalent cations, with Mg2+ ions more effectively contributing to core stability. Core structures showed high resistance to different concentrations of detergents, reducing agents, and urea and low concentrations of monovalent ions (<200 mM). Cores were composed of the nucleoprotein, RNA, and the C domain of the membrane (M) protein. At high salt concentrations (200 to 300 mM), the M protein was no longer associated with the nucleocapsid, which resulted in destruction of the core structure. A specific ionic interaction between the M protein carboxy terminus and the nucleocapsid was demonstrated using three complementary approaches: (i) a binding assay performed between a collection of M protein amino acid substitution or deletion mutants and purified nucleocapsids that led to the identification of a 16-amino-acid (aa) domain (aa 237 to 252) as being responsible for binding the M protein to the nucleocapsid; (ii) the specific inhibition of this binding by monoclonal antibodies (MAbs) binding to a carboxy-terminal M protein domain close to the indicated peptide but not by MAbs specific for the M protein amino terminus; and (iii) a 26-residue peptide, including the predicted sequence (aa 237 to 252), which specifically inhibited the binding. Direct binding of the M protein to the nucleoprotein was predicted, since degradation of the exposed RNA by RNase treatment did not affect the binding. It is proposed that the M protein is embedded within the virus membrane and that the C region, exposed to the interior face of the virion in a population of these molecules, interacts with the nucleocapsid to which it

  1. Effect of dietary protein restriction on renal ammonia metabolism

    Science.gov (United States)

    Lee, Hyun-Wook; Osis, Gunars; Handlogten, Mary E.; Guo, Hui; Verlander, Jill W.

    2015-01-01

    Dietary protein restriction has multiple benefits in kidney disease. Because protein intake is a major determinant of endogenous acid production, it is important that net acid excretion change in parallel during protein restriction. Ammonia is the primary component of net acid excretion, and inappropriate ammonia excretion can lead to negative nitrogen balance. Accordingly, we examined ammonia excretion in response to protein restriction and then we determined the molecular mechanism of the changes observed. Wild-type C57Bl/6 mice fed a 20% protein diet and then changed to 6% protein developed an 85% reduction in ammonia excretion within 2 days, which persisted during a 10-day study. The expression of multiple proteins involved in renal ammonia metabolism was altered, including the ammonia-generating enzymes phosphate-dependent glutaminase (PDG) and phosphoenolpyruvate carboxykinase (PEPCK) and the ammonia-metabolizing enzyme glutamine synthetase. Rhbg, an ammonia transporter, increased in expression in the inner stripe of outer medullary collecting duct intercalated cell (OMCDis-IC). However, collecting duct-specific Rhbg deletion did not alter the response to protein restriction. Rhcg deletion did not alter ammonia excretion in response to dietary protein restriction. These results indicate 1) dietary protein restriction decreases renal ammonia excretion through coordinated regulation of multiple components of ammonia metabolism; 2) increased Rhbg expression in the OMCDis-IC may indicate a biological role in addition to ammonia transport; and 3) Rhcg expression is not necessary to decrease ammonia excretion during dietary protein restriction. PMID:25925252

  2. Dietary protein, physiological condition and metabolic amino acid utilisation

    NARCIS (Netherlands)

    Weijs, P.J.M.

    1993-01-01

    This thesis describes the investigated effects of the level of dietary protein intake and the physiological condition of the animal on the percental oxidation of leucine. This measure reflects which part of the free leucine pool was used for protein and energy metabolism. The employed

  3. Comparative genome-scale metabolic modeling of actinomycetes : The topology of essential core metabolism

    NARCIS (Netherlands)

    Alam, Mohammad Tauqeer; Medema, Marnix H.; Takano, Eriko; Breitling, Rainer; Gojobori, Takashi

    2011-01-01

    Actinomycetes are highly important bacteria. On one hand, some of them cause severe human and plant diseases, on the other hand, many species are known for their ability to produce antibiotics. Here we report the results of a comparative analysis of genome-scale metabolic models of 37 species of

  4. Liver and muscle protein metabolism in cachexia

    NARCIS (Netherlands)

    Peters, J.A.C.

    2009-01-01

    Up to 50% of cancer patients suffer from progressive weight loss (cachexia). Cachexia is induced by proinflammatory mediators (cytokines), induced by the tumor’s presence. These cytokines induce so-called acute phase protein synthesis by the liver, followed by skeletal muscle protein breakdown.

  5. Amine Metabolism Is Influenced by Dietary Protein Source

    Directory of Open Access Journals (Sweden)

    Soumya K. Kar

    2017-09-01

    Full Text Available Growth in world population will inevitably leads to increased demand for protein for humans and animals. Protein from insects and blood plasma are being considered as possible alternatives, but more research on their nutritional quality and health effects is needed. Here, we studied the effect of dietary protein source on metabolism and metabolic amine profiles in serum and urine of mice. Groups of mice were fed semi-purified diets containing 300 g/kg of soybean meal, casein, partially delactosed whey powder, spray-dried plasma protein, wheat gluten meal, and yellow mealworm. Feed and water intake as well as body weight gain were measured for 28 days. After 14 and 28 days, serum and urine samples were collected for measurement of a large panel of amine metabolites. MetaboAnalyst 3.0 was used for analysis of the raw metabolic data. Out of 68 targeted amine metabolites, we could detect 54 in urine and 41 in blood serum. Dietary protein sources were found to have profound effects on host metabolism, particularly in systemic amine profiles, considered here as an endophenotype. We recommend serum over urine to screen for the amine metabolic endophenotype based on partial least squares discriminant analysis. We concluded that metabolites like alpha-aminobutyric acid and 1-methylhistidine are sensitive indicators of too much or too little availability of specific amino acids in the different protein diets. Furthermore, we concluded that amine metabolic profiles can be useful for assessing the nutritional quality of different protein sources.

  6. Amine Metabolism Is Influenced by Dietary Protein Source.

    Science.gov (United States)

    Kar, Soumya K; Jansman, Alfons J M; Schokker, Dirkjan; Kruijt, Leo; Harms, Amy C; Wells, Jerry M; Smits, Mari A

    2017-01-01

    Growth in world population will inevitably leads to increased demand for protein for humans and animals. Protein from insects and blood plasma are being considered as possible alternatives, but more research on their nutritional quality and health effects is needed. Here, we studied the effect of dietary protein source on metabolism and metabolic amine profiles in serum and urine of mice. Groups of mice were fed semi-purified diets containing 300 g/kg of soybean meal, casein, partially delactosed whey powder, spray-dried plasma protein, wheat gluten meal, and yellow mealworm. Feed and water intake as well as body weight gain were measured for 28 days. After 14 and 28 days, serum and urine samples were collected for measurement of a large panel of amine metabolites. MetaboAnalyst 3.0 was used for analysis of the raw metabolic data. Out of 68 targeted amine metabolites, we could detect 54 in urine and 41 in blood serum. Dietary protein sources were found to have profound effects on host metabolism, particularly in systemic amine profiles, considered here as an endophenotype. We recommend serum over urine to screen for the amine metabolic endophenotype based on partial least squares discriminant analysis. We concluded that metabolites like alpha-aminobutyric acid and 1-methylhistidine are sensitive indicators of too much or too little availability of specific amino acids in the different protein diets. Furthermore, we concluded that amine metabolic profiles can be useful for assessing the nutritional quality of different protein sources.

  7. HSC90 is required for nascent hepatitis C virus core protein stability in yeast cells.

    Science.gov (United States)

    Kubota, Naoko; Inayoshi, Yasutaka; Satoh, Naoko; Fukuda, Takashi; Iwai, Kenta; Tomoda, Hiroshi; Kohara, Michinori; Kataoka, Kazuhiro; Shimamoto, Akira; Furuichi, Yasuhiro; Nomoto, Akio; Naganuma, Akira; Kuge, Shusuke

    2012-07-30

    Hepatitis C virus core protein (Core) contributes to HCV pathogenicity. Here, we demonstrate that Core impairs growth in budding yeast. We identify HSP90 inhibitors as compounds that reduce intracellular Core protein level and restore yeast growth. Our results suggest that HSC90 (Hsc82) may function in the protection of the nascent Core polypeptide against degradation in yeast and the C-terminal region of Core corresponding to the organelle-interaction domain was responsible for Hsc82-dependent stability. The yeast system may be utilized to select compounds that can direct the C-terminal region to reduce the stability of Core protein. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  8. The role of whey acidic protein four-disulfide-core proteins in respiratory health and disease.

    Science.gov (United States)

    Small, Donna M; Doherty, Declan F; Dougan, Caoifa M; Weldon, Sinéad; Taggart, Clifford C

    2017-04-01

    Members of the whey acidic protein (WAP) or WAP four-disulfide-core (WFDC) family of proteins are a relatively under-explored family of low molecular weight proteins. The two most prominent WFDC proteins, secretory leukocyte protease inhibitor (SLPI) and elafin (or the precursor, trappin-2), have been shown to possess multiple functions including anti-protease, anti-bacterial, anti-viral and anti-inflammatory properties. It is therefore of no surprise that both SLPI and elafin/trappin-2 have been developed as potential therapeutics. Given the abundance of SLPI and elafin/trappin-2 in the human lung, most work in the area of WFDC research has focused on the role of WFDC proteins in protecting the lung from proteolytic attack. In this review, we will outline the current evidence regarding the expanding role of WFDC protein function with a focus on WFDC activity in lung disease as well as emerging data regarding the function of some of the more recently described WFDC proteins.

  9. Identification of a functional, CRM-1-dependent nuclear export signal in hepatitis C virus core protein.

    Directory of Open Access Journals (Sweden)

    Andrea Cerutti

    Full Text Available Hepatitis C virus (HCV infection is a major cause of chronic liver disease worldwide. HCV core protein is involved in nucleocapsid formation, but it also interacts with multiple cytoplasmic and nuclear molecules and plays a crucial role in the development of liver disease and hepatocarcinogenesis. The core protein is found mostly in the cytoplasm during HCV infection, but also in the nucleus in patients with hepatocarcinoma and in core-transgenic mice. HCV core contains nuclear localization signals (NLS, but no nuclear export signal (NES has yet been identified.We show here that the aa(109-133 region directs the translocation of core from the nucleus to the cytoplasm by the CRM-1-mediated nuclear export pathway. Mutagenesis of the three hydrophobic residues (L119, I123 and L126 in the identified NES or in the sequence encoding the mature core aa(1-173 significantly enhanced the nuclear localisation of the corresponding proteins in transfected Huh7 cells. Both the NES and the adjacent hydrophobic sequence in domain II of core were required to maintain the core protein or its fragments in the cytoplasmic compartment. Electron microscopy studies of the JFH1 replication model demonstrated that core was translocated into the nucleus a few minutes after the virus entered the cell. The blockade of nucleocytoplasmic export by leptomycin B treatment early in infection led to the detection of core protein in the nucleus by confocal microscopy and coincided with a decrease in virus replication.Our data suggest that the functional NLS and NES direct HCV core protein shuttling between the cytoplasmic and nuclear compartments, with at least some core protein transported to the nucleus. These new properties of HCV core may be essential for virus multiplication and interaction with nuclear molecules, influence cell signaling and the pathogenesis of HCV infection.

  10. Identification of a functional, CRM-1-dependent nuclear export signal in hepatitis C virus core protein.

    Science.gov (United States)

    Cerutti, Andrea; Maillard, Patrick; Minisini, Rosalba; Vidalain, Pierre-Olivier; Roohvand, Farzin; Pecheur, Eve-Isabelle; Pirisi, Mario; Budkowska, Agata

    2011-01-01

    Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. HCV core protein is involved in nucleocapsid formation, but it also interacts with multiple cytoplasmic and nuclear molecules and plays a crucial role in the development of liver disease and hepatocarcinogenesis. The core protein is found mostly in the cytoplasm during HCV infection, but also in the nucleus in patients with hepatocarcinoma and in core-transgenic mice. HCV core contains nuclear localization signals (NLS), but no nuclear export signal (NES) has yet been identified.We show here that the aa(109-133) region directs the translocation of core from the nucleus to the cytoplasm by the CRM-1-mediated nuclear export pathway. Mutagenesis of the three hydrophobic residues (L119, I123 and L126) in the identified NES or in the sequence encoding the mature core aa(1-173) significantly enhanced the nuclear localisation of the corresponding proteins in transfected Huh7 cells. Both the NES and the adjacent hydrophobic sequence in domain II of core were required to maintain the core protein or its fragments in the cytoplasmic compartment. Electron microscopy studies of the JFH1 replication model demonstrated that core was translocated into the nucleus a few minutes after the virus entered the cell. The blockade of nucleocytoplasmic export by leptomycin B treatment early in infection led to the detection of core protein in the nucleus by confocal microscopy and coincided with a decrease in virus replication.Our data suggest that the functional NLS and NES direct HCV core protein shuttling between the cytoplasmic and nuclear compartments, with at least some core protein transported to the nucleus. These new properties of HCV core may be essential for virus multiplication and interaction with nuclear molecules, influence cell signaling and the pathogenesis of HCV infection.

  11. The retrovirus MA and PreTM proteins follow immature MVL cores

    DEFF Research Database (Denmark)

    Andersen, Klaus Bahl

    2013-01-01

    Detergent can dissolve retrovirus, exept the immature core. Here we show that the Matrix protein (MA) and the Transmembrane protein in its immature form (PreTM) bind to the retrovirus core. These attachments explain the attachment in the virus particle and the dynamics of the ability to fuse...

  12. Regulation of intestinal protein metabolism by amino acids.

    Science.gov (United States)

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

    2013-09-01

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

  13. Efficient protein production by yeast requires global tuning of metabolism

    DEFF Research Database (Denmark)

    Huang, Mingtao; Bao, Jichen; Hallstrom, Bjorn M.

    2017-01-01

    intracellular processes with many underlying mechanisms still remaining unclear. Here, we use RNA-seq to study the genome-wide transcriptional response to protein secretion in mutant yeast strains. We find that many cellular processes have to be attuned to support efficient protein secretion. In particular......, altered energy metabolism resulting in reduced respiration and increased fermentation, as well as balancing of amino-acid biosynthesis and reduced thiamine biosynthesis seem to be particularly important. We confirm our findings by inverse engineering and physiological characterization and show...... that by tuning metabolism cells are able to efficiently secrete recombinant proteins. Our findings provide increased understanding of which cellular regulations and pathways are associated with efficient protein secretion....

  14. Protein acetylation and acetyl coenzyme a metabolism in budding yeast.

    Science.gov (United States)

    Galdieri, Luciano; Zhang, Tiantian; Rogerson, Daniella; Lleshi, Rron; Vancura, Ales

    2014-12-01

    Cells sense and appropriately respond to the physical conditions and availability of nutrients in their environment. This sensing of the environment and consequent cellular responses are orchestrated by a multitude of signaling pathways and typically involve changes in transcription and metabolism. Recent discoveries suggest that the signaling and transcription machineries are regulated by signals which are derived from metabolism and reflect the metabolic state of the cell. Acetyl coenzyme A (CoA) is a key metabolite that links metabolism with signaling, chromatin structure, and transcription. Acetyl-CoA is produced by glycolysis as well as other catabolic pathways and used as a substrate for the citric acid cycle and as a precursor in synthesis of fatty acids and steroids and in other anabolic pathways. This central position in metabolism endows acetyl-CoA with an important regulatory role. Acetyl-CoA serves as a substrate for lysine acetyltransferases (KATs), which catalyze the transfer of acetyl groups to the epsilon-amino groups of lysines in histones and many other proteins. Fluctuations in the concentration of acetyl-CoA, reflecting the metabolic state of the cell, are translated into dynamic protein acetylations that regulate a variety of cell functions, including transcription, replication, DNA repair, cell cycle progression, and aging. This review highlights the synthesis and homeostasis of acetyl-CoA and the regulation of transcriptional and signaling machineries in yeast by acetylation. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  15. The dynamics of fat, protein and sugar metabolism during walnut ...

    African Journals Online (AJOL)

    Walnut (Juglans regia L.) is named the “super food” in the 21st century. In this study, the 9-year-old precocious walnut cultivar 'Xiangling' were used to exam the developmental process, and the dynamics of fat, protein and sugar content in the fruit, and the activities of enzymes related with sugar metabolism were further ...

  16. Regulation of lipid metabolism by angiopoietin-like proteins

    NARCIS (Netherlands)

    Dijk, Wieneke; Kersten, Sander

    2016-01-01

    PURPOSE OF REVIEW: The angiopoietin-like proteins (ANGPTLs) 3, 4 and 8 have emerged as key regulators of plasma lipid metabolism by serving as potent inhibitors of the enzyme lipoprotein lipase (LPL). In this review, we provide an integrated picture of the role of ANGPTL3, ANGPTL4 and ANGPTL8 in

  17. HCV core protein promotes hepatocyte proliferation and chemoresistance by inhibiting NR4A1

    Energy Technology Data Exchange (ETDEWEB)

    Tan, Yongsheng, E-mail: yongshengtanwhu@126.com; Li, Yan, E-mail: liyansd2@163.com

    2015-10-23

    This study investigated the effect of HCV core protein on the proliferation of hepatocytes and hepatocellular carcinoma cells (HCC), the influence of HCV core protein on HCC apoptosis induced by the chemotherapeutic agent cisplatin, and the mechanism through which HCV core protein acts as a potential oncoprotein in HCV-related HCC by measuring the levels of NR4A1 and Runt-related transcription factor 3 (RUNX3), which are associated with tumor suppression and chemotherapy resistance. In the present study, PcDNA3.1-core and RUNX3 siRNA were transfected into LO2 and HepG2 cells using Lipofectamine 2000. LO2-core, HepG2-core, LO2-RUNX3 {sup low} and control cells were treated with different concentrations of cisplatin for 72 h, and cell proliferation and apoptosis were assayed using the CellTiter 96{sup ®}Aqueous Non-Radioactive Cell Proliferation Assay Kit. Western blot and real time PCR analyses were used to detect NR4A1, RUNX3, smad7, Cyclin D1 and BAX. Confocal microscopy was used to determine the levels of NR4A1 in HepG2 and HepG2-core cells. The growth rate of HepG2-core cells was considerably greater than that of HepG2 cells. HCV core protein increased the expression of cyclin D1 and decreased the expressions of NR4A1 and RUNX3. In LO2 – RUNX3 {sup low}, the rate of cell proliferation and the level of cisplatin resistance were the same as in the LO2 -core. These results suggest that HCV core protein decreases the sensitivity of hepatocytes to cisplatin by inhibiting the expression of NR4A1 and promoting the expression of smad7, which negatively regulates the TGF-β pathway. This effect results in down regulation of RUNX3, a target of the TGF-β pathway. Taken together, these findings indicate that in hepatocytes, HCV core protein increases drug resistance and inhibits cell apoptosis by inhibiting the expressions of NR4A1 and RUNX3. - Highlights: • HCV core protein inhibits HepG2 cell sensitivity to cisplatin. • Core expression in HepG2 decreases

  18. Hepatic inflammation mediated by hepatitis C virus core protein is ameliorated by blocking complement activation

    Directory of Open Access Journals (Sweden)

    Hsu Chen-Ming

    2009-08-01

    Full Text Available Abstract Background The pathogenesis of inflammation and fibrosis in chronic hepatitis C virus (HCV infection remains unclear. Transgenic mice with constitutive HCV core over-expression display steatosis only. While the reasons for this are unclear, it may be important that core protein production in these models begins during gestation, in contrast to human hepatitis C virus infection, which occurs post-natally and typically in adults. AIMS: To more realistically model the effect of core protein production in the adult liver, we developed a mouse with conditional expression of HCV core and examined the effect of core protein production in the adult liver. Methods Liver biopsy samples from transgenic mice with tetracycline(tet-regulated conditional core protein expression were evaluated immunohistologically. Microarray analysis of HCV core transgenic mice with steatohepatitis pointed to a role of the complement pathway. This was further explored by blocking complement activation by in vivo administration of CD55 (decay accelerating factor for complement, which inhibits activation of C3. Results Transgenic mice exhibited low, intermediate, or high HCV core protein expression when fed a permissive diet of standard chow. Aside from hepatic steatosis, hepatic inflammation and fibrosis were seen in mice with intermediate levels of core protein. Microarray analyses of inflamed liver demonstrated activation of both the complement (C3 up-regulation and coagulation pathways (fibrinogen B up-regulation. Administration of CD55 reduced hepatic inflammation. Conclusion Transgenic mice that conditionally express intermediate HCV core protein develop inflammation, steatosis, and fibrosis. These effects mediated by HCV core are reduced by administration of CD55, a regulator of the complement pathway. The model may be valuable in investigating the pathogenesis of liver inflammation in chronic hepatitis C.

  19. Bidirectional Lipid Droplet Velocities Are Controlled by Differential Binding Strengths of HCV Core DII Protein

    Science.gov (United States)

    Lyn, Rodney K.; Hope, Graham; Sherratt, Allison R.; McLauchlan, John; Pezacki, John Paul

    2013-01-01

    Host cell lipid droplets (LD) are essential in the hepatitis C virus (HCV) life cycle and are targeted by the viral capsid core protein. Core-coated LDs accumulate in the perinuclear region and facilitate viral particle assembly, but it is unclear how mobility of these LDs is directed by core. Herein we used two-photon fluorescence, differential interference contrast imaging, and coherent anti-Stokes Raman scattering microscopies, to reveal novel core-mediated changes to LD dynamics. Expression of core protein’s lipid binding domain II (DII-core) induced slower LD speeds, but did not affect directionality of movement on microtubules. Modulating the LD binding strength of DII-core further impacted LD mobility, revealing the temporal effects of LD-bound DII-core. These results for DII-core coated LDs support a model for core-mediated LD localization that involves core slowing down the rate of movement of LDs until localization at the perinuclear region is accomplished where LD movement ceases. The guided localization of LDs by HCV core protein not only is essential to the viral life cycle but also poses an interesting target for the development of antiviral strategies against HCV. PMID:24223760

  20. Metabolic engineering of Escherichia coli to improve recombinant protein production.

    Science.gov (United States)

    Liu, Min; Feng, Xinjun; Ding, Yamei; Zhao, Guang; Liu, Huizhou; Xian, Mo

    2015-12-01

    Escherichia coli is one of the most widely used strains for recombinant protein production. However, obstacles also exist in both academic researches and industrial applications, such as the metabolic burden, the carbon source waste, and the cells' physiological deterioration. This article reviews recent approaches for improving recombinant protein production in metabolic engineering, including workhorse selection, stress factor application, and carbon flux regulation. Selecting a suitable host is the first key point for recombinant protein production. In general, it all depends on characteristics of the strains and the target proteins. It will be triggered cells physiological deterioration when the medium is significantly different from the cell's natural environment. Coexpression of stress factors can help proteins to fold into their native conformation. Carbon flux regulation is a direct approach for redirecting more carbon flux toward the desirable pathways and products. However, some undesirable consequences are usually found in metabolic engineering, such as glucose transport inhibition, cell growth retardation, and useless metabolite accumulation. More efficient regulators and platform cell factories should be explored to meet a variety of production demands.

  1. FANCM-FAAP24 and FANCJ: FA proteins that metabolize DNA

    Energy Technology Data Exchange (ETDEWEB)

    Ali, Abdullah Mahmood; Singh, Thiyam Ramsing [Division of Experimental Hematology and Cancer Biology, Cincinnati Children' s Research Foundation, Cincinnati Children' s Hospital Medical Center, Cincinnati, OH 45229 (United States); Meetei, Amom Ruhikanta, E-mail: Ruhikanta.Meetei@cchmc.org [Division of Experimental Hematology and Cancer Biology, Cincinnati Children' s Research Foundation, Cincinnati Children' s Hospital Medical Center, Cincinnati, OH 45229 (United States); Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45229 (United States)

    2009-07-31

    Fanconi anemia (FA) is a rare autosomal recessive or X-linked disorder characterized by aplastic anemia, cancer susceptibility and cellular sensitivity to DNA-crosslinking agents. Eight FA proteins (FANCA, -B, -C, -E, -F, -G, -L and -M) and three non-FA proteins (FAAP100, FAAP24 and HES1) form the FA nuclear core complex that is required for monoubiquitination of the FANCD2-FANCI dimer upon DNA damage. The other three FA proteins, FANCD1/BRCA2, FANCJ/BACH1/BRIP1 and FANCN/PALB2, act in parallel or downstream of the FANCD2-FANCI dimer. Despite the isolation and characterization of several FA proteins, the mechanism by which these proteins protect cells from DNA interstrand crosslinking agents has been unclear. This is because a majority of the FA proteins lack any recognizable functional domains that can provide insight into their function. The recently discovered FANCM (Hef) and FANCJ (BRIP1/BACH1) proteins contain helicase domains, providing potential insight into the role of FA proteins in DNA repair. FANCM with its partner, FAAP24, and FANCJ bind and metabolize a variety of DNA substrates. In this review, we focus on the discovery, structure, and function of the FANCM-FAAP24 and FANCJ proteins.

  2. Both core and F proteins of hepatitis C virus could enhance cell proliferation in transgenic mice.

    Science.gov (United States)

    Hu, Wen-Ta; Li, Hui-Chun; Lee, Shen-Kao; Ma, Hsin-Chieh; Yang, Chee-Hing; Chen, Hung-Ling; Lo, Shih-Yen

    2013-05-24

    The role of the protein encoded by the alternative open reading frame (ARF/F/core+1) of the Hepatitis C virus (HCV) genome in viral pathogenesis remains unknown. The different forms of ARF/F/core+1 protein were labile in cultured cells, a myc-tag fused at the N-terminus of the F protein made it more stable. To determine the role of core and F proteins in HCV pathogenesis, transgenic mice with either protein expression under the control of Albumin promoter were generated. Expression of core protein and F protein with myc tag (myc-F) could be detected by Western blotting analysis in the livers of these mice. The ratio of liver to body weight is increased for both core and myc-F transgenic mice compared to that of wild type mice. Indeed, the proliferating cell nuclear antigen protein, a proliferation marker, was up-regulated in the transgenic mice with core or myc-F protein. Further analyses by microarray and Western blotting suggested that β-catenin signaling pathway was activated by either core or myc-F protein in the transgenic mice. These transgenic mice were further treated with either Diethynitrosamine (a tumor initiator) or Phenobarbital (a tumor promoter). Phenobarbital but not Diethynitrosamine treatment could increase the liver/body weight ratio of these mice. However, no tumor formation was observed in these mice. In conclusion, HCV core and myc-F proteins could induce hepatocyte proliferation in the transgenic mice possibly through β-catenin signaling pathway. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. Effects of hepatitis C virus core protein and nonstructural protein 4B on the Wnt/β-catenin pathway.

    Science.gov (United States)

    Jiang, Xiao-Hua; Xie, Yu-Tao; Cai, Ya-Ping; Ren, Jing; Ma, Tao

    2017-05-25

    Hepatitis C virus (HCV) core protein and nonstructural protein 4B (NS4B) are potentially oncogenic. Aberrant activation of the Wnt/β-catenin signaling pathway is closely associated with hepatocarcinogenesis. We investigated the effects of HCV type 1b core protein and NS4B on Wnt/β-catenin signaling in various liver cells, and explored the molecular mechanism underlying HCV-related hepatocarcinogenesis. Compared with the empty vector control, HCV core protein and NS4B demonstrated the following characteristics in the Huh7 cells: significantly enhanced β-catenin/Tcf-dependent transcriptional activity (F = 40.87, P  0.05), but they did significantly enhance Wnt3a-induced β-catenin/Tcf-dependent transcriptional activity (F = 64.25, P core protein than with NS4B (P core protein and NS4B directly activate the Wnt/β-catenin signaling pathway in Huh7 cells and LO2 cells induced by Wnt3a. These data suggest that HCV core protein and NS4B contribute to HCV-associated hepatocellular carcinogenesis.

  4. Hepatitis C virus core protein potentiates proangiogenic activity of hepatocellular carcinoma cells.

    Science.gov (United States)

    Shao, Yu-Yun; Hsieh, Min-Shu; Wang, Han-Yu; Li, Yong-Shi; Lin, Hang; Hsu, Hung-Wei; Huang, Chung-Yi; Hsu, Chih-Hung; Cheng, Ann-Lii

    2017-10-17

    Increased angiogenic activity has been demonstrated in hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC), but the mechanism was unclear. To study the role of HCV core protein, we used tube formation and Matrigel plug assays to assess the proangiogenic activity of an HCC cell line, HuH7, and 2 of its stable clones-HuH7-core-high and HuH7-core-low, with high and low HCV core protein expression, respectively. In both assays, HuH7-core-high and HuH7-core-low cells dose-dependently induced stronger angiogenesis than control cells. HuH7 cells with HCV core protein expression showed increased mRNA and protein expression of vascular endothelial growth factor (VEGF). VEGF inhibition by bevacizumab reduced the proangiogenic activity of HuH7-core-high cells. The promotor region of VEGF contains the binding site of activator protein-1 (AP-1). Compared with controls, HuH7-core-high cells had an increased AP-1 activity and nuclear localization of phospho-c-jun. AP-1 inhibition using either RNA knockdown or AP-1 inhibitors reduced the VEGF mRNA expression and the proangiogenic activity of HuH7-core-high cells. Among 131 tissue samples from HCC patients, HCV-related HCC revealed stronger VEGF expression than did hepatitis B virus-related HCC. In conclusion, increased VEGF expression through AP-1 activation is a crucial mechanism underlying the proangiogenic activity of the HCV core protein in HCC cells.

  5. The effect of HCV Core protein on the expression of miR-150

    Directory of Open Access Journals (Sweden)

    Sayad Khanizadeh

    2016-09-01

    Full Text Available Background : Hepatitis C virus (HCV is considered as one of the major pathogenic agents of chronic liver diseases. Previous studies have shown that HCV proteins can interaction with gene regulatory networks such as microRNAs. The aim of this study was to investigate the effect of HCV core protein on the expression of miR-150 in a cell culture model. Materials and Methods: Plasmids expressing full HCV core protein was transfected into Huh7 cell lines while a GFP expressing plasmid employed as negative control. Subsequently, total RNA extracted and Real-Time PCR performed to measure the expression level of miR-150 expression. Moreover, trypan blue exclusion assay was performed to investigate the effect of core protein on cell viability. Results: The gene expression analysis of miR-150 in Huh7 cells showed that endogenous HCV core protein could significantly down regulation of miR-150 when compared to GFP control plasmid and normal cells (P<0.01. Beside, core protein induced no significant proliferative or cytotoxic effects on hepatic cells as determined by trypan blue exclusion assay (P<0.05. Conclusion: Our study suggests that HCV core protein can led to down regulation of miR-150 expression. This data revealed that HCV protein interactions with cell regulatory machinery may contribute to pathogenesis of chronic liver diseases.

  6. Immunohistochemical localization of SNARE core proteins in intrapulpal and intradentinal nerve fibers of rat molar teeth.

    Science.gov (United States)

    Honma, Shiho; Kadono, Kohki; Kawano, Akiyo; Wakisaka, Satoshi

    2017-01-01

    The present study was designed to elucidate whether three soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) core proteins, syntaxin-1, synaptosomal-associated protein of 25kDa (SNAP-25), and vesicle-associated membrane protein-2 (VAMP-2), are present in the dental pulp of the rat molar at both the light and electron microscopic levels. Immunohistochemistry for protein gene product 9.5 (PGP 9.5), a pan-neuronal marker, syntaxin-1, SNAP-25, and VAMP-2 was performed on decalcified rat molars for light and electron microscopic analyses. Double-immunolabeling of PGP 9.5 and the SNARE core proteins, as well as combinations of the SNARE core proteins, was also carried out. PGP 9.5-immunoreactive nerve fibers ran toward the coronal region, ramified at the subodontoblast layer, and formed the subodontoblastic nerve plexus. Most nerve fibers penetrated the predentin and dentin along the dentinal tubules. Most, if not all, nerve fibers displayed immunoreactivity for syntaxin-1, SNAP-25, and VAMP-2. Immunoelectron microscopic analyses confirmed the presence of immunoreactivity for the SNARE core proteins within the intradental axonal elements. The present findings suggest that, since SNARE core proteins participate in the docking and exocytosis of synaptic vesicles in the central nervous system, they may contribute to vesicle exocytosis from the dental nerve fibers even though there are no apparent synapses. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Hepatitis B viral core protein disrupts human host gene expression by binding to promoter regions

    Directory of Open Access Journals (Sweden)

    Guo Yanhai

    2012-10-01

    Full Text Available Abstract Background The core protein (HBc of hepatitis B virus (HBV has been implicated in the malignant transformation of chronically-infected hepatocytes and displays pleiotropic functions, including RNA- and DNA-binding activities. However, the mechanism by which HBc interacts with the human genome to exert effects on hepatocyte function remains unknown. This study investigated the distribution of HBc binding to promoters in the human genome and evaluated its effects on the related genes’ expression. Results Whole-genome chromatin immunoprecipitation microarray (ChIP-on-chip analysis was used to identify HBc-bound human gene promoters. Gene Ontology and pathway analyses were performed on related genes. The quantitative polymerase chain reaction assay was used to verify ChIP-on-chip results. Five novel genes were selected for luciferase reporter assay evaluation to assess the influence of HBc promoter binding. The HBc antibody immunoprecipitated approximately 3100 human gene promoters. Among these, 1993 are associated with known biological processes, and 2208 regulate genes with defined molecular functions. In total, 1286 of the related genes mediate primary metabolic processes, and 1398 encode proteins with binding activity. Sixty-four of the promoters regulate genes related to the mitogen-activated protein kinase (MAPK pathways, and 41 regulate Wnt/beta-catenin pathway genes. The reporter gene assay indicated that HBc binding up-regulates proto-oncogene tyrosine-protein kinase (SRC, type 1 insulin-like growth factor receptor (IGF1R, and neurotrophic tyrosine kinase receptor 2 (NTRK2, and down-regulates v-Ha-ras Harvey rat sarcoma viral oncogene (HRAS. Conclusion HBc has the ability to bind a large number of human gene promoters, and can disrupt normal host gene expression. Manipulation of the transcriptional profile in HBV-infected hepatocytes may represent a key pathogenic mechanism of HBV infection.

  8. Protein and leucine metabolism in maple syrup urine disease

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, G.N.; Bresson, J.L.; Pacy, P.J.; Bonnefont, J.P.; Walter, J.H.; Leonard, J.V.; Saudubray, J.M.; Halliday, D. (Clinical Research Centre, Harrow (England))

    1990-04-01

    Constant infusions of (13C)leucine and (2H5)phenylalanine were used to trace leucine and protein kinetics, respectively, in seven children with maple syrup urine disease (MSUD) and eleven controls matched for age and dietary protein intake. Despite significant elevations of plasma leucine (mean 351 mumol/l, range 224-477) in MSUD subjects, mean whole body protein synthesis (3.78 +/- 0.42 (SD) g.kg-1. 24 h-1) and catabolism (4.07 +/- 0.46) were similar to control values (3.69 +/- 0.50 and 4.09 +/- 0.50, respectively). The relationship between phenylalanine and leucine fluxes was also similar in MSUD subjects (mean phenylalanine-leucine flux ratio 0.35 +/- 0.07) and previously reported adult controls (0.33 +/- 0.02). Leucine oxidation was undetectable in four of the MSUD subjects and very low in the other three (less than 4 mumol.kg-1.h-1; controls 13-20). These results show that persistent elevation in leucine concentration has no effect on protein synthesis. The marked disturbance in leucine metabolism in MSUD did not alter the relationship between rates of catabolism of protein to phenylalanine and leucine, which provides further support for the validity of the use of a single amino acid to trace whole body protein metabolism. The minimal leucine oxidation in MSUD differs from findings in other inborn metabolic errors and indicates that in patients with classical MSUD there is no significant route of leucine disposal other than through protein synthesis.

  9. Effect of soy protein/animal protein ratio on calcium metabolism of the rat.

    Science.gov (United States)

    Yoon, Gun Ae; Hwang, Hye Jin

    2006-04-01

    This study examined the effects of a ratio of soy protein to animal protein on bone metabolism of rats. Experimental groups were a high soy protein group (200 g of soy protein and 0 g of casein per kilogram of diet; HSoy), a middle soy protein group (100 g of soy protein and 100 g of casein per kilogram of diet; MSoy), a low soy protein group (50 g of soy protein and 150 g of casein per kilogram of diet; LSoy), and a no soy protein group (0 g of soy protein and 200 g of casein per kilogram of diet; NSoy). Calcium excretion and retention, biochemical parametrically related calcium metabolism, and bone mineral density were measured. Statistical analysis was performed with SAS software. Urinary excretion of calcium was significantly high in the LSoy and NSoy groups, and there was no difference in absorption of calcium across experimental groups. Calcium retention was significantly higher in the HSoy and MSoy groups than in the LSoy and NSoy groups. Experimental groups showed no differences in the activity of alkaline phosphatase. The casein group (NSoy) showed a remarkably lower degree of serum osteocalcin concentration. The concentration of deoxypyridinoline in urine showed an increasing tendency, i.e., HSoy and MSoy protein/soy proteins, its concentration increases. Wet weight of the femur appeared to be significantly greater in the MSoy and LSoy groups than in the NSoy group. Ash content of the femur and bone density were highest in the MSoy group, with the ratio of 1:1 between soy protein and animal protein. This study indicated that deoxypyridinoline concentration was lower and the density of osteocalcin was higher in the MSoy group than in the NSoy group, and that calcium retention was high and bone mineral density was the highest in the MSoy group. The ratio of soy to animal protein that seemed to have the most positively significant effect on calcium metabolism was 1:1.

  10. 3.5Å cryoEM structure of hepatitis B virus core assembled from full-length core protein.

    Directory of Open Access Journals (Sweden)

    Xuekui Yu

    Full Text Available The capsid shell of infectious hepatitis B virus (HBV is composed of 240 copies of a single protein called HBV core antigen (HBc. An atomic model of a core assembled from truncated HBc was determined previously by X-ray crystallography. In an attempt to obtain atomic structural information of HBV core in a near native, non-crystalline environment, we reconstructed a 3.5Å-resolution structure of a recombinant core assembled from full-length HBc by cryo electron microscopy (cryoEM and derived an atomic model. The structure shows that the 240 molecules of full-length HBc form a core with two layers. The outer layer, composed of the N-terminal assembly domain, is similar to the crystal structure of the truncated HBc, but has three differences. First, unlike the crystal structure, our cryoEM structure shows no disulfide bond between the Cys61 residues of the two subunits within the dimer building block, indicating such bond is not required for core formation. Second, our cryoEM structure reveals up to four more residues in the linker region (amino acids 140-149. Third, the loops in the cryoEM structures containing this linker region in subunits B and C are oriented differently (~30° and ~90° from their counterparts in the crystal structure. The inner layer, composed of the C-terminal arginine-rich domain (ARD and the ARD-bound RNAs, is partially-ordered and connected with the outer layer through linkers positioned around the two-fold axes. Weak densities emanate from the rims of positively charged channels through the icosahedral three-fold and local three-fold axes. We attribute these densities to the exposed portions of some ARDs, thus explaining ARD's accessibility by proteases and antibodies. Our data supports a role of ARD in mediating communication between inside and outside of the core during HBV maturation and envelopment.

  11. 3.5Å cryoEM Structure of Hepatitis B Virus Core Assembled from Full-Length Core Protein

    Science.gov (United States)

    Yu, Xuekui; Jin, Lei; Jih, Jonathan; Shih, Chiaho; Hong Zhou, Z.

    2013-01-01

    The capsid shell of infectious hepatitis B virus (HBV) is composed of 240 copies of a single protein called HBV core antigen (HBc). An atomic model of a core assembled from truncated HBc was determined previously by X-ray crystallography. In an attempt to obtain atomic structural information of HBV core in a near native, non-crystalline environment, we reconstructed a 3.5Å-resolution structure of a recombinant core assembled from full-length HBc by cryo electron microscopy (cryoEM) and derived an atomic model. The structure shows that the 240 molecules of full-length HBc form a core with two layers. The outer layer, composed of the N-terminal assembly domain, is similar to the crystal structure of the truncated HBc, but has three differences. First, unlike the crystal structure, our cryoEM structure shows no disulfide bond between the Cys61 residues of the two subunits within the dimer building block, indicating such bond is not required for core formation. Second, our cryoEM structure reveals up to four more residues in the linker region (amino acids 140-149). Third, the loops in the cryoEM structures containing this linker region in subunits B and C are oriented differently (~30° and ~90°) from their counterparts in the crystal structure. The inner layer, composed of the C-terminal arginine-rich domain (ARD) and the ARD-bound RNAs, is partially-ordered and connected with the outer layer through linkers positioned around the two-fold axes. Weak densities emanate from the rims of positively charged channels through the icosahedral three-fold and local three-fold axes. We attribute these densities to the exposed portions of some ARDs, thus explaining ARD’s accessibility by proteases and antibodies. Our data supports a role of ARD in mediating communication between inside and outside of the core during HBV maturation and envelopment. PMID:24039702

  12. Protein and metabolic engineering for the production of organic acids.

    Science.gov (United States)

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

    2017-09-01

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

  13. Fibroblast activation protein (FAP) as a novel metabolic target

    DEFF Research Database (Denmark)

    Sánchez-Garrido, Miguel Angel; Habegger, Kirk M; Clemmensen, Christoffer

    2016-01-01

    to block FAP enzymatic activity. RESULTS: TB administration to diet-induced obese (DIO) animals led to profound decreases in body weight, reduced food consumption and adiposity, increased energy expenditure, improved glucose tolerance and insulin sensitivity, and lowered cholesterol levels. Total...... on body weight or any other measures of metabolism. In support of these results we observed no enzymatic degradation of human FGF21 at either end of the protein when FAP was inhibited in vitro by TB. CONCLUSIONS: We conclude that pharmacological inhibition of FAP enhances levels of FGF21 in obese mice......OBJECTIVE: Fibroblast activation protein (FAP) is a serine protease belonging to a S9B prolyl oligopeptidase subfamily. This enzyme has been implicated in cancer development and recently reported to regulate degradation of FGF21, a potent metabolic hormone. Using a known FAP inhibitor, talabostat...

  14. The core protein of classical Swine Fever virus is dispensable for virus propagation in vitro.

    Directory of Open Access Journals (Sweden)

    Christiane Riedel

    Full Text Available Core protein of Flaviviridae is regarded as essential factor for nucleocapsid formation. Yet, core protein is not encoded by all isolates (GBV- A and GBV- C. Pestiviruses are a genus within the family Flaviviridae that affect cloven-hoofed animals, causing economically important diseases like classical swine fever (CSF and bovine viral diarrhea (BVD. Recent findings describe the ability of NS3 of classical swine fever virus (CSFV to compensate for disabling size increase of core protein (Riedel et al., 2010. NS3 is a nonstructural protein possessing protease, helicase and NTPase activity and a key player in virus replication. A role of NS3 in particle morphogenesis has also been described for other members of the Flaviviridae (Patkar et al., 2008; Ma et al., 2008. These findings raise questions about the necessity and function of core protein and the role of NS3 in particle assembly. A reverse genetic system for CSFV was employed to generate poorly growing CSFVs by modification of the core gene. After passaging, rescued viruses had acquired single amino acid substitutions (SAAS within NS3 helicase subdomain 3. Upon introduction of these SAAS in a nonviable CSFV with deletion of almost the entire core gene (Vp447(Δc, virus could be rescued. Further characterization of this virus with regard to its physical properties, morphology and behavior in cell culture did not reveal major differences between wildtype (Vp447 and Vp447(Δc. Upon infection of the natural host, Vp447(Δc was attenuated. Hence we conclude that core protein is not essential for particle assembly of a core-encoding member of the Flaviviridae, but important for its virulence. This raises questions about capsid structure and necessity, the role of NS3 in particle assembly and the function of core protein in general.

  15. Quantitative analysis of core fucosylation of serum proteins in liver diseases by LC-MS-MRM.

    Science.gov (United States)

    Ma, Junfeng; Sanda, Miloslav; Wei, Renhuizi; Zhang, Lihua; Goldman, Radoslav

    2018-02-07

    Aberrant core fucosylation of proteins has been linked to liver diseases. In this study, we carried out multiple reaction monitoring (MRM) quantification of core fucosylated N-glycopeptides of serum proteins partially deglycosylated by a combination of endoglycosidases (endoF1, endoF2, and endoF3). To minimize variability associated with the preparatory steps, the analysis was performed without enrichment of glycopeptides or fractionation of serum besides the nanoRP chromatography. Specifically, we quantified core fucosylation of 22 N-glycopeptides derived from 17 proteins together with protein abundance of these glycoproteins in a cohort of 45 participants (15 disease-free control, 15 fibrosis and 15 cirrhosis patients) using a multiplex nanoUPLC-MS-MRM workflow. We find increased core fucosylation of 5 glycopeptides at the stage of liver fibrosis (i.e., N630 of serotransferrin, N107 of alpha-1-antitrypsin, N253 of plasma protease C1 inhibitor, N397 of ceruloplasmin, and N86 of vitronectin), increase of additional 6 glycopeptides at the stage of cirrhosis (i.e., N138 and N762 of ceruloplasmin, N354 of clusterin, N187 of hemopexin, N71 of immunoglobulin J chain, and N127 of lumican), while the degree of core fucosylation of 10 glycopeptides did not change. Interestingly, although we observe an increase in the core fucosylation at N86 of vitronectin in liver fibrosis, core fucosylation decreases on the N169 glycopeptide of the same protein. Our results demonstrate that the changes in core fucosylation are protein and site specific during the progression of fibrotic liver disease and independent of the changes in the quantity of N-glycoproteins. It is expected that the fully optimized multiplex LC-MS-MRM assay of core fucosylated glycopeptides will be useful for the serologic assessment of the fibrosis of liver. We have quantified the difference in core fucosylation among three comparison groups (healthy control, fibrosis and cirrhosis patients) using a sensitive and

  16. Metatranscriptomic analysis of diverse microbial communities reveals core metabolic pathways and microbiome-specific functionality.

    Science.gov (United States)

    Jiang, Yue; Xiong, Xuejian; Danska, Jayne; Parkinson, John

    2016-01-12

    Metatranscriptomics is emerging as a powerful technology for the functional characterization of complex microbial communities (microbiomes). Use of unbiased RNA-sequencing can reveal both the taxonomic composition and active biochemical functions of a complex microbial community. However, the lack of established reference genomes, computational tools and pipelines make analysis and interpretation of these datasets challenging. Systematic studies that compare data across microbiomes are needed to demonstrate the ability of such pipelines to deliver biologically meaningful insights on microbiome function. Here, we apply a standardized analytical pipeline to perform a comparative analysis of metatranscriptomic data from diverse microbial communities derived from mouse large intestine, cow rumen, kimchi culture, deep-sea thermal vent and permafrost. Sequence similarity searches allowed annotation of 19 to 76% of putative messenger RNA (mRNA) reads, with the highest frequency in the kimchi dataset due to its relatively low complexity and availability of closely related reference genomes. Metatranscriptomic datasets exhibited distinct taxonomic and functional signatures. From a metabolic perspective, we identified a common core of enzymes involved in amino acid, energy and nucleotide metabolism and also identified microbiome-specific pathways such as phosphonate metabolism (deep sea) and glycan degradation pathways (cow rumen). Integrating taxonomic and functional annotations within a novel visualization framework revealed the contribution of different taxa to metabolic pathways, allowing the identification of taxa that contribute unique functions. The application of a single, standard pipeline confirms that the rich taxonomic and functional diversity observed across microbiomes is not simply an artefact of different analysis pipelines but instead reflects distinct environmental influences. At the same time, our findings show how microbiome complexity and availability of

  17. CORE

    DEFF Research Database (Denmark)

    Krigslund, Jeppe; Hansen, Jonas; Hundebøll, Martin

    2013-01-01

    different flows. Instead of maintaining these approaches separate, we propose a protocol (CORE) that brings together these coding mechanisms. Our protocol uses random linear network coding (RLNC) for intra- session coding but allows nodes in the network to setup inter- session coding regions where flows...... intersect. Routes for unicast sessions are agnostic to other sessions and setup beforehand, CORE will then discover and exploit intersecting routes. Our approach allows the inter-session regions to leverage RLNC to compensate for losses or failures in the overhearing or transmitting process. Thus, we...... increase the benefits of XORing by exploiting the underlying RLNC structure of individual flows. This goes beyond providing additional reliability to each individual session and beyond exploiting coding opportunistically. Our numerical results show that CORE outperforms both forwarding and COPE...

  18. Modulation of RANTES expression by HCV core protein in liver derived cell lines

    Directory of Open Access Journals (Sweden)

    Rapicetta Maria

    2007-06-01

    Full Text Available Abstract Background Hepatitis C virus (HCV infection is associated with high percentage of chronicity which implies the ability of the virus to evade or modulate host cell immune system. Modulation of chemokines, such as RANTES may be part of the virus induced pathogenicity. We examined the effect of core and structural proteins of HCV on RANTES expression in two liver derived cell lines, HepG2 and Chang Liver (CHL. Methods HepG2 and Chang Liver (CHL cell lines were established and selected for constitutive expression of HCV core and structural genes. Flow cytometry and quantitative RT-PCR analysis were performed to examine the effect of HCV core protein on RANTES expression. Luciferase analysis after RANTES-Luc-promoter transfection of established cell lines was assayed by luminometer measurements (RLU of RANTES promoter activity. IRF-1 and IRF-7 expression was then examined by immunoblotting analysis. Results Results of flow cytometry and RT-PCR analysis indicated that RANTES is differentially regulated by HCV core protein in the two cell lines examined as its expression was inhibited in HepG2 cells, by a reduction of RANTES promoter activity. Conversely, RANTES protein and mRNA were induced by the core protein in CHL cells, through the induction of the promoter. Since HCV genome modulates IRF-1 and IRF-7 in replicon system and IRF-1, IRF-3 and IRF-7 have been reported to regulate RANTES promoter in various cell systems, analysis of the mechanism underlying RANTES modulation by the core protein revealed that IRF-1 expression was induced in HepG2 cells by the core protein, whereas in CHL cells it was expressed at a very low level that was not influenced by transfection with the core protein construct. This suggested that IRF-1 level may mediate the expression of RANTES in cell lines of liver origin. The effect of the core protein on RANTES promoter was countered by co-transfection with NF90, a double-stranded-RNA binding protein that activates

  19. Metabolic impact assessment for heterologous protein production in Streptomyces lividans based on genome-scale metabolic network modeling.

    Science.gov (United States)

    Lule, Ivan; D'Huys, Pieter-Jan; Van Mellaert, Lieve; Anné, Jozef; Bernaerts, Kristel; Van Impe, Jan

    2013-11-01

    The metabolic impact exerted on a microorganism due to heterologous protein production is still poorly understood in Streptomyces lividans. In this present paper, based on exometabolomic data, a proposed genome-scale metabolic network model is used to assess this metabolic impact in S. lividans. Constraint-based modeling results obtained in this work revealed that the metabolic impact due to heterologous protein production is widely distributed in the genome of S. lividans, causing both slow substrate assimilation and a shift in active pathways. Exchange fluxes that are critical for model performance have been identified for metabolites of mouse tumor necrosis factor, histidine, valine and lysine, as well as biomass. Our results unravel the interaction of heterologous protein production with intracellular metabolism of S. lividans, thus, a possible basis for further studies in relieving the metabolic burden via metabolic or bioprocess engineering. Copyright © 2013. Published by Elsevier Inc.

  20. Hepatitis C virus core protein induces hepatic steatosis via Sirt1-dependent pathway.

    Science.gov (United States)

    Zhang, Chuanhai; Wang, Jingjing; Zhang, Hanlin; Liu, Shunai; Lee, Hyuek Jong; Jin, Wanzhu; Cheng, Jun

    2017-09-12

    Hepatic steatosis is a common feature of patients with chronic hepatitis C. Previous reports have shown that the overexpression of hepatitis C virus core-encoding sequences (hepatitis C virus genotypes 3a and 1b) significantly induces intracellular triglyceride accumulation. However, the underlying mechanism has not yet been revealed. To investigate whether Sirt1 is involved in hepatitis C virus-mediated hepatic steatosis, the overexpression of hepatitis C virus core 1b protein and Sirt1 and the knockdown of Sirt1 in HepG2 cells were performed. To confirm the results of the cellular experiment liver-specific Sirt1 KO mice with lentivirus-mediated hepatitis C virus core 1b overexpression were studied. Our results show that hepatitis C virus core 1b protein overexpression led to the accumulation of triglycerides in HepG2 cells. Notably the expression of PPARγ2 was dramatically increased at both the mRNA and protein levels by hepatitis C virus core 1b overexpression. The protein expression of Sirt1 is an upstream regulator of PPARγ2 and was also significantly increased after core 1b overexpression. In addition, the overexpression or knockdown of Sirt1 expression alone was sufficient to modulate p300-mediated PPARγ2 deacetylation. In vivo studies showed that hepatitis C virus core protein 1b-induced hepatic steatosis was attenuated in liver-specific Sirt1 KO mice by downregulation of PPARγ2 expression. Sirt1 mediates hepatitis C virus core protein 1b-induced hepatic steatosis by regulation of PPARγ2 expression. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  1. Identifying protein complex by integrating characteristic of core-attachment into dynamic PPI network.

    Directory of Open Access Journals (Sweden)

    Xianjun Shen

    Full Text Available How to identify protein complex is an important and challenging task in proteomics. It would make great contribution to our knowledge of molecular mechanism in cell life activities. However, the inherent organization and dynamic characteristic of cell system have rarely been incorporated into the existing algorithms for detecting protein complexes because of the limitation of protein-protein interaction (PPI data produced by high throughput techniques. The availability of time course gene expression profile enables us to uncover the dynamics of molecular networks and improve the detection of protein complexes. In order to achieve this goal, this paper proposes a novel algorithm DCA (Dynamic Core-Attachment. It detects protein-complex core comprising of continually expressed and highly connected proteins in dynamic PPI network, and then the protein complex is formed by including the attachments with high adhesion into the core. The integration of core-attachment feature into the dynamic PPI network is responsible for the superiority of our algorithm. DCA has been applied on two different yeast dynamic PPI networks and the experimental results show that it performs significantly better than the state-of-the-art techniques in terms of prediction accuracy, hF-measure and statistical significance in biology. In addition, the identified complexes with strong biological significance provide potential candidate complexes for biologists to validate.

  2. Effect of bacterial protein meal on protein and energy metabolism in growing chickens

    DEFF Research Database (Denmark)

    Hellwing, Anne Louise Frydendahl; Tauson, Anne-Helene; Skrede, Anders

    2006-01-01

    This experiment investigates the effect of increasing the dietary content of bacterial protein meal (BPM) on the protein and energy metabolism, and carcass chemical composition of growing chickens. Seventy-two Ross male chickens were allocated to four diets, each in three replicates with 0% (D0), 2......% (D2), 4% D4), and 6% BPM (D6), BPM providing up to 20% of total dietary N. Five balance experiments were conducted when the chickens were 3-7, 10-14, 17-21, 23-27, and 30-34 days old. During the same periods, 22-h respiration experiments (indirect calorimetry) were performed with troups of 6 chickens...... for protein and energy retention found in the balance and respiration experiments. It was concluded that the overall protein and energy metabolism as well as carcass composition were not influenced by a dietary content of up to 6% BPM corresponding to 20% of dietary N....

  3. Regulation of Lipid and Glucose Metabolism by Phosphatidylcholine Transfer Protein

    Science.gov (United States)

    Kang, Hye Won; Wei, Jie; Cohen, David E.

    2010-01-01

    Phosphatidylcholine transfer protein (PC-TP, a.k.a. StARD2) binds phosphatidylcholines and catalyzes their intermembrane transfer and exchange in vitro. The structure of PC-TP comprises a hydrophobic pocket and a well-defined head-group binding site, and its gene expression is regulated by peroxisome proliferator activated receptor α. Recent studies have revealed key regulatory roles for PC-TP in lipid and glucose metabolism. Notably, Pctp−/− mice are sensitized to insulin action and exhibit more efficient brown fat-mediated thermogenesis. PC-TP appears to limit access of fatty acids to mitochondria by stimulating the activity of thioesterase superfamily member 2, a newly characterized long-chain fatty acyl-CoA thioesterase. Because PC-TP discriminates among phosphatidylcholines within lipid bilayers, it may function as a sensor that links metabolic regulation to membrane composition. PMID:20338778

  4. Application of Divergence Entropy to Characterize the Structure of the Hydrophobic Core in DNA Interacting Proteins

    Directory of Open Access Journals (Sweden)

    Barbara Kalinowska

    2015-03-01

    Full Text Available The fuzzy oil drop model, a tool which can be used to study the structure of the hydrophobic core in proteins, has been applied in the analysis of proteins belonging to the jumonji group—JARID2, JARID1A, JARID1B and JARID1D—proteins that share the property of being able to interact with DNA. Their ARID and PHD domains, when analyzed in the context of the fuzzy oil drop model, are found to exhibit structural variability regarding the status of their secondary folds, including the β-hairpin which determines their biological function. Additionally, the structure of disordered fragments which are present in jumonji proteins (as confirmed by the DisProt database is explained on the grounds of the hydrophobic core model, suggesting that such fragments contribute to tertiary structural stabilization. This conclusion is supported by divergence entropy measurements, expressing the degree of ordering in each protein’s hydrophobic core.

  5. Discovery of protein complexes with core-attachment structures from Tandem Affinity Purification (TAP) data.

    Science.gov (United States)

    Wu, Min; Li, Xiao-Li; Kwoh, Chee-Keong; Ng, See-Kiong; Wong, Limsoon

    2012-09-01

    Many cellular functions involve protein complexes that are formed by multiple interacting proteins. Tandem Affinity Purification (TAP) is a popular experimental method for detecting such multi-protein interactions. However, current computational methods that predict protein complexes from TAP data require converting the co-complex relationships in TAP data into binary interactions. The resulting pairwise protein-protein interaction (PPI) network is then mined for densely connected regions that are identified as putative protein complexes. Converting the TAP data into PPI data not only introduces errors but also loses useful information about the underlying multi-protein relationships that can be exploited to detect the internal organization (i.e., core-attachment structures) of protein complexes. In this article, we propose a method called CACHET that detects protein complexes with Core-AttaCHment structures directly from bipartitETAP data. CACHET models the TAP data as a bipartite graph in which the two vertex sets are the baits and the preys, respectively. The edges between the two vertex sets represent bait-prey relationships. CACHET first focuses on detecting high-quality protein-complex cores from the bipartite graph. To minimize the effects of false positive interactions, the bait-prey relationships are indexed with reliability scores. Only non-redundant, reliable bicliques computed from the TAP bipartite graph are regarded as protein-complex cores. CACHET constructs protein complexes by including attachment proteins into the cores. We applied CACHET on large-scale TAP datasets and found that CACHET outperformed existing methods in terms of prediction accuracy (i.e., F-measure and functional homogeneity of predicted complexes). In addition, the protein complexes predicted by CACHET are equipped with core-attachment structures that provide useful biological insights into the inherent functional organization of protein complexes. Our supplementary material can

  6. Identification of Protein Complexes Using Weighted PageRank-Nibble Algorithm and Core-Attachment Structure.

    Science.gov (United States)

    Peng, Wei; Wang, Jianxin; Zhao, Bihai; Wang, Lusheng

    2015-01-01

    Protein complexes play a significant role in understanding the underlying mechanism of most cellular functions. Recently, many researchers have explored computational methods to identify protein complexes from protein-protein interaction (PPI) networks. One group of researchers focus on detecting local dense subgraphs which correspond to protein complexes by considering local neighbors. The drawback of this kind of approach is that the global information of the networks is ignored. Some methods such as Markov Clustering algorithm (MCL), PageRank-Nibble are proposed to find protein complexes based on random walk technique which can exploit the global structure of networks. However, these methods ignore the inherent core-attachment structure of protein complexes and treat adjacent node equally. In this paper, we design a weighted PageRank-Nibble algorithm which assigns each adjacent node with different probability, and propose a novel method named WPNCA to detect protein complex from PPI networks by using weighted PageRank-Nibble algorithm and core-attachment structure. Firstly, WPNCA partitions the PPI networks into multiple dense clusters by using weighted PageRank-Nibble algorithm. Then the cores of these clusters are detected and the rest of proteins in the clusters will be selected as attachments to form the final predicted protein complexes. The experiments on yeast data show that WPNCA outperforms the existing methods in terms of both accuracy and p-value. The software for WPNCA is available at "http://netlab.csu.edu.cn/bioinfomatics/weipeng/WPNCA/download.html".

  7. Changes in cardiac substrate transporters and metabolic proteins mirror the metabolic shift in patients with aortic stenosis.

    Science.gov (United States)

    Heather, Lisa C; Howell, Neil J; Emmanuel, Yaso; Cole, Mark A; Frenneaux, Michael P; Pagano, Domenico; Clarke, Kieran

    2011-01-01

    In the hypertrophied human heart, fatty acid metabolism is decreased and glucose utilisation is increased. We hypothesized that the sarcolemmal and mitochondrial proteins involved in these key metabolic pathways would mirror these changes, providing a mechanism to account for the modified metabolic flux measured in the human heart. Echocardiography was performed to assess in vivo hypertrophy and aortic valve impairment in patients with aortic stenosis (n = 18). Cardiac biopsies were obtained during valve replacement surgery, and used for western blotting to measure metabolic protein levels. Protein levels of the predominant fatty acid transporter, fatty acid translocase (FAT/CD36) correlated negatively with levels of the glucose transporters, GLUT1 and GLUT4. The decrease in FAT/CD36 was accompanied by decreases in the fatty acid binding proteins, FABPpm and H-FABP, the β-oxidation protein medium chain acyl-coenzyme A dehydrogenase, the Krebs cycle protein α-ketoglutarate dehydrogenase and the oxidative phosphorylation protein ATP synthase. FAT/CD36 and complex I of the electron transport chain were downregulated, whereas the glucose transporter GLUT4 was upregulated with increasing left ventricular mass index, a measure of cardiac hypertrophy. In conclusion, coordinated downregulation of sequential steps involved in fatty acid and oxidative metabolism occur in the human heart, accompanied by upregulation of the glucose transporters. The profile of the substrate transporters and metabolic proteins mirror the metabolic shift from fatty acid to glucose utilisation that occurs in vivo in the human heart.

  8. HCV Core Protein Uses Multiple Mechanisms to Induce Oxidative Stress in Human Hepatoma Huh7 Cells

    Science.gov (United States)

    Ivanov, Alexander V.; Smirnova, Olga A.; Petrushanko, Irina Y.; Ivanova, Olga N.; Karpenko, Inna L.; Alekseeva, Ekaterina; Sominskaya, Irina; Makarov, Alexander A.; Bartosch, Birke; Kochetkov, Sergey N.; Isaguliants, Maria G.

    2015-01-01

    Hepatitis C virus (HCV) infection is accompanied by the induction of oxidative stress, mediated by several virus proteins, the most prominent being the nucleocapsid protein (HCV core). Here, using the truncated forms of HCV core, we have delineated several mechanisms by which it induces the oxidative stress. The N-terminal 36 amino acids of HCV core induced TGFβ1-dependent expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases 1 and 4, both of which independently contributed to the production of reactive oxygen species (ROS). The same fragment also induced the expression of cyclo-oxygenase 2, which, however, made no input into ROS production. Amino acids 37–191 of HCV core up-regulated the transcription of a ROS generating enzyme cytochrome P450 2E1. Furthermore, the same fragment induced the expression of endoplasmic reticulum oxidoreductin 1α. The latter triggered efflux of Ca2+ from ER to mitochondria via mitochondrial Ca2+ uniporter, leading to generation of superoxide anions, and possibly also H2O2. Suppression of any of these pathways in cells expressing the full-length core protein led to a partial inhibition of ROS production. Thus, HCV core causes oxidative stress via several independent pathways, each mediated by a distinct region of the protein. PMID:26035647

  9. HCV Core Protein Uses Multiple Mechanisms to Induce Oxidative Stress in Human Hepatoma Huh7 Cells

    Directory of Open Access Journals (Sweden)

    Alexander V. Ivanov

    2015-05-01

    Full Text Available Hepatitis C virus (HCV infection is accompanied by the induction of oxidative stress, mediated by several virus proteins, the most prominent being the nucleocapsid protein (HCV core. Here, using the truncated forms of HCV core, we have delineated several mechanisms by which it induces the oxidative stress. The N-terminal 36 amino acids of HCV core induced TGF\\(\\upbeta\\1-dependent expression of nicotinamide adenine dinucleotide phosphate (NADPH oxidases 1 and 4, both of which independently contributed to the production of reactive oxygen species (ROS. The same fragment also induced the expression of cyclo-oxygenase 2, which, however, made no input into ROS production. Amino acids 37–191 of HCV core up-regulated the transcription of a ROS generating enzyme cytochrome P450 2E1. Furthermore, the same fragment induced the expression of endoplasmic reticulum oxidoreductin 1\\(\\upalpha\\. The latter triggered efflux of Ca2+ from ER to mitochondria via mitochondrial Ca2+ uniporter, leading to generation of superoxide anions, and possibly also H2O2. Suppression of any of these pathways in cells expressing the full-length core protein led to a partial inhibition of ROS production. Thus, HCV core causes oxidative stress via several independent pathways, each mediated by a distinct region of the protein.

  10. Leucine and protein metabolism in obese Zucker rats.

    Directory of Open Access Journals (Sweden)

    Pengxiang She

    Full Text Available Branched-chain amino acids (BCAAs are circulating nutrient signals for protein accretion, however, they increase in obesity and elevations appear to be prognostic of diabetes. To understand the mechanisms whereby obesity affects BCAAs and protein metabolism, we employed metabolomics and measured rates of [1-(14C]-leucine metabolism, tissue-specific protein synthesis and branched-chain keto-acid (BCKA dehydrogenase complex (BCKDC activities. Male obese Zucker rats (11-weeks old had increased body weight (BW, 53%, liver (107% and fat (∼300%, but lower plantaris and gastrocnemius masses (-21-24%. Plasma BCAAs and BCKAs were elevated 45-69% and ∼100%, respectively, in obese rats. Processes facilitating these rises appeared to include increased dietary intake (23%, leucine (Leu turnover and proteolysis [35% per g fat free mass (FFM, urinary markers of proteolysis: 3-methylhistidine (183% and 4-hydroxyproline (766%] and decreased BCKDC per g kidney, heart, gastrocnemius and liver (-47-66%. A process disposing of circulating BCAAs, protein synthesis, was increased 23-29% by obesity in whole-body (FFM corrected, gastrocnemius and liver. Despite the observed decreases in BCKDC activities per gm tissue, rates of whole-body Leu oxidation in obese rats were 22% and 59% higher normalized to BW and FFM, respectively. Consistently, urinary concentrations of eight BCAA catabolism-derived acylcarnitines were also elevated. The unexpected increase in BCAA oxidation may be due to a substrate effect in liver. Supporting this idea, BCKAs were elevated more in liver (193-418% than plasma or muscle, and per g losses of hepatic BCKDC activities were completely offset by increased liver mass, in contrast to other tissues. In summary, our results indicate that plasma BCKAs may represent a more sensitive metabolic signature for obesity than BCAAs. Processes supporting elevated BCAA]BCKAs in the obese Zucker rat include increased dietary intake, Leu and protein

  11. Brain metabolic dysfunction at the core of Alzheimer’s disease

    Science.gov (United States)

    de la Monte, Suzanne M.; Tong, Ming

    2015-01-01

    Growing evidence supports the concept that Alzheimer’s disease (AD) is fundamentally a metabolic disease with molecular and biochemical features that correspond with diabetes mellitus and other peripheral insulin resistance disorders. Brain insulin/IGF resistance and its consequences can readily account for most of the structural and functional abnormalities in AD. However, disease pathogenesis is complicated by the fact that AD can occur as a separate disease process, or arise in association with systemic insulin resistance diseases, including diabetes, obesity, and non-alcoholic fatty liver disease. Whether primary or secondary in origin, brain insulin/IGF resistance initiates a cascade of neurodegeneration that is propagated by metabolic dysfunction, increased oxidative and ER stress, neuro-inflammation, impaired cell survival, and dysregulated lipid metabolism. These injurious processes compromise neuronal and glial functions, reduce neurotransmitter homeostasis, and cause toxic oligomeric pTau and (amyloid beta peptide of amyloid beta precursor protein) AβPP-Aβ fibrils and insoluble aggregates (neurofibrillary tangles and plaques) to accumulate in brain. AD progresses due to: (1) activation of a harmful positive feedback loop that progressively worsens the effects of insulin resistance; and (2) the formation of ROS- and RNS-related lipid, protein, and DNA adducts that permanently damage basic cellular and molecular functions. Epidemiologic data suggest that insulin resistance diseases, including AD, are exposure-related in etiology. Furthermore, experimental and lifestyle trend data suggest chronic low-level nitrosamine exposures are responsible. These concepts offer opportunities to discover and implement new treatments and devise preventive measures to conquer the AD and other insulin resistance disease epidemics. PMID:24380887

  12. Hepatitis C virus core protein induces apoptosis-like caspase independent cell death

    Directory of Open Access Journals (Sweden)

    Gregor Michael

    2009-12-01

    Full Text Available Abstract Background Hepatitis C virus (HCV associated liver diseases may be related to apoptotic processes. Thus, we investigated the role of different HCV proteins in apoptosis induction as well as their potency to interact with different apoptosis inducing agents. Methods and Results The use of a tightly adjustable tetracycline (Tet-dependent HCV protein expression cell system with the founder osteosarcoma cell line U-2 OS allowed switch-off and on of the endogenous production of HCV proteins. Analyzed were cell lines expressing the HCV polyprotein, the core protein, protein complexes of the core, envelope proteins E1, E2 and p7, and non-structural proteins NS3 and NS4A, NS4B or NS5A and NS5B. Apoptosis was measured mainly by the detection of hypodiploid apoptotic nuclei in the absence or presence of mitomycin C, etoposide, TRAIL and an agonistic anti-CD95 antibody. To further characterize cell death induction, a variety of different methods like fluorescence microscopy, TUNEL (terminal deoxynucleotidyl transferase (TdT-catalyzed deoxyuridinephosphate (dUTP-nick end labeling assay, Annexin V staining, Western blot and caspase activation assays were included into our analysis. Two cell lines expressing the core protein but not the total polyprotein exerted a strong apoptotic effect, while the other cell lines did not induce any or only a slight effect by measuring the hypodiploid nuclei. Cell death induction was caspase-independent since it could not be blocked by zVAD-fmk. Moreover, caspase activity was absent in Western blot analysis and fluorometric assays while typical apoptosis-associated morphological features like the membrane blebbing and nuclei condensation and fragmentation could be clearly observed by microscopy. None of the HCV proteins influenced the apoptotic effect mediated via the mitochondrial apoptosis pathway while only the core protein enhanced death-receptor-mediated apoptosis. Conclusion Our data showed a caspase

  13. Sequence and structural implications of a bovine corneal keratan sulfate proteoglycan core protein. Protein 37B represents bovine lumican and proteins 37A and 25 are unique

    Science.gov (United States)

    Funderburgh, J. L.; Funderburgh, M. L.; Brown, S. J.; Vergnes, J. P.; Hassell, J. R.; Mann, M. M.; Conrad, G. W.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    Amino acid sequence from tryptic peptides of three different bovine corneal keratan sulfate proteoglycan (KSPG) core proteins (designated 37A, 37B, and 25) showed similarities to the sequence of a chicken KSPG core protein lumican. Bovine lumican cDNA was isolated from a bovine corneal expression library by screening with chicken lumican cDNA. The bovine cDNA codes for a 342-amino acid protein, M(r) 38,712, containing amino acid sequences identified in the 37B KSPG core protein. The bovine lumican is 68% identical to chicken lumican, with an 83% identity excluding the N-terminal 40 amino acids. Location of 6 cysteine and 4 consensus N-glycosylation sites in the bovine sequence were identical to those in chicken lumican. Bovine lumican had about 50% identity to bovine fibromodulin and 20% identity to bovine decorin and biglycan. About two-thirds of the lumican protein consists of a series of 10 amino acid leucine-rich repeats that occur in regions of calculated high beta-hydrophobic moment, suggesting that the leucine-rich repeats contribute to beta-sheet formation in these proteins. Sequences obtained from 37A and 25 core proteins were absent in bovine lumican, thus predicting a unique primary structure and separate mRNA for each of the three bovine KSPG core proteins.

  14. Conserved immunogenic region of a major core protein (p24) of human and simian immunodeficiency viruses.

    Science.gov (United States)

    Koito, A; Hattori, T; Matsushita, S; Maeda, Y; Nozaki, C; Sagawa, K; Takatsuki, K

    1988-12-01

    A murine monoclonal antibody (MoAb), VAK 4, has been known to specifically react with a major core protein (p24) as well as with its precursor (p55-57) and intermediate precursor (p40) of human immunodeficiency virus strain IIIB (HTLV-IIIB). Radioimmunoprecipitation assays revealed that VAK 4 MoAb precipitated a major core protein and its precursors from a variety of strains of HIV and also from simian immunodeficiency virus (SIV), although the molecular weights of the precursor proteins in each viral strain were slightly different. A protein synthesized by transfected Escherichia coli containing amino acid sequences corresponding to residues 121-436 of the HTLV-IIIB gag gene was reactive with VAK 4 MoAb, but the protein carrying only residues 121-309 was not reactive, suggesting that the epitope recognized by VAK 4 MoAb resides at the carboxyl terminus of p24 protein. A competitive enzyme-linked immunosorbent assay showed that patient sera containing anticore protein antibody inhibited the binding of VAK 4 to HTLV-IIIB. These findings suggested that VAK 4 MoAb recognized an immunogenic and conserved epitope belonging to a major core protein of HIV-related viruses.

  15. Changes in contralateral protein metabolism following unilateral sciatic nerve section

    Energy Technology Data Exchange (ETDEWEB)

    Menendez, J.A.; Cubas, S.C.

    1990-03-01

    Changes in nerve biochemistry, anatomy, and function following injuries to the contralateral nerve have been repeatedly reported, though their significance is unknown. The most likely mechanisms for their development are either substances carried by axoplasmic flow or electrically transmitted signals. This study analyzes which mechanism underlies the development of a contralateral change in protein metabolism. The incorporation of labelled amino acids (AA) into proteins of both sciatic nerves was assessed by liquid scintillation after an unilateral section. AA were offered locally for 30 min to the distal stump of the sectioned nerves and at homologous levels of the intact contralateral nerves. At various times, from 1 to 24 h, both sciatic nerves were removed and the proteins extracted with trichloroacetic acid (TCA). An increase in incorporation was found in both nerves 14-24 h after section. No difference existed between sectioned and intact nerves, which is consistent with the contralateral effect. Lidocaine, but not colchicine, when applied previously to the nerves midway between the sectioning site and the spinal cord, inhibited the contralateral increase in AA incorporation. It is concluded that electrical signals, crossing through the spinal cord, are responsible for the development of the contralateral effect. Both the nature of the proteins and the significance of the contralateral effect are matters for speculation.

  16. Protein and amino acid metabolism in skeletal muscle

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Guoyao.

    1989-01-01

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

  17. Mitochondrial iron accumulation exacerbates hepatic toxicity caused by hepatitis C virus core protein

    Energy Technology Data Exchange (ETDEWEB)

    Sekine, Shuichi; Ito, Konomi; Watanabe, Haruna; Nakano, Takafumi [Laboratory of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675 (Japan); Moriya, Kyoji; Shintani, Yoshizumi; Fujie, Hajime; Tsutsumi, Takeya; Miyoshi, Hideyuki; Fujinaga, Hidetake; Shinzawa, Seiko; Koike, Kazuhiko [Department of Internal Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655 (Japan); Horie, Toshiharu, E-mail: t.horie@thu.ac.jp [Laboratory of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675 (Japan)

    2015-02-01

    Patients with long-lasting hepatitis C virus (HCV) infection are at major risk of hepatocellular carcinoma (HCC). Iron accumulation in the livers of these patients is thought to exacerbate conditions of oxidative stress. Transgenic mice that express the HCV core protein develop HCC after the steatosis stage and produce an excess of hepatic reactive oxygen species (ROS). The overproduction of ROS in the liver is the net result of HCV core protein-induced dysfunction of the mitochondrial respiratory chain. This study examined the impact of ferric nitrilacetic acid (Fe-NTA)-mediated iron overload on mitochondrial damage and ROS production in HCV core protein-expressing HepG2 (human HCC) cells (Hep39b cells). A decrease in mitochondrial membrane potential and ROS production were observed following Fe-NTA treatment. After continuous exposure to Fe-NTA for six days, cell toxicity was observed in Hep39b cells, but not in mock (vector-transfected) HepG2 cells. Moreover, mitochondrial iron ({sup 59}Fe) uptake was increased in the livers of HCV core protein-expressing transgenic mice. This increase in mitochondrial iron uptake was inhibited by Ru360, a mitochondrial Ca{sup 2+} uniporter inhibitor. Furthermore, the Fe-NTA-induced augmentation of mitochondrial dysfunction, ROS production, and cell toxicity were also inhibited by Ru360 in Hep39b cells. Taken together, these results indicate that Ca{sup 2+} uniporter-mediated mitochondrial accumulation of iron exacerbates hepatocyte toxicity caused by the HCV core protein. - Highlights: • Iron accumulation in the livers of patients with hepatitis C virus (HCV) infection is thought to exacerbate oxidative stress. • The impact of iron overload on mitochondrial damage and ROS production in HCV core protein-expressing cells were examined. • Mitochondrial iron uptake was increased in the livers of HCV core protein-expressing transgenic mice. • Ca{sup 2+} uniporter-mediated mitochondrial accumulation of iron exacerbates

  18. Crystal structure of a core spliceosomal protein interface

    Science.gov (United States)

    Schellenberg, Matthew J.; Edwards, Ross A.; Ritchie, Dustin B.; Kent, Oliver A.; Golas, Monika M.; Stark, Holger; Lührmann, Reinhard; Glover, J. N. Mark; MacMillan, Andrew M.

    2006-01-01

    The precise excision of introns from precursor mRNAs (pre-mRNAs) in eukaryotes is accomplished by the spliceosome, a complex assembly containing five small nuclear ribonucleoprotein (snRNP) particles. Human p14, a component of the spliceosomal U2 and U11/U12 snRNPs, has been shown to associate directly with the pre-mRNA branch adenosine early in spliceosome assembly and within the fully assembled spliceosome. Here we report the 2.5-Å crystal structure of a complex containing p14 and a peptide derived from the p14-associated U2 snRNP component SF3b155. p14 contains an RNA recognition motif (RRM), the surface of which is largely occluded by a C-terminal α-helix and a portion of the SF3b155 peptide. An analysis of RNA·protein crosslinking to wild-type and mutant p14 shows that the branch adenosine directly interacts with a conserved aromatic within a pocket on the surface of the complex. This result, combined with a comparison of the structure with known RRMs and pseudoRRMs as well as model-building by using the electron cryomicroscopy structure of a spliceosomal U11/U12 di-snRNP, suggests that p14·SF3b155 presents a noncanonical surface for RNA recognition at the heart of the mammalian spliceosome. PMID:16432215

  19. Protein kinases responsible for the phosphorylation of the nuclear egress core complex of human cytomegalovirus.

    Science.gov (United States)

    Sonntag, Eric; Milbradt, Jens; Svrlanska, Adriana; Strojan, Hanife; Häge, Sigrun; Kraut, Alexandra; Hesse, Anne-Marie; Amin, Bushra; Sonnewald, Uwe; Couté, Yohann; Marschall, Manfred

    2017-10-01

    Nuclear egress of herpesvirus capsids is mediated by a multi-component nuclear egress complex (NEC) assembled by a heterodimer of two essential viral core egress proteins. In the case of human cytomegalovirus (HCMV), this core NEC is defined by the interaction between the membrane-anchored pUL50 and its nuclear cofactor, pUL53. NEC protein phosphorylation is considered to be an important regulatory step, so this study focused on the respective role of viral and cellular protein kinases. Multiply phosphorylated pUL50 varieties were detected by Western blot and Phos-tag analyses as resulting from both viral and cellular kinase activities. In vitro kinase analyses demonstrated that pUL50 is a substrate of both PKCα and CDK1, while pUL53 can also be moderately phosphorylated by CDK1. The use of kinase inhibitors further illustrated the importance of distinct kinases for core NEC phosphorylation. Importantly, mass spectrometry-based proteomic analyses identified five major and nine minor sites of pUL50 phosphorylation. The functional relevance of core NEC phosphorylation was confirmed by various experimental settings, including kinase knock-down/knock-out and confocal imaging, in which it was found that (i) HCMV core NEC proteins are not phosphorylated solely by viral pUL97, but also by cellular kinases; (ii) both PKC and CDK1 phosphorylation are detectable for pUL50; (iii) no impact of PKC phosphorylation on NEC functionality has been identified so far; (iv) nonetheless, CDK1-specific phosphorylation appears to be required for functional core NEC interaction. In summary, our findings provide the first evidence that the HCMV core NEC is phosphorylated by cellular kinases, and that the complex pattern of NEC phosphorylation has functional relevance.

  20. Mitochondrial energy metabolism is required for lifespan extension by the spastic paraplegia-associated protein spartin

    Directory of Open Access Journals (Sweden)

    Julia Ring

    2017-11-01

    Full Text Available Hereditary spastic paraplegias, a group of neurodegenerative disorders, can be caused by loss-of-function mutations in the protein spartin. However, the physiological role of spartin remains largely elusive. Here we show that heterologous expression of human or Drosophila spartin extends chronological lifespan of yeast, reducing age-associated ROS production, apoptosis, and necrosis. We demonstrate that spartin localizes to the proximity of mitochondria and physically interacts with proteins related to mitochondrial and respiratory metabolism. Interestingly, Nde1, the mitochondrial external NADH dehydrogenase, and Pda1, the core enzyme of the pyruvate dehydrogenase complex, are required for spartin-mediated cytoprotection. Furthermore, spartin interacts with the glycolysis enhancer phospo-fructo-kinase-2,6 (Pfk26 and is sufficient to complement for PFK26-deficiency at least in early aging. We conclude that mitochondria-related energy metabolism is crucial for spartin’s vital function during aging and uncover a network of specific interactors required for this function.

  1. EColiCore2: a reference network model of the central metabolism of Escherichia coli and relationships to its genome-scale parent model.

    Science.gov (United States)

    Hädicke, Oliver; Klamt, Steffen

    2017-01-03

    Genome-scale metabolic modeling has become an invaluable tool to analyze properties and capabilities of metabolic networks and has been particularly successful for the model organism Escherichia coli. However, for several applications, smaller metabolic (core) models are needed. Using a recently introduced reduction algorithm and the latest E. coli genome-scale reconstruction iJO1366, we derived EColiCore2, a model of the central metabolism of E. coli. EColiCore2 is a subnetwork of iJO1366 and preserves predefined phenotypes including optimal growth on different substrates. The network comprises 486 metabolites and 499 reactions, is accessible for elementary-modes analysis and can, if required, be further compressed to a network with 82 reactions and 54 metabolites having an identical solution space as EColiCore2. A systematic comparison of EColiCore2 with its genome-scale parent model iJO1366 reveals that several key properties (flux ranges, reaction essentialities, production envelopes) of the central metabolism are preserved in EColiCore2 while it neglects redundancies along biosynthetic routes. We also compare calculated metabolic engineering strategies in both models and demonstrate, as a general result, how intervention strategies found in a core model allow the identification of valid strategies in a genome-scale model. Overall, EColiCore2 holds promise to become a reference model of E. coli's central metabolism.

  2. Metabolism of minor isoforms of prion proteins: Cytosolic prion protein and transmembrane prion protein

    OpenAIRE

    Song, Zhiqi; Zhao, Deming; Yang, Lifeng

    2013-01-01

    Transmissible spongiform encephalopathy or prion disease is triggered by the conversion from cellular prion protein to pathogenic prion protein. Growing evidence has concentrated on prion protein configuration changes and their correlation with prion disease transmissibility and pathogenicity. In vivo and in vitro studies have shown that several cytosolic forms of prion protein with specific topological structure can destroy intracellular stability and contribute to prion protein pathogenicit...

  3. PROTEIN METABOLISM IN REGENERATING WOUND TISSUE: FUNCTION OF THE SULFUR AMINO ACIDS.

    Science.gov (United States)

    PROTEINS, *TISSUES(BIOLOGY), METABOLISM, TISSUES(BIOLOGY), REGENERATION(ENGINEERING), WOUNDS AND INJURIES, TISSUES(BIOLOGY), TRACER STUDIES, METHIONINE, COLLAGEN, TYROSINE, BIOSYNTHESIS, AMINO ACIDS .

  4. Preparation and recognition of surface molecularly imprinted core-shell microbeads for protein in aqueous solutions

    Energy Technology Data Exchange (ETDEWEB)

    Lu Yan, E-mail: yanlu2001@sohu.com [College of Chemistry and Environmental Science, Henan Normal University, 46 Jlanshe Road, Xinxiang 453007 (China); Yan Changling; Gao Shuyan [College of Chemistry and Environmental Science, Henan Normal University, 46 Jlanshe Road, Xinxiang 453007 (China)

    2009-04-01

    In this paper, a surface molecular imprinting technique was reported for preparing core-shell microbeads of protein imprinting, and bovine hemoglobin or bovine serum albumin were used as model proteins for studying the imprinted core-shell microbeads. 3-Aminophenylboronic acid (APBA) was polymerized onto the surface of polystyrene microbead in the presence of the protein templates to create protein-imprinted core-shell microbeads. The various samples were characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS) and Brunauer-Emmett-Teller (BET) methods. The effect of pH on rebinding of the template hemoglobin, the specific binding and selective recognition were studied for the imprinted microbeads. The results show that the bovine hemoglobin-imprinted core-shell microbeads were successfully created. The shell was a sort of imprinted thin films with porous structure and larger surface areas. The imprinted microbeads have good selectivity for templates and high stability. Due to the recognition sites locating at or closing to the surface, these imprinted microbeads have good property of mass-transport. Unfortunately, the imprint technology was not successfully applied to imprinting bovine serum albumin (BSA).

  5. Uncoupling proteins, dietary fat and the metabolic syndrome

    Directory of Open Access Journals (Sweden)

    Warden Craig H

    2006-09-01

    Full Text Available Abstract There has been intense interest in defining the functions of UCP2 and UCP3 during the nine years since the cloning of these UCP1 homologues. Current data suggest that both UCP2 and UCP3 proteins share some features with UCP1, such as the ability to reduce mitochondrial membrane potential, but they also have distinctly different physiological roles. Human genetic studies consistently demonstrate the effect of UCP2 alleles on type-2 diabetes. Less clear is whether UCP2 alleles influence body weight or body mass index (BMI with many studies showing a positive effect while others do not. There is strong evidence that both UCP2 and UCP3 protect against mitochondrial oxidative damage by reducing the production of reactive oxygen species. The evidence that UCP2 protein is a negative regulator of insulin secretion by pancreatic β-cells is also strong: increased UCP2 decreases glucose stimulated insulin secretion ultimately leading to β-cell dysfunction. UCP2 is also neuroprotective, reducing oxidative stress in neurons. UCP3 may also transport fatty acids out of mitochondria thereby protecting the mitochondria from fatty acid anions or peroxides. Current data suggest that UCP2 plays a role in the metabolic syndrome through down-regulation of insulin secretion and development of type-2 diabetes. However, UCP2 may protect against atherosclerosis through reduction of oxidative stress and both UCP2 and UCP3 may protect against obesity. Thus, these UCP1 homologues may both contribute to and protect from the markers of the metabolic syndrome.

  6. Metabolism of minor isoforms of prion proteins: Cytosolic prion protein and transmembrane prion protein

    Science.gov (United States)

    Song, Zhiqi; Zhao, Deming; Yang, Lifeng

    2013-01-01

    Transmissible spongiform encephalopathy or prion disease is triggered by the conversion from cellular prion protein to pathogenic prion protein. Growing evidence has concentrated on prion protein configuration changes and their correlation with prion disease transmissibility and pathogenicity. In vivo and in vitro studies have shown that several cytosolic forms of prion protein with specific topological structure can destroy intracellular stability and contribute to prion protein pathogenicity. In this study, the latest molecular chaperone system associated with endoplasmic reticulum-associated protein degradation, the endoplasmic reticulum resident protein quality-control system and the ubiquitination proteasome system, is outlined. The molecular chaperone system directly correlates with the prion protein degradation pathway. Understanding the molecular mechanisms will help provide a fascinating avenue for further investigations on prion disease treatment and prion protein-induced neurodegenerative diseases. PMID:25206608

  7. Characterization of the fusion core in zebrafish endogenous retroviral envelope protein

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Jian [State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei 430072 (China); State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071 (China); Zhang, Huaidong [CAS Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071 (China); Gong, Rui, E-mail: gongr@wh.iov.cn [CAS Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071 (China); Xiao, Gengfu, E-mail: xiaogf@wh.iov.cn [State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei 430072 (China); State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071 (China)

    2015-05-08

    Zebrafish endogenous retrovirus (ZFERV) is the unique endogenous retrovirus in zebrafish, as yet, containing intact open reading frames of its envelope protein gene in zebrafish genome. Similarly, several envelope proteins of endogenous retroviruses in human and other mammalian animal genomes (such as syncytin-1 and 2 in human, syncytin-A and B in mouse) were identified and shown to be functional in induction of cell–cell fusion involved in placental development. ZFERV envelope protein (Env) gene appears to be also functional in vivo because it is expressible. After sequence alignment, we found ZFERV Env shares similar structural profiles with syncytin and other type I viral envelopes, especially in the regions of N- and C-terminal heptad repeats (NHR and CHR) which were crucial for membrane fusion. We expressed the regions of N + C protein in the ZFERV Env (residues 459–567, including predicted NHR and CHR) to characterize the fusion core structure. We found N + C protein could form a stable coiled-coil trimer that consists of three helical NHR regions forming a central trimeric core, and three helical CHR regions packing into the grooves on the surface of the central core. The structural characterization of the fusion core revealed the possible mechanism of fusion mediated by ZFERV Env. These results gave comprehensive explanation of how the ancient virus infects the zebrafish and integrates into the genome million years ago, and showed a rational clue for discovery of physiological significance (e.g., medicate cell–cell fusion). - Highlights: • ZFERV Env shares similar structural profiles with syncytin and other type I viral envelopes. • The fusion core of ZFERV Env forms stable coiled-coil trimer including three NHRs and three CHRs. • The structural mechanism of viral entry mediated by ZFERV Env is disclosed. • The results are helpful for further discovery of physiological function of ZFERV Env in zebrafish.

  8. Systems biology definition of the core proteome of metabolism and expression is consistent with high-throughput data.

    Science.gov (United States)

    Yang, Laurence; Tan, Justin; O'Brien, Edward J; Monk, Jonathan M; Kim, Donghyuk; Li, Howard J; Charusanti, Pep; Ebrahim, Ali; Lloyd, Colton J; Yurkovich, James T; Du, Bin; Dräger, Andreas; Thomas, Alex; Sun, Yuekai; Saunders, Michael A; Palsson, Bernhard O

    2015-08-25

    Finding the minimal set of gene functions needed to sustain life is of both fundamental and practical importance. Minimal gene lists have been proposed by using comparative genomics-based core proteome definitions. A definition of a core proteome that is supported by empirical data, is understood at the systems-level, and provides a basis for computing essential cell functions is lacking. Here, we use a systems biology-based genome-scale model of metabolism and expression to define a functional core proteome consisting of 356 gene products, accounting for 44% of the Escherichia coli proteome by mass based on proteomics data. This systems biology core proteome includes 212 genes not found in previous comparative genomics-based core proteome definitions, accounts for 65% of known essential genes in E. coli, and has 78% gene function overlap with minimal genomes (Buchnera aphidicola and Mycoplasma genitalium). Based on transcriptomics data across environmental and genetic backgrounds, the systems biology core proteome is significantly enriched in nondifferentially expressed genes and depleted in differentially expressed genes. Compared with the noncore, core gene expression levels are also similar across genetic backgrounds (two times higher Spearman rank correlation) and exhibit significantly more complex transcriptional and posttranscriptional regulatory features (40% more transcription start sites per gene, 22% longer 5'UTR). Thus, genome-scale systems biology approaches rigorously identify a functional core proteome needed to support growth. This framework, validated by using high-throughput datasets, facilitates a mechanistic understanding of systems-level core proteome function through in silico models; it de facto defines a paleome.

  9. First principles design of a core bioenergetic transmembrane electron transfer protein

    Science.gov (United States)

    Goparaju, Geetha; Fry, Bryan A.; Chobot, Sarah E.; Wiedman, Gregory; Moser, Christopher C.; Dutton, P. Leslie; Discher, Bohdana M.

    2016-01-01

    Here we describe the design, E. coli expression and characterization of a simplified, adaptable and functionally transparent single chain 4-α-helix transmembrane protein frame that binds multiple heme and light activatable porphyrins. Such man-made cofactor-binding oxidoreductases, designed from first principles with minimal reference to natural protein sequences, are known as maquettes. This design is an adaptable frame aiming to uncover core engineering principles governing bioenergetic transmembrane electron-transfer function and recapitulate protein archetypes proposed to represent the origins of photosynthesis. PMID:26672896

  10. Lipoic acid entrains the hepatic circadian clock and lipid metabolic proteins that have been desynchronized with advanced age

    Energy Technology Data Exchange (ETDEWEB)

    Keith, Dove; Finlay, Liam; Butler, Judy [Linus Pauling Institute, Oregon State University (United States); Gómez, Luis; Smith, Eric [Linus Pauling Institute, Oregon State University (United States); Biochemistry Biophysics Department, Oregon State University (United States); Moreau, Régis [Linus Pauling Institute, Oregon State University (United States); Hagen, Tory, E-mail: Tory.Hagen@oregonstate.edu [Linus Pauling Institute, Oregon State University (United States); Biochemistry Biophysics Department, Oregon State University (United States)

    2014-07-18

    Highlights: • 24 month old rats were supplemented with 0.2% lipoic acid in the diet for 2 weeks. • Lipoic acid shifts phase of core circadian clock proteins. • Lipoic acid corrects age-induced desynchronized lipid metabolism rhythms. - Abstract: It is well established that lipid metabolism is controlled, in part, by circadian clocks. However, circadian clocks lose temporal precision with age and correlates with elevated incidence in dyslipidemia and metabolic syndrome in older adults. Because our lab has shown that lipoic acid (LA) improves lipid homeostasis in aged animals, we hypothesized that LA affects the circadian clock to achieve these results. We fed 24 month old male F344 rats a diet supplemented with 0.2% (w/w) LA for 2 weeks prior to sacrifice and quantified hepatic circadian clock protein levels and clock-controlled lipid metabolic enzymes. LA treatment caused a significant phase-shift in the expression patterns of the circadian clock proteins Period (Per) 2, Brain and Muscle Arnt-Like1 (BMAL1), and Reverse Erythroblastosis virus (Rev-erb) β without altering the amplitude of protein levels during the light phase of the day. LA also significantly altered the oscillatory patterns of clock-controlled proteins associated with lipid metabolism. The level of peroxisome proliferator-activated receptor (PPAR) α was significantly increased and acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) were both significantly reduced, suggesting that the LA-supplemented aged animals are in a catabolic state. We conclude that LA remediates some of the dyslipidemic processes associated with advanced age, and this mechanism may be at least partially through entrainment of circadian clocks.

  11. Hepatitis C Virus Core Protein Modulates Endoglin (CD105) Signaling Pathway for Liver Pathogenesis.

    Science.gov (United States)

    Kwon, Young-Chan; Sasaki, Reina; Meyer, Keith; Ray, Ranjit

    2017-11-01

    Endoglin is part of the TGF-β receptor complex and has a crucial role in fibrogenesis and angiogenesis. It is also an important protein for tumor growth, survival, and cancer cell metastasis. In a previous study, we have shown that hepatitis C virus (HCV) infection induces epithelial-mesenchymal transition (EMT) state and cancer stem-like cell (CSC) properties in human hepatocytes. Our array data suggested that endoglin (CD105) mRNA is significantly upregulated in HCV-associated CSCs. In this study, we have observed increased endoglin expression on the cell surface of an HCV core-expressing hepatocellular carcinoma (HepG2) cell line or immortalized human hepatocytes (IHH) and activation of its downstream signaling molecules. The status of phospho-SMAD1/5 and the expression of inhibitor of DNA binding protein 1 (ID1) were upregulated in HCV-infected cells or viral core gene-transfected cells. Additionally, we observed upregulation of endoglin/ID1 mRNA expression in chronic HCV patient liver biopsy samples. CSC generation by HCV core protein was dependent on the endoglin signaling pathway using activin receptor-like kinase 1 (ALK1) Fc blocking peptide and endoglin small interfering RNA (siRNA). Further, follow-up from in vitro analysis suggested that the antiapoptosis Bcl2 protein, proliferation-related cyclin D1 protein, and CSC-associated Hes1, Notch1, Nanog, and Sox2 proteins are enhanced during infection or ectopic expression of HCV core protein. IMPORTANCE Endoglin plays a crucial role in fibrogenesis and angiogenesis and is an important protein for tumor growth, survival, and cancer cell metastasis. Endoglin enhances ALK1-SMAD1/5 signaling in different cell types, leading to increased proliferation and migration responses. We have observed endoglin expression on the HCV core-expressing cell surface of human hepatocyte origin and activation of phospho-SMAD1/5 and ID1 downstream signaling molecules. ID1 protein plays a role in CSC properties, and we found that

  12. High prevalence of antibodies to core+1/ARF protein in HCV-infected patients with advanced cirrhosis.

    Science.gov (United States)

    Kassela, Katerina; Karakasiliotis, Ioannis; Charpantidis, Stefanos; Koskinas, John; Mylopoulou, Theodora; Mimidis, Konstantinos; Sarrazin, Christoph; Grammatikos, Georgios; Mavromara, Penelope

    2017-07-01

    Hepatitis C virus (HCV) possesses a second open reading frame (ORF) within the core gene encoding an additional protein, known as the alternative reading frame protein (ARFP), F or core+1. The biological significance of the core+1/ARF protein remains elusive. However, several independent studies have shown the presence of core+1/ARFP antibodies in chronically HCV-infected patients. Furthermore, a higher prevalence of core+1/ARFP antibodies was detected in patients with HCV-associated hepatocellular carcinoma (HCC). Here, we investigated the incidence of core+1/ARFPantibodies in chronically HCV-infected patients at different stages of cirrhosis in comparison to chronically HCV-infected patients at earlier stages of disease. Using ELISA, we assessed the prevalence of anti-core+1 antibodies in 30 patients with advanced cirrhosis [model for end-stage liver disease (MELD) ≥15] in comparison with 50 patients with mild cirrhosis (MELD core+1 antibodies, in contrast with 16.5 % of non-cirrhotic HCV patients. Moreover, there was significantly higher positivity for anti-core+1 antibodies in HCV patients with advanced cirrhosis (36.7 %) compared to those with early cirrhosis (24 %) (Pcore+1 antibodies in HCV patients with HCC, suggest that core+1 protein may have a role in virus-associated pathogenesis, and provide evidence to suggest that the levels of anti-core+1 antibodies may serve as a marker for disease progression.

  13. First principles design of a core bioenergetic transmembrane electron-transfer protein.

    Science.gov (United States)

    Goparaju, Geetha; Fry, Bryan A; Chobot, Sarah E; Wiedman, Gregory; Moser, Christopher C; Leslie Dutton, P; Discher, Bohdana M

    2016-05-01

    Here we describe the design, Escherichia coli expression and characterization of a simplified, adaptable and functionally transparent single chain 4-α-helix transmembrane protein frame that binds multiple heme and light activatable porphyrins. Such man-made cofactor-binding oxidoreductases, designed from first principles with minimal reference to natural protein sequences, are known as maquettes. This design is an adaptable frame aiming to uncover core engineering principles governing bioenergetic transmembrane electron-transfer function and recapitulate protein archetypes proposed to represent the origins of photosynthesis. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Protein and energy metabolism in two lines of chickens selected for growth on high or low protein diets

    DEFF Research Database (Denmark)

    Chwalibog, André; Eggum, B O; Sørensen, Peter

    1983-01-01

    Genetic adaptation was investigated in broilers selected for seven generations on a normal (A) or a low (B) protein diet. Protein and energy metabolism were studied in males from these selected lines fed on a diet of intermediate protein content. All selected birds retained more nitrogen than those...

  15. Impact of Dietary Carbohydrate and Protein Levels on Carbohydrate Metabolism

    Science.gov (United States)

    Lasker, Denise Ann

    2009-01-01

    The goal of this dissertation was to investigate the impact of changing dietary carbohydrate (CARB) intakes within recommended dietary guidelines on metabolic outcomes specifically associated with glycemic regulations and carbohydrate metabolism. This research utilized both human and animal studies to examine changes in metabolism across a wide…

  16. Mass spectrometry of Escherichia coli RNA polymerase: interactions of the core enzyme with sigma70 and Rsd protein.

    Science.gov (United States)

    Ilag, Leopold L; Westblade, Lars F; Deshayes, Caroline; Kolb, Annie; Busby, Stephen J W; Robinson, Carol V

    2004-02-01

    The E. coli RNA polymerase core enzyme is a multisubunit complex of 388,981 Da. To initiate transcription at promoters, the core enzyme associates with a sigma subunit to form holo RNA polymerase. Here we have used nanoflow electrospray mass spectrometry, coupled with tandem mass spectrometry, to probe the interaction of the RNA polymerase core enzyme with the most abundant sigma factor, sigma70. The results show remarkably well-resolved spectra for both the core and holo RNA polymerases. The regulator of sigma70, Rsd protein, has previously been identified as a protein that binds to free sigma70. We show that Rsd also interacts with core enzyme. In addition, by adding increasing amounts of Rsd, we show that sigma70 is displaced from holo RNA polymerase, resulting in complexes of Rsd with core and sigma70. The results argue for a model in which Rsd not only sequesters sigma70, but is also an effector of core RNA polymerase.

  17. A miniature protein stabilized by a cation-π interaction network core

    Science.gov (United States)

    Craven, Timothy W.; Cho, Min-Kyu; Traaseth, Nathaniel J.; Bonneau, Richard; Kirshenbaum, Kent

    2016-01-01

    The design of folded miniature proteins is predicated on establishing non-covalent interactions that direct the self-assembly of discrete thermo-stable tertiary structures. In this work, we describe how a network of cation-π interactions present in proteins containing “WSXWS motifs” can be emulated to stabilize the core of a miniature protein. This 19-residue protein sequence recapitulates a set of interdigitated arginine and tryptophan residues that stabilize a distinctive β-strand:loop:PPII-helix topology. Validation of the compact fold determined by NMR was carried out by mutagenesis of the cation-π network and by comparison to the corresponding disulfide-bridged structure. These results support the involvement of a coordinated set of cation-π interactions that stabilize the tertiary structure. PMID:26812069

  18. Comparative Proteomics Provides Insights into Metabolic Responses in Rat Liver to Isolated Soy and Meat Proteins.

    Science.gov (United States)

    Song, Shangxin; Hooiveld, Guido J; Zhang, Wei; Li, Mengjie; Zhao, Fan; Zhu, Jing; Xu, Xinglian; Muller, Michael; Li, Chunbao; Zhou, Guanghong

    2016-04-01

    It has been reported that isolated dietary soy and meat proteins have distinct effects on physiology and liver gene expression, but the impact on protein expression responses are unknown. Because these may differ from gene expression responses, we investigated dietary protein-induced changes in liver proteome. Rats were fed for 1 week semisynthetic diets that differed only regarding protein source; casein (reference) was fully replaced by isolated soy, chicken, fish, or pork protein. Changes in liver proteome were measured by iTRAQ labeling and LC-ESI-MS/MS. A robust set totaling 1437 unique proteins was identified and subjected to differential protein analysis and biological interpretation. Compared with casein, all other protein sources reduced the abundance of proteins involved in fatty acid metabolism and Pparα signaling pathway. All dietary proteins, except chicken, increased oxidoreductive transformation reactions but reduced energy and essential amino acid metabolic pathways. Only soy protein increased the metabolism of sulfur-containing and nonessential amino acids. Soy and fish proteins increased translation and mRNA processing, whereas only chicken protein increased TCA cycle but reduced immune responses. These findings were partially in line with previously reported transcriptome results. This study further shows the distinct effects of soy and meat proteins on liver metabolism in rats.

  19. Assessing the Metabolic Diversity of Streptococcus from a Protein Domain Point of View

    Science.gov (United States)

    Koehorst, Jasper J.; Martins dos Santos, Vitor A. P.; Schaap, Peter J.

    2015-01-01

    Understanding the diversity and robustness of the metabolism of bacteria is fundamental for understanding how bacteria evolve and adapt to different environments. In this study, we characterised 121 Streptococcus strains and studied metabolic diversity from a protein domain perspective. Metabolic pathways were described in terms of the promiscuity of domains participating in metabolic pathways that were inferred to be functional. Promiscuity was defined by adapting existing measures based on domain abundance and versatility. The approach proved to be successful in capturing bacterial metabolic flexibility and species diversity, indicating that it can be described in terms of reuse and sharing functional domains in different proteins involved in metabolic activity. Additionally, we showed striking differences among metabolic organisation of the pathogenic serotype 2 Streptococcus suis and other strains. PMID:26366735

  20. Magnetic resonance metabolic profiling of breast cancer tissue obtained with core needle biopsy for predicting pathologic response to neoadjuvant chemotherapy.

    Directory of Open Access Journals (Sweden)

    Ji Soo Choi

    Full Text Available The purpose of this study was to determine whether metabolic profiling of core needle biopsy (CNB samples using high-resolution magic angle spinning (HR-MAS magnetic resonance spectroscopy (MRS could be used for predicting pathologic response to neoadjuvant chemotherapy (NAC in patients with locally advanced breast cancer. After institutional review board approval and informed consent were obtained, CNB tissue samples were collected from 37 malignant lesions in 37 patients before NAC treatment. The metabolic profiling of CNB samples were performed by HR-MAS MRS. Metabolic profiles were compared according to pathologic response to NAC using the Mann-Whitney test. Multivariate analysis was performed with orthogonal projections to latent structure-discriminant analysis (OPLS-DA. Various metabolites including choline-containing compounds were identified and quantified by HR-MAS MRS in all 37 breast cancer tissue samples obtained by CNB. In univariate analysis, the metabolite concentrations and metabolic ratios of CNB samples obtained with HR-MAS MRS were not significantly different between different pathologic response groups. However, there was a trend of lower levels of phosphocholine/creatine ratio and choline-containing metabolite concentrations in the pathologic complete response group compared to the non-pathologic complete response group. In multivariate analysis, the OPLS-DA models built with HR-MAS MR metabolic profiles showed visible discrimination between the pathologic response groups. This study showed OPLS-DA multivariate analysis using metabolic profiles of pretreatment CNB samples assessed by HR- MAS MRS may be used to predict pathologic response before NAC, although we did not identify the metabolite showing statistical significance in univariate analysis. Therefore, our preliminary results raise the necessity of further study on HR-MAS MR metabolic profiling of CNB samples for a large number of cancers.

  1. Mapping the Differential Distribution of Proteoglycan Core Proteins in the Adult Human Retina, Choroid, and Sclera

    Science.gov (United States)

    Keenan, Tiarnan D. L.; Clark, Simon J.; Unwin, Richard D.; Ridge, Liam A.; Day, Anthony J.; Bishop, Paul N.

    2012-01-01

    Purpose. To examine the presence and distribution of proteoglycan (PG) core proteins in the adult human retina, choroid, and sclera. Methods. Postmortem human eye tissue was dissected into Bruch's membrane/choroid complex, isolated Bruch's membrane, or neurosensory retina. PGs were extracted and partially purified by anion exchange chromatography. Trypsinized peptides were analyzed by tandem mass spectrometry and PG core proteins identified by database search. The distribution of PGs was examined by immunofluorescence microscopy on human macular tissue sections. Results. The basement membrane PGs perlecan, agrin, and collagen-XVIII were identified in the human retina, and were present in the internal limiting membrane, blood vessel walls, and Bruch's membrane. The hyalectans versican and aggrecan were also detected. Versican was identified in Bruch's membrane, while aggrecan was distributed throughout the retina, choroid, and sclera. The cartilage link protein HAPLN1 was abundant in the interphotoreceptor matrix and sclera, while HAPLN4 (brain link protein 2) was found throughout the retina and choroid. The small leucine-rich repeat PG (SLRP) family members biglycan, decorin, fibromodulin, lumican, mimecan, opticin, and prolargin were present, with different patterns of distribution in the retina, choroid, and sclera. Conclusions. A combination of proteomics and immunohistochemistry approaches has provided for the first time a comprehensive analysis of the presence and distribution of PG core proteins throughout the human retina, choroid, and sclera. This complements our knowledge of glycosaminoglycan chain distribution in the human eye, and has important implications for understanding the structure and functional regulation of the eye in health and disease. PMID:23074202

  2. Differential Effects of Hydrophobic Core Packing Residues for Thermodynamic and Mechanical Stability of a Hyperthermophilic Protein.

    Science.gov (United States)

    Tych, Katarzyna M; Batchelor, Matthew; Hoffmann, Toni; Wilson, Michael C; Hughes, Megan L; Paci, Emanuele; Brockwell, David J; Dougan, Lorna

    2016-07-26

    Proteins from organisms that have adapted to environmental extremes provide attractive systems to explore and determine the origins of protein stability. Improved hydrophobic core packing and decreased loop-length flexibility can increase the thermodynamic stability of proteins from hyperthermophilic organisms. However, their impact on protein mechanical stability is not known. Here, we use protein engineering, biophysical characterization, single-molecule force spectroscopy (SMFS), and molecular dynamics (MD) simulations to measure the effect of altering hydrophobic core packing on the stability of the cold shock protein TmCSP from the hyperthermophilic bacterium Thermotoga maritima. We make two variants of TmCSP in which a mutation is made to reduce the size of aliphatic groups from buried hydrophobic side chains. In the first, a mutation is introduced in a long loop (TmCSP L40A); in the other, the mutation is introduced on the C-terminal β-strand (TmCSP V62A). We use MD simulations to confirm that the mutant TmCSP L40A shows the most significant increase in loop flexibility, and mutant TmCSP V62A shows greater disruption to the core packing. We measure the thermodynamic stability (ΔGD-N) of the mutated proteins and show that there is a more significant reduction for TmCSP L40A (ΔΔG = 63%) than TmCSP V62A (ΔΔG = 47%), as might be expected on the basis of the relative reduction in the size of the side chain. By contrast, SMFS measures the mechanical stability (ΔG*) and shows a greater reduction for TmCSP V62A (ΔΔG* = 8.4%) than TmCSP L40A (ΔΔG* = 2.5%). While the impact on the mechanical stability is subtle, the results demonstrate the power of tuning noncovalent interactions to modulate both the thermodynamic and mechanical stability of a protein. Such understanding and control provide the opportunity to design proteins with optimized thermodynamic and mechanical properties.

  3. Metabolic Turnover of Synaptic Proteins: Kinetics, Interdependencies and Implications for Synaptic Maintenance

    Science.gov (United States)

    Cohen, Laurie D.; Zuchman, Rina; Sorokina, Oksana; Müller, Anke; Dieterich, Daniela C.; Armstrong, J. Douglas; Ziv, Tamar; Ziv, Noam E.

    2013-01-01

    Chemical synapses contain multitudes of proteins, which in common with all proteins, have finite lifetimes and therefore need to be continuously replaced. Given the huge numbers of synaptic connections typical neurons form, the demand to maintain the protein contents of these connections might be expected to place considerable metabolic demands on each neuron. Moreover, synaptic proteostasis might differ according to distance from global protein synthesis sites, the availability of distributed protein synthesis facilities, trafficking rates and synaptic protein dynamics. To date, the turnover kinetics of synaptic proteins have not been studied or analyzed systematically, and thus metabolic demands or the aforementioned relationships remain largely unknown. In the current study we used dynamic Stable Isotope Labeling with Amino acids in Cell culture (SILAC), mass spectrometry (MS), Fluorescent Non–Canonical Amino acid Tagging (FUNCAT), quantitative immunohistochemistry and bioinformatics to systematically measure the metabolic half-lives of hundreds of synaptic proteins, examine how these depend on their pre/postsynaptic affiliation or their association with particular molecular complexes, and assess the metabolic load of synaptic proteostasis. We found that nearly all synaptic proteins identified here exhibited half-lifetimes in the range of 2–5 days. Unexpectedly, metabolic turnover rates were not significantly different for presynaptic and postsynaptic proteins, or for proteins for which mRNAs are consistently found in dendrites. Some functionally or structurally related proteins exhibited very similar turnover rates, indicating that their biogenesis and degradation might be coupled, a possibility further supported by bioinformatics-based analyses. The relatively low turnover rates measured here (∼0.7% of synaptic protein content per hour) are in good agreement with imaging-based studies of synaptic protein trafficking, yet indicate that the metabolic load

  4. Genome-scale metabolic model of Pichia pastoris with native and humanized glycosylation of recombinant proteins

    DEFF Research Database (Denmark)

    Irani, Zahra Azimzadeh; Kerkhoven, Eduard J.; Shojaosadati, Seyed Abbas

    2016-01-01

    native nor humanized N-glycosylation, and we therefore developed ihGlycopastoris, an extension to the iLC915 model with both native and humanized N-glycosylation for recombinant protein production, but also an estimation of N-glycosylation of P. pastoris native proteins. This new model gives a better......Pichia pastoris is used for commercial production of human therapeutic proteins, and genome-scale models of P. pastoris metabolism have been generated in the past to study the metabolism and associated protein production by this yeast. A major challenge with clinical usage of recombinant proteins...... produced by P. pastoris is the difference in N-glycosylation of proteins produced by humans and this yeast. However, through metabolic engineering, a P. pastoris strain capable of producing humanized N-glycosylated proteins was constructed. The current genome-scale models of P. pastoris do not address...

  5. Protein engineering for metabolic engineering: current and next-generation tools

    Science.gov (United States)

    Marcheschi, Ryan J.; Gronenberg, Luisa S.; Liao, James C.

    2014-01-01

    Protein engineering in the context of metabolic engineering is increasingly important to the field of industrial biotechnology. As the demand for biologically-produced food, fuels, chemicals, food additives, and pharmaceuticals continues to grow, the ability to design and modify proteins to accomplish new functions will be required to meet the high productivity demands for the metabolism of engineered organisms. This article reviews advances of selecting, modeling, and engineering proteins to improve or alter their activity. Some of the methods have only recently been developed for general use and are just beginning to find greater application in the metabolic engineering community. We also discuss methods of generating random and targeted diversity in proteins to generate mutant libraries for analysis. Recent uses of these techniques to alter cofactor use, produce non-natural amino acids, alcohols, and carboxylic acids, and alter organism phenotypes are presented and discussed as examples of the successful engineering of proteins for metabolic engineering purposes. PMID:23589443

  6. Inhibition of protein aggregation by zwitterionic polymer-based core-shell nanogels

    Science.gov (United States)

    Rajan, Robin; Matsumura, Kazuaki

    2017-04-01

    Protein aggregation is a process by which misfolded proteins polymerizes into aggregates and forms fibrous structures with a β-sheet conformation, known as amyloids. It is an undesired outcome, as it not only causes numerous neurodegenerative diseases, but is also a major deterrent in the development of protein biopharmaceuticals. Here, we report a rational design for the synthesis of novel zwitterionic polymer-based core-shell nanogels via controlled radical polymerization. Nanogels with different sizes and functionalities in the core and shell were prepared. The nanogels exhibit remarkable efficiency in the protection of lysozyme against aggregation. Addition of nanogels suppresses the formation of toxic fibrils and also enables lysozyme to retain its enzymatic activity. Increasing the molecular weight and degree of hydrophobicity markedly increases its overall efficiency. Investigation of higher order structures revealed that lysozyme when heated without any additive loses its secondary structure and transforms into a random coil conformation. In contrast, presence of nanogels facilitates the retention of higher order structures by acting as molecular chaperones, thereby reducing molecular collisions. The present study is the first to show that it is possible to design zwitterionic nanogels using appropriate polymerization techniques that will protect proteins under conditions of extreme stress and inhibit aggregation.

  7. On the mineral core of ferritin-like proteins: structural and magnetic characterization

    Science.gov (United States)

    García-Prieto, A.; Alonso, J.; Muñoz, D.; Marcano, L.; Abad Díaz de Cerio, A.; Fernández de Luis, R.; Orue, I.; Mathon, O.; Muela, A.; Fdez-Gubieda, M. L.

    2015-12-01

    It is generally accepted that the mineral core synthesized by ferritin-like proteins consists of a ferric oxy-hydroxide mineral similar to ferrihydrite in the case of horse spleen ferritin (HoSF) and an oxy-hydroxide-phosphate phase in plant and prokaryotic ferritins. The structure reflects a dynamic process of deposition and dissolution, influenced by different biological, chemical and physical variables. In this work we shed light on this matter by combining a structural (High Resolution Transmission Electron Microscopy (HRTEM) and Fe K-edge X-ray Absorption Spectroscopy (XAS)) and a magnetic study of the mineral core biomineralized by horse spleen ferritin (HoSF) and three prokaryotic ferritin-like proteins: bacterial ferritin (FtnA) and bacterioferritin (Bfr) from Escherichia coli and archaeal ferritin (PfFtn) from Pyrococcus furiosus. The prokaryotic ferritin-like proteins have been studied under native conditions and inside the cells for the sake of preserving their natural attributes. They share with HoSF a nanocrystalline structure rather than an amorphous one as has been frequently reported. However, the presence of phosphorus changes drastically the short-range order and magnetic response of the prokaryotic cores with respect to HoSF. The superparamagnetism observed in HoSF is absent in the prokaryotic proteins, which show a pure atomic-like paramagnetic behaviour attributed to phosphorus breaking the Fe-Fe exchange interaction.It is generally accepted that the mineral core synthesized by ferritin-like proteins consists of a ferric oxy-hydroxide mineral similar to ferrihydrite in the case of horse spleen ferritin (HoSF) and an oxy-hydroxide-phosphate phase in plant and prokaryotic ferritins. The structure reflects a dynamic process of deposition and dissolution, influenced by different biological, chemical and physical variables. In this work we shed light on this matter by combining a structural (High Resolution Transmission Electron Microscopy (HRTEM

  8. The origin of modern metabolic networks inferred from phylogenomic analysis of protein architecture.

    Science.gov (United States)

    Caetano-Anollés, Gustavo; Kim, Hee Shin; Mittenthal, Jay E

    2007-05-29

    Metabolism represents a complex collection of enzymatic reactions and transport processes that convert metabolites into molecules capable of supporting cellular life. Here we explore the origins and evolution of modern metabolism. Using phylogenomic information linked to the structure of metabolic enzymes, we sort out recruitment processes and discover that most enzymatic activities were associated with the nine most ancient and widely distributed protein fold architectures. An analysis of newly discovered functions showed enzymatic diversification occurred early, during the onset of the modern protein world. Most importantly, phylogenetic reconstruction exercises and other evidence suggest strongly that metabolism originated in enzymes with the P-loop hydrolase fold in nucleotide metabolism, probably in pathways linked to the purine metabolic subnetwork. Consequently, the first enzymatic takeover of an ancient biochemistry or prebiotic chemistry was related to the synthesis of nucleotides for the RNA world.

  9. TRF2 Protein Interacts with Core Histones to Stabilize Chromosome Ends*

    Science.gov (United States)

    Izumi, Takashi; Shimizu, Shigeomi

    2016-01-01

    Mammalian chromosome ends are protected by a specialized nucleoprotein complex called telomeres. Both shelterin, a telomere-specific multi-protein complex, and higher order telomeric chromatin structures combine to stabilize the chromosome ends. Here, we showed that TRF2, a component of shelterin, binds to core histones to protect chromosome ends from inappropriate DNA damage response and loss of telomeric DNA. The N-terminal Gly/Arg-rich domain (GAR domain) of TRF2 directly binds to the globular domain of core histones. The conserved arginine residues in the GAR domain of TRF2 are required for this interaction. A TRF2 mutant with these arginine residues substituted by alanine lost the ability to protect telomeres and induced rapid telomere shortening caused by the cleavage of a loop structure of the telomeric chromatin. These findings showed a previously unnoticed interaction between the shelterin complex and nucleosomal histones to stabilize the chromosome ends. PMID:27514743

  10. Changes in expression of proteoglycan core proteins and heparan sulfate enzymes in the developing and adult murine aorta.

    Science.gov (United States)

    Adhikari, Neeta; Carlson, Marjorie; Lerman, Ben; Hall, Jennifer L

    2011-06-01

    Proteoglycan core proteins are linked to four different classes of linear sugar chains referred to as glycosaminoglycans. Heparan sulfate constitutes one of these classes of glycosaminoglycans, and has been shown to be important in developmental processes as well as disease. We designed a low-density gene expression array to identify expression levels of heparan sulfate biosynthetic enzymes and proteoglycan core proteins in the aorta of late stage embryos (E18.5) and adult mice (12 weeks). Significant changes were found in mRNA expression of proteoglycan core proteins syndecan, glypican, decorin, perlecan, and versican from development to adulthood (n = 8, p proteoglycan core proteins and heparan sulfate biosynthetic enzymes in the aorta undergo significant changes in their expression from development to adulthood. These findings may have important biological significance in the specific cell-defined roles of proteoglycan and heparan sulfate related targets in vascular development, maintenance, and response to various perturbations.

  11. Advances in the Understanding of Protein-Protein Interactions in Drug Metabolizing Enzymes through the Use of Biophysical Techniques

    Directory of Open Access Journals (Sweden)

    Jed N. Lampe

    2017-08-01

    Full Text Available In recent years, a growing appreciation has developed for the importance of protein-protein interactions to modulate the function of drug metabolizing enzymes. Accompanied with this appreciation, new methods and technologies have been designed for analyzing protein-protein interactions both in vitro and in vivo. These technologies have been applied to several classes of drug metabolizing enzymes, including: cytochrome P450's (CYPs, monoamine oxidases (MAOs, UDP-glucuronosyltransferases (UGTs, glutathione S-transferases (GSTs, and sulfotransferases (SULTs. In this review, we offer a brief description and assessment of the impact of many of these technologies to the study of protein-protein interactions in drug disposition. The still expanding list of these techniques and assays has the potential to revolutionize our understanding of how these enzymes carry out their important functions in vivo.

  12. Analysis of the chondroitin sulfate proteoglycan core protein (CSPGCP) gene in achondroplasia and pseudoachondroplasia.

    Science.gov (United States)

    Finkelstein, J E; Doege, K; Yamada, Y; Pyeritz, R E; Graham, J M; Moeschler, J B; Pauli, R M; Hecht, J T; Francomano, C A

    1991-01-01

    Achondroplasia and pseudoachondroplasia are autosomal dominant skeletal dysplasias resulting in short-limbed dwarfism. Histologic and ultrastructural studies of the cartilage in pseudoachondroplasia and in homozygous achondroplasia have suggested a structural abnormality in chondroitin sulfate proteoglycan (CSPG), a major structural protein in the extra-cellular matrix. The gene encoding CSPG core protein (CSPGCP) is thus a logical "candidate gene" for analysis in these conditions. cDNA probes encoding CSPGCP were used to identify restriction fragment length polymorphisms (RFLPs) in DNA from a panel of control individuals. No gross alterations at the CSPGCP locus were noted in DNA from 37 individuals with achondroplasia and 5 individuals with pseudoachondroplasia. In addition, allelic frequencies of the RFLPs were not significantly different among controls and patients with either condition. In one three-generation family with achondroplasia, close linkage of the CSPGCP locus and the skeletal dysplasia was excluded using a Bgl II polymorphism. Similarly, in a three-generation family with pseudoachondroplasia, the CSPGCP gene was not tightly linked to the disease phenotype. These results indicate that mutations at the chondroitin sulfate proteoglycan core protein locus do not cause achondroplasia or pseudoachondroplasia in these families. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:1670752

  13. Study of Stationary Phase Metabolism Via Isotopomer Analysis of Amino Acids from an Isolated Protein

    Energy Technology Data Exchange (ETDEWEB)

    Shaikh, AfshanS.; Tang, YinjieJ.; Mukhopadhyay, Aindrila; Martin, Hector Garcia; Gin, Jennifer; Benke, Peter; Keasling, Jay D.

    2009-09-14

    Microbial production of many commercially important secondary metabolites occurs during stationary phase, and methods to measure metabolic flux during this growth phase would be valuable. Metabolic flux analysis is often based on isotopomer information from proteinogenic amino acids. As such, flux analysis primarily reflects the metabolism pertinent to the growth phase during which most proteins are synthesized. To investigate central metabolism and amino acids synthesis activity during stationary phase, addition of fully 13C-labeled glucose followed by induction of green fluorescent protein (GFP) expression during stationary phase was used. Our results indicate that Escherichia coli was able to produce new proteins (i.e., GFP) in the stationary phase, and the amino acids in GFP were mostly from degraded proteins synthesized during the exponential growth phase. Among amino acid biosynthetic pathways, only those for serine, alanine, glutamate/glutamine, and aspartate/asparagine had significant activity during the stationary phase.

  14. Effects of Quercetin Supplementation on Lipid and Protein Metabolism after Classic Boxing Training

    Science.gov (United States)

    Demirci, Nevzat

    2017-01-01

    The metabolic fitness (MF) is a component of athletes' physical conditioning. This study aims to investigate the effects of quercetin supplementation on Turkish Junior athletes' lipid and protein metabolism relating to MF after one month classic boxing training. Totally 20 voluntary junior male athletes were separated into two equal groups as the…

  15. The Rab-GTPase-activating protein TBC1D1 regulates skeletal muscle glucose metabolism

    DEFF Research Database (Denmark)

    Szekeres, Ferenc; Chadt, Alexandra; Tom, Robby Z

    2012-01-01

    The Rab-GTPase-activating protein TBC1D1 has emerged as a novel candidate involved in metabolic regulation. Our aim was to determine whether TBC1D1 is involved in insulin as well as energy-sensing signals controlling skeletal muscle metabolism. TBC1D1-deficient congenic B6.SJL-Nob1.10 (Nob1.10(SJ...

  16. Circulating adipocyte fatty acid-binding protein, juvenile obesity, and metabolic syndrome

    NARCIS (Netherlands)

    Krzystek-Korpacka, Malgorzata; Patryn, Eliza; Bednarz-Misa, Iwona; Mierzchala, Magdalena; Hotowy, Katarzyna; Czapinska, Elzbieta; Kustrzeba-Wojcicka, Irena; Gamian, Andrzej; Noczynska, Anna

    2011-01-01

    Adipocyte fatty acid-binding protein (A-FABP) links obesity and metabolic syndrome (MetS) and might be targeted in future therapies. Its utility as a MetS biomarker has been suggested in adults but has not been examined in children/adolescents. Our objectives were to identify metabolic parameters

  17. Use of Designer G Protein-Coupled Receptors to Dissect Metabolic Pathways.

    Science.gov (United States)

    Wess, Jürgen

    2016-09-01

    G protein-coupled receptors (GPCRs) regulate virtually all metabolic processes, including glucose and energy homeostasis. Recently, the use of designer GPCRs referred to as designer receptors exclusively activated by designer drug (DREADDs) has made it possible to dissect metabolically relevant GPCR signaling pathways in a temporally and spatially controlled fashion in vivo. Published by Elsevier Ltd.

  18. HBV core protein allosteric modulators differentially alter cccDNA biosynthesis from de novo infection and intracellular amplification pathways

    Science.gov (United States)

    Guo, Fang; Zhao, Qiong; Cheng, Junjun; Qi, Yonghe; Su, Qing; Wei, Lai; Li, Wenhui; Chang, Jinhong

    2017-01-01

    Hepatitis B virus (HBV) core protein assembles viral pre-genomic (pg) RNA and DNA polymerase into nucleocapsids for reverse transcriptional DNA replication to take place. Several chemotypes of small molecules, including heteroaryldihydropyrimidines (HAPs) and sulfamoylbenzamides (SBAs), have been discovered to allosterically modulate core protein structure and consequentially alter the kinetics and pathway of core protein assembly, resulting in formation of irregularly-shaped core protein aggregates or “empty” capsids devoid of pre-genomic RNA and viral DNA polymerase. Interestingly, in addition to inhibiting nucleocapsid assembly and subsequent viral genome replication, we have now demonstrated that HAPs and SBAs differentially modulate the biosynthesis of covalently closed circular (ccc) DNA from de novo infection and intracellular amplification pathways by inducing disassembly of nucleocapsids derived from virions as well as double-stranded DNA-containing progeny nucleocapsids in the cytoplasm. Specifically, the mistimed cuing of nucleocapsid uncoating prevents cccDNA formation during de novo infection of hepatocytes, while transiently accelerating cccDNA synthesis from cytoplasmic progeny nucleocapsids. Our studies indicate that elongation of positive-stranded DNA induces structural changes of nucleocapsids, which confers ability of mature nucleocapsids to bind CpAMs and triggers its disassembly. Understanding the molecular mechanism underlying the dual effects of the core protein allosteric modulators on nucleocapsid assembly and disassembly will facilitate the discovery of novel core protein-targeting antiviral agents that can more efficiently suppress cccDNA synthesis and cure chronic hepatitis B. PMID:28945802

  19. Functional analysis of thermostable proteins involved in carbohydrate metabolism

    NARCIS (Netherlands)

    Akerboom, A.P.

    2007-01-01

    Thermostable proteins can resist temperature stress whilst keeping their integrity and functionality. In many cases,  thermostable proteins originate from hyperthermophilic microorganisms that thrive in extreme environments. These systems are generally located close to geothermal (volcanic)

  20. Effect of ultrasound on protein metabolism in the silkworm, Bombyx ...

    African Journals Online (AJOL)

    Ultrasound was found to promote the accumulation of proteins, which include silk proteins as well, while retarding proteolysis and turnover of proteins towards the release of amino acids, keto-acids etc. Changes in the levels of these biochemical constituents are correlated with the events of histogenesis and histolysis ...

  1. Integrating the protein and metabolic engineering toolkits for next-generation chemical biosynthesis.

    Science.gov (United States)

    Pirie, Christopher M; De Mey, Marjan; Jones Prather, Kristala L; Ajikumar, Parayil Kumaran

    2013-04-19

    Through microbial engineering, biosynthesis has the potential to produce thousands of chemicals used in everyday life. Metabolic engineering and synthetic biology are fields driven by the manipulation of genes, genetic regulatory systems, and enzymatic pathways for developing highly productive microbial strains. Fundamentally, it is the biochemical characteristics of the enzymes themselves that dictate flux through a biosynthetic pathway toward the product of interest. As metabolic engineers target sophisticated secondary metabolites, there has been little recognition of the reduced catalytic activity and increased substrate/product promiscuity of the corresponding enzymes compared to those of central metabolism. Thus, fine-tuning these enzymatic characteristics through protein engineering is paramount for developing high-productivity microbial strains for secondary metabolites. Here, we describe the importance of protein engineering for advancing metabolic engineering of secondary metabolism pathways. This pathway integrated enzyme optimization can enhance the collective toolkit of microbial engineering to shape the future of chemical manufacturing.

  2. Pulsatile protein release from monodisperse liquid-core microcapsules of controllable shell thickness

    Science.gov (United States)

    Xia, Yujie; Pack, Daniel W.

    2014-01-01

    Purpose Pulsatile delivery of proteins, in which release occurs over a short time after a period of little or no release, is desirable for many applications. This paper investigates the effect of biodegradable polymer shell thickness on pulsatile protein release from biodegradable polymer microcapsules. Methods Using precision particle fabrication (PPF) technology, monodisperse microcapsules were fabricated encapsulating bovine serum albumin (BSA) in a liquid core surrounded by a drug-free poly(lactide-co-glycolide) (PLG) shell of uniform, controlled thickness from 14 to 19 μm. Results When using high molecular weight PLG (Mw 88 kDa), microparticles exhibited the desired core-shell structure with high BSA loading and encapsulation efficiency (55-65%). These particles exhibited very slow release of BSA for several weeks followed by rapid release of 80-90% of the encapsulated BSA within seven days. Importantly, with increasing shell thickness the starting time of the pulsatile release could be controlled from 25 to 35 days. Conclusions Biodegradable polymer microcapsules with precisely controlled shell thickness provide pulsatile release with enhanced control of release profiles. PMID:24831313

  3. Simple, direct conjugation of bacterial O-SP-core antigens to proteins: development of cholera conjugate vaccines.

    Science.gov (United States)

    Xu, Peng; Alam, Mohammad Murshid; Kalsy, Anuj; Charles, Richelle C; Calderwood, Stephen B; Qadri, Firdausi; Ryan, Edward T; Kováč, Pavol

    2011-10-19

    Bacterial O-SP-core antigens can be conjugated to proteins in the same, simple way as synthetic, linker-equipped carbohydrates by applying squaric acid chemistry. Introduction of spacers (linkers) to either O-SP-core antigens or protein carriers, which is involved in commonly applied protocols, is not required. The newly developed method described here consists of preparation of a squaric acid monoester derivative of O-SP-core antigen, utilizing the amino group inherent in the core, and reaction of the monoester with the carrier protein. The intermediate monoester can be easily purified; its conjugation can be monitored by SELDI-TOF mass spectrometry and, thus, readily controlled, since the conjugation can be terminated when the desired carbohydrate-protein ratio is reached. Here, we describe production of conjugates containing the O-SP-core antigen of Vibrio cholerae O1, the major cause of cholera, a severe dehydrating diarrheal disease of humans. The resultant products are recognized by convalescent phase sera from patients recovering from cholera in Bangladesh, and anti-O-SP-core-protein responses correlate with plasma antilipopolysaccharide and vibriocidal responses, which are the primary markers of protection from cholera. The results suggest that such conjugates have potential as vaccines for cholera and other bacterial diseases.

  4. A Simple, Direct Conjugation of Bacterial O-SP–Core Antigens to Proteins: Development of Cholera Conjugate Vaccines

    Science.gov (United States)

    Xu, Peng; Alam, Mohammad Murshid; Kalsy, Anuj; Charles, Richelle C.; Calderwood, Stephen B.; Qadri, Firdausi; Ryan, Edward T.; Kováč, Pavol

    2011-01-01

    Bacterial O-SP–core antigens can be conjugated to proteins in the same, simple way as synthetic, linker-equipped carbohydrates by applying squaric acid chemistry. Introduction of spacers (linkers) to either O-SP–core antigens or protein carriers, which is involved in commonly applied protocols, is not required. The newly developed method here described consists of preparation of a squaric acid monoester derivative of O-SP–core antigen, utilizing the amino group inherent in the core, and reaction of the monoester with the carrier protein. The intermediate monoester can be easily purified, its conjugation can be monitored by SELDI-TOF mass spectrometry and, thus, readily controlled, since the conjugation can be terminated when the desired carbohydrate–protein ratio is reached. Here we describe production of conjugates containing the O-SP-core antigen of Vibrio cholerae O1, the major cause of cholera, a severe dehydrating diarrheal disease of humans. The resultant products are recognized by convalescent phase sera from patients recovering from cholera in Bangladesh, and anti-O-SP-core-protein responses correlate with plasma anti-lipopolysaccharide and vibriocidal responses, which are the primary markers of protection from cholera. The results suggest that such conjugates have potential as vaccines for cholera and other bacterial diseases. PMID:21899371

  5. An evolutionarily conserved glycine-tyrosine motif forms a folding core in outer membrane proteins.

    Directory of Open Access Journals (Sweden)

    Marcin Michalik

    Full Text Available An intimate interaction between a pair of amino acids, a tyrosine and glycine on neighboring β-strands, has been previously reported to be important for the structural stability of autotransporters. Here, we show that the conservation of this interacting pair extends to nearly all major families of outer membrane β-barrel proteins, which are thought to have originated through duplication events involving an ancestral ββ hairpin. We analyzed the function of this motif using the prototypical outer membrane protein OmpX. Stopped-flow fluorescence shows that two folding processes occur in the millisecond time regime, the rates of which are reduced in the tyrosine mutant. Folding assays further demonstrate a reduction in the yield of folded protein for the mutant compared to the wild-type, as well as a reduction in thermal stability. Taken together, our data support the idea of an evolutionarily conserved 'folding core' that affects the folding, membrane insertion, and thermal stability of outer membrane protein β-barrels.

  6. Cannibalism Affects Core Metabolic Processes in Helicoverpa armigera Larvae—A 2D NMR Metabolomics Study

    Directory of Open Access Journals (Sweden)

    Fredd Vergara

    2016-09-01

    Full Text Available Cannibalism is known in many insect species, yet its impact on insect metabolism has not been investigated in detail. This study assessed the effects of cannibalism on the metabolism of fourth-instar larvae of the non-predatory insect Helicoverpa armigera (Lepidotera: Noctuidea. Two groups of larvae were analyzed: one group fed with fourth-instar larvae of H. armigera (cannibal, the other group fed with an artificial plant diet. Water-soluble small organic compounds present in the larvae were analyzed using two-dimensional nuclear magnetic resonance (NMR and principal component analysis (PCA. Cannibalism negatively affected larval growth. PCA of NMR spectra showed that the metabolic profiles of cannibal and herbivore larvae were statistically different with monomeric sugars, fatty acid- and amino acid-related metabolites as the most variable compounds. Quantitation of 1H-13C HSQC (Heteronuclear Single Quantum Coherence signals revealed that the concentrations of glucose, glucono-1,5-lactone, glycerol phosphate, glutamine, glycine, leucine, isoleucine, lysine, ornithine, proline, threonine and valine were higher in the herbivore larvae.

  7. Cannibalism Affects Core Metabolic Processes in Helicoverpa armigera Larvae-A 2D NMR Metabolomics Study.

    Science.gov (United States)

    Vergara, Fredd; Shino, Amiu; Kikuchi, Jun

    2016-09-02

    Cannibalism is known in many insect species, yet its impact on insect metabolism has not been investigated in detail. This study assessed the effects of cannibalism on the metabolism of fourth-instar larvae of the non-predatory insect Helicoverpa armigera (Lepidotera: Noctuidea). Two groups of larvae were analyzed: one group fed with fourth-instar larvae of H. armigera (cannibal), the other group fed with an artificial plant diet. Water-soluble small organic compounds present in the larvae were analyzed using two-dimensional nuclear magnetic resonance (NMR) and principal component analysis (PCA). Cannibalism negatively affected larval growth. PCA of NMR spectra showed that the metabolic profiles of cannibal and herbivore larvae were statistically different with monomeric sugars, fatty acid- and amino acid-related metabolites as the most variable compounds. Quantitation of ¹H-(13)C HSQC (Heteronuclear Single Quantum Coherence) signals revealed that the concentrations of glucose, glucono-1,5-lactone, glycerol phosphate, glutamine, glycine, leucine, isoleucine, lysine, ornithine, proline, threonine and valine were higher in the herbivore larvae.

  8. Fibroblast activation protein (FAP as a novel metabolic target

    Directory of Open Access Journals (Sweden)

    Miguel Angel Sánchez-Garrido

    2016-10-01

    Conclusions: We conclude that pharmacological inhibition of FAP enhances levels of FGF21 in obese mice to provide robust metabolic benefits not observed in lean animals, thus validating this enzyme as a novel drug target for the treatment of obesity and diabetes.

  9. Metabolic flux profiling of recombinant protein secreting Pichia pastoris growing on glucose:methanol mixtures

    Directory of Open Access Journals (Sweden)

    Jordà Joel

    2012-05-01

    Full Text Available Abstract Background The methylotrophic yeast Pichia pastoris has emerged as one of the most promising yeast hosts for the production of heterologous proteins. Mixed feeds of methanol and a multicarbon source instead of methanol as sole carbon source have been shown to improve product productivities and alleviate metabolic burden derived from protein production. Nevertheless, systematic quantitative studies on the relationships between the central metabolism and recombinant protein production in P. pastoris are still rather limited, particularly when growing this yeast on mixed carbon sources, thus hampering future metabolic network engineering strategies for improved protein production. Results The metabolic flux distribution in the central metabolism of P. pastoris growing on a mixed feed of glucose and methanol was analyzed by Metabolic Flux Analysis (MFA using 13C-NMR-derived constraints. For this purpose, we defined new flux ratios for methanol assimilation pathways in P. pastoris cells growing on glucose:methanol mixtures. By using this experimental approach, the metabolic burden caused by the overexpression and secretion of a Rhizopus oryzae lipase (Rol in P. pastoris was further analyzed. This protein has been previously shown to trigger the unfolded protein response in P. pastoris. A series of 13C-tracer experiments were performed on aerobic chemostat cultivations with a control and two different Rol producing strains growing at a dilution rate of 0.09 h−1 using a glucose:methanol 80:20 (w/w mix as carbon source. The MFA performed in this study reveals a significant redistristribution of carbon fluxes in the central carbon metabolism when comparing the two recombinant strains vs the control strain, reflected in increased glycolytic, TCA cycle and NADH regeneration fluxes, as well as higher methanol dissimilation rates. Conclusions Overall, a further 13C-based MFA development to characterise the central metabolism of methylotrophic

  10. Metabolic flux profiling of recombinant protein secreting Pichia pastoris growing on glucose:methanol mixtures

    Science.gov (United States)

    2012-01-01

    Background The methylotrophic yeast Pichia pastoris has emerged as one of the most promising yeast hosts for the production of heterologous proteins. Mixed feeds of methanol and a multicarbon source instead of methanol as sole carbon source have been shown to improve product productivities and alleviate metabolic burden derived from protein production. Nevertheless, systematic quantitative studies on the relationships between the central metabolism and recombinant protein production in P. pastoris are still rather limited, particularly when growing this yeast on mixed carbon sources, thus hampering future metabolic network engineering strategies for improved protein production. Results The metabolic flux distribution in the central metabolism of P. pastoris growing on a mixed feed of glucose and methanol was analyzed by Metabolic Flux Analysis (MFA) using 13C-NMR-derived constraints. For this purpose, we defined new flux ratios for methanol assimilation pathways in P. pastoris cells growing on glucose:methanol mixtures. By using this experimental approach, the metabolic burden caused by the overexpression and secretion of a Rhizopus oryzae lipase (Rol) in P. pastoris was further analyzed. This protein has been previously shown to trigger the unfolded protein response in P. pastoris. A series of 13C-tracer experiments were performed on aerobic chemostat cultivations with a control and two different Rol producing strains growing at a dilution rate of 0.09 h−1 using a glucose:methanol 80:20 (w/w) mix as carbon source. The MFA performed in this study reveals a significant redistristribution of carbon fluxes in the central carbon metabolism when comparing the two recombinant strains vs the control strain, reflected in increased glycolytic, TCA cycle and NADH regeneration fluxes, as well as higher methanol dissimilation rates. Conclusions Overall, a further 13C-based MFA development to characterise the central metabolism of methylotrophic yeasts when growing on mixed

  11. Synergizing metabolic flux analysis and nucleotide sugar metabolism to understand the control of glycosylation of recombinant protein in CHO cells

    LENUS (Irish Health Repository)

    Burleigh, Susan C

    2011-10-18

    Abstract Background The glycosylation of recombinant proteins can be altered by a range of parameters including cellular metabolism, metabolic flux and the efficiency of the glycosylation process. We present an experimental set-up that allows determination of these key processes associated with the control of N-linked glycosylation of recombinant proteins. Results Chinese hamster ovary cells (CHO) were cultivated in shake flasks at 0 mM glutamine and displayed a reduced growth rate, glucose metabolism and a slower decrease in pH, when compared to other glutamine-supplemented cultures. The N-linked glycosylation of recombinant human chorionic gonadotrophin (HCG) was also altered under these conditions; the sialylation, fucosylation and antennarity decreased, while the proportion of neutral structures increased. A continuous culture set-up was subsequently used to understand the control of HCG glycosylation in the presence of varied glutamine concentrations; when glycolytic flux was reduced in the absence of glutamine, the glycosylation changes that were observed in shake flask culture were similarly detected. The intracellular content of UDP-GlcNAc was also reduced, which correlated with a decrease in sialylation and antennarity of the N-linked glycans attached to HCG. Conclusions The use of metabolic flux analysis illustrated a case of steady state multiplicity, where use of the same operating conditions at each steady state resulted in altered flux through glycolysis and the TCA cycle. This study clearly demonstrated that the control of glycoprotein microheterogeneity may be examined by use of a continuous culture system, metabolic flux analysis and assay of intracellular nucleotides. This system advances our knowledge of the relationship between metabolic flux and the glycosylation of biotherapeutics in CHO cells and will be of benefit to the bioprocessing industry.

  12. Human Adenovirus Core Protein V Is Targeted by the Host SUMOylation Machinery To Limit Essential Viral Functions.

    Science.gov (United States)

    Freudenberger, Nora; Meyer, Tina; Groitl, Peter; Dobner, Thomas; Schreiner, Sabrina

    2018-02-15

    Human adenoviruses (HAdV) are nonenveloped viruses containing a linear, double-stranded DNA genome surrounded by an icosahedral capsid. To allow proper viral replication, the genome is imported through the nuclear pore complex associated with viral core proteins. Until now, the role of these incoming virion proteins during the early phase of infection was poorly understood. The core protein V is speculated to bridge the core and the surrounding capsid. It binds the genome in a sequence-independent manner and localizes in the nucleus of infected cells, accumulating at nucleoli. Here, we show that protein V contains conserved SUMO conjugation motifs (SCMs). Mutation of these consensus motifs resulted in reduced SUMOylation of the protein; thus, protein V represents a novel target of the host SUMOylation machinery. To understand the role of protein V SUMO posttranslational modification during productive HAdV infection, we generated a replication-competent HAdV with SCM mutations within the protein V coding sequence. Phenotypic analyses revealed that these SCM mutations are beneficial for adenoviral replication. Blocking protein V SUMOylation at specific sites shifts the onset of viral DNA replication to earlier time points during infection and promotes viral gene expression. Simultaneously, the altered kinetics within the viral life cycle are accompanied by more efficient proteasomal degradation of host determinants and increased virus progeny production than that observed during wild-type infection. Taken together, our studies show that protein V SUMOylation reduces virus growth; hence, protein V SUMOylation represents an important novel aspect of the host antiviral strategy to limit virus replication and thereby points to potential intervention strategies. IMPORTANCE Many decades of research have revealed that HAdV structural proteins promote viral entry and mainly physical stability of the viral genome in the capsid. Our work over the last years showed that this

  13. Amino acid metabolism and whole-body protein turnover in lambs ...

    African Journals Online (AJOL)

    The effect of protein supplementation of a wheat straw diet on the metabolism of lysine, leucine, methionine and urea, and on whole-body ... nitrogen content with a source of protein which is resistant to degradation in the rumen has ... are well established for use in humans (Waterlow et ai., 1978), and have been success- ...

  14. Reconstruction of the yeast protein-protein interaction network involved in nutrient sensing and global metabolic regulation

    DEFF Research Database (Denmark)

    Nandy, Subir Kumar; Jouhten, Paula; Nielsen, Jens

    2010-01-01

    BACKGROUND: Several protein-protein interaction studies have been performed for the yeast Saccharomyces cerevisiae using different high-throughput experimental techniques. All these results are collected in the BioGRID database and the SGD database provide detailed annotation of the different......-sensing and metabolic regulatory signal transduction pathways (STP) operating in Saccharomyces cerevisiae. The reconstructed STP network includes a full protein-protein interaction network including the key nodes Snf1, Tor1, Hog1 and Pka1. The network includes a total of 623 structural open reading frames (ORFs...

  15. The mitochondrial H(+)-ATP synthase and the lipogenic switch: new core components of metabolic reprogramming in induced pluripotent stem (iPS) cells.

    Science.gov (United States)

    Vazquez-Martin, Alejandro; Corominas-Faja, Bruna; Cufi, Sílvia; Vellon, Luciano; Oliveras-Ferraros, Cristina; Menendez, Octavio J; Joven, Jorge; Lupu, Ruth; Menendez, Javier A

    2013-01-15

    Induced pluripotent stem (iPS) cells share some basic properties, such as self-renewal and pluripotency, with cancer cells, and they also appear to share several metabolic alterations that are commonly observed in human tumors. The cancer cells' glycolytic phenotype, first reported by Otto Warburg, is necessary for the optimal routing of somatic cells to pluripotency. However, how iPS cells establish a Warburg-like metabolic phenotype and whether the metabolic pathways that support the bioenergetics of iPS cells are produced by the same mechanisms that are selected during the tumorigenic process remain largely unexplored. We recently investigated whether the reprogramming-competent metabotype of iPS cells involves changes in the activation/expression status of the H(+)-ATPase, which is a core component of mitochondrial oxidative phosphorylation that is repressed at both the activity and protein levels in human carcinomas, and of the lipogenic switch, which refers to a marked overexpression and hyperactivity of the acetyl-CoA carboxylase (ACACA) and fatty acid synthase (FASN) lipogenic enzymes that has been observed in nearly all examined cancer types. A comparison of a starting population of mouse embryonic fibroblasts and their iPS cell progeny revealed that somatic cell reprogramming involves a significant increase in the expression of ATPase inhibitor factor 1 (IF1), accompanied by extremely low expression levels of the catalytic β-F1-ATPase subunit. The pharmacological inhibition of ACACA and FASN activities markedly decreases reprogramming efficiency, and ACACA and FASN expression are notably upregulated in iPS cells. Importantly, iPS cells exhibited a significant intracellular accumulation of neutral lipid bodies; however, these bodies may be a reflection of intense lysosomal/autophagocytic activity rather than bona fide lipid droplet formation in iPS cells, as they were largely unresponsive to pharmacological modulation of PPARgamma and FASN activities. The

  16. The Protein Cost of Metabolic Fluxes: Prediction from Enzymatic Rate Laws and Cost Minimization

    Science.gov (United States)

    Noor, Elad; Flamholz, Avi; Bar-Even, Arren; Davidi, Dan; Milo, Ron; Liebermeister, Wolfram

    2016-01-01

    Bacterial growth depends crucially on metabolic fluxes, which are limited by the cell’s capacity to maintain metabolic enzymes. The necessary enzyme amount per unit flux is a major determinant of metabolic strategies both in evolution and bioengineering. It depends on enzyme parameters (such as kcat and KM constants), but also on metabolite concentrations. Moreover, similar amounts of different enzymes might incur different costs for the cell, depending on enzyme-specific properties such as protein size and half-life. Here, we developed enzyme cost minimization (ECM), a scalable method for computing enzyme amounts that support a given metabolic flux at a minimal protein cost. The complex interplay of enzyme and metabolite concentrations, e.g. through thermodynamic driving forces and enzyme saturation, would make it hard to solve this optimization problem directly. By treating enzyme cost as a function of metabolite levels, we formulated ECM as a numerically tractable, convex optimization problem. Its tiered approach allows for building models at different levels of detail, depending on the amount of available data. Validating our method with measured metabolite and protein levels in E. coli central metabolism, we found typical prediction fold errors of 4.1 and 2.6, respectively, for the two kinds of data. This result from the cost-optimized metabolic state is significantly better than randomly sampled metabolite profiles, supporting the hypothesis that enzyme cost is important for the fitness of E. coli. ECM can be used to predict enzyme levels and protein cost in natural and engineered pathways, and could be a valuable computational tool to assist metabolic engineering projects. Furthermore, it establishes a direct connection between protein cost and thermodynamics, and provides a physically plausible and computationally tractable way to include enzyme kinetics into constraint-based metabolic models, where kinetics have usually been ignored or oversimplified

  17. The Protein Cost of Metabolic Fluxes: Prediction from Enzymatic Rate Laws and Cost Minimization.

    Directory of Open Access Journals (Sweden)

    Elad Noor

    2016-11-01

    Full Text Available Bacterial growth depends crucially on metabolic fluxes, which are limited by the cell's capacity to maintain metabolic enzymes. The necessary enzyme amount per unit flux is a major determinant of metabolic strategies both in evolution and bioengineering. It depends on enzyme parameters (such as kcat and KM constants, but also on metabolite concentrations. Moreover, similar amounts of different enzymes might incur different costs for the cell, depending on enzyme-specific properties such as protein size and half-life. Here, we developed enzyme cost minimization (ECM, a scalable method for computing enzyme amounts that support a given metabolic flux at a minimal protein cost. The complex interplay of enzyme and metabolite concentrations, e.g. through thermodynamic driving forces and enzyme saturation, would make it hard to solve this optimization problem directly. By treating enzyme cost as a function of metabolite levels, we formulated ECM as a numerically tractable, convex optimization problem. Its tiered approach allows for building models at different levels of detail, depending on the amount of available data. Validating our method with measured metabolite and protein levels in E. coli central metabolism, we found typical prediction fold errors of 4.1 and 2.6, respectively, for the two kinds of data. This result from the cost-optimized metabolic state is significantly better than randomly sampled metabolite profiles, supporting the hypothesis that enzyme cost is important for the fitness of E. coli. ECM can be used to predict enzyme levels and protein cost in natural and engineered pathways, and could be a valuable computational tool to assist metabolic engineering projects. Furthermore, it establishes a direct connection between protein cost and thermodynamics, and provides a physically plausible and computationally tractable way to include enzyme kinetics into constraint-based metabolic models, where kinetics have usually been ignored or

  18. Dietary Proteins as Determinants of Metabolic and Physiologic Functions of the Gastrointestinal Tract

    Directory of Open Access Journals (Sweden)

    G. Harvey Anderson

    2011-05-01

    Full Text Available Dietary proteins elicit a wide range of nutritional and biological functions. Beyond their nutritional role as the source of amino acids for protein synthesis, they are instrumental in the regulation of food intake, glucose and lipid metabolism, blood pressure, bone metabolism and immune function. The interaction of dietary proteins and their products of digestion with the regulatory functions of the gastrointestinal (GI tract plays a dominant role in determining the physiological properties of proteins. The site of interaction is widespread, from the oral cavity to the colon. The characteristics of proteins that influence their interaction with the GI tract in a source-dependent manner include their physico-chemical properties, their amino acid composition and sequence, their bioactive peptides, their digestion kinetics and also the non-protein bioactive components conjugated with them. Within the GI tract, these products affect several regulatory functions by interacting with receptors releasing hormones, affecting stomach emptying and GI transport and absorption, transmitting neural signals to the brain, and modifying the microflora. This review discusses the interaction of dietary proteins during digestion and absorption with the physiological and metabolic functions of the GI tract, and illustrates the importance of this interaction in the regulation of amino acid, glucose, lipid metabolism, and food intake.

  19. Colorful Packages: Encapsulation of Fluorescent Proteins in Complex Coacervate Core Micelles

    Directory of Open Access Journals (Sweden)

    Antsje Nolles

    2017-07-01

    Full Text Available Encapsulation of proteins can be beneficial for food and biomedical applications. To study their biophysical properties in complex coacervate core micelles (C3Ms, we previously encapsulated enhanced green fluorescent protein (EGFP and its monomeric variant, mEGFP, with the cationic-neutral diblock copolymer poly(2-methyl-vinyl-pyridiniumn-b-poly(ethylene-oxidem (P2MVPn-b-PEOm as enveloping material. C3Ms with high packaging densities of fluorescent proteins (FPs were obtained, resulting in a restricted orientational freedom of the protein molecules, influencing their structural and spectral properties. To address the generality of this behavior, we encapsulated seven FPs with P2MVP41-b-PEO205 and P2MVP128-b-PEO477. Dynamic light scattering and fluorescence correlation spectroscopy showed lower encapsulation efficiencies for members of the Anthozoa class (anFPs than for Hydrozoa FPs derived from Aequorea victoria (avFPs. Far-UV CD spectra of the free FPs showed remarkable differences between avFPs and anFPs, caused by rounder barrel structures for avFPs and more elliptic ones for anFPs. These structural differences, along with the differences in charge distribution, might explain the variations in encapsulation efficiency between avFPs and anFPs. Furthermore, the avFPs remain monomeric in C3Ms with minor spectral and structural changes. In contrast, the encapsulation of anFPs gives rise to decreased quantum yields (monomeric Kusabira Orange 2 (mKO2 and Tag red fluorescent protein (TagRFP or to a pKa shift of the chromophore (FP variant mCherry.

  20. Dietary protein for athletes: from requirements to metabolic advantage

    National Research Council Canada - National Science Library

    Phillips, Stuart M

    2006-01-01

    ... . This Recommended Dietary Allowance (RDA) is cited as adequate for all persons. This amount of protein would be considered by many athletes as the amount to be consumed in a single meal, particularly for strength-training athletes...

  1. Effect of dietary protein on lipid and glucose metabolism: implications for metabolic health

    NARCIS (Netherlands)

    Rietman, A.

    2015-01-01

    Abstract Background: Diet is an important factor in the development of the Metabolic Syndrome (Mets) and type 2 Diabetes Mellitus. Accumulation of intra hepatic lipid (IHL) can result in non-alcoholic fatty liver disease (NAFLD), which is sometimes considered the

  2. Identification of protein W, the elusive sixth subunit of the Rhodopseudomonas palustris reaction center-light harvesting 1 core complex.

    Science.gov (United States)

    Jackson, Philip J; Hitchcock, Andrew; Swainsbury, David J K; Qian, Pu; Martin, Elizabeth C; Farmer, David A; Dickman, Mark J; Canniffe, Daniel P; Neil Hunter, C

    2017-11-07

    The X-ray crystal structure of the Rhodopseudomonas (Rps.) palustris reaction center-light harvesting 1 (RC-LH1) core complex revealed the presence of a sixth protein component, variably referred to in the literature as helix W, subunit W or protein W. The position of this protein prevents closure of the LH1 ring, possibly to allow diffusion of ubiquinone/ubiquinol between the RC and the cytochrome bc1 complex in analogous fashion to the well-studied PufX protein from Rhodobacter sphaeroides. The identity and function of helix W have remained unknown for over 13 years; here we use a combination of biochemistry, mass spectrometry, molecular genetics and electron microscopy to identify this protein as RPA4402 in Rps. palustris CGA009. Protein W shares key conserved sequence features with PufX homologs, and although a deletion mutant was able to grow under photosynthetic conditions with no discernible phenotype, we show that a tagged version of protein W pulls down the RC-LH1 complex. Protein W is not encoded in the photosynthesis gene cluster and our data indicate that only approximately 10% of wild-type Rps. palustris core complexes contain this non-essential subunit; functional and evolutionary consequences of this observation are discussed. The ability to purify uniform RC-LH1 and RC-LH1-protein W preparations will also be beneficial for future structural studies of these bacterial core complexes. Copyright © 2017. Published by Elsevier B.V.

  3. TRF2 Protein Interacts with Core Histones to Stabilize Chromosome Ends.

    Science.gov (United States)

    Konishi, Akimitsu; Izumi, Takashi; Shimizu, Shigeomi

    2016-09-23

    Mammalian chromosome ends are protected by a specialized nucleoprotein complex called telomeres. Both shelterin, a telomere-specific multi-protein complex, and higher order telomeric chromatin structures combine to stabilize the chromosome ends. Here, we showed that TRF2, a component of shelterin, binds to core histones to protect chromosome ends from inappropriate DNA damage response and loss of telomeric DNA. The N-terminal Gly/Arg-rich domain (GAR domain) of TRF2 directly binds to the globular domain of core histones. The conserved arginine residues in the GAR domain of TRF2 are required for this interaction. A TRF2 mutant with these arginine residues substituted by alanine lost the ability to protect telomeres and induced rapid telomere shortening caused by the cleavage of a loop structure of the telomeric chromatin. These findings showed a previously unnoticed interaction between the shelterin complex and nucleosomal histones to stabilize the chromosome ends. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Improving Antigenicity of the Recombinant Hepatitis C Virus Core Protein via Random Mutagenesis

    Directory of Open Access Journals (Sweden)

    Chen-Ji Huang

    2011-01-01

    Full Text Available In order to enhance the sensitivity of diagnosis, a recombinant clone containing domain I of HCV core (amino acid residues 1 to 123 was subjected to random mutagenesis. Five mutants with higher sensitivity were obtained by colony screening of 616 mutants using reverse ELISA. Sequence analysis of these mutants revealed alterations focusing on W84, P95, P110, or V129. The inclusion bodies of these recombinant proteins overexpressed in E. coli BL21(DE3 were subsequently dissolved using 6 M urea and then refolded by stepwise dialysis. Compared to the unfolded wild-type antigen, the refolded M3b antigen (W84S, P110S and V129L exhibited an increase of 66% antigenicity with binding capacity of 0.96 and affinity of 113 μM−1. Moreover, the 33% decrease of the production demand suggests that M3b is a potential substitute for anti-HCV antibody detection.

  5. PTEN Mediates Activation of Core Clock Protein BMAL1 and Accumulation of Epidermal Stem Cells

    Directory of Open Access Journals (Sweden)

    Chiara Zagni

    2017-07-01

    Full Text Available Tissue integrity requires constant maintenance of a quiescent, yet responsive, population of stem cells. In the skin, hair follicle stem cells (HFSCs that reside within the bulge maintain tissue homeostasis in response to activating cues that occur with each new hair cycle or upon injury. We found that PTEN, a major regulator of the PI3K-AKT pathway, controlled HFSC number and size in the bulge and maintained genomically stable pluripotent cells. This regulatory function is central for HFSC quiescence, where PTEN-deficiency phenotype is in part regulated by BMAL1. Furthermore, PTEN ablation led to downregulation of BMI-1, a critical regulator of adult stem cell self-renewal, and elevated senescence, suggesting the presence of a protective system that prevents transformation. We found that short- and long-term PTEN depletion followed by activated BMAL1, a core clock protein, contributed to accumulation of HFSC.

  6. Apolipoprotein A-IV: a protein intimately involved in metabolism

    OpenAIRE

    Wang, Fei; Kohan, Alison B.; Lo, Chun-Min; Liu, Min; Howles, Philip; Tso, Patrick

    2015-01-01

    The purpose of this review is to summarize our current understanding of the physiological roles of apoA-IV in metabolism, and to underscore the potential for apoA-IV to be a focus for new therapies aimed at the treatment of diabetes and obesity-related disorders. ApoA-IV is primarily synthesized by the small intestine, attached to chylomicrons by enterocytes, and secreted into intestinal lymph during fat absorption. In circulation, apoA-IV is associated with HDL and chylomicron remnants, but ...

  7. A Core Outcome Set for the Benefits and Adverse Events of Bariatric and Metabolic Surgery: The BARIACT Project.

    Directory of Open Access Journals (Sweden)

    Karen D Coulman

    2016-11-01

    Full Text Available Bariatric and metabolic surgery is used as a treatment for patients with severe and complex obesity. However, there is a need to improve outcome selection and reporting in bariatric surgery trials. A Core Outcome Set (COS, an agreed minimum set of outcomes reported in all studies of a specific condition, may achieve this. Here, we present the development of a COS for BARIAtric and metabolic surgery Clinical Trials-the BARIACT Study.Outcomes identified from systematic reviews and patient interviews informed a questionnaire survey. Patients and health professionals were surveyed three times and asked to rate the importance of each item on a 1-9 scale. Delphi methods provided anonymised feedback to participants. Items not meeting predefined criteria were discarded between rounds. Remaining items were discussed at consensus meetings, held separately with patients and professionals, where the COS was agreed. Data sources identified 2,990 outcomes, which were used to develop a 130-item questionnaire. Round 1 response rates were moderate but subsequently improved to above 75% for other rounds. After rounds 2 and 3, 81 and 14 items were discarded, respectively, leaving 35 items for discussion at consensus meetings. The final COS included nine items: "weight," "diabetes status," "cardiovascular risk," "overall quality of life (QOL," "mortality," "technical complications of the specific operation," "any re-operation/re-intervention," "dysphagia/regurgitation," and "micronutrient status." The main limitation of this study was that it was based in the United Kingdom only.The COS is recommended to be used as a minimum in all trials of bariatric and metabolic surgery. Adoption of the COS will improve data synthesis and the value of research data. Future work will establish methods for the measurement of the outcomes in the COS.

  8. A Core Outcome Set for the Benefits and Adverse Events of Bariatric and Metabolic Surgery: The BARIACT Project

    Science.gov (United States)

    Hopkins, James; Brookes, Sara T.; Main, Barry; Owen-Smith, Amanda; Andrews, Robert C.; Byrne, James; Mazza, Graziella; Welbourn, Richard; Wordsworth, Sarah; Blazeby, Jane M.

    2016-01-01

    Background Bariatric and metabolic surgery is used as a treatment for patients with severe and complex obesity. However, there is a need to improve outcome selection and reporting in bariatric surgery trials. A Core Outcome Set (COS), an agreed minimum set of outcomes reported in all studies of a specific condition, may achieve this. Here, we present the development of a COS for BARIAtric and metabolic surgery Clinical Trials—the BARIACT Study. Methods and Findings Outcomes identified from systematic reviews and patient interviews informed a questionnaire survey. Patients and health professionals were surveyed three times and asked to rate the importance of each item on a 1–9 scale. Delphi methods provided anonymised feedback to participants. Items not meeting predefined criteria were discarded between rounds. Remaining items were discussed at consensus meetings, held separately with patients and professionals, where the COS was agreed. Data sources identified 2,990 outcomes, which were used to develop a 130-item questionnaire. Round 1 response rates were moderate but subsequently improved to above 75% for other rounds. After rounds 2 and 3, 81 and 14 items were discarded, respectively, leaving 35 items for discussion at consensus meetings. The final COS included nine items: “weight,” “diabetes status,” “cardiovascular risk,” “overall quality of life (QOL),” “mortality,” “technical complications of the specific operation,” “any re-operation/re-intervention,” “dysphagia/regurgitation,” and “micronutrient status.” The main limitation of this study was that it was based in the United Kingdom only. Conclusions The COS is recommended to be used as a minimum in all trials of bariatric and metabolic surgery. Adoption of the COS will improve data synthesis and the value of research data. Future work will establish methods for the measurement of the outcomes in the COS. PMID:27898680

  9. Expression of HCV Alternative Reading Frame Protein (Core+1/F) in Baculovirus Expression System and its Evaluation for Assessment of Specific Anti-core+1 Antibody in Iranian HCV Infected Patients.

    Science.gov (United States)

    Rahimi, Pooneh; Vahabpour, Rouhollah; Fard, Farideh Sadat Sajadian; Motevalli, Fatemeh; Chahouki, Fatemeh Fotouhi; Aghasadeghi, Mohammad Reza; Bolhassani, Azam; Sadat, Seyed Mehdi; Mostafavi, Ehsan; Mohajel, Nasir; Jahanian-Najafabadi, Ali; Amiran, Mohammad Reza

    2016-10-01

    Hepatitis C virus (HCV) genome contains an overlapping reading frame which results in alternative core protein (ARFP). Baculovirus expression system was used as a powerful eukaryotic vector system to express core+1/F protein for the first time. This recombinant core+1/F protein was used to assess the anti-core+1 antibody in anti-HCV drug resistant and sustained virologic response (SVR) patients. The core+1 coding sequence from HCV genotype 1 was designed and synthesized in pUC57 vector. It was subcloned into baculovirus donor plasmid pFastBacTM HTA and transposed into baculovirus shuttle vector (bacmid) to transfect Sf9 cells. Recombinant core+1 protein was purified using Ni-NTA agarose under native condition and verified using SDS-PAGE electrophoresis and Western blotting. An enzyme-linked immunosorbent assay (ELISA) was developed using this purified protein to assess anti-core+1 antibody in 28 anti-HCV drug resistant patients and in 34 patients with sustained virologic response (SVR) in comparison with 31 healthy volunteers used as the negative control. Expression of HCV core+1 protein in Sf9 cells was confirmed by using SDS-PAGE and Western blotting. Antibody titer against core+1 protein in anti-HCV drug resistant patients was significantly higher than that in both the healthy volunteers and SVR patients (p < 0.0001). HCV core+1 protein was expressed successfully in a baculovirus expression system in high yield in order to develop an ELISA to assess the anti-core+1 antibody. Further studies are needed to reveal the potential application of core+1 protein in anti-HCV treatment prognosis.

  10. Data set of interactomes and metabolic pathways of proteins differentially expressed in brains with Alzheimer׳s disease

    Directory of Open Access Journals (Sweden)

    Benito Minjarez

    2016-06-01

    Full Text Available Alzheimer׳s disease is one of the main causes of dementia in the elderly and its frequency is on the rise worldwide. It is considered the result of complex interactions between genetic and environmental factors, being many of them unknown. Therefore, there is a dire necessity for the identification of novel molecular players for the understanding of this disease. In this data article we determined the protein expression profiles of whole protein extracts from cortex regions of brains from patients with Alzheimer׳s disease in comparison to a normal brain. We identified 721 iTRAQ-labeled polypeptides with more than 95% in confidence. We analyzed all proteins that changed in their expression level and located them in the KEGG metabolic pathways, as well as in the mitochondrial complexes of the electron transport chain and ATP synthase. In addition, we analyzed the over- and sub-expressed polypeptides through IPA software, specifically Core I and Biomarkers I modules. Data in this article is related to the research article “Identification of proteins that are differentially expressed in brains with Alzheimer’s disease using iTRAQ labeling and tandem mass spectrometry” (Minjarez et al., 2016 [1].

  11. Effect of Prolonged Simulated Microgravity on Metabolic Proteins in Rat Hippocampus: Steps toward Safe Space Travel.

    Science.gov (United States)

    Wang, Yun; Javed, Iqbal; Liu, Yahui; Lu, Song; Peng, Guang; Zhang, Yongqian; Qing, Hong; Deng, Yulin

    2016-01-04

    Mitochondria are not only the main source of energy in cells but also produce reactive oxygen species (ROS), which result in oxidative stress when in space. This oxidative stress is responsible for energy imbalances and cellular damage. In this study, a rat tail suspension model was used in individual experiments for 7 and 21 days to explore the effect of simulated microgravity (SM) on metabolic proteins in the hippocampus, a vital brain region involved in learning, memory, and navigation. A comparative (18)O-labeled quantitative proteomic strategy was used to observe the differential expression of metabolic proteins. Forty-two and sixty-seven mitochondrial metabolic proteins were differentially expressed after 21 and 7 days of SM, respectively. Mitochondrial Complex I, III, and IV, isocitrate dehydrogenase and malate dehydrogenase were down-regulated. Moreover, DJ-1 and peroxiredoxin 6, which defend against oxidative damage, were up-regulated in the hippocampus. Western blot analysis of proteins DJ-1 and COX 5A confirmed the mass spectrometry results. Despite these changes in mitochondrial protein expression, no obvious cell apoptosis was observed after 21 days of SM. The results of this study indicate that the oxidative stress induced by SM has profound effects on metabolic proteins.

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

    Science.gov (United States)

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

    2015-06-09

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

  13. Enhanced-Transient Expression of Hepatitis C Virus Core Protein in Nicotiana tabacum, a Protein With Potential Clinical Applications.

    Science.gov (United States)

    Mohammadzadeh, Sara; Khabiri, Alireza; Roohvand, Farzin; Memarnejadian, Arash; Salmanian, Ali Hatef; Ajdary, Soheila; Ehsani, Parastoo

    2014-11-01

    Hepatitis C virus (HCV) is major cause of liver cirrhosis in humans. HCV capsid (core) protein (HCVcp) is a highly demanded antigen for various diagnostic, immunization and pathogenesis studies. Plants are considered as an expression system for producing safe and inexpensive biopharmaceutical proteins. Although invention of transgenic (stable) tobacco plants expressing HCVcp with proper antigenic properties was recently reported, no data for "transient-expression" that is currently the method of choice for rapid, simple and lower-priced protein expression in plants is available for HCVcp. The purpose of this study was to design a highly codon-optimized HCVcp gene for construction of an efficient transient-plant expression system for production of HCVcp with proper antigenic properties in a regional tobacco plant (Iranian Jafarabadi-cultivar) by evaluation of different classes of vectors and suppression of gene-silencing in tobacco. A codon-optimized gene encoding the Kozak sequence, 6xHis-tag, HCVcp (1-122) and KDEL peptide in tandem (from N- to C-terminal) was designed and inserted into potato virus-X (PVX) and classic pBI121 binary vectors in separate cloning reactions. The resulted recombinant plasmids were transferred into Agrobacterium tumefaciens and vacuum infiltrated into tobacco leaves. The effect of gene silencing suppressor P19 protein derived from tomato bushy stunt virus on the expression yield of HCVcp by each construct was also evaluated by co-infiltration in separate groups. The expressed HCVcp was evaluated by dot and western blotting and ELISA assays. The codon-optimized gene had an increased adaptation index value (from 0.65 to 0.85) and reduced GC content (from 62.62 to 51.05) in tobacco and removed the possible deleterious effect of "GGTAAG" splice site in native HCVcp. Blotting assays via specific antibodies confirmed the expression of the 15 kDa HCVcp. The expression level of HCVcp was enhanced by 4-5 times in P19 co-agroinfiltrated plants

  14. Spaceflight and protein metabolism, with special reference to humans

    Science.gov (United States)

    Stein, T. P.; Gaprindashvili, T.

    1994-01-01

    Human space missions have shown that human spaceflight is associated with a loss of body protein. Specific changes include a loss of lean body mass, decreased muscle mass in the calves, decreased muscle strength, and changes in plasma proteins and amino acids. The major muscle loss is believed to be associated with the antigravity (postural) muscle. The most significant loss of protein appears to occur during the first month of flight. The etiology is believed to be multifactorial with contributions from disuse atrophy, undernutrition, and a stress type of response. This article reviews the results of American and Russian space missions to investigate this problem in humans, monkeys, and rats. The relationship of the flight results with ground-based models including bedrest for humans and hindlimb unweighting for rats is also discussed. The results suggest that humans adapt to spaceflight much better than either monkeys or rats.

  15. Role of Heme and Heme-Proteins in Trypanosomatid Essential Metabolic Pathways

    Directory of Open Access Journals (Sweden)

    Karina E. J. Tripodi

    2011-01-01

    Full Text Available Around the world, trypanosomatids are known for being etiological agents of several highly disabling and often fatal diseases like Chagas disease (Trypanosoma cruzi, leishmaniasis (Leishmania spp., and African trypanosomiasis (Trypanosoma brucei. Throughout their life cycle, they must cope with diverse environmental conditions, and the mechanisms involved in these processes are crucial for their survival. In this review, we describe the role of heme in several essential metabolic pathways of these protozoans. Notwithstanding trypanosomatids lack of the complete heme biosynthetic pathway, we focus our discussion in the metabolic role played for important heme-proteins, like cytochromes. Although several genes for different types of cytochromes, involved in mitochondrial respiration, polyunsaturated fatty acid metabolism, and sterol biosynthesis, are annotated at the Tritryp Genome Project, the encoded proteins have not yet been deeply studied. We pointed our attention into relevant aspects of these protein functions that are amenable to be considered for rational design of trypanocidal agents.

  16. Expression of sarcosine metabolism-related proteins in invasive lobular carcinoma: comparison to invasive ductal carcinoma.

    Science.gov (United States)

    Cha, Yoon Jin; Jung, Woo Hee; Cho, Nam Hoon; Koo, Ja Seung

    2015-05-01

    The aims of this study were to compare the expression of sarcosine metabolism-related proteins between invasive lobular carcinoma (ILC) and invasive ductal carcinoma (IDC) and to determine the implications of these results. Tissue microarrays were constructed, containing 30 samples from normal breast tissue, 114 samples from patients with ILC, and 692 samples from patients with IDC. Immunohistochemical staining was performed to examine the expression of sarcosine metabolism-related proteins [glycine N-methyltransferase, sarcosine dehydrogenase, and l-pipecolic acid oxidase (PIPOX)]. The sarcosine metabolic phenotype differed between ILC and IDC (plow sarcosine type (30.4%)>high sarcosine type (5.0%)>intermediate type (2.9%). However, in ILC, the sarcosine metabolic phenotype was distributed as low sarcosine type (61.4%)>null type (32.5%)>intermediate type (5.3%)>high sarcosine type (0.9%). PIPOX showed higher expression in ILC than in IDC (p<0.001) and correlated with androgen receptor (AR) positivity (p=0.001) in ILC. Expression of sarcosine metabolism-related proteins differed between ILC and IDC. Low sarcosine type was the majority sarcosine metabolic phenotype of ILC. PIPOX expression was predominant in ILC and correlated with AR positivity.

  17. The Role of Maternal Dietary Proteins in Development of Metabolic Syndrome in Offspring

    Directory of Open Access Journals (Sweden)

    Alireza Jahan-Mihan

    2015-11-01

    Full Text Available The prevalence of metabolic syndrome and obesity has been increasing. Pre-natal environment has been suggested as a factor influencing the risk of metabolic syndrome in adulthood. Both observational and experimental studies showed that maternal diet is a major modifier of the development of regulatory systems in the offspring in utero and post-natally. Both protein content and source in maternal diet influence pre- and early post-natal development. High and low protein dams’ diets have detrimental effect on body weight, blood pressure191 and metabolic and intake regulatory systems in the offspring. Moreover, the role of the source of protein in a nutritionally adequate maternal diet in programming of food intake regulatory system, body weight, glucose metabolism and blood pressure in offspring is studied. However, underlying mechanisms are still elusive. The purpose of this review is to examine the current literature related to the role of proteins in maternal diets in development of characteristics of the metabolic syndrome in offspring.

  18. The Role of Maternal Dietary Proteins in Development of Metabolic Syndrome in Offspring

    Science.gov (United States)

    Jahan-Mihan, Alireza; Rodriguez, Judith; Christie, Catherine; Sadeghi, Marjan; Zerbe, Tara

    2015-01-01

    The prevalence of metabolic syndrome and obesity has been increasing. Pre-natal environment has been suggested as a factor influencing the risk of metabolic syndrome in adulthood. Both observational and experimental studies showed that maternal diet is a major modifier of the development of regulatory systems in the offspring in utero and post-natally. Both protein content and source in maternal diet influence pre- and early post-natal development. High and low protein dams’ diets have detrimental effect on body weight, blood pressure191 and metabolic and intake regulatory systems in the offspring. Moreover, the role of the source of protein in a nutritionally adequate maternal diet in programming of food intake regulatory system, body weight, glucose metabolism and blood pressure in offspring is studied. However, underlying mechanisms are still elusive. The purpose of this review is to examine the current literature related to the role of proteins in maternal diets in development of characteristics of the metabolic syndrome in offspring. PMID:26561832

  19. A liver stress-endocrine nexus promotes metabolic integrity during dietary protein dilution

    Science.gov (United States)

    Maida, Adriano; Zota, Annika; Sjøberg, Kim A.; Sijmonsma, Tjeerd P.; Pfenninger, Anja; Christensen, Marie M.; Gantert, Thomas; Fuhrmeister, Jessica; Rothermel, Ulrike; Schmoll, Dieter; Heikenwälder, Mathias; Iovanna, Juan L.; Stemmer, Kerstin; Herzig, Stephan; Rose, Adam J.

    2016-01-01

    Dietary protein intake is linked to an increased incidence of type 2 diabetes (T2D). Although dietary protein dilution (DPD) can slow the progression of some aging-related disorders, whether this strategy affects the development and risk for obesity-associated metabolic disease such as T2D is unclear. Here, we determined that DPD in mice and humans increases serum markers of metabolic health. In lean mice, DPD promoted metabolic inefficiency by increasing carbohydrate and fat oxidation. In nutritional and polygenic murine models of obesity, DPD prevented and curtailed the development of impaired glucose homeostasis independently of obesity and food intake. DPD-mediated metabolic inefficiency and improvement of glucose homeostasis were independent of uncoupling protein 1 (UCP1), but required expression of liver-derived fibroblast growth factor 21 (FGF21) in both lean and obese mice. FGF21 expression and secretion as well as the associated metabolic remodeling induced by DPD also required induction of liver-integrated stress response–driven nuclear protein 1 (NUPR1). Insufficiency of select nonessential amino acids (NEAAs) was necessary and adequate for NUPR1 and subsequent FGF21 induction and secretion in hepatocytes in vitro and in vivo. Taken together, these data indicate that DPD promotes improved glucose homeostasis through an NEAA insufficiency–induced liver NUPR1/FGF21 axis. PMID:27548521

  20. The dynamics of fat, protein and sugar metabolism during walnut ...

    African Journals Online (AJOL)

    Yomi

    Key words: Walnut (Juglans regia L.), fruit development, sugar, fat, protein, invertase. INTRODUCTION. The 'hidden hunger' .... mixture, and then determined by KDY-9820 Kjeldahl apparatus. (Beijing Huawei Science and ... Where, V2, Vl, is the measure value at two measure time; T2 – T1is the days between two measure ...

  1. Current issues in determining dietary protein quality and metabolic utilization

    Science.gov (United States)

    In resource-limited settings, poor dietary quality has a marked negative impact on health, especially during the sensitive periods of pregnancy and first 2 years of life (the first 1000 days) when stunting, poor development and increased risk of later disease develop. Protein quality is often poor o...

  2. Metabolic responses to high protein diet in Korean elite bodybuilders with high-intensity resistance exercise

    Directory of Open Access Journals (Sweden)

    Choue Ryowon

    2011-07-01

    Full Text Available Abstract Background High protein diet has been known to cause metabolic acidosis, which is manifested by increased urinary excretion of nitrogen and calcium. Bodybuilders habitually consumed excessive dietary protein over the amounts recommended for them to promote muscle mass accretion. This study investigated the metabolic response to high protein consumption in the elite bodybuilders. Methods Eight elite Korean bodybuilders within the age from 18 to 25, mean age 21.5 ± 2.6. For data collection, anthropometry, blood and urinary analysis, and dietary assessment were conducted. Results They consumed large amounts of protein (4.3 ± 1.2 g/kg BW/day and calories (5,621.7 ± 1,354.7 kcal/day, as well as more than the recommended amounts of vitamins and minerals, including potassium and calcium. Serum creatinine (1.3 ± 0.1 mg/dl and potassium (5.9 ± 0.8 mmol/L, and urinary urea nitrogen (24.7 ± 9.5 mg/dl and creatinine (2.3 ± 0.7 mg/dl were observed to be higher than the normal reference ranges. Urinary calcium (0.3 ± 0.1 mg/dl, and phosphorus (1.3 ± 0.4 mg/dl were on the border of upper limit of the reference range and the urine pH was in normal range. Conclusions Increased urinary excretion of urea nitrogen and creatinine might be due to the high rates of protein metabolism that follow high protein intake and muscle turnover. The obvious evidence of metabolic acidosis in response to high protein diet in the subjects with high potassium intake and intensive resistance exercise were not shown in this study results. However, this study implied that resistance exercise with adequate mineral supplementation, such as potassium and calcium, could reduce or offset the negative effects of protein-generated metabolic changes. This study provides preliminary information of metabolic response to high protein intake in bodybuilders who engaged in high-intensity resistance exercise. Further studies will be needed to determine the effects of the intensity

  3. Protein homeostasis disorders of key enzymes of amino acids metabolism: mutation-induced protein kinetic destabilization and new therapeutic strategies.

    Science.gov (United States)

    Pey, Angel L

    2013-12-01

    Many inborn errors of amino acids metabolism are caused by single point mutations affecting the ability of proteins to fold properly (i.e., protein homeostasis), thus leading to enzyme loss-of-function. Mutations may affect protein homeostasis by altering intrinsic physical properties of the polypeptide (folding thermodynamics, and rates of folding/unfolding/misfolding) as well as the interaction of partially folded states with elements of the protein homeostasis network (such as molecular chaperones and proteolytic machineries). Understanding these mutational effects on protein homeostasis is required to develop new therapeutic strategies aimed to target specific features of the mutant polypeptide. Here, I review recent work in three different diseases of protein homeostasis associated to inborn errors of amino acids metabolism: phenylketonuria, inherited homocystinuria and primary hyperoxaluria type I. These three different genetic disorders involve proteins operating in different cell organelles and displaying different structural complexities. Mutations often decrease protein kinetic stability of the native state (i.e., its half-life for irreversible denaturation), which can be studied using simple kinetic models amenable to biophysical and biochemical characterization. Natural ligands and pharmacological chaperones are shown to stabilize mutant enzymes, thus supporting their therapeutic application to overcome protein kinetic destabilization. The role of molecular chaperones in protein folding and misfolding is also discussed as well as their potential pharmacological modulation as promising new therapeutic approaches. Since current available treatments for these diseases are either burdening or only successful in a fraction of patients, alternative treatments must be considered covering studies from protein structure and biophysics to studies in animal models and patients.

  4. Unravelling core microbial metabolisms in the hypersaline microbial mats of Shark Bay using high-throughput metagenomics

    Energy Technology Data Exchange (ETDEWEB)

    Ruvindy, Rendy; White III, Richard Allen; Neilan, Brett Anthony; Burns, Brendan Paul

    2015-05-29

    Modern microbial mats are potential analogues of some of Earth’s earliest ecosystems. Excellent examples can be found in Shark Bay, Australia, with mats of various morphologies. To further our understanding of the functional genetic potential of these complex microbial ecosystems, we conducted for the first time shotgun metagenomic analyses. We assembled metagenomic nextgeneration sequencing data to classify the taxonomic and metabolic potential across diverse morphologies of marine mats in Shark Bay. The microbial community across taxonomic classifications using protein-coding and small subunit rRNA genes directly extracted from the metagenomes suggests that three phyla Proteobacteria, Cyanobacteria and Bacteriodetes dominate all marine mats. However, the microbial community structure between Shark Bay and Highbourne Cay (Bahamas) marine systems appears to be distinct from each other. The metabolic potential (based on SEED subsystem classifications) of the Shark Bay and Highbourne Cay microbial communities were also distinct. Shark Bay metagenomes have a metabolic pathway profile consisting of both heterotrophic and photosynthetic pathways, whereas Highbourne Cay appears to be dominated almost exclusively by photosynthetic pathways. Alternative non-rubisco-based carbon metabolism including reductive TCA cycle and 3-hydroxypropionate/4-hydroxybutyrate pathways is highly represented in Shark Bay metagenomes while not represented in Highbourne Cay microbial mats or any other mat forming ecosystems investigated to date. Potentially novel aspects of nitrogen cycling were also observed, as well as putative heavy metal cycling (arsenic, mercury, copper and cadmium). Finally, archaea are highly represented in Shark Bay and may have critical roles in overall ecosystem function in these modern microbial mats.

  5. In situ preparation and protein delivery of silicate-alginate composite microspheres with core-shell structure.

    Science.gov (United States)

    Wu, Chengtie; Fan, Wei; Gelinsky, Michael; Xiao, Yin; Chang, Jiang; Friis, Thor; Cuniberti, Gianaurelio

    2011-12-07

    The efficient loading and sustained release of proteins from bioactive microspheres remain a significant challenge. In this study, we have developed bioactive microspheres which can be loaded with protein and then have a controlled rate of protein release into a surrounding medium. This was achieved by preparing a bioactive microsphere system with core-shell structure, combining a calcium silicate (CS) shell with an alginate (A) core by a one-step in situ method. The result was to improve the microspheres' protein adsorption and release, which yielded a highly bioactive material with potential uses in bone repair applications. The composition and the core-shell structure, as well as the formation mechanism of the obtained CS-A microspheres, were investigated by X-ray diffraction, optical microscopy, scanning electron microscopy, energy dispersive spectrometer dot and line-scanning analysis. The protein loading efficiency reached 75 per cent in CS-A microspheres with a core-shell structure by the in situ method. This is significantly higher than that of pure A or CS-A microspheres prepared by non-in situ method, which lack a core-shell structure. CS-A microspheres with a core-shell structure showed a significant decrease in the burst release of proteins, maintaining sustained release profile in phosphate-buffered saline (PBS) at both pH 7.4 and 4.3, compared with the controls. The protein release from CS-A microspheres is predominantly controlled by a Fickian diffusion mechanism. The CS-A microspheres with a core-shell structure were shown to have improved apatite-mineralization in simulated body fluids compared with the controls, most probably owing to the existence of bioactive CS shell on the surface of the microspheres. Our results indicate that the core-shell structure of CS-A microspheres play an important role in enhancing protein delivery and mineralization, which makes these composite materials promising candidates for application in bone tissue

  6. The contribution of proteins with binding activity and specific metabolic pathways in tolerating abiotic stress by canola: An in silico study

    Directory of Open Access Journals (Sweden)

    Reza Shokri Gharelo

    2016-12-01

    Full Text Available Plants indicate different degrees of sensitivity and of tolerance upon encountering stressful conditions. In molecular level, plants respond in complex manner to stressful conditions. Obviously, the tolerant plants recruit molecular mechanisms that enable them to trigger appropriate reactions. To investigate molecular mechanisms involved in the response of Canola to stress conditions, two cultivars of Canola including Sarigol (as a sensitive cultivar and Hyola308 (as a tolerant cultivar were in silico studied based on their identified proteins with significantly expression changes under abiotic stresses. The results indicated that in Hyola308, the genes/proteins that are mostly involved in the response to stress and with functions related to binding activity are overrepresented. In term of Sarigol, the number of the genes/proteins involved in response to stress were the low and the most of its induced genes/proteins were involvement in catalytic and antioxidant activities. Five metabolic pathways including protein processing in endoplasmic reticulum, carbon fixation in photosynthetic organisms, endocytosis, spliceosome, and fructose and mannose metabolism were uniquely observed in Hyola308, which could be in relation with the tolerance of Hyola308. These pathways could be supposed as key metabolic pathways in Canola under stress. Finally, the genes UGD2, TPI, and AT3G09440 are introduced as potential candidate to be regarded in genetic engineering of Canola due to their core central roles in PPI network.

  7. Expression of cardiac proteins in neonatal cardiomyocytes on PGS/fibrinogen core/shell substrate for Cardiac tissue engineering.

    Science.gov (United States)

    Ravichandran, Rajeswari; Venugopal, Jayarama Reddy; Sundarrajan, Subramanian; Mukherjee, Shayanti; Sridhar, Radhakrishnan; Ramakrishna, Seeram

    2013-08-20

    Heart failure due to myocardial infarction remains the leading cause of death worldwide owing to the inability of myocardial tissue regeneration. The aim of this study is to develop a core/shell fibrous cardiac patch having desirable mechanical properties and biocompatibility to engineer the infarcted myocardium. We fabricated poly(glycerol sebacate)/fibrinogen (PGS/fibrinogen) core/shell fibers with core as elastomeric PGS provides suitable mechanical properties comparable to that of native tissue and shell as fibrinogen to promote cell-biomaterial interactions. The PGS/fibrinogen core/shell fibers and fibrinogen nanofibers were characterized by SEM, contact angle and tensile testing to analyze the fiber morphology, wettability, and mechanical properties of the scaffold. The cell-scaffold interactions were analyzed using isolated neonatal cardiomyocytes for cell proliferation, confocal analysis for the expression of marker proteins α-actinin, Troponin-T, β-myosin heavy chain and connexin 43 and SEM analysis for cell morphology. We observed PGS/fibrinogen core/shell fibers had a Young's modulus of about 3.28 ± 1.7 MPa, which was comparable to that of native myocardium. Neonatal cardiomyocytes cultured on these scaffolds showed normal expression of cardiac specific marker proteins α-actinin, Troponin, β-myosin heavy chain and connexin 43 to prove PGS/fibrinogen core/shell fibers have potential for cardiac tissue engineering. Results indicated that neonatal cardiomyocytes formed predominant gap junctions and expressed cardiac specific marker proteins on PGS/fibrinogen core/shell fibers compared to fibrinogen nanofibers, indicating PGS/fibrinogen core/shell fibers may serve as a suitable cardiac patch for the regeneration of infarcted myocardium. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  8. Pathway analysis of Pichia pastoris to elucidate methanol metabolism and its regulation for production of recombinant proteins.

    Science.gov (United States)

    Unrean, Pornkamol

    2014-01-01

    This research rationally analyzes metabolic pathways of Pichia pastoris to study the metabolic flux responses of this yeast under methanol metabolism. A metabolic model of P. pastoris was constructed and analyzed by elementary mode analysis (EMA). EMA was used to comprehensively identify the cell's metabolic flux profiles and its underlying regulation mechanisms for the production of recombinant proteins from methanol. Change in phenotypes and flux profiles during methanol adaptation with varying feed mixture of glycerol and methanol was examined. EMA identified increasing and decreasing fluxes during the glycerol-methanol metabolic shift, which well agreed with experimental observations supporting the validity of the metabolic network model. Analysis of all the identified pathways also led to the determination of the metabolic capacities as well as the optimum metabolic pathways for recombinant protein synthesis during methanol induction. The network sensitivity analysis revealed that the production of proteins can be improved by manipulating the flux ratios at the pyruvate branch point. In addition, EMA suggested that protein synthesis is optimum under hypoxic culture conditions. The metabolic modeling and analysis presented in this study could potentially form a valuable knowledge base for future research on rational design and optimization of P. pastoris by determining target genes, pathways, and culture conditions for enhanced recombinant protein synthesis. The metabolic pathway analysis is also of considerable value for production of therapeutic proteins by P. pastoris in biopharmaceutical applications. © 2013 American Institute of Chemical Engineers.

  9. Motile hepatocellular carcinoma cells preferentially secret sugar metabolism regulatory proteins via exosomes.

    Science.gov (United States)

    Zhang, Jing; Lu, Shaohua; Zhou, Ye; Meng, Kun; Chen, Zhipeng; Cui, Yizhi; Shi, Yunfeng; Wang, Tong; He, Qing-Yu

    2017-07-01

    Exosomes are deliverers of critically functional proteins, capable of transforming target cells in numerous cancers, including hepatocellular carcinoma (HCC). We hypothesize that the motility of HCC cells can be featured by comparative proteome of exosomes. Hence, we performed the super-SILAC-based MS analysis on the exosomes secreted by three human HCC cell lines, including the non-motile Hep3B cell, and the motile 97H and LM3 cells. More than 1400 exosomal proteins were confidently quantified in each MS analysis with highly biological reproducibility. We justified that 469 and 443 exosomal proteins represented differentially expressed proteins (DEPs) in the 97H/Hep3B and LM3/Hep3B comparisons, respectively. These DEPs focused on sugar metabolism-centric canonical pathways per ingenuity pathway analysis, which was consistent with the gene ontology analysis on biological process enrichment. These pathways included glycolysis I, gluconeogenesis I and pentose phosphate pathways; and the DEPs enriched in these pathways could form a tightly connected network. By analyzing the relative abundance of proteins and translating mRNAs, we found significantly positive correlation between exosomes and cells. The involved exosomal proteins were again focusing on sugar metabolism. In conclusion, motile HCC cells tend to preferentially export more sugar metabolism-associated proteins via exosomes that differentiate them from non-motile HCC cells. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Female hormones: do they influence muscle and tendon protein metabolism?

    DEFF Research Database (Denmark)

    Hansen, Mette

    2018-01-01

    (or lack of female hormones) on skeletal muscle protein turnover at rest and in response to exercise. This review is primarily based on data from human trials. Many elderly post-menopausal women experience physical disabilities and loss of independence related to sarcopenia, which reduces life quality...... and is associated with substantial financial costs. Resistance training and dietary optimisation can counteract or at least decelerate the degenerative ageing process, but lack of oestrogen in post-menopausal women may reduce their sensitivity to these anabolic stimuli and accelerate muscle loss. Tendons...... oestrogen seems to play a significant role with regard to skeletal muscle protein turnover. Therefore, oestrogen/hormonal replacement therapy may counteract the degenerative changes in skeletal muscle. Nevertheless, there is a need for greater insight into the direct and indirect mechanistic effects...

  11. Conformational stability of mammalian prion protein amyloid fibrils is dictated by a packing polymorphism within the core region.

    Science.gov (United States)

    Cobb, Nathan J; Apostol, Marcin I; Chen, Shugui; Smirnovas, Vytautas; Surewicz, Witold K

    2014-01-31

    Mammalian prion strains are believed to arise from the propagation of distinct conformations of the misfolded prion protein PrP(Sc). One key operational parameter used to define differences between strains has been conformational stability of PrP(Sc) as defined by resistance to thermal and/or chemical denaturation. However, the structural basis of these stability differences is unknown. To bridge this gap, we have generated two strains of recombinant human prion protein amyloid fibrils that show dramatic differences in conformational stability and have characterized them by a number of biophysical methods. Backbone amide hydrogen/deuterium exchange experiments revealed that, in sharp contrast to previously studied strains of infectious amyloid formed from the yeast prion protein Sup35, differences in β-sheet core size do not underlie differences in conformational stability between strains of mammalian prion protein amyloid. Instead, these stability differences appear to be dictated by distinct packing arrangements (i.e. steric zipper interfaces) within the amyloid core, as indicated by distinct x-ray fiber diffraction patterns and large strain-dependent differences in hydrogen/deuterium exchange kinetics for histidine side chains within the core region. Although this study was limited to synthetic prion protein amyloid fibrils, a similar structural basis for strain-dependent conformational stability may apply to brain-derived PrP(Sc), especially because large strain-specific differences in PrP(Sc) stability are often observed despite a similar size of the PrP(Sc) core region.

  12. Structural Rearrangement upon Fragmentation of the Stability Core of the ALS-Linked Protein TDP-43.

    Science.gov (United States)

    Morgan, Brittany R; Zitzewitz, Jill A; Massi, Francesca

    2017-08-08

    Amyotrophic lateral sclerosis (ALS) is the most common adult degenerative motor neuron disease. Experimental evidence indicates a direct role of transactive-response DNA-binding protein 43 (TDP-43) in the pathology of ALS and other neurodegenerative diseases. TDP-43 has been identified as a major component of cytoplasmic inclusions in patients with sporadic ALS; however, the molecular basis of the disease mechanism is not yet fully understood. Fragmentation within the second RNA recognition motif (RRM2) of TDP-43 has been observed in patient tissues and may play a role in the formation of aggregates in disease. To determine the structural and dynamical changes resulting from the truncation that could lead to aggregation and toxicity, we performed molecular dynamics simulations of the full-length RRM2 domain (the stability core of TDP-43) and of a truncated variant (where residues 189-207 are deleted to mimic a site of cleavage within RRM2 found in ALS patients). Our simulations show heterogeneous structural reorganization and decreased stability of the truncated RRM2 domain compared to the full-length domain, consistent with previous experimental results. The decreased stability and structural reorganization in the truncated RRM2 result in a higher probability of protein-protein interactions through altered electrostatic surface charges and increased accessibility of hydrophobic residues (including the nuclear export sequence), providing a rationale for the increased cytoplasmic aggregation of RRM2 fragments seen in sporadic ALS patients. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  13. [New theory of holistic integrative physiology and medicine. III: New insight of neurohumoral mechanism and pattern of control and regulation for core axe of respiration, circulation and metabolism].

    Science.gov (United States)

    Sun, Xing-guo

    2015-07-01

    Systemic mechanism of neurohumoral control and regulation for human is limited. We used the new theory of holistic integrative physiology and medicine to approach the mechanism and pattern of neurohumoral control and regulation for life. As the core of human life, there are two core axes of functions. The first one is the common goal of respiration and circulation to transport oxygen and carbon dioxide for cells, and the second one is the goal of gastrointestinal tract and circulation to transport energy material and metabolic product for cells. These two core axes maintain the metabolism. The neurohumoral regulation is holistically integrated and unified for all functions in human body. We simplified explain the mechanism of neurohumoral control and regulation life (respiration and circulation) as the example pattern of sound system. Based upon integrated regulation of life, we described the neurohumoral pattern to control respiration and circulation.

  14. Identification of Proteins Involved in Carbohydrate Metabolism and Energy Metabolism Pathways and Their Regulation of Cytoplasmic Male Sterility in Wheat

    Directory of Open Access Journals (Sweden)

    Xingxia Geng

    2018-01-01

    Full Text Available Cytoplasmic male sterility (CMS where no functional pollen is produced has important roles in wheat breeding. The anther is a unique organ for male gametogenesis and its abnormal development can cause male sterility. However, the mechanisms and regulatory networks related to plant male sterility are poorly understood. In this study, we conducted comparative analyses using isobaric tags for relative and absolute quantification (iTRAQ of the pollen proteins in a CMS line and its wheat maintainer. Differentially abundant proteins (DAPs were analyzed based on Gene Ontology classifications, metabolic pathways and transcriptional regulation networks using Blast2GO. We identified 5570 proteins based on 23,277 peptides, which matched with 73,688 spectra, including proteins in key pathways such as glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase and 6-phosphofructokinase 1 in the glycolysis pathway, isocitrate dehydrogenase and citrate synthase in the tricarboxylic acid cycle and nicotinamide adenine dinucleotide (NADH-dehydrogenase and adenosine-triphosphate (ATP synthases in the oxidative phosphorylation pathway. These proteins may comprise a network that regulates male sterility in wheat. Quantitative real time polymerase chain reaction (qRT-PCR analysis, ATP assays and total sugar assays validated the iTRAQ results. These DAPs could be associated with abnormal pollen grain formation and male sterility. Our findings provide insights into the molecular mechanism related to male sterility in wheat.

  15. Production of biopharmaceutical proteins by yeast: Advances through metabolic engineering

    DEFF Research Database (Denmark)

    Nielsen, Jens

    2013-01-01

    by yeast are human serum albumin, hepatitis vaccines and virus like particles used for vaccination against human papillomavirus. Here is given a brief overview of biopharmaceutical production by yeast and it is discussed how the secretory pathway can be engineered to ensure more efficient protein...... for production of several large volume products. Insulin and insulin analogs are by far the dominating biopharmaceuticals produced by yeast, and this will increase as the global insulin market is expected to grow from USD12B in 2011 to more than USD32B by 2018. Other important biopharmaceuticals produced...

  16. Computational interaction analysis of organophosphorus pesticides with different metabolic proteins in humans

    OpenAIRE

    Sharma, Amit Kumar; Gaur, Karuna; Tiwari, Rajeev Kumar; Gaur, Mulayam Singh

    2011-01-01

    Pesticides have the potential to leave harmful effects on humans, animals, other living organisms, and the environment. Several human metabolic proteins inhibited after exposure to organophosphorus pesticides absorbed through the skin, inhalation, eyes and oral mucosa, are most important targets for this interaction study. The crystal structure of five different proteins, PDBIDs: 3LII, 3NXU, 4GTU, 2XJ1 and 1YXA in Homo sapiens (H. sapiens), interact with organophosphorus pesticides at the mol...

  17. A liver stress-endocrine nexus promotes metabolic integrity during dietary protein dilution

    DEFF Research Database (Denmark)

    Maida, Adriano; Zota, Annika; Sjøberg, Kim Anker

    2016-01-01

    Dietary protein intake is linked to an increased incidence of type 2 diabetes (T2D). Although dietary protein dilution (DPD) can slow the progression of some aging-related disorders, whether this strategy affects the development and risk for obesity-associated metabolic disease such as T2D...... and secretion in hepatocytes in vitro and in vivo. Taken together, these data indicate that DPD promotes improved glucose homeostasis through an NEAA insufficiency-induced liver NUPR1/FGF21 axis....

  18. Data of the interacting protein networks and nucleotide metabolism pathways related to NDK and NT5

    Directory of Open Access Journals (Sweden)

    Dan Zhang

    2016-12-01

    Full Text Available The data presented in this article are related to the research article entitled “Antibacterial mechanism of daptomycin antibiotic against Staphylococcus aureus based on a quantitative bacterial proteome analysis” (Ma et al., 2016 [1]. Nucleoside diphosphate kinase (NDK and 5′-nucleotidase (NT5 are two proteins related to bacterial growth. Here, a bioinformatics analysis was presented to explore NDK and NT5-invovled in the interacting protein network and purine metabolism.

  19. Is impaired energy regulation the core of the metabolic syndrome in various ethnic groups of the USA and Taiwan?

    Directory of Open Access Journals (Sweden)

    Wang Wuan-Szu

    2010-06-01

    Full Text Available Abstract Background The metabolic syndrome (MetS concept is widely used in public health and clinical settings without an agreed pathophysiology. We have re-examined the MetS in terms of body fuels, so as to provide a coherent cross-cultural pathogenesis. Methods National Health and Nutrition Examination Survey (NHANES 2001-2 with n = 2254 and Taiwanese National Health Interview Survey (NHIS sub-set for hypertension, hyperglycemia and hyperlipidemia assessment (TwSHHH 2002, n = 5786, were used to compare different ethnicities according to NCEP-ATPIII (NCEP-tw criteria for METS. Exploratory factor analysis (EFA using principal components (PC was employed to differentiate and unify MetS components across four ethnicities, gender, age-strata, and urban-rural settings. Results The first two factors from the PC analysis (PCA accounted for from 55.2% (non-Hispanic white to 63.7% (Taiwanese of the variance. Rotated factor loadings showed that the six MetS components provided three clusters: the impaired energy regulation (IER components (waist circumference, WC, fasting triglycerides, TG, and fasting plasma glucose, FPG, systolic and diastolic blood pressures (BPs, and HDL-cholesterol, where the IER components accounted for 25-26% of total variance of MetS components. For the three US ethnic subgroups, factor 1 was mainly determined by IER and HDL-cholesterol, and factor 2 was related to the BP components. For Taiwanese, IER was determinant for both factors, and BPs and HDL-cholesterol were related to factors 1 and 2 respectively. Conclusions There is a MetS core which unifies populations. It comprises WC, TG and FPG as a core, IER, which may be expressed and modulated in various second order ways.

  20. Effect of Mutant p53 Proteins on Glycolysis and Mitochondrial Metabolism.

    Science.gov (United States)

    Eriksson, Matilda; Ambroise, Gorbatchev; Ouchida, Amanda Tomie; Lima Queiroz, Andre; Smith, Dominique; Gimenez-Cassina, Alfredo; Iwanicki, Marcin P; Muller, Patricia A; Norberg, Erik; Vakifahmetoglu-Norberg, Helin

    2017-12-15

    TP53 is one of the most commonly mutated genes in human cancers. Unlike other tumor suppressors that are frequently deleted or acquire loss-of-function mutations, the majority of TP53 mutations in tumors are missense substitutions, which lead to the expression of full-length mutant proteins that accumulate in cancer cells and may confer unique gain-of-function (GOF) activities to promote tumorigenic events. Recently, mutant p53 proteins have been shown to mediate metabolic changes as a novel GOF to promote tumor development. There is a strong rationale that the GOF activities, including alterations in cellular metabolism, might vary between the different p53 mutants. Accordingly, the effect of different mutant p53 proteins on cancer cell metabolism is largely unknown. In this study, we have metabolically profiled several individual frequently occurring p53 mutants in cancers, focusing on glycolytic and mitochondrial oxidative phosphorylation pathways. Our investigation highlights the diversity of different p53 mutants in terms of their effect on metabolism, which might provide a foundation for the development of more effective targeted pharmacological approaches toward variants of mutant p53. Copyright © 2017 American Society for Microbiology.

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

    Science.gov (United States)

    Ney, Denise Marie; Etzel, Mark Raymond

    2017-04-01

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

  2. Metabolic Effects of Dietary Proteins, Amino Acids and The Other Amine Consisting Compounds on Cardiovascular System.

    Directory of Open Access Journals (Sweden)

    Elif Uğur

    2017-01-01

    Full Text Available During the prevention and treatment of cardiovascular diseases, first cause of deaths in the world, diet has a vital role. While nutrition programs for the cardiovascular health generally focus on lipids and carbohydrates, effects of proteins are not well concerned. Thus this review is written in order to examine effect of proteins, amino acids, and the other amine consisting compounds on cardiovascular system. Because of that animal or plant derived proteins have different protein composition in different foods such as dairy products, egg, meat, chicken, fish, pulse and grains, their effects on blood pressure and regulation of lipid profile are unlike. In parallel amino acids made up proteins have different effect on cardiovascular system. From this point, sulfur containing amino acids, branched chain amino acids, aromatic amino acids, arginine, ornithine, citrulline, glycine, and glutamine may affect cardiovascular system in different metabolic pathways. In this context, one carbon metabolism, synthesis of hormone, stimulation of signaling pathways and effects of intermediate and final products that formed as a result of amino acids metabolism is determined. Despite the protein and amino acids, some other amine consisting compounds in diet include trimethylamine N-oxide, heterocyclic aromatic amines, polycyclic aromatic hydrocarbons and products of Maillard reaction. These amine consisting compounds generally increase the risk for cardiovascular diseases by stimulating oxidative stress, inflammation, and formation of atherosclerotic plaque.

  3. Magnetic core/shell Fe3O4/Au nanoparticles for studies of quinolones binding to protein by fluorescence spectroscopy.

    Science.gov (United States)

    Jin, Rui; Song, Daqian; Xiong, Huixia; Ai, Lisha; Ma, Pinyi; Sun, Ying

    2016-03-01

    Magnetic core/shell Fe3O4/Au nanoparticles were used in the determination of drug binding to bovine serum albumin (BSA) using a fluorescence spectroscopic method. The binding constants and number of binding sites for protein with drugs were calculated using the Scatchard equation. Because of their superparamagnetic and biocompatible characteristics, magnetic core/shell Fe3O4/Au nanoparticles served as carrier proteins for fixing proteins. After binding of the protein to a drug, the magnetic core/shell Fe3O4/Au nanoparticles-protein-drug complex was separated from the free drug using an applied magnetic field. The free drug concentration was obtained directly by fluorescence spectrometry and the proteins did not influence the drug determination. So, the achieved number of binding sites should be reliable. The binding constant and site number for ciprofloxacin (CPFX) binding to BSA were 2.055 × 10(5) L/mol and 31.7, and the corresponding values for norfloxacin (NOR) binding to BSA were 1.383 × 10(5) L/mol and 38.8. Based on the achieved results, a suitable method was proposed for the determination of binding constants and the site number for molecular interactions. The method was especially suitable for studies on the interactions of serum albumin with the active ingredients of Chinese medicine. Copyright © 2015 John Wiley & Sons, Ltd.

  4. Association between C-reactive protein and features of the metabolic syndrome

    DEFF Research Database (Denmark)

    Fröhlich, M; Imhof, A; Berg, Gabriele

    2000-01-01

    OBJECTIVE: To assess the association of circulating levels of C-reactive protein, a sensitive systemic marker of inflammation, with different components of the metabolic syndrome. RESEARCH DESIGN AND METHODS: Total cholesterol (TC), HDL cholesterol, triglycerides, uric acid, BMI , and prevalence...... C-reactive protein and TC (R = 0.19), TG (R = 0.29), BMI (R = 0.32), glucose (R = 0.11), and uric acid (R = 0.14) (all P ... concentrations in subjects grouped according to the presence of 0-1, 2-3, and > or =4 features of the metabolic syndrome were 1.11, 1.27, and 2.16 mg/l, respectively, with a statistically highly significant trend (P metabolic syndrome...

  5. Riboflavin carrier protein-targeted fluorescent USPIO for the assessment of vascular metabolism in tumors

    NARCIS (Netherlands)

    Jayapaul, J.; Arns, S.; Lederle, W.; Lammers, Twan Gerardus Gertudis Maria; Comba, P.; Gätjens, J.; Kiessling, F.

    2012-01-01

    Abstract Riboflavin (Rf) and its metabolic analogs flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are essential for normal cellular growth and function. Their intracellular transport is regulated by the riboflavin carrier protein (RCP), which has been shown to be over-expressed by

  6. Cercal sensory regulation of ganglionic protein metabolism in the field cricket, Gryllotalpa africana.

    Science.gov (United States)

    Sekhar, V; Dayanand, Y; Madhusudhuna, L; Srinivasulu, Y; Reddy, G R

    1991-07-01

    The effect of cercal deafferentation (cercectomy) on the ganglionic protein metabolism of the cricket, Gryllotalpa africana was studied. Significant changes in the activities of the enzymes acetylcholinesterase, glutamate dehydrogenase, alanine aminotransferase and aspartate aminotransferase were observed in the terminal ganglion following unilateral and bilateral cercectomy.

  7. Heme metabolism in stress regulation and protein production: from Cinderella to a key player

    DEFF Research Database (Denmark)

    Martinez Ruiz, José Luis; Petranovic, D.; Nielsen, Jens

    2016-01-01

    Heme biosynthesis is a highly conserved pathway which is present in all kingdoms, from Archaea to higher organisms such as plants and mammals. The heme molecule acts as a prosthetic group for different proteins and enzymes involved in energy metabolism and reactions involved in electron transfer....

  8. Regulatory mechanism of protein metabolic pathway during the differentiation process of chicken male germ cell.

    Science.gov (United States)

    Li, Dong; Zuo, Qisheng; Lian, Chao; Zhang, Lei; Shi, Qingqing; Zhang, Zhentao; Wang, Yingjie; Ahmed, Mahmoud F; Tang, Beibei; Xiao, Tianrong; Zhang, Yani; Li, Bichun

    2015-08-01

    We explored the regulatory mechanism of protein metabolism during the differentiation process of chicken male germ cells and provide a basis for improving the induction system of embryonic stem cell differentiation to male germ cells in vitro. We sequenced the transcriptome of embryonic stem cells, primordial germ cells, and spermatogonial stem cells with RNA sequencing (RNA-Seq), bioinformatics analysis methods, and detection of the key genes by quantitative reverse transcription PCR (qRT-PCR). Finally, we found 16 amino acid metabolic pathways enriched in the biological metabolism during the differentiation process of embryonic stem cells to primordial germ cells and 15 amino acid metabolic pathways enriched in the differentiation stage of primordial germ cells to spermatogonial stem cells. We found three pathways, arginine-proline metabolic pathway, tyrosine metabolic pathway, and tryptophan metabolic pathway, significantly enriched in the whole differentiation process of embryonic stem cells to spermatogonial stem cells. Moreover, for these three pathways, we screened key genes such as NOS2, ADC, FAH, and IDO. qRT-PCR results showed that the expression trend of these genes were the same to RNA-Seq. Our findings showed that the three pathways and these key genes play an important role in the differentiation process of embryonic stem cells to male germ cells. These results provide basic information for improving the induction system of embryonic stem cell differentiation to male germ cells in vitro.

  9. Regulation of lifespan, metabolism, and stress responses by the Drosophila SH2B protein, Lnk.

    Directory of Open Access Journals (Sweden)

    Cathy Slack

    2010-03-01

    Full Text Available Drosophila Lnk is the single ancestral orthologue of a highly conserved family of structurally-related intracellular adaptor proteins, the SH2B proteins. As adaptors, they lack catalytic activity but contain several protein-protein interaction domains, thus playing a critical role in signal transduction from receptor tyrosine kinases to form protein networks. Physiological studies of SH2B function in mammals have produced conflicting data. However, a recent study in Drosophila has shown that Lnk is an important regulator of the insulin/insulin-like growth factor (IGF-1 signaling (IIS pathway during growth, functioning in parallel to the insulin receptor substrate, Chico. As this pathway also has an evolutionary conserved role in the determination of organism lifespan, we investigated whether Lnk is required for normal lifespan in Drosophila. Phenotypic analysis of mutants for Lnk revealed that loss of Lnk function results in increased lifespan and improved survival under conditions of oxidative stress and starvation. Starvation resistance was found to be associated with increased metabolic stores of carbohydrates and lipids indicative of impaired metabolism. Biochemical and genetic data suggest that Lnk functions in both the IIS and Ras/Mitogen activated protein Kinase (MapK signaling pathways. Microarray studies support this model, showing transcriptional feedback onto genes in both pathways as well as indicating global changes in both lipid and carbohydrate metabolism. Finally, our data also suggest that Lnk itself may be a direct target of the IIS responsive transcription factor, dFoxo, and that dFoxo may repress Lnk expression. We therefore describe novel functions for a member of the SH2B protein family and provide the first evidence for potential mechanisms of SH2B regulation. Our findings suggest that IIS signaling in Drosophila may require the activity of a second intracellular adaptor, thereby yielding fundamental new insights into the

  10. Impact of weight loss and maintenance with ad libitum diets varying in protein and glycemic index content on metabolic syndrome

    DEFF Research Database (Denmark)

    Papadaki, Angeliki; Linardakis, Manolis; Plada, Maria

    2014-01-01

    We investigated the effects of weight loss and maintenance with diets that varied with regard to protein content and glycemic index (GI) on metabolic syndrome (MetSyn) status.......We investigated the effects of weight loss and maintenance with diets that varied with regard to protein content and glycemic index (GI) on metabolic syndrome (MetSyn) status....

  11. pH/sugar dual responsive core-cross-linked PIC micelles for enhanced intracellular protein delivery.

    Science.gov (United States)

    Ren, Jie; Zhang, Yanxin; Zhang, Ju; Gao, Hongjun; Liu, Gan; Ma, Rujiang; An, Yingli; Kong, Deling; Shi, Linqi

    2013-10-14

    Herein, a series of biocompatible, robust, pH/sugar-sensitive, core-cross-linked, polyion complex (PIC) micelles based on phenylboronic acid-catechol interaction were developed for protein intracellular delivery. The rationally designed poly(ethylene glycol)-b-poly(glutamic acid-co-glutamicamidophenylboronic acid) (PEG-b-P(Glu-co-GluPBA)) and poly(ethylene glycol)-b-poly(l-lysine-co-ε-3,4-dihydroxyphenylcarboxyl-L-lysine) (PEG-b-P(Lys-co-LysCA)) copolymers were successfully synthesized and self-assembled under neutral aqueous condition to form uniform micelles. These micelles possessed a distinct core-cross-linked core-shell structure comprised of the PEG outer shell and the PGlu/PLys polyion complex core bearing boronate ester cross-linking bonds. The cross-linked micelles displayed superior physiological stabilities compared with their non-cross-linked counterparts while swelling and disassembling in the presence of excess fructose or at endosomal pH. Notably, either negatively or positively charged proteins can be encapsulated into the micelles efficiently under mild conditions. The in vitro release studies showed that the release of protein cargoes under physiological conditions was minimized, while a burst release occurred in response to excess fructose or endosomal pH. The cytotoxicity of micelles was determined by cck-8 assay in HepG2 cells. The cytochrome C loaded micelles could efficiently delivery proteins into HepG2 cells and exhibited enhanced apoptosis ability. Hence, this type of core-cross-linked PIC micelles has opened a new avenue to intracellular protein delivery.

  12. Cloning and sequence analysis of a partial cDNA for chicken cartilage proteoglycan core protein.

    Science.gov (United States)

    Sai, S; Tanaka, T; Kosher, R A; Tanzer, M L

    1986-01-01

    A chicken embryo sternal cartilage cDNA library, created in the plasmid expression vector pUC9, was screened for sequences coding for immunologically detectable core protein of the large, major proteoglycan of cartilage. A 1229-base-pair cDNA clone was isolated that contained only one extended open reading frame, which had sequences coding for a polypeptide of 379 amino acid residues. These deduced sequences corresponded to those anticipated from current models of proteoglycan structure; a deduced sequence encompassing 21 amino acids was almost identical to a known sequence of bovine nasal cartilage proteoglycan. Significant homology was found between the deduced amino acid sequence of the proteoglycan and two regions of a chicken hepatic lectin. Immunoprecipitation of the products of cell-free translation yielded a component of about 340 kDa, and transfer blot hybridization of sternal cartilage RNA showed a single mRNA of about 8.1 kilobases. Hybridizable mRNA sequences were readily detectable by dot-blot analyses of the cytoplasm of cartilaginous tissues of the chicken embryo, whereas similar analyses of prechondrogenic limb mesenchymal cells did not demonstrate such hybridizable mRNA signals. Images PMID:3460082

  13. Hepatitis C virus core protein epigenetically silences SFRP1 and enhances HCC aggressiveness by inducing epithelial-mesenchymal transition.

    Science.gov (United States)

    Quan, H; Zhou, F; Nie, D; Chen, Q; Cai, X; Shan, X; Zhou, Z; Chen, K; Huang, A; Li, S; Tang, N

    2014-05-29

    Hepatocellular carcinoma (HCC) is one of the most deadly cancers. Aberrant oncogenic activation of the Wnt/β-catenin signaling pathway contributes to hepatocellular carcinogenesis. Various epigenetic modifications of the Wnt antagonist secreted frizzled-related protein (SFRP) family have been implicated in regulating Wnt signaling. Here, we report that Hepatitis C virus (HCV) core protein downregulates SFRP1 expression when it is expressed in Huh7 and HepG2 cells. SFRP1 expression can be effectively restored by using either a DNA methylation inhibitor alone or in combination with a histone deacetylase inhibitor. DNA methylation analysis of the SFRP1 promoter revealed that cytosine-phosphate-guanine (CpG) islands close to the transcriptional start site (TSS) in the SFRP1 promoter were hypermethylated in core-expressing Huh7 cells, suggesting that HCV core protein may downregulate SFRP1 expression by inducing hypermethylation of the SFRP1 promoter. Chromatin immunoprecipitation revealed that HCV core protein markedly increased the expression level and binding of DNA methyltransferase-1 (Dnmt1) and histone deacetylase-1 (HDAC1) to the TSS of the SFRP1 promoter region, resulting in repression of acetyl-histone H3-binding capacity to SFRP1 promoter and the eventual epigenetic silencing of SFRP1 expression. Furthermore, the core protein-promoted cell proliferation, migration and invasiveness were effectively abrogated either by Dnmt1 knockdown or restoration of SFRP1 expression in hepatoma cells. Dnmt1 knockdown or SFRP1 overexpression also inhibited HCV core-induced epithelial-mesenchymal transition (EMT) and significantly decreased the expression levels of activated β-catenin and Wnt/β-catenin target genes, c-Myc and cyclin D1. We further showed that knockdown of Dnmt1 and restoration of SFRP1 inhibited core-induced in vivo tumor growth and aggressiveness in a xenograft HCC model. Taken together, our results strongly suggest that the HCV core-induced epigenetic

  14. Mesitylene-Cored Glucoside Amphiphiles (MGAs) for Membrane Protein Studies: Importance of Alkyl Chain Density in Detergent Efficacy

    DEFF Research Database (Denmark)

    Cho, Kyung Ho; Ribeiro, Orquidea; Du, Yang

    2016-01-01

    Detergents serve as useful tools for membrane protein structural and functional studies. Their amphipathic nature allows detergents to associate with the hydrophobic regions of membrane proteins whilst maintaining the proteins in aqueous solution. However, widely used conventional detergents...... are limited in their ability to maintain the structural integrity of membrane proteins and thus there are major efforts underway to develop novel agents with improved properties. We prepared mesitylene-cored glucoside amphiphiles (MGAs) with three alkyl chains and compared these agents with previously...... developed xylene-linked maltoside agents (XMAs) with two alkyl chains and a conventional detergent (DDM). When these agents were evaluated for four membrane proteins including a G protein-coupled receptor (GPCR), some agents such as MGA-C13 and MGA-C14 resulted in markedly enhanced stability of membrane...

  15. Genes regulating lipid and protein metabolism are highly expressed in mammary gland of lactating dairy goats.

    Science.gov (United States)

    Shi, Hengbo; Zhu, Jiangjiang; Luo, Jun; Cao, Wenting; Shi, Huaiping; Yao, Dawei; Li, Jun; Sun, Yuting; Xu, Huifen; Yu, Kang; Loor, Juan J

    2015-05-01

    Dairy goats serve as an important source of milk and also fulfill agricultural and economic roles in developing countries. Understanding the genetic background of goat mammary gland is important for research on the regulatory mechanisms controlling tissue function and the synthesis of milk components. We collected tissue at four different stages of goat mammary gland development and generated approximately 25 GB of data from Illumina de novo RNA sequencing. The combined reads were assembled into 51,361 unigenes, and approximately 60.07 % of the unigenes had homology to other proteins in the NCBI non-redundant protein database (NR). Functional classification through eukaryotic Ortholog Groups of Protein (KOG), gene ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that the unigenes from goat mammary glands are involved in a wide range of biological processes and metabolic pathways, including lipid metabolism and lactose metabolism. The results of qPCR revealed that genes encoding FABP3, FASN, SCD, PLIN2, whey proteins (LALBA and BLG), and caseins (CSN1S1, CSN1S2, CSN2 and CSN3) at 100 and 310 days postpartum increased significantly compared with the non-lactating period. In addition to their role in lipid and protein synthesis, the higher expression at 310 days postpartum could contribute to mammary cell turnover during pregnancy. In conclusion, this is the first study to characterize the complete transcriptome of goat mammary glands and constitutes a comprehensive genomic resource available for further studies of ruminant lactation.

  16. Stability of Retroviral Vectors Against Ultracentrifugation Is Determined by the Viral Internal Core and Envelope Proteins Used for Pseudotyping.

    Science.gov (United States)

    Kim, Soo-Hyun; Lim, Kwang-Il

    2017-05-31

    Retroviral and lentiviral vectors are mostly pseudotyped and often purified and concentrated via ultracentrifugation. In this study, we quantified and compared the stabilities of retroviral [murine leukemia virus (MLV)-based] and lentiviral [human immunodeficiency virus (HIV)-1-based] vectors pseudotyped with relatively mechanically stable envelope proteins, vesicular stomatitis virus glycoproteins (VSVGs), and the influenza virus WSN strain envelope proteins against ultracentrifugation. Lentiviral genomic and functional particles were more stable than the corresponding retroviral particles against ultracentrifugation when pseudotyped with VSVGs. However, both retroviral and lentiviral particles were unstable when pseudotyped with the influenza virus WSN strain envelope proteins. Therefore, the stabilities of pseudotyped retroviral and lentiviral vectors against ultracentrifugation process are a function of not only the type of envelope proteins, but also the type of viral internal core (MLV or HIV-1 core). In addition, the fraction of functional viral particles among genomic viral particles greatly varied at times during packaging, depending on the type of envelope proteins used for pseudotyping and the viral internal core.

  17. Small Heat Shock Proteins in Redox Metabolism: Implications for Cardiovascular Diseases

    Science.gov (United States)

    Christians, Elisabeth S.; Ishiwata, Takahiro; Benjamin, Ivor J.

    2012-01-01

    A timely review series on small heat shock proteins has to appropriately examine their fundamental properties and implications in the cardiovascular system since several members of this chaperone family exhibit robust expression in the myocardium and blood vessels. Due to energetic and metabolic demands, the cardiovascular system maintains a high mitochondrial activity but irreversible oxidative damage might ensue from increased production of reactive oxygen species. How equilibrium between their production and scavenging is achieved becomes paramount for physiological maintenance. For example, heat shock protein B1 (HSPB1) is implicated in maintaining this equilibrium or redox homeostasis by upholding the level of glutathione, a major redox mediator. Studies of gain or loss of function achieved by genetic manipulations have been highly informative for understanding the roles of those proteins. For example, genetic deficiency of several small heat shock proteins such as HSPB5 and HSPB2 is well-tolerated in heart cells whereas a single missense mutation causes human pathology. Such evidence highlights both the profound genetic redundancy observed among the multigene family of small heat shock proteins while underscoring the role proteotoxicity plays in driving disease pathogenesis. We will discuss the available data on small heat shock proteins in the cardiovascular system, redox metabolism and human diseases. From the medical perspective, we envision that such emerging knowledge of the multiple roles small heat shock proteins exert in the cardiovascular system will undoubtedly open new avenues for their identification and possible therapeutic targeting in humans. PMID:22710345

  18. The N-terminus of murine leukaemia virus p12 protein is required for mature core stability.

    Science.gov (United States)

    Wight, Darren J; Boucherit, Virginie C; Wanaguru, Madushi; Elis, Efrat; Hirst, Elizabeth M A; Li, Wilson; Ehrlich, Marcelo; Bacharach, Eran; Bishop, Kate N

    2014-10-01

    The murine leukaemia virus (MLV) gag gene encodes a small protein called p12 that is essential for the early steps of viral replication. The N- and C-terminal regions of p12 are sequentially acting domains, both required for p12 function. Defects in the C-terminal domain can be overcome by introducing a chromatin binding motif into the protein. However, the function of the N-terminal domain remains unknown. Here, we undertook a detailed analysis of the effects of p12 mutation on incoming viral cores. We found that both reverse transcription complexes and isolated mature cores from N-terminal p12 mutants have altered capsid complexes compared to wild type virions. Electron microscopy revealed that mature N-terminal p12 mutant cores have different morphologies, although immature cores appear normal. Moreover, in immunofluorescent studies, both p12 and capsid proteins were lost rapidly from N-terminal p12 mutant viral cores after entry into target cells. Importantly, we determined that p12 binds directly to the MLV capsid lattice. However, we could not detect binding of an N-terminally altered p12 to capsid. Altogether, our data imply that p12 stabilises the mature MLV core, preventing premature loss of capsid, and that this is mediated by direct binding of p12 to the capsid shell. In this manner, p12 is also retained in the pre-integration complex where it facilitates tethering to mitotic chromosomes. These data also explain our previous observations that modifications to the N-terminus of p12 alter the ability of particles to abrogate restriction by TRIM5alpha and Fv1, factors that recognise viral capsid lattices.

  19. The N-terminus of murine leukaemia virus p12 protein is required for mature core stability.

    Directory of Open Access Journals (Sweden)

    Darren J Wight

    2014-10-01

    Full Text Available The murine leukaemia virus (MLV gag gene encodes a small protein called p12 that is essential for the early steps of viral replication. The N- and C-terminal regions of p12 are sequentially acting domains, both required for p12 function. Defects in the C-terminal domain can be overcome by introducing a chromatin binding motif into the protein. However, the function of the N-terminal domain remains unknown. Here, we undertook a detailed analysis of the effects of p12 mutation on incoming viral cores. We found that both reverse transcription complexes and isolated mature cores from N-terminal p12 mutants have altered capsid complexes compared to wild type virions. Electron microscopy revealed that mature N-terminal p12 mutant cores have different morphologies, although immature cores appear normal. Moreover, in immunofluorescent studies, both p12 and capsid proteins were lost rapidly from N-terminal p12 mutant viral cores after entry into target cells. Importantly, we determined that p12 binds directly to the MLV capsid lattice. However, we could not detect binding of an N-terminally altered p12 to capsid. Altogether, our data imply that p12 stabilises the mature MLV core, preventing premature loss of capsid, and that this is mediated by direct binding of p12 to the capsid shell. In this manner, p12 is also retained in the pre-integration complex where it facilitates tethering to mitotic chromosomes. These data also explain our previous observations that modifications to the N-terminus of p12 alter the ability of particles to abrogate restriction by TRIM5alpha and Fv1, factors that recognise viral capsid lattices.

  20. Suitability of magnetic single- and multi-core nanoparticles to detect protein binding with dynamic magnetic measurement techniques

    Energy Technology Data Exchange (ETDEWEB)

    Remmer, Hilke, E-mail: h.remmer@tu-bs.de; Dieckhoff, Jan; Schilling, Meinhard; Ludwig, Frank

    2015-04-15

    We investigated the binding of biotinylated proteins to various streptavidin functionalized magnetic nanoparticles with different dynamic magnetic measurement techniques to examine their potential for homogeneous bioassays. As particle systems, single-core nanoparticles with a nominal core diameter of 30 nm as well as multi-core nanoparticles with hydrodynamic sizes varying between nominally 60 nm and 100 nm were chosen. As experimental techniques, fluxgate magnetorelaxometry (MRX), complex ac susceptibility (ACS) and measurements of the phase lag between rotating field and sample magnetization are applied. MRX measurements are only suited for the detection of small analytes if the multivalency of functionalized nanoparticles and analytes causes cross-linking, thus forming larger aggregates. ACS measurements showed for all nanoparticle systems a shift of the imaginary part's maximum towards small frequencies. In rotating field measurements only the single-core nanoparticle systems with dominating Brownian mechanism exhibit an increase of the phase lag upon binding in the investigated frequency range. The coexistence of Brownian and Néel relaxation processes can cause a more complex phase lag change behavior, as demonstrated for multi-core nanoparticle systems. - Highlights: • Cealization of homogeneous magnetic bioassays using different magnetic techniques. • Comparison of single- and multi-core nanoparticle systems. • ac Susceptibility favorable for detection of small analytes. • Magnetorelaxometry favorable for detection of large analytes or cross-linking assays.

  1. Progressive dry-core-wet-rim hydration trend in a nested-ring topology of protein binding interfaces

    Directory of Open Access Journals (Sweden)

    Li Zhenhua

    2012-03-01

    Full Text Available Abstract Background Water is an integral part of protein complexes. It shapes protein binding sites by filling cavities and it bridges local contacts by hydrogen bonds. However, water molecules are usually not included in protein interface models in the past, and few distribution profiles of water molecules in protein binding interfaces are known. Results In this work, we use a tripartite protein-water-protein interface model and a nested-ring atom re-organization method to detect hydration trends and patterns from an interface data set which involves immobilized interfacial water molecules. This data set consists of 206 obligate interfaces, 160 non-obligate interfaces, and 522 crystal packing contacts. The two types of biological interfaces are found to be drier than the crystal packing interfaces in our data, agreeable to a hydration pattern reported earlier although the previous definition of immobilized water is pure distance-based. The biological interfaces in our data set are also found to be subject to stronger water exclusion in their formation. To study the overall hydration trend in protein binding interfaces, atoms at the same burial level in each tripartite protein-water-protein interface are organized into a ring. The rings of an interface are then ordered with the core atoms placed at the middle of the structure to form a nested-ring topology. We find that water molecules on the rings of an interface are generally configured in a dry-core-wet-rim pattern with a progressive level-wise solvation towards to the rim of the interface. This solvation trend becomes even sharper when counterexamples are separated. Conclusions Immobilized water molecules are regularly organized in protein binding interfaces and they should be carefully considered in the studies of protein hydration mechanisms.

  2. Labeling Cell Surface GPIs and GPI-Anchored Proteins through Metabolic Engineering with Artificial Inositol Derivatives.

    Science.gov (United States)

    Lu, Lili; Gao, Jian; Guo, Zhongwu

    2015-08-10

    Glycosylphosphatidylinositol (GPI) anchoring of proteins to the cell surface is important for various biological processes, but GPI-anchored proteins are difficult to study. An effective strategy was developed for the metabolic engineering of cell-surface GPIs and GPI-anchored proteins by using inositol derivatives carrying an azido group. The azide-labeled GPIs and GPI-anchored proteins were then tagged with biotin on live cells through a click reaction, which allows further elaboration with streptavidin-conjugated dyes or other molecules. The strategy can be used to label GPI-anchored proteins with various tags for biological studies. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Expression of Lipid Metabolism-Related Proteins Differs between Invasive Lobular Carcinoma and Invasive Ductal Carcinoma.

    Science.gov (United States)

    Cha, Yoon Jin; Kim, Hye Min; Koo, Ja Seung

    2017-01-23

    We comparatively investigated the expression and clinical implications of lipid metabolism-related proteins in invasive lobular carcinoma (ILC) and invasive ductal carcinoma (IDC) of the breast. A total of 584 breast cancers (108 ILC and 476 IDC) were subjected to tissue microarray and immunohistochemical analysis for lipid metabolism-related proteins including hormone-sensitive lipase (HSL), perilipin A, fatty acid binding protein (FABP)4, carnitine palmitoyltransferase (CPT)-1, acyl-CoA oxidase 1, and fatty acid synthetase (FASN). HSL, perilipin A, and FABP4 expression (all p invasive cancers, HSL and FABP4 were highly expressed in luminal A-type ILC (p < 0.001) and perilipin A in luminal A-type IDC (p = 0.007). Among luminal B-type cancers, HSL and FABP4 were more highly expressed in ILC (p < 0.001). Univariate analysis found associations of shorter disease-free survival with CPT-1 positivity (p = 0.004) and acyl-CoA oxidase 1 positivity (p = 0.032) and of shorter overall survival with acyl-CoA oxidase 1 positivity (p = 0.027). In conclusion, ILC and IDC exhibited different immunohistochemical lipid metabolism-related protein expression profiles. Notably, ILC exhibited high HSL and FABP4 and low perilipin A expression.

  4. Expression of Lipid Metabolism-Related Proteins Differs between Invasive Lobular Carcinoma and Invasive Ductal Carcinoma

    Directory of Open Access Journals (Sweden)

    Yoon Jin Cha

    2017-01-01

    Full Text Available We comparatively investigated the expression and clinical implications of lipid metabolism-related proteins in invasive lobular carcinoma (ILC and invasive ductal carcinoma (IDC of the breast. A total of 584 breast cancers (108 ILC and 476 IDC were subjected to tissue microarray and immunohistochemical analysis for lipid metabolism-related proteins including hormone-sensitive lipase (HSL, perilipin A, fatty acid binding protein (FABP4, carnitine palmitoyltransferase (CPT-1, acyl-CoA oxidase 1, and fatty acid synthetase (FASN. HSL, perilipin A, and FABP4 expression (all p < 0.001 differed significantly: HSL and FABP4 were more frequently present in ILC, whereas perilipin A was more frequently detected in IDC. Among all invasive cancers, HSL and FABP4 were highly expressed in luminal A-type ILC (p < 0.001 and perilipin A in luminal A-type IDC (p = 0.007. Among luminal B-type cancers, HSL and FABP4 were more highly expressed in ILC (p < 0.001. Univariate analysis found associations of shorter disease-free survival with CPT-1 positivity (p = 0.004 and acyl-CoA oxidase 1 positivity (p = 0.032 and of shorter overall survival with acyl-CoA oxidase 1 positivity (p = 0.027. In conclusion, ILC and IDC exhibited different immunohistochemical lipid metabolism-related protein expression profiles. Notably, ILC exhibited high HSL and FABP4 and low perilipin A expression.

  5. Type and amount of dietary protein in the treatment of metabolic syndrome: a randomized controlled trial.

    Science.gov (United States)

    Hill, Alison M; Harris Jackson, Kristina A; Roussell, Michael A; West, Sheila G; Kris-Etherton, Penny M

    2015-10-01

    Food-based dietary patterns emphasizing plant protein that were evaluated in the Dietary Approaches to Stop Hypertension (DASH) and OmniHeart trials are recommended for the treatment of metabolic syndrome (MetS). However, the contribution of plant protein to total protein in these diets is proportionally less than that of animal protein. This study compared 3 diets varying in type (animal compared with plant) and amount of protein on MetS criteria. Sixty-two overweight adults with MetS consumed a healthy American diet for 2 wk before being randomly allocated to either a modified DASH diet rich in plant protein (18% protein, two-thirds plant sources, n = 9 males, 12 females), a modified DASH diet rich in animal protein (Beef in an Optimal Lean Diet: 18.4% protein, two-thirds animal sources, n = 9 males, 11 females), or a moderate-protein diet (Beef in an Optimal Lean Diet Plus Protein: 27% protein, two-thirds animal sources, n = 10 males, 11 females). Diets were compared across 3 phases of energy balance: 5 wk of controlled (all foods provided) weight maintenance (WM), 6 wk of controlled weight loss (minimum 500-kcal/d deficit) including exercise (WL), and 12 wk of prescribed, free-living weight loss (FL). The primary endpoint was change in MetS criteria. All groups achieved ∼5% weight loss at the end of the WL phase and maintained it through FL, with no between-diet differences (WM compared with WL, FL, P protein source or amount. Our findings demonstrate that heart-healthy weight-loss dietary patterns that emphasize either animal or plant protein improve MetS criteria similarly. This study was registered at clinicaltrials.gov as NCT00937638. © 2015 American Society for Nutrition.

  6. Reciprocal regulation of protein synthesis and carbon metabolism for thylakoid membrane biogenesis.

    Directory of Open Access Journals (Sweden)

    Alexandra-Viola Bohne

    Full Text Available Metabolic control of gene expression coordinates the levels of specific gene products to meet cellular demand for their activities. This control can be exerted by metabolites acting as regulatory signals and/or a class of metabolic enzymes with dual functions as regulators of gene expression. However, little is known about how metabolic signals affect the balance between enzymatic and regulatory roles of these dual functional proteins. We previously described the RNA binding activity of a 63 kDa chloroplast protein from Chlamydomonas reinhardtii, which has been implicated in expression of the psbA mRNA, encoding the D1 protein of photosystem II. Here, we identify this factor as dihydrolipoamide acetyltransferase (DLA2, a subunit of the chloroplast pyruvate dehydrogenase complex (cpPDC, which is known to provide acetyl-CoA for fatty acid synthesis. Analyses of RNAi lines revealed that DLA2 is involved in the synthesis of both D1 and acetyl-CoA. Gel filtration analyses demonstrated an RNP complex containing DLA2 and the chloroplast psbA mRNA specifically in cells metabolizing acetate. An intrinsic RNA binding activity of DLA2 was confirmed by in vitro RNA binding assays. Results of fluorescence microscopy and subcellular fractionation experiments support a role of DLA2 in acetate-dependent localization of the psbA mRNA to a translation zone within the chloroplast. Reciprocally, the activity of the cpPDC was specifically affected by binding of psbA mRNA. Beyond that, in silico analysis and in vitro RNA binding studies using recombinant proteins support the possibility that RNA binding is an ancient feature of dihydrolipoamide acetyltransferases. Our results suggest a regulatory function of DLA2 in response to growth on reduced carbon energy sources. This raises the intriguing possibility that this regulation functions to coordinate the synthesis of lipids and proteins for the biogenesis of photosynthetic membranes.

  7. Hepatitis B Virus Core Protein Phosphorylation Sites Affect Capsid Stability and Transient Exposure of the C-terminal Domain.

    Science.gov (United States)

    Selzer, Lisa; Kant, Ravi; Wang, Joseph C-Y; Bothner, Brian; Zlotnick, Adam

    2015-11-20

    Hepatitis B virus core protein has 183 amino acids divided into an assembly domain and an arginine-rich C-terminal domain (CTD) that regulates essential functions including genome packaging, reverse transcription, and intracellular trafficking. Here, we investigated the CTD in empty hepatitis B virus (HBV) T=4 capsids. We examined wild-type core protein (Cp183-WT) and a mutant core protein (Cp183-EEE), in which three CTD serines are replaced with glutamate to mimic phosphorylated protein. We found that Cp183-WT capsids were less stable than Cp183-EEE capsids. When we tested CTD sensitivity to trypsin, we detected two different populations of CTDs differentiated by their rate of trypsin cleavage. Interestingly, CTDs from Cp183-EEE capsids exhibited a much slower rate of proteolytic cleavage when compared with CTDs of Cp183-WT capsids. Cryo-electron microscopy studies of trypsin-digested capsids show that CTDs at five-fold symmetry vertices are most protected. We hypothesize that electrostatic interactions between glutamates and arginines in Cp183-EEE, particularly at five-fold, increase capsid stability and reduce CTD exposure. Our studies show that quasi-equivalent CTDs exhibit different rates of exposure and thus might perform distinct functions during the hepatitis B virus lifecycle. Our results demonstrate a structural role for CTD phosphorylation and indicate crosstalk between CTDs within a capsid particle. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. An alternative outer membrane secretion mechanism for an autotransporter protein lacking a C-terminal stable core.

    Science.gov (United States)

    Besingi, Richard N; Chaney, Julie L; Clark, Patricia L

    2013-12-01

    Autotransporter (AT) proteins are a broad class of virulence factors from Gram-negative pathogens. AT outer membrane (OM) secretion appears simple in many regards, yet the mechanism that enables transport of the central AT 'passenger' across the OM remains unclear. OM secretion efficiency for two AT passengers is enhanced by approximately 20 kDa stable core at the C-terminus of the passenger, but studies on a broader range of AT proteins are needed in order to determine whether a stability difference between the passenger N- and C-terminus represents a truly common mechanistic feature. Yersinia pestis YapV is homologous to Shigella flexneri IcsA, and like IcsA, YapV recruits mammalian neural Wiskott-Aldrich syndrome protein (N-WASP). In vitro, the purified YapV passenger is functional and rich in β-sheet structure, but lacks a approximately 20 kDa C-terminal stable core. However, the N-terminal 49 residues of the YapV passenger globally destabilize the entire YapV passenger, enhancing its OM secretion efficiency. These results indicate that the contributions of AT passenger sequences to OM secretion efficiency extend beyond a C-terminal stable core, and highlight a role of the passenger N-terminus in reducing passenger stability in order to facilitate OM secretion of some AT proteins. © 2013 John Wiley & Sons Ltd.

  9. Decorin Core Protein (Decoron) Shape Complements Collagen Fibril Surface Structure and Mediates Its Binding

    Energy Technology Data Exchange (ETDEWEB)

    Orgel, Joseph P.R.O.; Eid, Aya; Antipova, Olga; Bella, Jordi; Scott, John E.; (IIT); (Manchester)

    2010-02-11

    Decorin is the archetypal small leucine rich repeat proteoglycan of the vertebrate extracellular matrix (ECM). With its glycosaminoglycuronan chain, it is responsible for stabilizing inter-fibrillar organization. Type I collagen is the predominant member of the fibrillar collagen family, fulfilling both organizational and structural roles in animal ECMs. In this study, interactions between decoron (the decorin core protein) and binding sites in the d and e1 bands of the type I collagen fibril were investigated through molecular modeling of their respective X-ray diffraction structures. Previously, it was proposed that a model-based, highly curved concave decoron interacts with a single collagen molecule, which would form extensive van der Waals contacts and give rise to strong non-specific binding. However, the large well-ordered aggregate that is the collagen fibril places significant restraints on modes of ligand binding and necessitates multi-collagen molecular contacts. We present here a relatively high-resolution model of the decoron-fibril collagen complex. We find that the respective crystal structures complement each other well, although it is the monomeric form of decoron that shows the most appropriate shape complementarity with the fibril surface and favorable calculated energies of interaction. One molecule of decoron interacts with four to six collagen molecules, and the binding specificity relies on a large number of hydrogen bonds and electrostatic interactions, primarily with the collagen motifs KXGDRGE and AKGDRGE (d and e{sub 1} bands). This work helps us to understand collagen-decorin interactions and the molecular architecture of the fibrillar ECM in health and disease.

  10. Metabolic adaptation of Escherichia coli during temperature-induced recombinant protein production: 1. Readjustment of metabolic enzyme synthesis.

    Science.gov (United States)

    Hoffmann, Frank; Weber, Jan; Rinas, Ursula

    2002-11-05

    The metabolic burden and the stress load resulting from temperature-induced production of human basic fibroblast growth factor is connected to an increase in the respiratory activity of recombinant Escherichia coli, thereby reducing the biomass yield. To study the underlying changes in metabolic enzyme synthesis rates, the radiolabeled proteom was subjected to two-dimen- sional gel electrophoresis. After temperature-induction, the cAMP-CRP controlled dehydrogenases of the pyruvate dehydrogenase complex and the tricarboxylic acid cycle (LpdA and SdhA) were induced four times, reaching a maximum 1 h after the temperature upshift. The more abundant tricarboxylic acid cycle dehydrogenases (Icd and Mdh) were initially produced at reduced rates but regained preshift rates within 30 min. The adenylate energy charge dropped immediately after the temperature upshift but recovered within 1 h. Similar profiles in dehydrogenase synthesis rates and adenylate energy charge were found in a control cultivation of a strain carrying the "empty" parental expression vector. Although both strains exhibited significant differences in growth pattern and respiration rates after the temperature upshift, the adaptation of the energetic state of the cells and the synthesis of enzymes from the energy-generating catabolic pathway did not seem to be affected by the strong overproduction of the recombinant growth factor. In contrast, the synthesis rates of enzymes belonging to the biosynthetic machinery, e.g., translational elongation factors, decreased more strongly in the culture synthesizing the recombinant protein. In control and producing culture, synthesis rates of elongation factors paralleled the respective growth rate profiles. Thus, cells seem to readjust their metabolic activities according to their energetic requirements and, if necessary, at the cost of their biosynthetic capabilities. Copyright 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 313-319, 2002.

  11. Analysis of hepatitis C virus core/NS5A protein co-localization using novel cell culture systems expressing core-NS2 and NS5A of genotypes 1-7

    DEFF Research Database (Denmark)

    Galli, Andrea; Scheel, Troels K H; Prentoe, Jannick C

    2013-01-01

    Hepatitis C virus (HCV) is an important human pathogen infecting hepatocytes. With the advent of infectious cell culture systems, the HCV particle assembly and release processes are finally being uncovered. The HCV core and NS5A proteins co-localize on cytoplasmic lipid droplets (cLDs) or on the ......Hepatitis C virus (HCV) is an important human pathogen infecting hepatocytes. With the advent of infectious cell culture systems, the HCV particle assembly and release processes are finally being uncovered. The HCV core and NS5A proteins co-localize on cytoplasmic lipid droplets (c...... JFH1-based recombinants expressing core-NS2 and NS5A from genotypes 1-7, and analysed core and NS5A co-localization in infected cells. Huh7.5 cells were transfected with RNA of core-NS2/NS5A recombinants and putative adaptive mutations were analysed by reverse genetics. Adapted core-NS2/NS5A...... recombinants produced infectivity titres of 10(2.5)-10(4.5) f.f.u. ml(-1). Co-localization analysis demonstrated that the core and NS5A proteins from all genotypes co-localized extensively, and there was no significant difference in protein co-localization among genotypes. In addition, we found that the core...

  12. Comparative Proteome Analysis in Schizosaccharomyces pombe Identifies Metabolic Targets to Improve Protein Production and Secretion.

    Science.gov (United States)

    Hung, Chien-Wen; Klein, Tobias; Cassidy, Liam; Linke, Dennis; Lange, Sabrina; Anders, Uwe; Bureik, Matthias; Heinzle, Elmar; Schneider, Konstantin; Tholey, Andreas

    2016-10-01

    Protein secretion in yeast is a complex process and its efficiency depends on a variety of parameters. We performed a comparative proteome analysis of a set of Schizosaccharomyces pombe strains producing the α-glucosidase maltase in increasing amounts to investigate the overall proteomic response of the cell to the burden of protein production along the various steps of protein production and secretion. Proteome analysis of these strains, utilizing an isobaric labeling/two dimensional LC-MALDI MS approach, revealed complex changes, from chaperones and secretory transport machinery to proteins controlling transcription and translation. We also found an unexpectedly high amount of changes in enzyme levels of the central carbon metabolism and a significant up-regulation of several amino acid biosyntheses. These amino acids were partially underrepresented in the cellular protein compared with the composition of the model protein. Additional feeding of these amino acids resulted in a 1.5-fold increase in protein secretion. Membrane fluidity was identified as a second bottleneck for high-level protein secretion and addition of fluconazole to the culture caused a significant decrease in ergosterol levels, whereas protein secretion could be further increased by a factor of 2.1. In summary, we show that high level protein secretion causes global changes of protein expression levels in the cell and that precursor availability and membrane composition limit protein secretion in this yeast. In this respect, comparative proteome analysis is a powerful tool to identify targets for an efficient increase of protein production and secretion in S. pombe Data are available via ProteomeXchange with identifiers PXD002693 and PXD003016. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. In vitro influence of dietary protein and fructooligosaccharides on metabolism of canine fecal microbiota.

    Science.gov (United States)

    Pinna, Carlo; Vecchiato, Carla Giuditta; Zaghini, Giuliano; Grandi, Monica; Nannoni, Eleonora; Stefanelli, Claudio; Biagi, Giacomo

    2016-03-12

    The present in vitro study investigated whether the utilization of fructooligosaccharides (FOS) may influence canine fecal microbial population in presence of diets differing in their protein content and digestibility. Fresh fecal samples were collected from five adult dogs, pooled, and incubated for 24 h with the undigested residue of three diets: 1, Low protein high digestibility diet (LP HD, crude protein (CP) 229 g/kg); 2, High protein high digestibility diet (HP HD, CP 304 g/kg); 3, High protein low digestibility diet (HP LD, CP 303 g/kg) that had been previously subjected to enzymatic digestion. In the in vitro fermentation study, there were six treatments: 1) LP HD; 2) HP HD 3) HP LD; 4) LP HD + FOS; 5) HP HD + FOS; 6) HP LD + FOS. Fructooligosaccharides were added at the final concentration of 1.5 g/L. Samples of fermentation fluid were collected at 6 and 24 h of incubation. Values of pH were reduced by FOS at 6 and 24 h (P protein digestibility and high dietary protein level resulted in higher pH at both sampling times (P protein digestibility resulted (P protein digestibility (+21 and 22%, respectively; P dietary protein level resulted in lower counts of lactobacilli and enterococci (-0.5 and -0.7 log cells/mL, respectively; P protein digestibility tended to increase counts of C. perfringens (+0.2 log cells/mL; P = 0.07). Results from the present study showed that diets rich in protein may exert negative influences on the canine intestinal ecosystem, slightly increasing the presence of ammonia and reducing counts of lactobacilli and enterococci. Moreover, the presence of poorly digestible protein resulted in lower concentrations of VFA. Conversely, administration of FOS may improve metabolism of canine intestinal microbiota, reducing ammonia concentrations and enhancing VFA production.

  14. The Copper Metabolism MURR1 Domain Protein 1 (COMMD1) Modulates the Aggregation of Misfolded Protein Species in a Client-Specific Manner

    NARCIS (Netherlands)

    Vonk, Willianne I. M.; Kakkar, Vaishali; Bartuzi, Paulina; Jaarsma, Dick; Berger, Ruud; Hofker, Marten H.; Klomp, Leo W. J.; Wijmenga, Cisca; Kampinga, Harm H.; van de Sluis, Bart

    2014-01-01

    The Copper Metabolism MURR1 domain protein 1 (COMMD1) is a protein involved in multiple cellular pathways, including copper homeostasis, NF-kappa B and hypoxia signalling. Acting as a scaffold protein, COMMD1 mediates the levels, stability and proteolysis of its substrates (e.g. the

  15. The Copper Metabolism MURR1 Domain protein 1 (COMMD1) modulates the aggregation of misfolded protein species in a client-specific manner

    NARCIS (Netherlands)

    W.I.M. Vonk (Willianne I.); V. Kakkar (Vaishali); P. Bartuzi (Paulina); D. Jaarsma (Dick); R. Berger (Ruud); M.A. Hofker (Marten); L.W.J. Klomp (Leo W.); C. Wijmenga (Cisca); H. Kampinga (Harm); B. van de Sluis (Bart)

    2014-01-01

    textabstractThe Copper Metabolism MURR1 domain protein 1 (COMMD1) is a protein involved in multiple cellular pathways, including copper homeostasis, NF-κB and hypoxia signalling. Acting as a scaffold protein, COMMD1 mediates the levels, stability and proteolysis of its substrates (e.g. the

  16. The Effect of Casein Protein Prior to Sleep on Fat Metabolism in Obese Men

    Directory of Open Access Journals (Sweden)

    Amber W. Kinsey

    2016-07-01

    Full Text Available We have previously shown that ingesting protein at night before sleep is either beneficial or non-detrimental to metabolism, health, and body composition in obese women. However, the overnight protein-induced lipolytic actions and mechanism for improved metabolism and body composition have not been fully established. Therefore, in a crossover design, twelve obese men (age, 27.0 ± 2.2 years were randomly assigned to ingest (within 30 min of sleep casein protein (CAS, 120 kcal or a non-nutritive placebo (PLA before going to sleep. Markers of fat metabolism (lipolysis, substrate utilization, growth hormone, insulin, glucose, resting energy expenditure (REE, and appetite (questionnaire and ghrelin were measured. During sleep and the next morning, interstitial glycerol from the subcutaneous abdominal adipose tissue (SCAAT was measured using microdialysis. There were no differences in SCAAT glycerol (overnight: CAS, 177.4 ± 26.7; PLA, 183.8 ± 20.2 μmol/L; morning: CAS, 171.6 ± 19.1; PLA, 161.5 ± 18.6 μmol/L, substrate utilization, REE, or any blood markers between CAS and PLA. Desire to eat was greater for CAS compared to baseline (p = 0.03, but not different from PLA (baseline: 39 ± 6, CAS: 62 ± 8, PLA: 55 ± 5 mm. CAS consumption before sleep did not affect fat or glucose metabolism, REE, or suppress appetite in hyperinsulemic obese men. CAS may be consumed before sleep without impeding overnight or morning fat metabolism in young, obese men.

  17. Effect of protein provision via milk replacer or solid feed on protein metabolism in veal calves

    DEFF Research Database (Denmark)

    Berends, H.; van den Borne, J. J G C; Røjen, B. A.

    2015-01-01

    The current study evaluated the effects of protein provision to calves fed a combination of solid feed (SF) and milk replacer (MR) at equal total N intake on urea recycling and N retention. Nitrogen balance traits and [15N2]urea kinetics were measured in 30 calves (23 wk of age, 180±3.7kg of body......, increasing low-N SF intake at the expense of N intake from MR, did not affect protein retention efficiency in calves. Increasing the N content of SF at equal total N intake decreased urea production, increased protein retention, and coincided with improved fiber degradation. Therefore, results suggest...

  18. Chronic Ethanol Consumption Disrupts the Core Molecular Clock and Diurnal Rhythms of Metabolic Genes in the Liver without Affecting the Suprachiasmatic Nucleus

    Science.gov (United States)

    Filiano, Ashley N.; Millender-Swain, Telisha; Johnson, Russell; Young, Martin E.; Gamble, Karen L.; Bailey, Shannon M.

    2013-01-01

    Chronic ethanol consumption disrupts several metabolic pathways including β-oxidation and lipid biosynthesis, facilitating the development of alcoholic fatty liver disease. Many of these same metabolic pathways are directly regulated by cell autonomous circadian clocks, and recent studies suggest that disruption of daily rhythms in metabolism contributes to multiple common cardiometabolic diseases (including non-alcoholic fatty liver disease). However, it is not known whether ethanol disrupts the core molecular clock in the liver, nor whether this, in turn, alters rhythms in lipid metabolism. Herein, we tested the hypothesis that chronic ethanol consumption disrupts the molecular circadian clock in the liver and potentially changes the diurnal expression patterns of lipid metabolism genes. Consistent with previous studies, male C57BL/6J mice fed an ethanol-containing diet exhibited higher levels of liver triglycerides compared to control mice, indicating hepatic steatosis. Further, the diurnal oscillations of core clock genes (Bmal1, Clock, Cry1, Cry2, Per1, and Per2) and clock-controlled genes (Dbp, Hlf, Nocturnin, Npas2, Rev-erbα, and Tef) were altered in livers from ethanol-fed mice. In contrast, ethanol had only minor effects on the expression of core clock genes in the suprachiasmatic nucleus (SCN). These results were confirmed in Per2Luciferase knock-in mice, in which ethanol induced a phase advance in PER2::LUC bioluminescence oscillations in liver, but not SCN. Further, there was greater variability in the phase of PER2::LUC oscillations in livers from ethanol-fed mice. Ethanol consumption also affected the diurnal oscillations of metabolic genes, including Adh1, Cpt1a, Cyp2e1, Pck1, Pdk4, Ppargc1a, Ppargc1b and Srebp1c, in the livers of C57BL/6J mice. In summary, chronic ethanol consumption alters the function of the circadian clock in liver. Importantly, these results suggest that chronic ethanol consumption, at levels sufficient to cause steatosis

  19. Conformational Changes in the Hepatitis B Virus Core Protein Are Consistent with a Role for Allostery in Virus Assembly

    Energy Technology Data Exchange (ETDEWEB)

    Packianathan, Charles; Katen, Sarah P.; Dann, III, Charles E.; Zlotnick, Adam (Indiana)

    2010-01-12

    In infected cells, virus components must be organized at the right place and time to ensure assembly of infectious virions. From a different perspective, assembly must be prevented until all components are available. Hypothetically, this can be achieved by allosterically controlling assembly. Consistent with this hypothesis, here we show that the structure of the hepatitis B virus (HBV) core protein dimer, which can spontaneously self-assemble, is incompatible with capsid assembly. Systematic differences between core protein dimer and capsid conformations demonstrate linkage between the intradimer interface and interdimer contact surface. These structures also provide explanations for the capsid-dimer selectivity of some antibodies and the activities of assembly effectors. Solution studies suggest that the assembly-inactive state is more accurately an ensemble of conformations. Simulations show that allostery supports controlled assembly and results in capsids that are resistant to dissociation. We propose that allostery, as demonstrated in HBV, is common to most self-assembling viruses.

  20. Integrated multilaboratory systems biology reveals differences in protein metabolism between two reference yeast strains

    DEFF Research Database (Denmark)

    Canelas, Andre B.; Harrison, Nicola; Fazio, Alessandro

    2010-01-01

    , under two standard growth conditions. We ensured the high quality of the experimental data by evaluating a wide range of sampling and analytical techniques. Here we show significant differences in the maximum specific growth rate and biomass yield between the two strains. On the basis of the integrated...... analysis of the high-throughput data, we hypothesize that differences in phenotype are due to differences in protein metabolism....

  1. Heme metabolism in stress regulation and protein production: From Cinderella to a key player.

    Science.gov (United States)

    Martínez, J L; Petranovic, D; Nielsen, J

    2016-04-02

    Heme biosynthesis is a highly conserved pathway which is present in all kingdoms, from Archaea to higher organisms such as plants and mammals. The heme molecule acts as a prosthetic group for different proteins and enzymes involved in energy metabolism and reactions involved in electron transfer. Based on our recent findings and other recent reports, we here illustrate that heme is more than a co-factor. We also discuss the necessity to gain more insight into the heme biosynthesis pathway regulation, as this interacts closely with overall stress control. Understanding heme biosynthesis and its regulation could impact our ability to develop more efficient yeast cell factories for heterologous protein production.

  2. The Roles of Vitamin A in the Regulation of Carbohydrate, Lipid, and Protein Metabolism

    Directory of Open Access Journals (Sweden)

    Wei Chen

    2014-05-01

    Full Text Available Currently, two-thirds of American adults are overweight or obese. This high prevalence of overweight/obesity negatively affects the health of the population, as obese individuals tend to develop several chronic diseases, such as type 2 diabetes and cardiovascular diseases. Due to obesity’s impact on health, medical costs, and longevity, the rise in the number of obese people has become a public health concern. Both genetic and environmental/dietary factors play a role in the development of metabolic diseases. Intuitively, it seems to be obvious to link over-nutrition to the development of obesity and other metabolic diseases. However, the underlying mechanisms are still unclear. Dietary nutrients not only provide energy derived from macronutrients, but also factors such as micronutrients with regulatory roles. How micronutrients, such as vitamin A (VA; retinol, regulate macronutrient homeostasis is still an ongoing research topic. As an essential micronutrient, VA plays a key role in the general health of an individual. This review summarizes recent research progress regarding VA’s role in carbohydrate, lipid, and protein metabolism. Due to the large amount of information regarding VA functions, this review focusses on metabolism in metabolic active organs and tissues. Additionally, some perspectives for future studies will be provided.

  3. The Role of Carbohydrate Response Element Binding Protein in Intestinal and Hepatic Fructose Metabolism

    Directory of Open Access Journals (Sweden)

    Katsumi Iizuka

    2017-02-01

    Full Text Available Many articles have discussed the relationship between fructose consumption and the incidence of obesity and related diseases. Fructose is absorbed in the intestine and metabolized in the liver to glucose, lactate, glycogen, and, to a lesser extent, lipids. Unabsorbed fructose causes bacterial fermentation, resulting in irritable bowl syndrome. Therefore, understanding the mechanisms underlying intestinal and hepatic fructose metabolism is important for the treatment of metabolic syndrome and fructose malabsorption. Carbohydrate response element binding protein (ChREBP is a glucose-activated transcription factor that controls approximately 50% of de novo lipogenesis in the liver. ChREBP target genes are involved in glycolysis (Glut2, liver pyruvate kinase, fructolysis (Glut5, ketohexokinase, and lipogenesis (acetyl CoA carboxylase, fatty acid synthase. ChREBP gene deletion protects against high sucrose diet-induced and leptin-deficient obesity, because Chrebp−/− mice cannot consume fructose or sucrose. Moreover, ChREBP contributes to some of the physiological effects of fructose on sweet taste preference and glucose production through regulation of ChREBP target genes, such as fibroblast growth factor-21 and glucose-6-phosphatase catalytic subunits. Thus, ChREBP might play roles in fructose metabolism. Restriction of excess fructose intake will be beneficial for preventing not only metabolic syndrome but also irritable bowl syndrome.

  4. The Role of Carbohydrate Response Element Binding Protein in Intestinal and Hepatic Fructose Metabolism.

    Science.gov (United States)

    Iizuka, Katsumi

    2017-02-22

    Many articles have discussed the relationship between fructose consumption and the incidence of obesity and related diseases. Fructose is absorbed in the intestine and metabolized in the liver to glucose, lactate, glycogen, and, to a lesser extent, lipids. Unabsorbed fructose causes bacterial fermentation, resulting in irritable bowl syndrome. Therefore, understanding the mechanisms underlying intestinal and hepatic fructose metabolism is important for the treatment of metabolic syndrome and fructose malabsorption. Carbohydrate response element binding protein (ChREBP) is a glucose-activated transcription factor that controls approximately 50% of de novo lipogenesis in the liver. ChREBP target genes are involved in glycolysis (Glut2, liver pyruvate kinase), fructolysis (Glut5, ketohexokinase), and lipogenesis (acetyl CoA carboxylase, fatty acid synthase). ChREBP gene deletion protects against high sucrose diet-induced and leptin-deficient obesity, because Chrebp(-/-) mice cannot consume fructose or sucrose. Moreover, ChREBP contributes to some of the physiological effects of fructose on sweet taste preference and glucose production through regulation of ChREBP target genes, such as fibroblast growth factor-21 and glucose-6-phosphatase catalytic subunits. Thus, ChREBP might play roles in fructose metabolism. Restriction of excess fructose intake will be beneficial for preventing not only metabolic syndrome but also irritable bowl syndrome.

  5. Predictive association of copper metabolism proteins with Alzheimer's disease and Parkinson's disease: a preliminary perspective.

    Science.gov (United States)

    Pal, Amit; Kumar, Ashok; Prasad, Rajendra

    2014-02-01

    Neurodegenerative diseases, Alzheimer's disease (AD) and Parkinson's disease (PD), constitute a major worldwide health problem. Several hypothesis have been put forth to elucidate the basis of onset and pathogenesis of AD and PD; however, till date, none of these seems to clearly elucidate the complex pathoetiology of these disorders. Notably, copper dyshomeostasis has been shown to underlie the pathophysiology of several neurodegenerative diseases including AD and PD. Numerous studies have concluded beyond doubt that imbalance in copper homeostatic mechanisms in conjunction with aging causes an acceleration in the copper toxicity elicited oxidative stress, which is detrimental to the central nervous system. Amyloid precursor protein and α-synuclein protein involved in AD and PD are copper binding proteins, respectively. In this review, we have discussed the possible association of copper metabolism proteins with AD and PD along with briefly outlining the expanding proportion of "copper interactome" in human biology. Using network biology, we found that copper metabolism proteins, superoxide dismutase 1 and ceruloplasmin may represent direct and indirect link with AD and PD, respectively.

  6. Protein acetylation affects acetate metabolism, motility and acid stress response in Escherichia coli.

    Science.gov (United States)

    Castaño-Cerezo, Sara; Bernal, Vicente; Post, Harm; Fuhrer, Tobias; Cappadona, Salvatore; Sánchez-Díaz, Nerea C; Sauer, Uwe; Heck, Albert J R; Altelaar, A F Maarten; Cánovas, Manuel

    2014-11-27

    Although protein acetylation is widely observed, it has been associated with few specific regulatory functions making it poorly understood. To interrogate its functionality, we analyzed the acetylome in Escherichia coli knockout mutants of cobB, the only known sirtuin-like deacetylase, and patZ, the best-known protein acetyltransferase. For four growth conditions, more than 2,000 unique acetylated peptides, belonging to 809 proteins, were identified and differentially quantified. Nearly 65% of these proteins are related to metabolism. The global activity of CobB contributes to the deacetylation of a large number of substrates and has a major impact on physiology. Apart from the regulation of acetyl-CoA synthetase, we found that CobB-controlled acetylation of isocitrate lyase contributes to the fine-tuning of the glyoxylate shunt. Acetylation of the transcription factor RcsB prevents DNA binding, activating flagella biosynthesis and motility, and increases acid stress susceptibility. Surprisingly, deletion of patZ increased acetylation in acetate cultures, which suggests that it regulates the levels of acetylating agents. The results presented offer new insights into functional roles of protein acetylation in metabolic fitness and global cell regulation. © 2014 The Authors. Published under the terms of the CC BY 4.0 license.

  7. Association among Fibrinolytic Proteins, Metabolic Syndrome Components, Insulin Secretion, and Resistance in Schoolchildren

    Directory of Open Access Journals (Sweden)

    Jin-Shuen Chen

    2015-01-01

    Full Text Available We investigated the role of urokinase plasminogen activator (uPA and its soluble receptors (suPAR and plasminogen activator inhibitor-1 (PAI-1 in metabolic syndrome (MetS components, insulin secretion, and resistance in schoolchildren. We enrolled 387 children, aged 10.3 ± 1.5 years, in Taipei. Anthropometry, fibrinolytic proteins, MetS components, insulin secretion, and resistance were measured. Subjects were divided into normal, overweight, and obese groups. Finally, the relationship between fibrinolytic proteins and metabolic syndrome in boys and girls was analyzed. In boys, PAI-1 was positively associated with body mass index (BMI percentile, hypertriglyceride, insulin secretion, and resistance. In girls, PAI-1 was positively associated with obesity, hypertriglyceridemia, and insulin secretion. In girls, uPA was positively associated with insulin secretion. suPAR was positively associated with high-sensitivity C-reactive protein in both boys and girls, and with BMI percentile and body fat in girls. The obese boys had higher suPAR and PAI-1 levels than the normal group. The obese girls had higher uPA, suPAR, and PAI-1 than the normal group. Boys and girls with MetS had higher PAI-1. Fibrinolytic proteins, especially PAI-1, are associated with MetS components and insulin secretion in children. Fibrinolytic proteins changes were more likely to occur in girls than in boys.

  8. Mitochondrial Genome Encoded Proteins Expression Disorder, the Possible Mechanism of the Heart Disease in Metabolic Syndrome

    Directory of Open Access Journals (Sweden)

    Ningning Zheng

    2017-09-01

    Full Text Available Background/Aims: The direct consequence of metabolic syndrome (MS is the increased morbidity and mortality caused by the heart disease. We tried to explain why the heart is more severely damaged during MS from the point of mitochondria, the center of cellular metabolism. Methods: 1. The classic diet induced MS rat model was used to observe the morphological changes of mitochondria by transmission electron microscope (TEM; 2. The expression of mitochondrial DNA (mt-DNA encoded proteins was observed by immunohistochemistry and Western blot; 3. The expression of mitochondrial ribosomal proteins (MRPs was observed by real-time PCR. Results: 1. The mitochondrial volume increased but the number was normal in myocardial cells of the MS rats. But in the hepatocytes and skeletal muscle cells, the mitochondrial number decreased; 2.The mt-DNA encoded protein cytochrome b increased significantly in heart but decreased in liver and the ATPase6 increased in liver but decreased in heart of the MS rats; 3. The mRNA levels of MRPS23, MRPL27, MRPL45 and MRPL48 elevated in heart but down-regulated in liver of the MS rats. Conclusion: The morphologic and functional alterations of mitochondrion in MS were tissue specific. Heart displays a distinctive pattern of mitochondrial metabolic status compared with other tissues.

  9. An alternative outer membrane secretion mechanism for an autotransporter protein lacking a C-terminal stable core

    OpenAIRE

    Besingi, Richard N.; Chaney, Julie L.; Clark, Patricia L.

    2013-01-01

    Autotransporter (AT) proteins are a broad class of virulence factors from Gram-negative pathogens. AT outer membrane (OM) secretion appears simple in many regards, yet the mechanism that enables transport of the central AT “passenger” across the OM remains unclear. OM secretion efficiency for two AT passengers is enhanced by a ~20 kDa stable core at the C-terminus of the passenger, but studies on a broader range of AT proteins are needed in order to determine whether a stability difference be...

  10. Crystallization of the avian reovirus double-stranded RNA-binding and core protein σA

    Energy Technology Data Exchange (ETDEWEB)

    Hermo-Parrado, X. Lois; Guardado-Calvo, Pablo [Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain); Llamas-Saiz, Antonio L. [Unidad de Difracción de Rayos X, Laboratorio Integral de Dinámica y Estructura de Biomoléculas José R. Carracido, Edificio CACTUS, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain); Fox, Gavin C. [Spanish CRG Beamline BM16, European Synchrotron Radiation Facility (ESRF), 6 Rue Jules Horowitz, BP 220, F-38043 Grenoble (France); Vazquez-Iglesias, Lorena; Martínez-Costas, José; Benavente, Javier [Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain); Raaij, Mark J. van, E-mail: vanraaij@usc.es [Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain); Unidad de Difracción de Rayos X, Laboratorio Integral de Dinámica y Estructura de Biomoléculas José R. Carracido, Edificio CACTUS, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain)

    2007-05-01

    The avian reovirus double-stranded RNA-binding and core protein σA has been crystallized in space group P1, with unit-cell parameters a = 103.2, b = 129.9, c = 144.0 Å, α = 93.8, β = 105.1, γ = 98.2°. A complete data set has been collected to 2.3 Å resolution and analyzed. The avian reovirus protein σA plays a dual role: it is a structural protein forming part of the transcriptionally active core, but it has also been implicated in the resistance of the virus to interferon by strongly binding double-stranded RNA and thus inhibiting the double-stranded RNA-dependent protein kinase. The σA protein has been crystallized from solutions containing ammonium sulfate at pH values around 6. Crystals belonging to space group P1, with unit-cell parameters a = 103.2, b = 129.9, c = 144.0 Å, α = 93.8, β = 105.1, γ = 98.2° were grown and a complete data set has been collected to 2.3 Å resolution. The self-rotation function suggests that σA may form symmetric arrangements in the crystals.

  11. Exercise training and work task induced metabolic and stress-related mRNA and protein responses in myalgic muscles

    DEFF Research Database (Denmark)

    Sjøgaard, Gisela; Zebis, Mette K; Kiilerich, Kristian

    2013-01-01

    The aim was to assess mRNA and/or protein levels of heat shock proteins, cytokines, growth regulating, and metabolic proteins in myalgic muscle at rest and in response to work tasks and prolonged exercise training. A randomized controlled trial included 28 females with trapezius myalgia and 16 he...

  12. Effects of Dietary Protein Source and Quantity during Weight Loss on Appetite, Energy Expenditure, and Cardio-Metabolic Responses

    Directory of Open Access Journals (Sweden)

    Jia Li

    2016-01-01

    Full Text Available Higher protein meals increase satiety and the thermic effect of feeding (TEF in acute settings, but it is unclear whether these effects remain after a person becomes acclimated to energy restriction or a given protein intake. This study assessed the effects of predominant protein source (omnivorous, beef/pork vs. lacto-ovo vegetarian, soy/legume and quantity (10%, 20%, or 30% of energy from protein on appetite, energy expenditure, and cardio-metabolic indices during energy restriction (ER in overweight and obese adults. Subjects were randomly assigned to one protein source and then consumed diets with different quantities of protein (4 weeks each in a randomized crossover manner. Perceived appetite ratings (free-living and in-lab, TEF, and fasting cardio-metabolic indices were assessed at the end of each 4-week period. Protein source and quantity did not affect TEF, hunger, or desire to eat, other than a modestly higher daily composite fullness rating with 30% vs. 10% protein diet (p = 0.03. While the 20% and 30% protein diets reduced cholesterol, triacylglycerol, and APO-B vs. 10% protein (p < 0.05, protein source did not affect cardio-metabolic indices. In conclusion, diets varying in protein quantity with either beef/pork or soy/legume as the predominant source have minimal effects on appetite control, energy expenditure and cardio-metabolic risk factors during ER-induced weight loss.

  13. Systems biology definition of the core proteome of metabolism and expression is consistent with high-throughput data

    DEFF Research Database (Denmark)

    Yang, Laurence; Tan, Justin; O'Brien, Edward J.

    2015-01-01

    Finding the minimal set of gene functions needed to sustain life is of both fundamental and practical importance. Minimal gene lists have been proposed by using comparative genomics-based core proteome definitions. A definition of a core proteome that is supported by empirical data, is understood...... based on proteomics data. This systems biology core proteome includes 212 genes not found in previous comparative genomics-based core proteome definitions, accounts for 65% of known essential genes in E. coli, and has 78% gene function overlap with minimal genomes (Buchnera aphidicola and Mycoplasma...... genitalium). Based on transcriptomics data across environmental and genetic backgrounds, the systems biology core proteome is significantly enriched in nondifferentially expressed genes and depleted in differentially expressed genes. Compared with the noncore, core gene expression levels are also similar...

  14. Mesitylene-Cored Glucoside Amphiphiles (MGAs) for Membrane Protein Studies: Importance of Alkyl Chain Density in Detergent Efficacy

    Science.gov (United States)

    Cho, Kyung Ho; Ribeiro, Orquidea; Du, Yang; Tikhonova, Elena; Mortensen, Jonas S.; Markham, Kelsey; Hariharan, Parameswaran; Loland, Claus J.; Guan, Lan; Kobilka, Brian K.; Byrne, Bernadette

    2017-01-01

    Detergents serve as useful tools for membrane protein structural and functional studies. Their amphipathic nature allows detergents to associate with the hydrophobic regions of membrane proteins whilst maintaining the proteins in aqueous solution. However, widely used conventional detergents are limited in their ability to maintain the structural integrity of membrane proteins and thus there are major efforts underway to develop novel agents with improved properties. We prepared mesitylene-cored glucoside amphiphiles (MGAs) with three alkyl chains and compared these agents with previously developed xylene-linked maltoside agents (XMAs) with two alkyl chains and a conventional detergent (DDM). When these agents were evaluated for four membrane proteins including a G protein-coupled receptor (GPCR), some agents such as MGA-C13 and MGA-C14 resulted in markedly enhanced stability of membrane proteins compared to both DDM and the XMAs. This favourable behaviour is due likely to the increased hydrophobic density provided by the extra alkyl chain. Thus, this study not only describes new glucoside agents with potential for membrane protein research, but also introduces a new detergent design principle for future development. PMID:27743406

  15. Single molecule force spectroscopy reveals critical roles of hydrophobic core packing in determining the mechanical stability of protein GB1.

    Science.gov (United States)

    Bu, Tianjia; Wang, Hui-Chuan Eileen; Li, Hongbin

    2012-08-21

    Understanding molecular determinants of protein mechanical stability is important not only for elucidating how elastomeric proteins are designed and functioning in biological systems but also for designing protein building blocks with defined nanomechanical properties for constructing novel biomaterials. GB1 is a small α/β protein and exhibits significant mechanical stability. It is thought that the shear topology of GB1 plays an important role in determining its mechanical stability. Here, we combine single molecule atomic force microscopy and protein engineering techniques to investigate the effect of side chain reduction and hydrophobic core packing on the mechanical stability of GB1. We engineered seven point mutants and carried out mechanical φ-value analysis of the mechanical unfolding of GB1. We found that three mutations, which are across the surfaces of two subdomains that are to be sheared by the applied stretching force, in the hydrophobic core (F30L, Y45L, and F52L) result in significant decrease in mechanical unfolding force of GB1. The mechanical unfolding force of these mutants drop by 50-90 pN compared with wild-type GB1, which unfolds at around 180 pN at a pulling speed of 400 nm/s. These results indicate that hydrophobic core packing plays an important role in determining the mechanical stability of GB1 and suggest that optimizing hydrophobic interactions across the surfaces that are to be sheared will likely be an efficient method to enhance the mechanical stability of GB1 and GB1 homologues.

  16. Effect of protein provision via milk replacer or solid feed on protein metabolism in veal calves

    NARCIS (Netherlands)

    Berends, H.; Borne, van den J.J.G.C.; Røjen, B.A.; Hendriks, W.H.; Gerrits, W.J.J.

    2015-01-01

    The current study evaluated the effects of protein provision to calves fed a combination of solid feed (SF) and milk replacer (MR) at equal total N intake on urea recycling and N retention. Nitrogen balance traits and [15N2]urea kinetics were measured in 30 calves (23 wk of age, 180 ± 3.7 kg of body

  17. The Effect of Oral Leucine on Protein Metabolism in Adolescents with Type 1 Diabetes Mellitus

    Directory of Open Access Journals (Sweden)

    Wilson ThomasA

    2010-11-01

    Full Text Available Lack of insulin results in a catabolic state in subjects with insulin-dependent diabetes mellitus which is reversed by insulin treatment. Amino acid supply, especially branched chain amino acids such as leucine, enhances protein synthesis in both animal and human studies. This small study was undertaken to assess the acute effect of supplemental leucine on protein metabolism in adolescents with type 1 diabetes. L-[1-13C] Leucine was used to assess whole-body protein metabolism in six adolescent females (16–18 yrs with type 1 diabetes during consumption of a basal diet (containing 58 μmoles leucine/kg/h and the basal diet with supplemental leucine (232 μmoles leucine/kg/h. Net leucine balance was significantly higher with supplemental leucine ( μmoles leucine/kg body weight/hr than with the basal diet (, due to reduced protein degradation ( μmoles leucine/kg body weight/hr compared to the basal diet (, .

  18. From endosymbiont to host-controlled organelle: the hijacking of mitochondrial protein synthesis and metabolism.

    Directory of Open Access Journals (Sweden)

    Toni Gabaldón

    2007-11-01

    Full Text Available Mitochondria are eukaryotic organelles that originated from the endosymbiosis of an alpha-proteobacterium. To gain insight into the evolution of the mitochondrial proteome as it proceeded through the transition from a free-living cell to a specialized organelle, we compared a reconstructed ancestral proteome of the mitochondrion with the proteomes of alpha-proteobacteria as well as with the mitochondrial proteomes in yeast and man. Overall, there has been a large turnover of the mitochondrial proteome during the evolution of mitochondria. Early in the evolution of the mitochondrion, proteins involved in cell envelope synthesis have virtually disappeared, whereas proteins involved in replication, transcription, cell division, transport, regulation, and signal transduction have been replaced by eukaryotic proteins. More than half of what remains from the mitochondrial ancestor in modern mitochondria corresponds to translation, including post-translational modifications, and to metabolic pathways that are directly, or indirectly, involved in energy conversion. Altogether, the results indicate that the eukaryotic host has hijacked the proto-mitochondrion, taking control of its protein synthesis and metabolism.

  19. Effects of supplemental protein provided to postpartum beef cows in liquid or cube form on metabolic, endocrine and reproductive functions

    National Research Council Canada - National Science Library

    Kane, K.K; Schafer, T.P; Harris, J.M; Hallford, D.M; Remmenga, M.D; Hawkins, D.E

    2001-01-01

    Metabolic, endocrine and reproductive functions in postpartum beef cows, supplemented with protein supplied as range cubes or liquid, were evaluated. Angus cows (n = 60; 2 to 8 yr; mean BCS 4.6 [+ or -] 0.1...

  20. Perplexing cooperative folding and stability of a low-sequence complexity, polyproline 2 protein lacking a hydrophobic core.

    Science.gov (United States)

    Gates, Zachary P; Baxa, Michael C; Yu, Wookyung; Riback, Joshua A; Li, Hui; Roux, Benoît; Kent, Stephen B H; Sosnick, Tobin R

    2017-02-28

    The burial of hydrophobic side chains in a protein core generally is thought to be the major ingredient for stable, cooperative folding. Here, we show that, for the snow flea antifreeze protein (sfAFP), stability and cooperativity can occur without a hydrophobic core, and without α-helices or β-sheets. sfAFP has low sequence complexity with 46% glycine and an interior filled only with backbone H-bonds between six polyproline 2 (PP2) helices. However, the protein folds in a kinetically two-state manner and is moderately stable at room temperature. We believe that a major part of the stability arises from the unusual match between residue-level PP2 dihedral angle bias in the unfolded state and PP2 helical structure in the native state. Additional stabilizing factors that compensate for the dearth of hydrophobic burial include shorter and stronger H-bonds, and increased entropy in the folded state. These results extend our understanding of the origins of cooperativity and stability in protein folding, including the balance between solvent and polypeptide chain entropies.

  1. Relationship of C-reactive protein with components of the metabolic syndrome in normal-weight and overweight elderly

    NARCIS (Netherlands)

    Hoekstra, T.; Geleijnse, J.M.; Schouten, E.G.; Kluft, C.; Kok, F.J.

    2005-01-01

    C-reactive protein (CRP) is known to be elevated in the metabolic syndrome. We aimed to explore in more detail the relationship between CRP and other components of the metabolic syndrome in a general population of 605 Dutch elderly individuals aged 65¿84 years. Methods and results Data were

  2. Whey Protein Supplementation Enhances Whole Body Protein Metabolism and Performance Recovery after Resistance Exercise: A Double-Blind Crossover Study

    Directory of Open Access Journals (Sweden)

    Daniel W. D. West

    2017-07-01

    Full Text Available No study has concurrently measured changes in free-living whole body protein metabolism and exercise performance during recovery from an acute bout of resistance exercise. We aimed to determine if whey protein ingestion enhances whole body net protein balance and recovery of exercise performance during overnight (10 h and 24 h recovery after whole body resistance exercise in trained men. In a double-blind crossover design, 12 trained men (76 ± 8 kg, 24 ± 4 years old, 14% ± 5% body fat; means ± standard deviation (SD performed resistance exercise in the evening prior to consuming either 25 g of whey protein (PRO; MuscleTech 100% Whey or an energy-matched placebo (CHO immediately post-exercise (0 h, and again the following morning (~10 h of recovery. A third randomized trial, completed by the same participants, involving no exercise and no supplement served as a rested control trial (Rest. Participants ingested [15N]glycine to determine whole body protein kinetics and net protein balance over 10 and 24 h of recovery. Performance was assessed pre-exercise and at 0, 10, and 24 h of recovery using a battery of tests. Net protein balance tended to improve in PRO (P = 0.064; effect size (ES = 0.61, PRO vs. CHO during overnight recovery. Over 24 h, net balance was enhanced in PRO (P = 0.036 but not in CHO (P = 0.84; ES = 0.69, PRO vs. CHO, which was mediated primarily by a reduction in protein breakdown (PRO < CHO; P < 0.01. Exercise decreased repetitions to failure (REP, maximal strength (MVC, peak and mean power, and countermovement jump performance (CMJ at 0 h (all P < 0.05 vs. Pre. At 10 h, there were small-to-moderate effects for enhanced recovery of the MVC (ES = 0.56, mean power (ES = 0.49, and CMJ variables (ES: 0.27–0.49 in PRO. At 24 h, protein supplementation improved MVC (ES = 0.76, REP (ES = 0.44, and peak power (ES = 0.55. In conclusion, whey protein supplementation enhances whole body anabolism, and may improve acute recovery of

  3. Localization of a brain protein metabolically linked with behavioral plasticity in the goldfish.

    Science.gov (United States)

    Benowitz, L I; Shashoua, V E

    1977-11-11

    Immunofluorescent methods were used to map the distribution of a soluble, 32,000 dalton brain protein, the synthesis of which increases when goldfish master a vestibulomotor task. The protein was found to be associated with a family of approximately 15,000 cells which vary in diameter from 8 to 15 micrometer and possess multiple processes. These cells are distributed near periventricular and external surfaces from the spinal cord up through the forebrain, but are most abundant in the broad ependymal zones of the optic tectum and vagal lobes. This localization pattern suggests that one of the major changes in brain protein metabolism associated with performance of the 'float-training' task in goldfish may reflect the activity of a non-neuronal population of brain cells.

  4. Metabolic adaptation to chronic inhibition of mitochondrial protein synthesis in acute myeloid leukemia cells.

    Directory of Open Access Journals (Sweden)

    Bozhena Jhas

    Full Text Available Recently, we demonstrated that the anti-bacterial agent tigecycline preferentially induces death in leukemia cells through the inhibition of mitochondrial protein synthesis. Here, we sought to understand mechanisms of resistance to tigecycline by establishing a leukemia cell line resistant to the drug. TEX leukemia cells were treated with increasing concentrations of tigecycline over 4 months and a population of cells resistant to tigecycline (RTEX+TIG was selected. Compared to wild type cells, RTEX+TIG cells had undetectable levels of mitochondrially translated proteins Cox-1 and Cox-2, reduced oxygen consumption and increased rates of glycolysis. Moreover, RTEX+TIG cells were more sensitive to inhibitors of glycolysis and more resistant to hypoxia. By electron microscopy, RTEX+TIG cells had abnormally swollen mitochondria with irregular cristae structures. RNA sequencing demonstrated a significant over-representation of genes with binding sites for the HIF1α:HIF1β transcription factor complex in their promoters. Upregulation of HIF1α mRNA and protein in RTEX+TIG cells was confirmed by Q-RTPCR and immunoblotting. Strikingly, upon removal of tigecycline from RTEX+TIG cells, the cells re-established aerobic metabolism. Levels of Cox-1 and Cox-2, oxygen consumption, glycolysis, mitochondrial mass and mitochondrial membrane potential returned to wild type levels, but HIF1α remained elevated. However, upon re-treatment with tigecycline for 72 hours, the glycolytic phenotype was re-established. Thus, we have generated cells with a reversible metabolic phenotype by chronic treatment with an inhibitor of mitochondrial protein synthesis. These cells will provide insight into cellular adaptations used to cope with metabolic stress.

  5. Elevated advanced oxidation protein products (AOPPs) indicate metabolic risk in severely obese children.

    Science.gov (United States)

    Codoñer-Franch, P; Tavárez-Alonso, S; Murria-Estal, R; Tortajada-Girbés, M; Simó-Jordá, R; Alonso-Iglesias, E

    2012-03-01

    The assessment of oxidative stress may aid in the identification of subsequent metabolic risk in obese children. The objective of this study was to determine whether the plasma level of advanced oxidation protein products, analyzed with a recently proposed modified assay that involves a delipidation step (mAOPPs), was related to metabolic risk factors (MRFs) in severely obese children. The plasma levels of mAOPPs were determined by spectrophotometry in 54 severely obese and 44 healthy children. We also measured lipid peroxidation biomarkers (thiobarbituric acid-reactive substances, malondialdehyde, and 8-isoprotane F(2α)) and sulfhydryl groups, a marker of antioxidant defense. Protein oxidation and lipid peroxidation markers were higher and sulfhydryl levels were lower in obese children compared with controls. Taking metabolic risk into account, obese children were subdivided according to the cutoff point (53.2 μmol/L) obtained for their mAOPPs values from the ROC curve. Anthropometric measures and the existence of hypertension did not differ between groups. The presence of dyslipidemia and insulin resistance was significantly higher in the group with higher mAOPPs levels. The highest levels of mAOPPs were found in the children with ≥3 MRFs. The level of mAOPPs was positively correlated with triglycerides and negatively correlated with high-density lipoprotein cholesterol. There was no correlation of this marker of protein oxidation with biomarkers of lipid peroxidation. The determination of mAOPPs in delipidated plasma is an easy way to evaluate protein oxidation. It may be useful in severely obese children for better cardiovascular risk assessment. Copyright © 2010 Elsevier B.V. All rights reserved.

  6. Historical and contemporary stable isotope tracer approaches to studying mammalian protein metabolism

    Science.gov (United States)

    2016-01-01

    Over a century ago, Frederick Soddy provided the first evidence for the existence of isotopes; elements that occupy the same position in the periodic table are essentially chemically identical but differ in mass due to a different number of neutrons within the atomic nucleus. Allied to the discovery of isotopes was the development of some of the first forms of mass spectrometers, driven forward by the Nobel laureates JJ Thomson and FW Aston, enabling the accurate separation, identification, and quantification of the relative abundance of these isotopes. As a result, within a few years, the number of known isotopes both stable and radioactive had greatly increased and there are now over 300 stable or radioisotopes presently known. Unknown at the time, however, was the potential utility of these isotopes within biological disciplines, it was soon discovered that these stable isotopes, particularly those of carbon (13C), nitrogen (15N), oxygen (18O), and hydrogen (2H) could be chemically introduced into organic compounds, such as fatty acids, amino acids, and sugars, and used to “trace” the metabolic fate of these compounds within biological systems. From this important breakthrough, the age of the isotope tracer was born. Over the following 80 yrs, stable isotopes would become a vital tool in not only the biological sciences, but also areas as diverse as forensics, geology, and art. This progress has been almost exclusively driven through the development of new and innovative mass spectrometry equipment from IRMS to GC‐MS to LC‐MS, which has allowed for the accurate quantitation of isotopic abundance within samples of complex matrices. This historical review details the development of stable isotope tracers as metabolic tools, with particular reference to their use in monitoring protein metabolism, highlighting the unique array of tools that are now available for the investigation of protein metabolism in vivo at a whole body down to a single protein level

  7. Arabidopsis ribosomal proteins control vacuole trafficking and developmental programs through the regulation of lipid metabolism.

    Science.gov (United States)

    Li, Ruixi; Sun, Ruobai; Hicks, Glenn R; Raikhel, Natasha V

    2015-01-06

    The vacuole is the most prominent compartment in plant cells and is important for ion and protein storage. In our effort to search for key regulators in the plant vacuole sorting pathway, ribosomal large subunit 4 (rpl4d) was identified as a translational mutant defective in both vacuole trafficking and normal development. Polysome profiling of the rpl4d mutant showed reduction in polysome-bound mRNA compared with wild-type, but no significant change in the general mRNA distribution pattern. Ribsomal profiling data indicated that genes in the lipid metabolism pathways were translationally down-regulated in the rpl4d mutant. Live imaging studies by Nile red staining suggested that both polar and nonpolar lipid accumulation was reduced in meristem tissues of rpl4d mutants. Pharmacological evidence showed that sterol and sphingolipid biosynthetic inhibitors can phenocopy the defects of the rpl4d mutant, including an altered vacuole trafficking pattern. Genetic evidence from lipid biosynthetic mutants indicates that alteration in the metabolism of either sterol or sphingolipid biosynthesis resulted in vacuole trafficking defects, similar to the rpl4d mutant. Tissue-specific complementation with key enzymes from lipid biosynthesis pathways can partially rescue both vacuole trafficking and auxin-related developmental defects in the rpl4d mutant. These results indicate that lipid metabolism modulates auxin-mediated tissue differentiation and endomembrane trafficking pathways downstream of ribosomal protein function.

  8. Hepatitis C virus core protein overcomes H2O2-induced apoptosis by downregulating p14 expression via DNA methylation.

    Science.gov (United States)

    Seo, Young Lan; Heo, Shinhee; Jang, Kyung Lib

    2015-04-01

    Infection with hepatitis C virus (HCV) is characterized by systemic oxidative stress that is caused by either viral core protein or chronic inflammation. It is well recognized that reactive oxygen species (ROS) such as H2O2 can induce apoptotic cell death and can therefore function as anti-tumorigenic species. However, the detailed mechanisms by which ROS induce apoptotic cell death and HCV copes with the oxidative conditions are largely unknown. In the present study, we found that H2O2 induced apoptotic cell death in p53-positive human hepatocytes, but not in p53-negative human hepatocytes. For this effect, H2O2 upregulated levels of p14, increased ubiquitin-dependent degradation of mouse double minute 2 (MDM2), and reduced the interaction between MDM2 and p53 to prevent p53 degradation, resulting in accumulation of p53 and subsequent activation of p53-dependent apoptotic pathways. Interestingly, HCV core repressed p14 expression via promoter hypermethylation to abolish the potential of H2O2 to activate the p14-MDM2-p53 pathway. As a consequence, HCV core-expressing cells could overcome p53-mediated apoptosis provoked by H2O2. Taken together, HCV core could contribute to hepatocellular carcinoma formation by removing deleterious roles of ROS inducing cell death. © 2015 The Authors.

  9. Comparative proteome analysis of metabolic proteins from seeds of durum wheat (cv. Svevo) subjected to heat stress

    DEFF Research Database (Denmark)

    Laino, Paolo; Shelton, Dale; Finnie, Christine

    2010-01-01

    include proteins with metabolic activity or structural function. In order to investigate the consequences of heat stress on the accumulation of nonprolamin proteins in mature durum wheat kernels, the Italian cultivar Svevo was subjected to two thermal regimes (heat stress versus control). The 2-D patterns...... polypeptides, 47 of which were identified by MALDI-TOF and MALDI-TOF-TOF MS and included HSPs, proteins involved in the glycolysis and carbohydrate metabolism, as well as stress-related proteins. Many of the heat-induced polypeptides are considered to be allergenic for sensitive individuals....

  10. High-sensitivity C-reactive protein predicts target organ damage in Chinese patients with metabolic syndrome

    DEFF Research Database (Denmark)

    Zhao, Zhigang; Nie, Hai; He, Hongbo

    2007-01-01

    with metabolic syndrome. A total of 1082 consecutive patients of Chinese origin were screened for the presence of metabolic syndrome according to the National Cholesterol Education Program's Adult Treatment Panel III. High-sensitivity C-reactive protein and target organ damage, including cardiac hypertrophy...... with the elevated high-sensitivity C-reactive protein had a higher percentage of target organ damage than those with lower high-sensitivity C-reactive protein. Stepwise multiple logistic regression confirmed that high-sensitivity C-reactive protein was significantly associated with cardiac hypertrophy, carotid...

  11. Perturbed rhythmic activation of signaling pathways in mice deficient for Sterol Carrier Protein 2-dependent diurnal lipid transport and metabolism.

    Science.gov (United States)

    Jouffe, Céline; Gobet, Cédric; Martin, Eva; Métairon, Sylviane; Morin-Rivron, Delphine; Masoodi, Mojgan; Gachon, Frédéric

    2016-04-21

    Through evolution, most of the living species have acquired a time keeping system to anticipate daily changes caused by the rotation of the Earth. In all of the systems this pacemaker is based on a molecular transcriptional/translational negative feedback loop able to generate rhythmic gene expression with a period close to 24 hours. Recent evidences suggest that post-transcriptional regulations activated mostly by systemic cues play a fundamental role in the process, fine tuning the time keeping system and linking it to animal physiology. Among these signals, we consider the role of lipid transport and metabolism regulated by SCP2. Mice harboring a deletion of the Scp2 locus present a modulated diurnal accumulation of lipids in the liver and a perturbed activation of several signaling pathways including PPARα, SREBP, LRH-1, TORC1 and its upstream regulators. This defect in signaling pathways activation feedbacks upon the clock by lengthening the circadian period of animals through post-translational regulation of core clock regulators, showing that rhythmic lipid transport is a major player in the establishment of rhythmic mRNA and protein expression landscape.

  12. Proteomic analysis reveals novel proteins associated with the Plasmodium protein exporter PTEX and a loss of complex stability upon truncation of the core PTEX component, PTEX150.

    Science.gov (United States)

    Elsworth, Brendan; Sanders, Paul R; Nebl, Thomas; Batinovic, Steven; Kalanon, Ming; Nie, Catherine Q; Charnaud, Sarah C; Bullen, Hayley E; de Koning Ward, Tania F; Tilley, Leann; Crabb, Brendan S; Gilson, Paul R

    2016-11-01

    The Plasmodium translocon for exported proteins (PTEX) has been established as the machinery responsible for the translocation of all classes of exported proteins beyond the parasitophorous vacuolar membrane of the intraerythrocytic malaria parasite. Protein export, particularly in the asexual blood stage, is crucial for parasite survival as exported proteins are involved in remodelling the host cell, an essential process for nutrient uptake, waste removal and immune evasion. Here, we have truncated the conserved C-terminus of one of the essential PTEX components, PTEX150, in Plasmodium falciparum in an attempt to create mutants of reduced functionality. Parasites tolerated C-terminal truncations of up to 125 amino acids with no reduction in growth, protein export or the establishment of new permeability pathways. Quantitative proteomic approaches however revealed a decrease in other PTEX subunits associating with PTEX150 in truncation mutants, suggesting a role for the C-terminus of PTEX150 in regulating PTEX stability. Our analyses also reveal three previously unreported PTEX-associated proteins, namely PV1, Pf113 and Hsp70-x (respective PlasmoDB numbers; PF3D7_1129100, PF3D7_1420700 and PF3D7_0831700) and demonstrate that core PTEX proteins exist in various distinct multimeric forms outside the major complex. © 2016 John Wiley & Sons Ltd.

  13. Multidomain Carbohydrate-binding Proteins Involved in Bacteroides thetaiotaomicron Starch Metabolism*

    Science.gov (United States)

    Cameron, Elizabeth A.; Maynard, Mallory A.; Smith, Christopher J.; Smith, Thomas J.; Koropatkin, Nicole M.; Martens, Eric C.

    2012-01-01

    Human colonic bacteria are necessary for the digestion of many dietary polysaccharides. The intestinal symbiont Bacteroides thetaiotaomicron uses five outer membrane proteins to bind and degrade starch. Here, we report the x-ray crystallographic structures of SusE and SusF, two outer membrane proteins composed of tandem starch specific carbohydrate-binding modules (CBMs) with no enzymatic activity. Examination of the two CBMs in SusE and three CBMs in SusF reveals subtle differences in the way each binds starch and is reflected in their Kd values for both high molecular weight starch and small maltooligosaccharides. Thus, each site seems to have a unique starch preference that may enable these proteins to interact with different regions of starch or its breakdown products. Proteins similar to SusE and SusF are encoded in many other polysaccharide utilization loci that are possessed by human gut bacteria in the phylum Bacteroidetes. Thus, these proteins are likely to play an important role in carbohydrate metabolism in these abundant symbiotic species. Understanding structural changes that diversify and adapt related proteins in the human gut microbial community will be critical to understanding the detailed mechanistic roles that they perform in the complex digestive ecosystem. PMID:22910908

  14. Multidomain Carbohydrate-binding Proteins Involved in Bacteroides thetaiotaomicron Starch Metabolism.

    Science.gov (United States)

    Cameron, Elizabeth A; Maynard, Mallory A; Smith, Christopher J; Smith, Thomas J; Koropatkin, Nicole M; Martens, Eric C

    2012-10-05

    Human colonic bacteria are necessary for the digestion of many dietary polysaccharides. The intestinal symbiont Bacteroides thetaiotaomicron uses five outer membrane proteins to bind and degrade starch. Here, we report the x-ray crystallographic structures of SusE and SusF, two outer membrane proteins composed of tandem starch specific carbohydrate-binding modules (CBMs) with no enzymatic activity. Examination of the two CBMs in SusE and three CBMs in SusF reveals subtle differences in the way each binds starch and is reflected in their K(d) values for both high molecular weight starch and small maltooligosaccharides. Thus, each site seems to have a unique starch preference that may enable these proteins to interact with different regions of starch or its breakdown products. Proteins similar to SusE and SusF are encoded in many other polysaccharide utilization loci that are possessed by human gut bacteria in the phylum Bacteroidetes. Thus, these proteins are likely to play an important role in carbohydrate metabolism in these abundant symbiotic species. Understanding structural changes that diversify and adapt related proteins in the human gut microbial community will be critical to understanding the detailed mechanistic roles that they perform in the complex digestive ecosystem.

  15. Nitrogen metabolism, digestive parameters, and protein requirements for the maintenance of buffalo growth.

    Science.gov (United States)

    Machado, Erica; Yoshimura, Emerson Henri; Santos, Nadine Woruby; Agustinho, Bruna Calvo; Pereira, Lucelia de Moura; Samensari, Rafael Barreiros; de Aguiar, Silvia Cristina; Zeoula, Lucia Maria

    2016-02-01

    The objectives of this study were to evaluate the effect of crude protein (CP) levels in the diet of growing female buffaloes on nitrogen metabolism and estimate protein requirements for maintenance. Four female buffaloes were used, cannulated in the rumen, with an average initial body weight (BW) of 355 ± 3.5 kg, in a Latin square (4 × 4) with four animals and four levels of CP in the diet (70, 90, 110, and 130 g/kg dry matter (DM)) composed of corn silage and concentrate. The increase in protein intake with increasing levels of dietary CP resulted in a higher concentration of ammonia in the rumen and higher ruminal disappearance of PB. However, omasal flow of protein increased linearly as did the efficiency of microbial protein synthesis. The CP levels affected DM intake and other nutrients positively, but there was no effect on nutrient total digestibility. Nitrogen (N) balance, when expressed relative to N intake, had an average value of 48.5 % observed across. The protein requirement for the maintenance of growing female buffaloes was 4.6 g CP/kg BW(0.75).

  16. Myostatin promotes distinct responses on protein metabolism of skeletal and cardiac muscle fibers of rodents

    Directory of Open Access Journals (Sweden)

    L.H. Manfredi

    2017-10-01

    Full Text Available Myostatin is a novel negative regulator of skeletal muscle mass. Myostatin expression is also found in heart in a much less extent, but it can be upregulated in pathological conditions, such as heart failure. Myostatin may be involved in inhibiting protein synthesis and/or increasing protein degradation in skeletal and cardiac muscles. Herein, we used cell cultures and isolated muscles from rats to determine protein degradation and synthesis. Muscles incubated with myostatin exhibited an increase in proteolysis with an increase of Atrogin-1, MuRF1 and LC3 genes. Extensor digitorum longus muscles and C2C12 myotubes exhibited a reduction in protein turnover. Cardiomyocytes showed an increase in proteolysis by activating autophagy and the ubiquitin proteasome system, and a decrease in protein synthesis by decreasing P70S6K. The effect of myostatin on protein metabolism is related to fiber type composition, which may be associated to the extent of atrophy mediated effect of myostatin on muscle.

  17. Structure and Characterization of Proteins and Enzymes Involved in Nucleotide Metabolism and Iron-Sulfur Proteins

    DEFF Research Database (Denmark)

    Løvgreen, Monika Nøhr; Ooi, Bee Lean

    , a program named MyCrystals has been developed to keep track of crystallization trials and results. The program combines pictures with crystallization conditions and is able to sort the pictures based on selected conditions. MyCrystals was used extensively throughout this work and allows for an overview...... extended β-sheet dimers. These dimers were not observed in solution and were likely a result of the high protein concentration in the crystals. WT, A115V and A115G Mt DCD-DUT were successfully purified, and the crystal structure of the A115V variant with dTTP bound was solved. The variants were created...

  18. Effects of Radiation and Dietary Iron on Expression of Genes and Proteins Involved in Drug Metabolism

    Science.gov (United States)

    Faust, K. M.; Wotring, V. E.

    2014-01-01

    Liver function, especially the rate of metabolic enzyme activities, determines the concentration of circulating drugs and the duration of their efficacy. Most pharmaceuticals are metabolized by the liver, and clinically-used medication doses are given with normal liver function in mind. A drug overdose can result in the case of a liver that is damaged and removing pharmaceuticals from the circulation at a rate slower than normal. Alternatively, if liver function is elevated and removing drugs from the system more quickly than usual, it would be as if too little drug had been given for effective treatment. Because of the importance of the liver in drug metabolism, we want to understand any effects of spaceflight on the enzymes of the liver. Dietary factors and exposure to radiation are aspects of spaceflight that are potential oxidative stressors and both can be modeled in ground experiments. In this experiment, we examined the effects of high dietary iron and low dose gamma radiation (individually and combined) on the gene expression of enzymes involved in drug metabolism, redox homeostasis, and DNA repair. METHODS All procedures were approved by the JSC Animal Care and Use Committee. Male Sprague-Dawley rats were divided into 4 groups (n=8); control, high Fe diet (650 mg iron/kg), radiation (fractionated 3 Gy exposure from a Cs- 137 source) and combined high Fe diet + radiation exposure. Animals were euthanized 24h after the last treatment of radiation; livers were removed immediately and flash -frozen in liquid nitrogen. Expression of genes thought to be involved in redox homeostasis, drug metabolism and DNA damage repair was measured by RT-qPCR. Where possible, protein expression of the same genes was measured by western blotting. All data are expressed as % change in expression normalized to reference gene expression; comparisons were then made of each treatment group to the sham exposed/ normal diet control group. Data was considered significant at p< 0

  19. Highly sensitive SERS detection of cancer proteins in low sample volume using hollow core photonic crystal fiber.

    Science.gov (United States)

    U S, Dinish; Fu, Chit Yaw; Soh, Kiat Seng; Ramaswamy, Bhuvaneswari; Kumar, Anil; Olivo, Malini

    2012-03-15

    Enzyme-linked immunosorbent assays (ELISA) are commonly used for detecting cancer proteins at concentration in the range of about ng-μg/mL. Hence it often fails to detect tumor markers at the early stages of cancer and other diseases where the amount of protein is extremely low. Herein, we report a novel photonic crystal fiber (PCF) based surface enhanced Raman scattering (SERS) sensing platform for the ultrasensitive detection of cancer proteins in an extremely low sample volume. As a proof of concept, epidermal growth factor receptors (EGFRs) in a lysate solution from human epithelial carcinoma cells were immobilized into the hollow core PCF. Highly sensitive detection of protein was achieved using anti-EGFR antibody conjugated SERS nanotag. This SERS nanotag probe was realized by anchoring highly active Raman molecules onto the gold nanoparticles followed by bioconjugation. The proposed sensing method can detect low amount of proteins at ∼100 pg in a sample volume of ∼10 nL. Our approach may lead to the highly sensitive protein sensing methodology for the early detection of diseases. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. Carbohydrate, protein and glycoprotein metabolism by the guinea-pig liver in chronic metribuzin poisoning.

    Science.gov (United States)

    Tomaszewski, J; Zwierz, K; Mnich, Z; Jezyna, C

    1985-01-01

    Male guinea-pigs weighing 400-600 g, 8 months old, were given metribuzin directly into the gastric lumen over a period of 30 days (20 animals) or 90 days (20 animals) 6 times a week. In the liver of the poisoned animals, the glycogen level and the AspAT and AlAT activities, while in the serum the total protein and the fractions albumin, alpha 1-globulin and gamma-globulin significantly decreased; serum glucose and the serum fractions alpha 2-globulin and beta-globulin, each showed an increase. The glycogen level in the liver, total protein, glucose as well as the alpha 1 and alpha 2 globulin fractions in the serum showed not appreciable difference between 30 and 90 days of intoxication. After 90 days of metribuzin treatment AspAT and AlAT dropped in the liver and rose in the serum, in comparison to the 30-day values. As to the parameters of glycoprotein metabolism, the intoxicated animals showed a significant decrease and increase in concentration of hexosamines and sialic acids in the liver and serum, respectively. Metribuzin intoxication also cause a significant decrease in activity of glucosamine phosphate isomerase and significant increase in activity of glycosidases in the liver. The results suggest that metribuzin disturbs the metabolism of carbohydrates, proteins and glycoproteins in the guinea-pig liver.

  1. Alcohol induced hepatic degeneration in a hepatitis C virus core protein transgenic mouse model.

    Science.gov (United States)

    Noh, Dong-Hyung; Lee, Eun-Joo; Kim, Ah-Young; Lee, Eun-Mi; Min, Chang-Woo; Kang, Kyung-Ku; Lee, Myeong-Mi; Kim, Sang-Hyeob; Sung, Soo-Eun; Hwang, Meeyul; Yu, Dae-Yeul; Jeong, Kyu-Shik

    2014-03-07

    Hepatitis C virus (HCV) has become a major public health issue. It is prevalent in most countries. HCV infection frequently begins without clinical symptoms, before progressing to persistent viremia, chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC) in the majority of patients (70% to 80%). Alcohol is an independent cofactor that accelerates the development of HCC in chronic hepatitis C patients. The purpose of the current study was to evaluate ethanol-induced hepatic changes in HCV core-Tg mice and mutant core Tg mice. Wild type (NTG), core wild-Tg mice (TG-K), mutant core 116-Tg mice (TG-116) and mutant core 99-Tg mice (TG-99) were used in this investigation. All groups were given drinking water with 10% ethanol and 5% sucrose for 13 weeks. To observe liver morphological changes, we performed histopathological and immunohistochemical examinations. Histopathologically, NTG, TG-K and TG-116 mice showed moderate centrilobular necrosis, while severe centrilobular necrosis and hepatocyte dissociation were observed in TG-99 mice with increasing lymphocyte infiltration and piecemeal necrosis. In all groups, a small amount of collagen fiber was found, principally in portal areas. None of the mice were found to have myofibroblasts based on immunohistochemical staining specific for α-SMA. CYP2E1-positive cells were clearly detected in the centrilobular area in all groups. In the TG-99 mice, we also observed cells positive for CK8/18, TGF-β1 and phosphorylated (p)-Smad2/3 and p21 around the necrotic hepatocytes in the centrilobular area (p alcohol intake induced piecemeal necrosis and hepatocyte dissociation in the TG-99 mice. These phenomena involved activation of the TGF-β1/p-Smad2/3/p21 signaling pathway in hepatocytes. Data from this study will be useful for elucidating the association between alcohol intake and HCV infection.

  2. Effects of rumen undegradable protein supplementation on productive performance and indicators of protein and energy metabolism in Holstein fresh cows.

    Science.gov (United States)

    Amanlou, H; Farahani, T Amirabadi; Farsuni, N Eslamian

    2017-05-01

    The objective of this study was to determine the effects of feeding increased dietary crude protein (CP) on productive performance and indicators of protein and energy metabolism during 21 d postpartum. Thirty multiparous Holstein dairy cows were balanced by previous lactation milk yield, body condition score (BCS) at calving, and parity and randomly allocated to 1 of 3 dietary treatments from calving until 21 d postpartum. Dietary treatments were 16.0% CP with 5.0% rumen undegradable protein (RUP) based on dry matter (DM) (16CP), 18.7% CP with 7.0% RUP based on DM (19CP), and 21.4% CP with 9.0% RUP based on DM (21CP). Diets were similar in net energy for lactation (approximately 1.7 Mcal/kg of DM) and CP levels were increased with corn gluten meal and fish meal. Dry matter intake (DMI) was increased by increasing dietary CP levels from 16.0 to 19.0% of DM, but dietary CP beyond 19.0% had no effect on DMI. Milk yields were 4.7 and 6.5 kg/d greater in cows fed the 19CP and 21CP diets versus those fed the 16CP diet, whereas 4% fat-corrected milk was greater for cows fed the 21CP than the 16CP diet (36.0 vs. 31.4 kg/d). Milk protein content and yield, lactose yield, and milk urea nitrogen were elevated by increased dietary CP. Milk lactose content and fat yield were not different among dietary treatments, but milk fat content tended to decline with increasing content of CP in diets. High CP levels increased milk N secretion but decreased milk N efficiency. Apparent digestibility of DM, CP, and neutral detergent fiber was greater on the 19CP and 21CP diets compared with the 16CP diet. Cows fed the 19CP and 21CP diets lost less body condition relative to those fed the 16CP diet over 21 d postpartum. Feeding higher CP levels increased the concentrations of serum albumin, albumin to globulin ratio, and urea nitrogen and decreased aspartate aminotransferase, nonesterified fatty acids, and β-hydroxybutyrate, but had no effect on globulin, glucose, cholesterol, or

  3. The presence of a helix breaker in the hydrophobic core of signal sequences of secretory proteins prevents recognition by the signal-recognition particle in Escherichia coli.

    NARCIS (Netherlands)

    Adams, H; Scottie, P.A.; Cock, H.; Luirink, S.; Tommassen, J.

    2002-01-01

    Signal sequences often contain α-helix-destabilizing amino acids within the hydrophobic core. In the precursor of the Escherichia coli outer-membrane protein PhoE, the glycine residue at position -10 (Gly

  4. Biochemical and cellular characterization of the plant ortholog of PYM, a protein that interacts with the exon junction complex core proteins Mago and Y14.

    Science.gov (United States)

    Park, Nam-il; Muench, Douglas G

    2007-02-01

    The exon junction complex (EJC) plays an important role in post-transcriptional control of gene expression. Mago nashi (Mago) and Y14 are core EJC proteins that operate as a functional unit in animal cells, and the Mago-Y14 heterodimer interacts with other EJC core and peripheral proteins. Little is known about the biochemical and cellular characteristics of the EJC and its orthologs in plants. Here, we demonstrate that Arabidopsis Mago and Y14 form a ternary complex with PYM, an RNA-binding protein that was previously shown to interact with the Mago-Y14 heterodimer in Drosophila. Fluorescence microscopy indicated that Arabidopsis Mago and Y14 are localized primarily in the nucleus, whereas PYM is mostly cytoplasmic. In vitro pull-down assays using recombinant proteins showed that the amino-terminal region of the Arabidopsis PYM interacts with the Mago-Y14 heterodimer, a similar observation to that previously reported for the animal versions of these proteins. However, we demonstrated also that Arabidopsis PYM has the ability to interact with monomeric Mago and monomeric Y14. Immunoprecipitation and tandem affinity purification from whole cell extracts detected a subtle interaction between the Arabidopsis Mago-Y14 heterodimer and PYM in flowers, indicating that the ternary complex is not abundant in plant cells. The regions of the polypeptide responsible for nuclear import and export were defined using protein truncations and site-directed mutagenesis. This study identifies unique characteristics of Arabidopsis Mago, Y14 and PYM compared to those observed in animal cells. These are predicted to have important functional implications associated with post-transcriptional regulation of gene expression in plant cells.

  5. Pharmacokinetics, metabolism, and excretion of torcetrapib, a cholesteryl ester transfer protein inhibitor, in humans.

    Science.gov (United States)

    Dalvie, Deepak; Chen, Weichao; Zhang, Chenghong; Vaz, Alfin D; Smolarek, Teresa A; Cox, Loretta M; Lin, Jian; Obach, R Scott

    2008-11-01

    The pharmacokinetics, metabolism, and excretion of torcetrapib, a selective inhibitor of human cholesteryl ester transfer protein, were investigated in healthy human male volunteers after oral administration of [(14)C]torcetrapib (120-mg dose). The total mean recovery of radiolabeled dose after 21 days was 75.7%, and most of the dose (63%) was excreted in the urine. The total circulating radioactivity and unchanged torcetrapib plasma concentrations increased over the first 6 h and then declined slowly with mean terminal elimination half-lives of 373 and 211 h. Metabolism of torcetrapib was extensive in humans. Only 5.2% of the total dose constituted unchanged torcetrapib in the feces, whereas no parent was excreted unchanged in the urine. Similarly, pharmacokinetic analysis of total radioactivity and unchanged torcetrapib revealed that the area under the concentration versus time curve from zero to infinity of torcetrapib accounted for approximately 7.0% of the circulating radioactivity. Torcetrapib was metabolized to numerous metabolites via oxidation. The primary metabolic pathway involved initial oxidative decarbamoylation followed by extensive further oxidation, resulting in the formation of bistrifluoromethylbenzoic acid (M1) and quinaldic acid (M4) metabolites. A mean 40% of the total dose was excreted in the urine as M4 (and its glucuronide and urea conjugates), whereas 7.0% of the total dose was excreted as M1. In vitro studies using human subcellular fractions suggested that the initial metabolism of torcetrapib proceeds via CYP3A-mediated decarbamoylation. Subsequent oxidations lead to the major circulating and excretory metabolites M1 and M4.

  6. Adherence issues in inherited metabolic disorders treated by low natural protein diets

    DEFF Research Database (Denmark)

    MaCdonald, A; van Rijn, M; Feillet, F

    2012-01-01

    Common inborn errors of metabolism treated by low natural protein diets [amino acid (AA) disorders, organic acidemias and urea cycle disorders] are responsible for a collection of diverse clinical symptoms, each condition presenting at different ages with variable severity. Precursor...... on their neuropsychological profile. There are little data about their ability to self-manage their own diet or the success of any formal educational programs that may have been implemented. Trials conducted in non-phenylketonuria (PKU) patients are rare, and the development of specialist L-AAs for non-PKU AA disorders has...

  7. The effect of hypodynamia on mineral and protein metabolism in calcified tissues of the maxillodental system (experimental radioisotope study)

    Science.gov (United States)

    Prokhonchukov, A. A.; Kovalenko, Y. A.; Kolesnik, A. G.; Kondratyev, Y. I.; Ilyushko, N. A.

    1980-01-01

    Mineral and protein metabolism was studied in experiments on 60 white rats, using P-32 and Ca-45 uptake in the mineral fractions, 2C-14-glycine in the protein fractions, and P-32 in both fractions of calcified tissues as indices over a 100 day period of experimental hypodynamia. Combined alterations in mineral and protein metabolism occurred in the calcified tissues of the experimental animals. The most pronounced changes were found in P-32 and 2C-14-glycine metabolism. In the incisors and femoral bones, these alterations occurred in two phases: P-32 and 2C-14-glycine uptake first increased, then decreased. Changes in Ca-45 metabolism were less pronounced, particularly in the initial period of the experiment. A marked reduction in P-32, Ca-45, and 2C-14-glycine uptake was found in various fractions of the calcified tissues on the 100th day of experimental hypodynamia.

  8. Soy Germ Protein With or Without-Zn Improve Plasma Lipid Profile in Metabolic Syndrome Women

    Directory of Open Access Journals (Sweden)

    HERY WINARSI

    2012-03-01

    Full Text Available The aim of this research was to determine the effect of soy germ protein on lipid profile of metabolic syndrome (MetS patients. Respondents were 30 women with criteria, i.e. blood glucose level > normal, body mass index > 25 kg/m2, hypertriglyceridemia, low cholesterol-HDL level, 40-65 years old, living in Purwokerto, and signed the informed consent. The project was approved by the ethics committee of the Medical Faculty from Gadjah Mada University-Yogyakarta. Respondents were divided into three randomly chosen groups consisting of ten women each. The first, second, and third groups were treated, respectively, with milk enriched soy germ protein plus Zn, milk enriched soy germ protein (without Zn, and placebo for two months. Blood samples were taken at baseline, one and two months after observation. Two months after observation the groups consuming milk enriched with soy germ protein, both with or without Zn, had their level of cholesterol-total decrease from 215.8 to 180.2 mg/dl (P = 0.03, triglyceride from 240.2 to 162.5 mg/dl (P = 0.02, and LDL from 154.01 to 93.85 mg/dl (P = 0.03. In contrast, HDL increased from 38.91 to 49.49 mg/dl (P = 0.0008. In conclusion, soy germ protein can improve lipid profile, thus it can inhibit atherosclerosis incident.

  9. Aminoacylase 3 binds to and cleaves the N-terminus of the hepatitis C virus core protein

    Science.gov (United States)

    Tsirulnikov, Kirill; Abuladze, Natalia; Vahi, Ritu; Hasnain, Huma; Phillips, Martin; Ryan, Christopher M.; Atanasov, Ivo; Faull, Kym F.; Kurtz, Ira; Pushkin, Alexander

    2012-01-01

    Aminoacylase 3 (AA3) mediates deacetylation of N-acetyl aromatic amino acids and mercapturic acids. Deacetylation of mercapturic acids of exo- and endobiotics are likely involved in their toxicity. AA3 is predominantly expressed in kidney, and to a lesser extent in liver, brain, and blood. AA3 has been recently reported to interact with the hepatitis C virus core protein (HCVCP) in the yeast two-hybrid system. Here we demonstrate that AA3 directly binds to HCVCP (Kd~10 μM) that may by implicated in HCV pathogenesis. AA3 also revealed a weak endopeptidase activity towards the N-terminus of HCVCP. PMID:23010594

  10. Ultrastructural localization of the core protein of a basement membrane-specific chondroitin sulfate proteoglycan in adult rat skin

    DEFF Research Database (Denmark)

    McCarthy, K J; Horiguchi, Y; Couchman, J R

    1990-01-01

    , fibronectin, and entactin/nidogen. IN this paper we show, using core protein-specific antibodies, the presence of a newly described basement membrane-specific chondroitin sulfate proteoglycan at the epithelial/mesenchymal interface of adult rat skin. Ultrastructurally, this antigen was proven to reside......Basement membranes are complex extracellular matrices present at epithelial/mesenchymal interfaces of tissues. The dermal-epidermal junction has been shown to contain numerous components, some of the most well known being laminin, types IV and VII collagens, heparan sulfate proteoglycan...

  11. Aberrant expression of mucin core proteins and o-linked glycans associated with progression of pancreatic cancer

    DEFF Research Database (Denmark)

    Remmers, Neeley; Anderson, Judy M; Linde, Erin M

    2013-01-01

    Mucin expression is a common feature of most adenocarcinomas and features prominently in current attempts to improve diagnosis and therapy for pancreatic cancer and other adenocarcinomas. We investigated the expression of a number of mucin core proteins and associated O-linked glycans expressed...... in pancreatic adenocarcinoma-sialyl Tn (STn), Tn, T antigen, sialyl Lewis A (CA19-9), sialyl Lewis C (SLeC), Lewis X (LeX), and sialyl LeX (SLeX)-during the progression of pancreatic cancer from early stages to metastatic disease....

  12. Quantitative Metabolomics and Instationary 13C-Metabolic Flux Analysis Reveals Impact of Recombinant Protein Production on Trehalose and Energy Metabolism in Pichia pastoris

    NARCIS (Netherlands)

    Jorda, J.; Cueto Rojas, H.F.; Carnicer, M.; Wahl, S.A.; Ferrer, P.; Albiol, J.

    2014-01-01

    Pichia pastoris has been recognized as an effective host for recombinant protein production. In this work, we combine metabolomics and instationary 13C metabolic flux analysis (INST 13C-MFA) using GC-MS and LC-MS/MS to evaluate the potential impact of the production of a Rhizopus oryzae lipase (Rol)

  13. Getting to the core of protein pharmaceuticals – comprehensive structure analysis by mass spectrometry

    DEFF Research Database (Denmark)

    Leurs, Ulrike; Mistarz, Ulrik Hvid; Rand, Kasper Dyrberg

    2015-01-01

    Protein pharmaceuticals are the fastest growing class of novel therapeutic agents, and have been a major research and development focus in the (bio)pharmaceutical industry. Due to their large size and structural diversity, biopharmaceuticals represent a formidable challenge regarding analysis...... and characterization compared to traditional small molecule drugs. Any changes to the primary, secondary, tertiary or quaternary structure of a protein can potentially impact its function, efficacy and safety. The analysis and characterization of (structural) protein heterogeneity is therefore of utmost importance....... Mass spectrometry has evolved as a powerful tool for the characterization of both primary and higher order structures of protein pharmaceuticals. Furthermore, the chemical and physical stability of protein drugs, as well as their pharmacokinetics are nowadays routinely determined by mass spectrometry...

  14. Role of protein farnesylation in burn-induced metabolic derangements and insulin resistance in mouse skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Harumasa Nakazawa

    Full Text Available OBJECTIVE: Metabolic derangements, including insulin resistance and hyperlactatemia, are a major complication of major trauma (e.g., burn injury and affect the prognosis of burn patients. Protein farnesylation, a posttranslational lipid modification of cysteine residues, has been emerging as a potential component of inflammatory response in sepsis. However, farnesylation has not yet been studied in major trauma. To study a role of farnesylation in burn-induced metabolic aberration, we examined the effects of farnesyltransferase (FTase inhibitor, FTI-277, on burn-induced insulin resistance and metabolic alterations in mouse skeletal muscle. METHODS: A full thickness burn (30% total body surface area was produced under anesthesia in male C57BL/6 mice at 8 weeks of age. After the mice were treated with FTI-277 (5 mg/kg/day, IP or vehicle for 3 days, muscle insulin signaling, metabolic alterations and inflammatory gene expression were evaluated. RESULTS: Burn increased FTase expression and farnesylated proteins in mouse muscle compared with sham-burn at 3 days after burn. Simultaneously, insulin-stimulated phosphorylation of insulin receptor (IR, insulin receptor substrate (IRS-1, Akt and GSK-3β was decreased. Protein expression of PTP-1B (a negative regulator of IR-IRS-1 signaling, PTEN (a negative regulator of Akt-mediated signaling, protein degradation and lactate release by muscle, and plasma lactate levels were increased by burn. Burn-induced impaired insulin signaling and metabolic dysfunction were associated with increased inflammatory gene expression. These burn-induced alterations were reversed or ameliorated by FTI-277. CONCLUSIONS: Our data demonstrate that burn increased FTase expression and protein farnesylation along with insulin resistance, metabolic alterations and inflammatory response in mouse skeletal muscle, all of which were prevented by FTI-277 treatment. These results indicate that increased protein farnesylation plays a

  15. A metabolic core model elucidates how enhanced utilization of glucose and glutamine, with enhanced glutamine-dependent lactate production, promotes cancer cell growth: The WarburQ effect.

    Science.gov (United States)

    Damiani, Chiara; Colombo, Riccardo; Gaglio, Daniela; Mastroianni, Fabrizia; Pescini, Dario; Westerhoff, Hans Victor; Mauri, Giancarlo; Vanoni, Marco; Alberghina, Lilia

    2017-09-01

    Cancer cells share several metabolic traits, including aerobic production of lactate from glucose (Warburg effect), extensive glutamine utilization and impaired mitochondrial electron flow. It is still unclear how these metabolic rearrangements, which may involve different molecular events in different cells, contribute to a selective advantage for cancer cell proliferation. To ascertain which metabolic pathways are used to convert glucose and glutamine to balanced energy and biomass production, we performed systematic constraint-based simulations of a model of human central metabolism. Sampling of the feasible flux space allowed us to obtain a large number of randomly mutated cells simulated at different glutamine and glucose uptake rates. We observed that, in the limited subset of proliferating cells, most displayed fermentation of glucose to lactate in the presence of oxygen. At high utilization rates of glutamine, oxidative utilization of glucose was decreased, while the production of lactate from glutamine was enhanced. This emergent phenotype was observed only when the available carbon exceeded the amount that could be fully oxidized by the available oxygen. Under the latter conditions, standard Flux Balance Analysis indicated that: this metabolic pattern is optimal to maximize biomass and ATP production; it requires the activity of a branched TCA cycle, in which glutamine-dependent reductive carboxylation cooperates to the production of lipids and proteins; it is sustained by a variety of redox-controlled metabolic reactions. In a K-ras transformed cell line we experimentally assessed glutamine-induced metabolic changes. We validated computational results through an extension of Flux Balance Analysis that allows prediction of metabolite variations. Taken together these findings offer new understanding of the logic of the metabolic reprogramming that underlies cancer cell growth.

  16. Localization of Core Planar Cell Polarity Proteins, PRICKLEs, in Ameloblasts of Rat Incisors: Possible Regulation of Enamel Rod Decussation.

    Science.gov (United States)

    Nishikawa, Sumio; Kawamoto, Tadafumi

    2015-04-28

    To confirm the possible involvement of planar cell polarity proteins in odontogenesis, one group of core proteins, PRICKLE1, PRICKLE2, PRICKLE3, and PRICKLE4, was examined in enamel epithelial cells and ameloblasts by immunofluorescence microscopy. PRICKLE1 and PRICKLE2 showed similar localization in the proliferation and secretory zones of the incisor. Immunoreactive dots and short rods in ameloblasts and stratum intermedium cells were evident in the proliferation to differentiation zone, but in the secretion zone, cytoplasmic dots decreased and the distal terminal web was positive for PRICKLE1 and PRICKLE2. PRICKLE3 and PRICKLE4 showed cytoplasmic labeling in ameloblasts and other enamel epithelial cells. Double labeling of PRICKLE2 with VANGL1, which is another planar cell polarity protein, showed partial co-localization. To examine the transport route of PRICKLE proteins, PRICKLE1 localization was examined after injection of a microtubule-disrupting reagent, colchicine, and was compared with CX43, which is a membrane protein transported as vesicles via microtubules. The results confirmed the retention of immunoreactive dots for PRICKLE1 in the cytoplasm of secretory ameloblasts of colchicine-injected animals, but fewer dots were observed in control animals. These results suggest that PRICKLE1 and PRICKLE2 are transported as vesicles to the junctional area, and are involved in pattern formation of distal junctional complexes and terminal webs of ameloblasts, further implying a role in the formed enamel rod arrangement.

  17. Soy protein diet alters expression of hepatic genes regulating fatty acid and thyroid hormone metabolism in the male rat

    Science.gov (United States)

    We determined effects of soy protein (SPI) and the isoflavone genistein (GEN) on mRNA expression of key lipid metabolism and thyroid hormone system genes in young adult, male Sprague-Dawley rats. SPI-fed rats had less retroperitoneal fat and less hepato-steatosis than casein (CAS, control protein)-...

  18. The interplay between lnRNAs, SNPs, and protein complexes - what does it mean for cancer metabolism?

    Science.gov (United States)

    Redis, Roxana S; Calin, George A

    2016-07-01

    Long non-coding RNAs (lncRNAs) exert most of their functions through protein interactions. A better understanding of these interactions will facilitate the development of novel therapeutics. Recently, we described how the lncRNA CCAT2 located at the 8q24 cancer amplicon reprograms cancer metabolism by directly interacting in an allele-specific manner with a protein complex.

  19. Abomasal protein infusion in postpartum transition dairy cows: Effect on performance and mammary metabolism

    DEFF Research Database (Denmark)

    Larsen, Mogens; Lapierre, H; Kristensen, Niels Bastian

    2014-01-01

    The effect of increasing the postpartum metabolizable protein (MP) supply on performance and mammary metabolism was studied using 8 Holstein cows in a complete randomized design. At parturition, cows were assigned to abomasal infusion of water (CTRL) or casein (CAS). Arterial and epigastric venous...... blood samples were taken 14 d before expected parturition and at 4, 15, and 29 d in milk (DIM). To compensate previously estimated deficiency of essential AA and to avoid oversupply, casein protein infusion was graduated with 696 ± 1, 490 ± 9, and 212 ± 10 g/d at 4, 15 and 29 DIM, respectively. Dry...... indicated that Lys, Leu, and Tyr were the first-limiting AA at 4 DIM with CTRL. Mammary plasma flow was unaffected by treatment, indicating similar perfusion of mammary tissue. The greater milk yield with CAS was associated with greater mammary uptake of individual essential AA, tendencies to greater uptake...

  20. The effect of milk and milk proteins on risk factors of metabolic syndrome in overweight adolecents

    DEFF Research Database (Denmark)

    Arnberg, Karina

    in the adolescents. The analyses showed that increased physical activity was related to an improved arterial function whereas central adiposity and a high protein intake were related to increased arterial stiffness. In the intervention study, the adolescents with habitual low milk intakes were randomized to drink 1L...... factors than children having a high milk intake. The aim of the intervention study was to examine whether it is beneficial for overweight adolescents with a habitual low milk intake to increase the consumption of low fat milk and whether a potential beneficial effect is caused by whey or casein. The data......This PhD is based on data from an intervention study with milk and milk proteins conducted in Danish adolescents with overweight. There is a high prevalence of overweight in Danish adolescents. Metabolic syndrome is a cluster of risk factors related to overweight and believed to increase the risk...

  1. The effect of milk and milk proteins on risk factors of metabolic syndrome in overweight adolecents

    DEFF Research Database (Denmark)

    Arnberg, Karina

    and casein for prevention of cardiovascular diseases in overweight children. Also, this study indicates that increasing the intake of skimmed milk to 1L per day is not advice-able due to the apparent increase in body weight. Drinking water showed to be harmless and may even be beneficial in overweight...... skimmed milk, whey, casein or water for three months. The background for the intervention is that milk is an important source of protein in the Western diet and epidemiological studies in children have shown that children drinking low amounts of milk have higher concentrations of the metabolic risk...... in the adolescents. The analyses showed that increased physical activity was related to an improved arterial function whereas central adiposity and a high protein intake were related to increased arterial stiffness. In the intervention study, the adolescents with habitual low milk intakes were randomized to drink 1L...

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

    Energy Technology Data Exchange (ETDEWEB)

    Binas, B. [Max Delbrueck Center for Molecular Medicine, Berlin-Buch (Germany)

    1998-12-31

    Fatty acid-binding proteins (FABPs) are major targets for specific binding of fatty acids in vivo. They constitute a widely expressed family of genetically related, small cytosolic proteins which very likely mediate intracellular transport of free long chain fatty acids. Genetic inhibition of FABP expression in vivo should therefore provide a useful tool to investigate and engineer fatty acid metabolism. (orig.) [Deutsch] Fettsaeurebindungsproteine (FABPs) sind wichtige Bindungsstellen fuer Fettsaeuren in vivo; sie bilden eine breit exprimierte Familie genetisch verwandter kleiner Zytosoleiweisse, die sehr wahrscheinlich den intrazellulaeren Transport unveresterter langkettiger Fettsaeuren vermitteln. Die genetische Hemmung der FABP-Expanssion in vivo bietet sich deshalb als Werkzeug zur Erforschung und gezielten Veraenderung des Fettsaeurestoffwechsels an. (orig.)

  3. A new fluorescence-based method identifies protein phosphatases regulating lipid droplet metabolism.

    Directory of Open Access Journals (Sweden)

    Bruno L Bozaquel-Morais

    Full Text Available In virtually every cell, neutral lipids are stored in cytoplasmic structures called lipid droplets (LDs and also referred to as lipid bodies or lipid particles. We developed a rapid high-throughput assay based on the recovery of quenched BODIPY-fluorescence that allows to quantify lipid droplets. The method was validated by monitoring lipid droplet turnover during growth of a yeast culture and by screening a group of strains deleted in genes known to be involved in lipid metabolism. In both tests, the fluorimetric assay showed high sensitivity and good agreement with previously reported data using microscopy. We used this method for high-throughput identification of protein phosphatases involved in lipid droplet metabolism. From 65 yeast knockout strains encoding protein phosphatases and its regulatory subunits, 13 strains revealed to have abnormal levels of lipid droplets, 10 of them having high lipid droplet content. Strains deleted for type I protein phosphatases and related regulators (ppz2, gac1, bni4, type 2A phosphatase and its related regulator (pph21 and sap185, type 2C protein phosphatases (ptc1, ptc4, ptc7 and dual phosphatases (pps1, msg5 were catalogued as high-lipid droplet content strains. Only reg1, a targeting subunit of the type 1 phosphatase Glc7p, and members of the nutrient-sensitive TOR pathway (sit4 and the regulatory subunit sap190 were catalogued as low-lipid droplet content strains, which were studied further. We show that Snf1, the homologue of the mammalian AMP-activated kinase, is constitutively phosphorylated (hyperactive in sit4 and sap190 strains leading to a reduction of acetyl-CoA carboxylase activity. In conclusion, our fast and highly sensitive method permitted us to catalogue protein phosphatases involved in the regulation of LD metabolism and present evidence indicating that the TOR pathway and the SNF1/AMPK pathway are connected through the Sit4p-Sap190p pair in the control of lipid droplet biogenesis.

  4. Digestibility of extruded proteins and metabolic transit of N ε -carboxymethyllysine in rats.

    Science.gov (United States)

    Alamir, Issam; Niquet-Leridon, Céline; Jacolot, Philippe; Rodriguez, Camille; Orosco, Martine; Anton, Pauline M; Tessier, Frédéric J

    2013-06-01

    Milk proteins are frequently used as supplements in fortified foods. However, processing produces chemical changes which likely affect the nutritional advantage. This study was intended to explore the possible difference in digestibility between extruded and non-extruded caseins and how the dietary N (ε) -carboxymethyllysine (CML) is metabolised. Normal rats were randomized into either an extruded protein diet (EP) or the same with unextruded proteins (UEP), for two periods of 2 weeks at 7 to 9 and 11 to 13 weeks of age. However, no difference in protein digestibility was detected between the two diets, either in young or in adult animals, despite a 9.4-fold higher level of CML and an 8.5-fold higher level of lysinoalanine in the EP than in the UEP. No diet-related changes were observed in plasma CML, either protein bound or free. Amounts of 38 and 48 % of the orally absorbed CML were excreted in urine and faeces, respectively, in UEP-fed rats. Lower rates of excretion were found in the EP-fed rats (23 and 37 %, respectively). A second animal study using a single oral dose of free CML (400 μg/rat) was set up to measure the systemic concentration of CML every hour from 0 to 4 h. It revealed that protein-bound CML was not affected by the oral dose of CML, and the highest free CML level found in the circulation was 600 ng/mL. Extruded proteins, therefore, appear to be well digested, and CML rapidly eliminated. Since its elimination is, however, incomplete, the question of its biodistribution and metabolism remains open.

  5. Preparation of Core-Shell Hybrid Materials by Producing a Protein Corona Around Magnetic Nanoparticles.

    Science.gov (United States)

    Weidner, A; Gräfe, C; von der Lühe, M; Remmer, H; Clement, J H; Eberbeck, D; Ludwig, F; Müller, R; Schacher, F H; Dutz, S

    2015-12-01

    Nanoparticles experience increasing interest for a variety of medical and pharmaceutical applications. When exposing nanomaterials, e.g., magnetic iron oxide nanoparticles (MNP), to human blood, a protein corona consisting of various components is formed immediately. The composition of the corona as well as its amount bound to the particle surface is dependent on different factors, e.g., particle size and surface charge. The actual composition of the formed protein corona might be of major importance for cellular uptake of magnetic nanoparticles. The aim of the present study was to analyze the formation of the protein corona during in vitro serum incubation in dependency of incubation time and temperature. For this, MNP with different shells were incubated in fetal calf serum (FCS, serving as protein source) within a water bath for a defined time and at a defined temperature. Before and after incubation the particles were characterized by a variety of methods. It was found that immediately (seconds) after contact of MNP and FCS, a protein corona is formed on the surface of MNP. This formation led to an increase of particle size and a slight agglomeration of the particles, which was relatively constant during the first minutes of incubation. A longer incubation (from hours to days) resulted in a stronger agglomeration of the FCS incubated MNP. Quantitative analysis (gel electrophoresis) of serum-incubated particles revealed a relatively constant amount of bound proteins during the first minutes of serum incubation. After a longer incubation (>20 min), a considerably higher amount of surface proteins was determined for incubation temperatures below 40 °C. For incubation temperatures above 50 °C, the influence of time was less significant which might be attributed to denaturation of proteins during incubation. Overall, analysis of the molecular weight distribution of proteins found in the corona revealed a clear influence of incubation time and temperature on

  6. Preparation of Core-Shell Hybrid Materials by Producing a Protein Corona Around Magnetic Nanoparticles

    Science.gov (United States)

    Weidner, A.; Gräfe, C.; von der Lühe, M.; Remmer, H.; Clement, J. H.; Eberbeck, D.; Ludwig, F.; Müller, R.; Schacher, F. H.; Dutz, S.

    2015-07-01

    Nanoparticles experience increasing interest for a variety of medical and pharmaceutical applications. When exposing nanomaterials, e.g., magnetic iron oxide nanoparticles (MNP), to human blood, a protein corona consisting of various components is formed immediately. The composition of the corona as well as its amount bound to the particle surface is dependent on different factors, e.g., particle size and surface charge. The actual composition of the formed protein corona might be of major importance for cellular uptake of magnetic nanoparticles. The aim of the present study was to analyze the formation of the protein corona during in vitro serum incubation in dependency of incubation time and temperature. For this, MNP with different shells were incubated in fetal calf serum (FCS, serving as protein source) within a water bath for a defined time and at a defined temperature. Before and after incubation the particles were characterized by a variety of methods. It was found that immediately (seconds) after contact of MNP and FCS, a protein corona is formed on the surface of MNP. This formation led to an increase of particle size and a slight agglomeration of the particles, which was relatively constant during the first minutes of incubation. A longer incubation (from hours to days) resulted in a stronger agglomeration of the FCS incubated MNP. Quantitative analysis (gel electrophoresis) of serum-incubated particles revealed a relatively constant amount of bound proteins during the first minutes of serum incubation. After a longer incubation (>20 min), a considerably higher amount of surface proteins was determined for incubation temperatures below 40 °C. For incubation temperatures above 50 °C, the influence of time was less significant which might be attributed to denaturation of proteins during incubation. Overall, analysis of the molecular weight distribution of proteins found in the corona revealed a clear influence of incubation time and temperature on corona

  7. Energy requirements, protein-energy metabolism and balance, and carbohydrates in preterm infants.

    Science.gov (United States)

    Hay, William W; Brown, Laura D; Denne, Scott C

    2014-01-01

    Energy is necessary for all vital functions of the body at molecular, cellular, organ, and systemic levels. Preterm infants have minimum energy requirements for basal metabolism and growth, but also have requirements for unique physiology and metabolism that influence energy expenditure. These include body size, postnatal age, physical activity, dietary intake, environmental temperatures, energy losses in the stool and urine, and clinical conditions and diseases, as well as changes in body composition. Both energy and protein are necessary to produce normal rates of growth. Carbohydrates (primarily glucose) are principle sources of energy for the brain and heart until lipid oxidation develops over several days to weeks after birth. A higher protein/energy ratio is necessary in most preterm infants to approximate normal intrauterine growth rates. Lean tissue is predominantly produced during early gestation, which continues through to term. During later gestation, fat accretion in adipose tissue adds increasingly large caloric requirements to the lean tissue growth. Once protein intake is sufficient to promote net lean body accretion, additional energy primarily produces more body fat, which increases almost linearly at energy intakes >80-90 kcal/kg/day in normal, healthy preterm infants. Rapid gains in adiposity have the potential to produce later life obesity, an increasingly recognized risk of excessive energy intake. In addition to fundamental requirements for glucose, protein, and fat, a variety of non-glucose carbohydrates found in human milk may have important roles in promoting growth and development, as well as production of a gut microbiome that could protect against necrotizing enterocolitis. © 2014 S. Karger AG, Basel.

  8. Cocoa and Whey Protein Differentially Affect Markers of Lipid and Glucose Metabolism and Satiety.

    Science.gov (United States)

    Campbell, Caroline L; Foegeding, E Allen; Harris, G Keith

    2016-03-01

    Food formulation with bioactive ingredients is a potential strategy to promote satiety and weight management. Whey proteins are high in leucine and are shown to decrease hunger ratings and increase satiety hormone levels; cocoa polyphenolics moderate glucose levels and slow digestion. This study examined the effects of cocoa and whey proteins on lipid and glucose metabolism and satiety in vitro and in a clinical trial. In vitro, 3T3-L1 preadipocytes were treated with 0.5-100 μg/mL cocoa polyphenolic extract (CPE) and/or 1-15 mM leucine (Leu) and assayed for lipid accumulation and leptin production. In vivo, a 6-week clinical trial consisted of nine panelists (age: 22.6 ± 1.7; BMI: 22.3 ± 2.1) consuming chocolate-protein beverages once per week, including placebo, whey protein isolate (WPI), low polyphenolic cocoa (LP), high polyphenolic cocoa (HP), LP-WPI, and HP-WPI. Measurements included blood glucose and adiponectin levels, and hunger ratings at baseline and 0.5-4.0 h following beverage consumption. At levels of 50 and 100 μg/mL, CPE significantly inhibited preadipocyte lipid accumulation by 35% and 50%, respectively, and by 22% and 36% when combined with 15 mM Leu. Leu treatment increased adipocyte leptin production by 26-37%. In the clinical trial, all beverages significantly moderated blood glucose levels 30 min postconsumption. WPI beverages elicited lowest peak glucose levels and HP levels were significantly lower than LP. The WPI and HP beverage treatments significantly increased adiponectin levels, but elicited no significant changes in hunger ratings. These trends suggest that combinations of WPI and cocoa polyphenols may improve markers of metabolic syndrome and satiety.

  9. [Differential display of messenger RNA and identification of selenocysteine lyase gene in hepatocellular carcinoma cells transiently expressing hepatitis C virus core protein].

    Science.gov (United States)

    Yepes, Jesús Orlando; Luz Gunturiz, María; Henao, Luis Felipe; Navas, María Cristina; Balcázar, Norman; Gómez, Luis Alberto

    2006-06-01

    Hepatitis C virus is associated with diverse liver diseases including acute and chronic hepatitis, steatosis, cirrhosis and hepatocellular carcinoma. Several studies have explored viral mechanisms involved in the establishment of persistent infection and oncogenic Hepatitis C virus. Expression assays of Hepatitis C virus core protein suggest that this protein has transforming and carcinogenic properties with multifunctional activities in host cells. Characterization of expressed genes in cells expressing Core protein is important in order to identify candidate genes responsible for these pathogenic alterations. To compare and identify gene expression profiles in the human hepatocarcinoma derived cell line, HepG2, with transient expression of Hepatitis C virus Core protein. We have used comparative PCR-mediated differential display of mRNA from HepG2 hepatocarcinoma with and without transient expression of HCV Core protein or green fluorescent protein, previously obtained using the Semliki Forest Virus-based expression, through transduction of recombinant particles, rSFV-Core and rSFV-GFP, respectively. We observed differences in band intensities of mRNA in HepG2 cells transduced with rSFV-Core compared with those detected in cells without transduction, and transduced with rSFV-GFP. Cloning and sequencing of a gene fragment (258 bp) that was expressed differentially in HepG2 cells transduced with rSFV-Core, was identified as selenocystein lyase. The results confirm that HCV Core protein expressed in HepG2 is associated with specific changes in mRNA expression, including the gene for selenocystein lyase. This gene may be involved in the pathophysiology of hepatocellular carcinoma.

  10. Energy metabolism in young mink kits (Neovison vison) affected by protein and carbohydrate level in the diet

    DEFF Research Database (Denmark)

    Hellwing, Anne Louise Frydendahl; Hansen, NE; Tauson, A-H

    The mink is a strict carnivore and mink diets usually have a high content of protein. The energy metabolism in young minks in the transition period from milk to solid food is not investigated in detail, and the protein requirement is poorly defined. The substrate oxidation can give useful...... information about the relative contribution of different nutrients to the total heat production (HE; Tauson et al. 1997). The aim of the study was to examine the effect of different provision of protein and carbohydrate on the energy metabolism and substrate oxidation of mink kits between 6 and 12 weeks...

  11. Role of the Irr protein in the regulation of iron metabolism in Rhodobacter sphaeroides.

    Directory of Open Access Journals (Sweden)

    Verena Peuser

    Full Text Available In Rhizobia the Irr protein is an important regulator for iron-dependent gene expression. We studied the role of the Irr homolog RSP_3179 in the photosynthetic alpha-proteobacterium Rhodobacter sphaeroides. While Irr had little effect on growth under iron-limiting or non-limiting conditions its deletion resulted in increased resistance to hydrogen peroxide and singlet oxygen. This correlates with an elevated expression of katE for catalase in the Irr mutant compared to the wild type under non-stress conditions. Transcriptome studies revealed that Irr affects the expression of genes for iron metabolism, but also has some influence on genes involved in stress response, citric acid cycle, oxidative phosphorylation, transport, and photosynthesis. Most genes showed higher expression levels in the wild type than in the mutant under normal growth conditions indicating an activator function of Irr. Irr was however not required to activate genes of the iron metabolism in response to iron limitation, which showed even stronger induction in the absence of Irr. This was also true for genes mbfA and ccpA, which were verified as direct targets for Irr. Our results suggest that in R. sphaeroides Irr diminishes the strong induction of genes for iron metabolism under iron starvation.

  12. Glucagon-Like Peptide 2 Stimulates Postresection Intestinal Adaptation in Preterm Pigs by Affecting Proteins Related to Protein, Carbohydrate, and Sulphur Metabolism

    DEFF Research Database (Denmark)

    Jiang, Pingping; Vegge, Andreas; Thymann, Thomas

    2017-01-01

    BACKGROUND: Exogenous glucagon-like peptide 2 (GLP-2) stimulates intestinal adaptation after resection in animal models of pediatric short bowel syndrome (SBS). It is unknown whether the molecular mechanisms of such GLP-2 effects are similar to those of postresection spontaneous adaptation. Using...... cellular structural proteins, while the added GLP-2 treatment affected proteins involved in protein processing and the metabolism of protein, carbohydrate, and sulphur. CONCLUSION: In the first days following resection, proteins affected by resection plus GLP-2 treatment differed markedly from those...... affected by the spontaneous intestinal adaptation following resection alone. Whether more long-term GLP-2 treatment may affect the intestinal proteome following intestinal resection remains unknown....

  13. Insight into the Unfolding Properties of Chd64, a Small, Single Domain Protein with a Globular Core and Disordered Tails.

    Directory of Open Access Journals (Sweden)

    Aneta Tarczewska

    Full Text Available Two major lipophilic hormones, 20-hydroxyecdysone (20E and juvenile hormone (JH, govern insect development and growth. While the mode of action of 20E is well understood, some understanding of JH-dependent signalling has been attained only in the past few years, and the crosstalk of the two hormonal pathways remains unknown. Two proteins, the calponin-like Chd64 and immunophilin FKBP39 proteins, have recently been found to play pivotal roles in the formation of dynamic, multiprotein complex that cross-links these two signalling pathways. However, the molecular mechanism of the interaction remains unexplored. The aim of this work was to determine structural elements of Chd64 to provide an understanding of molecular basis of multiple interactions. We analysed Chd64 in two unrelated insect species, Drosophila melanogaster (DmChd64 and Tribolium castaneum (TcChd64. Using hydrogen-deuterium exchange mass spectrometry (HDX-MS, we showed that both Chd64 proteins have disordered tails that outflank the globular core. The folds of the globular cores of both Chd64 resemble the calponin homology (CH domain previously resolved by crystallography. Monitoring the unfolding of DmChd64 and TcChd64 by far-ultraviolet (UV circular dichroism (CD spectroscopy, fluorescence spectroscopy and size-exclusion chromatography (SEC revealed a highly complex process. Chd64 unfolds and forms of a molten globule (MG-like intermediate state. Furthermore, our data indicate that in some conditions, Chd64 may exists in discrete structural forms, indicating that the protein is pliable and capable of easily acquiring different conformations. The plasticity of Chd64 and the existence of terminal intrinsically disordered regions (IDRs may be crucial for multiple interactions with many partners.

  14. Sm-like protein-mediated RNA metabolism is required for heat stress tolerance in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Masanori Okamoto

    2016-07-01

    Full Text Available Sm-like proteins play multiple functions in RNA metabolism, which is essential for biological processes such as stress responses in eukaryotes. The Arabidopsis thaliana sad1 mutant has a mutation of sm-like protein 5 (LSM5 and shows impaired drought and salt stress tolerances. The lsm5/sad1 mutant also showed hypersensitivity to heat stress. GFP-fused LSM5/SAD1 was localized in the nucleus under optimal growth conditions. After heat stress treatment, GFP-fused LSM5/SAD1 fluorescence was also observed as small cytoplasmic dots, in addition to nuclear localization. Whole genome transcriptome analysis revealed that many genes in Arabidopsis were drastically changed in response to heat stress. More heat-responsive genes were highly expressed in lsm5/sad1 mutant at both 2 h and 6 h after heat stress treatment. Additionally, intron-retained and capped transcripts accumulated in the lsm5/sad1 mutant after heat stress treatment. In this study, we also identified non-Arabidopsis Genome Initiative (AGI transcripts that were expressed from unannotated regions. Most of these transcripts were antisense transcripts, and many capped non-AGI transcripts accumulated in the lsm5/sad1 mutant during heat stress treatment. These results indicated that LSM5/SAD1 functions to degrade aberrant transcripts through appropriate mRNA splicing and decapping, and precise RNA metabolic machinery is required for heat stress tolerance.

  15. Lack of the Lysosomal Membrane Protein, GLMP, in Mice Results in Metabolic Dysregulation in Liver.

    Directory of Open Access Journals (Sweden)

    Xiang Yi Kong

    Full Text Available Ablation of glycosylated lysosomal membrane protein (GLMP, formerly known as NCU-G1 has been shown to cause chronic liver injury which progresses into liver fibrosis in mice. Both lysosomal dysfunction and chronic liver injury can cause metabolic dysregulation. Glmp gt/gt mice (formerly known as Ncu-g1gt/gt mice were studied between 3 weeks and 9 months of age. Body weight gain and feed efficiency of Glmp gt/gt mice were comparable to wild type siblings, only at the age of 9 months the Glmp gt/gt siblings had significantly reduced body weight. Reduced size of epididymal fat pads was accompanied by hepatosplenomegaly in Glmp gt/gt mice. Blood analysis revealed reduced levels of blood glucose, circulating triacylglycerol and non-esterified fatty acids in Glmp gt/gt mice. Increased flux of glucose, increased de novo lipogenesis and lipid accumulation were detected in Glmp gt/gt primary hepatocytes, as well as elevated triacylglycerol levels in Glmp gt/gt liver homogenates, compared to hepatocytes and liver from wild type mice. Gene expression analysis showed an increased expression of genes involved in fatty acid uptake and lipogenesis in Glmp gt/gt liver compared to wild type. Our findings are in agreement with the metabolic alterations observed in other mouse models lacking lysosomal proteins, and with alterations characteristic for advanced chronic liver injury.

  16. Osteocalcin: The extra-skeletal role of a vitamin K-dependent protein in glucose metabolism

    Directory of Open Access Journals (Sweden)

    Eibhlís M. O'Connor

    2017-03-01

    Full Text Available The role of vitamin K in the body has long been associated with blood clotting and coagulation. In more recent times, its role in a range of physiological processes has been described including the regulation of bone and soft tissue calcification, cell growth and proliferation, cognition, inflammation, various oxidative processes and fertility, where osteocalcin is thought to up-regulate the synthesis of the enzymes needed for the biosynthesis of testosterone thereby increasing male fertility. Vitamin K dependent proteins (VKDP contain γ-carboxyglutamic acid residues which require post-translational, gamma-glutamyl carboxylation by the vitamin K-dependent (VKD gamma-glutamyl carboxylase enzyme for full functionality. These proteins are present both hepatically and extrahepatically. The role of bone-derived osteocalcin has many physiological roles including, maintenance of bone mass with more recent links to energy metabolism due to the role of the skeleton as an endocrine organ. It has been proposed that insulin binds to bone forming cells (osteoblasts promoting osteocalcin production which in turn promotes β-cell proliferation, insulin secretion and glucose control. However much of this research has been conducted in animal models with equivocal findings in human studies. This review will discuss the role of osteocalcin in relation to its role in human health, focusing specifically on glucose metabolism.

  17. Metabolic Basis for Thyroid Hormone Liver Preconditioning: Upregulation of AMP-Activated Protein Kinase Signaling

    Directory of Open Access Journals (Sweden)

    Luis A. Videla

    2012-01-01

    Full Text Available The liver is a major organ responsible for most functions of cellular metabolism and a mediator between dietary and endogenous sources of energy for extrahepatic tissues. In this context, adenosine-monophosphate- (AMP- activated protein kinase (AMPK constitutes an intrahepatic energy sensor regulating physiological energy dynamics by limiting anabolism and stimulating catabolism, thus increasing ATP availability. This is achieved by mechanisms involving direct allosteric activation and reversible phosphorylation of AMPK, in response to signals such as energy status, serum insulin/glucagon ratio, nutritional stresses, pharmacological and natural compounds, and oxidative stress status. Reactive oxygen species (ROS lead to cellular AMPK activation and downstream signaling under several experimental conditions. Thyroid hormone (L-3,3′,5-triiodothyronine, T3 administration, a condition that enhances liver ROS generation, triggers the redox upregulation of cytoprotective proteins affording preconditioning against ischemia-reperfusion (IR liver injury. Data discussed in this work suggest that T3-induced liver activation of AMPK may be of importance in the promotion of metabolic processes favouring energy supply for the induction and operation of preconditioning mechanisms. These include antioxidant, antiapoptotic, and anti-inflammatory mechanisms, repair or resynthesis of altered biomolecules, induction of the homeostatic acute-phase response, and stimulation of liver cell proliferation, which are required to cope with the damaging processes set in by IR.

  18. A Protein Scaffold Coordinates SRC-Mediated JNK Activation in Response to Metabolic Stress.

    Science.gov (United States)

    Kant, Shashi; Standen, Claire L; Morel, Caroline; Jung, Dae Young; Kim, Jason K; Swat, Wojciech; Flavell, Richard A; Davis, Roger J

    2017-09-19

    Obesity is a major risk factor for the development of metabolic syndrome and type 2 diabetes. How obesity contributes to metabolic syndrome is unclear. Free fatty acid (FFA) activation of a non-receptor tyrosine kinase (SRC)-dependent cJun NH 2 -terminal kinase (JNK) signaling pathway is implicated in this process. However, the mechanism that mediates SRC-dependent JNK activation is unclear. Here, we identify a role for the scaffold protein JIP1 in SRC-dependent JNK activation. SRC phosphorylation of JIP1 creates phosphotyrosine interaction motifs that bind the SH2 domains of SRC and the guanine nucleotide exchange factor VAV. These interactions are required for SRC-induced activation of VAV and the subsequent engagement of a JIP1-tethered JNK signaling module. The JIP1 scaffold protein, therefore, plays a dual role in FFA signaling by coordinating upstream SRC functions together with downstream effector signaling by the JNK pathway. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  19. Synthetic protein scaffolds based on peptide motifs and cognate adaptor domains for improving metabolic productivity

    Directory of Open Access Journals (Sweden)

    Anselm H.C. Horn

    2015-11-01

    Full Text Available The efficiency of many cellular processes relies on the defined interaction among different proteins within the same metabolic or signaling pathway. Consequently, a spatial colocalization of functionally interacting proteins has frequently emerged during evolution. This concept has been adapted within the synthetic biology community for the purpose of creating artificial scaffolds. A recent advancement of this concept is the use of peptide motifs and their cognate adaptor domains. SH2, SH3, GBD, and PDZ domains have been used most often in research studies to date. The approach has been successfully applied to the synthesis of a variety of target molecules including catechin, D-glucaric acid, H2, hydrochinone, resveratrol, butyrate, gamma-aminobutyric acid, and mevalonate. Increased production levels of up to 77-fold have been observed compared to non-scaffolded systems. A recent extension of this concept is the creation of a covalent linkage between peptide motifs and adaptor domains, which leads to a more stable association of the scaffolded systems and thus bears the potential to further enhance metabolic productivity.

  20. Biochemical and clinical aspects of advanced oxidation protein products in kidney diseases and metabolic disturbances

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    Agnieszka Piwowar

    2014-02-01

    Full Text Available Intensified oxidative modification of proteins and increased concentration of advanced oxidation protein products (AOPPs are confirmed by many experimental investigations in different pathological states, especially these with well-known participation of oxidative stress (OS in etiopathogenesis but also these with not well recognized its role. Presented data indicate that AOPPs play a significant role in many disorders with chronic background, because of they reflect both intensification of OS and the degree of pathological changes connected with OS in these diseases. This review sets out the clinical and diagnostic aspects of AOPPs in these diseases such as: renal diseases with different etiology, cardiovascular diseases, as well as connected with metabolic disturbances – e.g. diabetes, atherosclerosis or metabolic syndrome. Moreover results of investigation about utility of AOPPs measurement, mainly in plasma/serum, in these diseases are presented. The review and evaluation of application of AOPPs as useful marker in diagnosis, prognosis and monitoring the course of these diseases were performed. This paper also describes the suggested mechanisms of their action which contribute to biochemical and clinic changes undergoing in the condition of increased OS. Diagnostic or prognostic utility of AOPPs are especially indicated in the course of diabetes and its complications (diabetic nephropahy and cardiovascular diseases.

  1. Hepatitis C virus core protein targets 4E-BP1 expression and phosphorylation and potentiates Myc-induced liver carcinogenesis in transgenic mice.

    Science.gov (United States)

    Abdallah, Cosette; Lejamtel, Charlène; Benzoubir, Nassima; Battaglia, Serena; Sidahmed-Adrar, Nazha; Desterke, Christophe; Lemasson, Matthieu; Rosenberg, Arielle R; Samuel, Didier; Bréchot, Christian; Pflieger, Delphine; Le Naour, François; Bourgeade, Marie-Françoise

    2017-08-22

    Hepatitis C virus (HCV) is a leading cause of liver diseases including the development of hepatocellular carcinoma (HCC). Particularly, core protein has been involved in HCV-related liver pathologies. However, the impact of HCV core on signaling pathways supporting the genesis of HCC remains largely elusive. To decipher the host cell signaling pathways involved in the oncogenic potential of HCV core, a global quantitative phosphoproteomic approach was carried out. This study shed light on novel differentially phosphorylated proteins, in particular several components involved in translation. Among the eukaryotic initiation factors that govern the translational machinery, 4E-BP1 represents a master regulator of protein synthesis that is associated with the development and progression of cancers due to its ability to increase protein expression of oncogenic pathways. Enhanced levels of 4E-BP1 in non-modified and phosphorylated forms were validated in human hepatoma cells and in mouse primary hepatocytes expressing HCV core, in the livers of HCV core transgenic mice as well as in HCV-infected human primary hepatocytes. The contribution of HCV core in carcinogenesis and the status of 4E-BP1 expression and phosphorylation were studied in HCV core/Myc double transgenic mice. HCV core increased the levels of 4E-BP1 expression and phosphorylation and significantly accelerated the onset of Myc-induced tumorigenesis in these double transgenic mice. These results reveal a novel function of HCV core in liver carcinogenesis potentiation. They position 4E-BP1 as a tumor-specific target of HCV core and support the involvement of the 4E-BP1/eIF4E axis in hepatocarcinogenesis.

  2. Pokemon siRNA Delivery Mediated by RGD-Modified HBV Core Protein Suppressed the Growth of Hepatocellular Carcinoma.

    Science.gov (United States)

    Kong, Jing; Liu, Xiaoping; Jia, Jianbo; Wu, Jinsheng; Wu, Ning; Chen, Jun; Fang, Fang

    2015-10-01

    Hepatocellular carcinoma (HCC) is a deadly human malignant tumor that is among the most common cancers in the world, especially in Asia. Hepatitis B virus (HBV) infection has been well established as a high risk factor for hepatic malignance. Studies have shown that Pokemon is a master oncogene for HCC growth, suggesting it as an ideal therapeutic target. However, efficient delivery system is still lacking for Pokemon targeting treatment. In this study, we used core proteins of HBV, which is modified with RGD peptides, to construct a biomimetic vector for the delivery of Pokemon siRNAs (namely, RGD-HBc-Pokemon siRNA). Quantitative PCR and Western blot assays revealed that RGD-HBc-Pokemon siRNA possessed the highest efficiency of Pokemon suppression in HCC cells. In vitro experiments further indicated that RGD-HBc-Pokemon-siRNA exerted a higher tumor suppressor activity on HCC cell lines, evidenced by reduced proliferation and attenuated invasiveness, than Pokemon-siRNA or RGD-HBc alone. Finally, animal studies demonstrated that RGD-HBc-Pokemon siRNA suppressed the growth of HCC xenografts in mice by a greater extent than Pokemon-siRNA or RGD-HBc alone. Based on the above results, Pokemon siRNA delivery mediated by RGD-modified HBV core protein was shown to be an effective strategy of HCC gene therapy.

  3. The Conserved VPS-50 Protein Functions in Dense-Core Vesicle Maturation and Acidification and Controls Animal Behavior.

    Science.gov (United States)

    Paquin, Nicolas; Murata, Yasunobu; Froehlich, Allan; Omura, Daniel T; Ailion, Michael; Pender, Corinne L; Constantine-Paton, Martha; Horvitz, H Robert

    2016-04-04

    The modification of behavior in response to experience is crucial for animals to adapt to environmental changes. Although factors such as neuropeptides and hormones are known to function in the switch between alternative behavioral states, the mechanisms by which these factors transduce, store, retrieve, and integrate environmental signals to regulate behavior are poorly understood. The rate of locomotion of the nematode Caenorhabditis elegans depends on both current and past food availability. Specifically, C. elegans slows its locomotion when it encounters food, and animals in a food-deprived state slow even more than animals in a well-fed state. The slowing responses of well-fed and food-deprived animals in the presence of food represent distinct behavioral states, as they are controlled by different sets of genes, neurotransmitters, and neurons. Here we describe an evolutionarily conserved C. elegans protein, VPS-50, that is required for animals to assume the well-fed behavioral state. Both VPS-50 and its murine homolog mVPS50 are expressed in neurons, are associated with synaptic and dense-core vesicles, and control vesicle acidification and hence synaptic function, likely through regulation of the assembly of the V-ATPase complex. We propose that dense-core vesicle acidification controlled by the evolutionarily conserved protein VPS-50/mVPS50 affects behavioral state by modulating neuropeptide levels and presynaptic neuronal function in both C. elegans and mammals. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Human Cytomegalovirus Nuclear Capsids Associate with the Core Nuclear Egress Complex and the Viral Protein Kinase pUL97.

    Science.gov (United States)

    Milbradt, Jens; Sonntag, Eric; Wagner, Sabrina; Strojan, Hanife; Wangen, Christina; Lenac Rovis, Tihana; Lisnic, Berislav; Jonjic, Stipan; Sticht, Heinrich; Britt, William J; Schlötzer-Schrehardt, Ursula; Marschall, Manfred

    2018-01-13

    The nuclear phase of herpesvirus replication is regulated through the formation of regulatory multi-component protein complexes. Viral genomic replication is followed by nuclear capsid assembly, DNA encapsidation and nuclear egress. The latter has been studied intensely pointing to the formation of a viral core nuclear egress complex (NEC) that recruits a multimeric assembly of viral and cellular factors for the reorganization of the nuclear envelope. To date, the mechanism of the association of human cytomegalovirus (HCMV) capsids with the NEC, which in turn initiates the specific steps of nuclear capsid budding, remains undefined. Here, we provide electron microscopy-based data demonstrating the association of both nuclear capsids and NEC proteins at nuclear lamina budding sites. Specifically, immunogold labelling of the core NEC constituent pUL53 and NEC-associated viral kinase pUL97 suggested an intranuclear NEC-capsid interaction. Staining patterns with phospho-specific lamin A/C antibodies are compatible with earlier postulates of targeted capsid egress at lamina-depleted areas. Important data were provided by co-immunoprecipitation and in vitro kinase analyses using lysates from HCMV-infected cells, nuclear fractions, or infectious virions. Data strongly suggest that nuclear capsids interact with pUL53 and pUL97. Combined, the findings support a refined concept of HCMV nuclear trafficking and NEC-capsid interaction.

  5. A Systematic Review of the Effects of Plant Compared with Animal Protein Sources on Features of Metabolic Syndrome.

    Science.gov (United States)

    Chalvon-Demersay, Tristan; Azzout-Marniche, Dalila; Arfsten, Judith; Egli, Léonie; Gaudichon, Claire; Karagounis, Leonidas G; Tomé, Daniel

    2017-03-01

    Dietary protein may play an important role in the prevention of metabolic dysfunctions. However, the way in which the protein source affects these dysfunctions has not been clearly established. The aim of the current systematic review was to compare the impact of plant- and animal-sourced dietary proteins on several features of metabolic syndrome in humans. The PubMed database was searched for both chronic and acute interventional studies, as well as observational studies, in healthy humans or those with metabolic dysfunctions, in which the impact of animal and plant protein intake was compared while using the following variables: cholesterolemia and triglyceridemia, blood pressure, glucose homeostasis, and body composition. Based on data extraction, we observed that soy protein consumption (with isoflavones), but not soy protein alone (without isoflavones) or other plant proteins (pea and lupine proteins, wheat gluten), leads to a 3% greater decrease in both total and LDL cholesterol compared with animal-sourced protein ingestion, especially in individuals with high fasting cholesterol concentrations. This observation was made when animal proteins were provided as a whole diet rather than given supplementally. Some observational studies reported an inverse association between plant protein intake and systolic and diastolic blood pressure, but this was not confirmed by intervention studies. Moreover, plant protein (wheat gluten, soy protein) intake as part of a mixed meal resulted in a lower postprandial insulin response than did whey. This systematic review provides some evidence that the intake of soy protein associated with isoflavones may prevent the onset of risk factors associated with cardiovascular disease, i.e., hypercholesterolemia and hypertension, in humans. However, we were not able to draw any further conclusions from the present work on the positive effects of plant proteins relating to glucose homeostasis and body composition. © 2017 American

  6. Presence of early stage cancer does not impair the early protein metabolic response to major surgery.

    Science.gov (United States)

    Engelen, Mariëlle P K J; Klimberg, V Suzanne; Allasia, Arianna; Deutz, Nicolaas Ep

    2017-06-01

    Combined bilateral mastectomy and reconstruction is a common major surgical procedure in women with breast cancer and in those with a family history of breast cancer. As this large surgical procedure induces muscle protein loss, a preserved anabolic response to nutrition is warranted for optimal recovery. It is unclear whether the presence of early stage cancer negatively affects the protein metabolic response to major surgery as this would mandate perioperative nutritional support. In nine women with early stage (Stage II) breast malignancy and nine healthy women with a genetic predisposition to breast cancer undergoing the same large surgical procedure, we examined whether surgery influences the catabolic response to overnight fasting and the anabolic response to nutrition differently. Prior to and within 24 h after combined bilateral mastectomy and reconstruction surgery, whole body protein synthesis and breakdown rates were assessed after overnight fasting and after meal intake by stable isotope methodology to enable the calculation of net protein catabolism in the post-absorptive state and net protein anabolic response to a meal. Major surgery resulted in an up-regulation of post-absorptive protein synthesis and breakdown rates (P early stage breast cancer or surgery. The presence of early stage breast cancer does not enhance the normal catabolic response to major surgery or further attenuates the anabolic response to meal intake within 24 h after major surgery in patients with non-cachectic breast cancer. This indicates that the acute anabolic potential to conventional feeding is maintained in non-cachectic early stage breast cancer after major surgery. © 2017 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of the Society on Sarcopenia, Cachexia and Wasting Disorders.

  7. Integration and Validation of the Genome-Scale Metabolic Models of Pichia pastoris: A Comprehensive Update of Protein Glycosylation Pathways, Lipid and Energy Metabolism.

    Science.gov (United States)

    Tomàs-Gamisans, Màrius; Ferrer, Pau; Albiol, Joan

    2016-01-01

    Genome-scale metabolic models (GEMs) are tools that allow predicting a phenotype from a genotype under certain environmental conditions. GEMs have been developed in the last ten years for a broad range of organisms, and are used for multiple purposes such as discovering new properties of metabolic networks, predicting new targets for metabolic engineering, as well as optimizing the cultivation conditions for biochemicals or recombinant protein production. Pichia pastoris is one of the most widely used organisms for heterologous protein expression. There are different GEMs for this methylotrophic yeast of which the most relevant and complete in the published literature are iPP668, PpaMBEL1254 and iLC915. However, these three models differ regarding certain pathways, terminology for metabolites and reactions and annotations. Moreover, GEMs for some species are typically built based on the reconstructed models of related model organisms. In these cases, some organism-specific pathways could be missing or misrepresented. In order to provide an updated and more comprehensive GEM for P. pastoris, we have reconstructed and validated a consensus model integrating and merging all three existing models. In this step a comprehensive review and integration of the metabolic pathways included in each one of these three versions was performed. In addition, the resulting iMT1026 model includes a new description of some metabolic processes. Particularly new information described in recently published literature is included, mainly related to fatty acid and sphingolipid metabolism, glycosylation and cell energetics. Finally the reconstructed model was tested and validated, by comparing the results of the simulations with available empirical physiological datasets results obtained from a wide range of experimental conditions, such as different carbon sources, distinct oxygen availability conditions, as well as producing of two different recombinant proteins. In these simulations, the

  8. Combined transcript, proteome, and metabolite analysis of transgenic maize seeds engineered for enhanced carotenoid synthesis reveals pleotropic effects in core metabolism.

    Science.gov (United States)

    Decourcelle, Mathilde; Perez-Fons, Laura; Baulande, Sylvain; Steiger, Sabine; Couvelard, Linhdavanh; Hem, Sonia; Zhu, Changfu; Capell, Teresa; Christou, Paul; Fraser, Paul; Sandmann, Gerhard

    2015-06-01

    The aim of this study was to assess whether endosperm-specific carotenoid biosynthesis influenced core metabolic processes in maize embryo and endosperm and how global seed metabolism adapted to this expanded biosynthetic capacity. Although enhancement of carotenoid biosynthesis was targeted to the endosperm of maize kernels, a concurrent up-regulation of sterol and fatty acid biosynthesis in the embryo was measured. Targeted terpenoid analysis, and non-targeted metabolomic, proteomic, and transcriptomic profiling revealed changes especially in carbohydrate metabolism in the transgenic line. In-depth analysis of the data, including changes of metabolite pools and increased enzyme and transcript concentrations, gave a first insight into the metabolic variation precipitated by the higher up-stream metabolite demand by the extended biosynthesis capacities for terpenoids and fatty acids. An integrative model is put forward to explain the metabolic regulation for the increased provision of terpenoid and fatty acid precursors, particularly glyceraldehyde 3-phosphate and pyruvate or acetyl-CoA from imported fructose and glucose. The model was supported by higher activities of fructokinase, glucose 6-phosphate isomerase, and fructose 1,6-bisphosphate aldolase indicating a higher flux through the glycolytic pathway. Although pyruvate and acetyl-CoA utilization was higher in the engineered line, pyruvate kinase activity was lower. A sufficient provision of both metabolites may be supported by a by-pass in a reaction sequence involving phosphoenolpyruvate carboxylase, malate dehydrogenase, and malic enzyme. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  9. Association of cancer metabolism-related proteins with oral carcinogenesis – indications for chemoprevention and metabolic sensitizing of oral squamous cell carcinoma?

    Science.gov (United States)

    2014-01-01

    Background Tumor metabolism is a crucial factor for the carcinogenesis of oral squamous cell carcinoma (OSCC). Methods Expression of IGF-R1, glycolysis-related proteins (GLUT-1, HK 2, PFK-1, LDHA, TKTL1), mitochondrial enzymes (SDHA, SDHB, ATP synthase) were analyzed in normal oral mucosa (n = 5), oral precursor lesions (simple hyperplasia, n = 11; squamous intraepithelial neoplasia, SIN I-III, n = 35), and OSCC specimen (n = 42) by immunohistochemistry and real-time polymerase chain reaction (qPCR) analysis in OSCC cell lines. Metabolism-related proteins were correlated with proliferation activity (Ki-67) and apoptotic properties (TUNEL assay) in OSCC. Specificity of antibodies was confirmed by western blotting in cancer cell lines. Results Expression of IGF-R1, glycolysis-related proteins (GLUT-1, HK 2, LDHA, TKTL1), and mitochondrial enzymes (SDHA, SDHB, ATP synthase) were significantly increased in the carcinogenesis of OSCC. Metabolic active regions of OSCC were strongly correlated with proliferating cancer (Ki-67+) cells without detection of apoptosis (TUNEL assay). Conclusions This study provides the first evidence of the expression of IGF-R1, glycolysis-related proteins GLUT-1, HK 2, PFK-1, LDHA, and TKTL1, as well as mitochondrial enzymes SDHA, SDHB, and ATP synthase in the multi-step carcinogenesis of OSCC. Both, hypoxia-related glucose metabolism and mitochondrial oxidative phosphorylation characteristics are associated with the carcinogenesis of OSCC. Acidosis and OXPHOS may drive a metabolic shift towards the pentose phosphate pathway (PPP). Therefore, inhibition of the PPP, glycolysis, and targeted anti-mitochondrial therapies (ROS generation) by natural compounds or synthetic vitamin derivatives may act as sensitizer for apoptosis in cancer cells mediated by adjuvant therapies in OSCC. PMID:25048361

  10. Association of cancer metabolism-related proteins with oral carcinogenesis - indications for chemoprevention and metabolic sensitizing of oral squamous cell carcinoma?

    Science.gov (United States)

    Grimm, Martin; Cetindis, Marcel; Lehmann, Max; Biegner, Thorsten; Munz, Adelheid; Teriete, Peter; Kraut, Wiebke; Reinert, Siegmar

    2014-07-21

    Tumor metabolism is a crucial factor for the carcinogenesis of oral squamous cell carcinoma (OSCC). Expression of IGF-R1, glycolysis-related proteins (GLUT-1, HK 2, PFK-1, LDHA, TKTL1), mitochondrial enzymes (SDHA, SDHB, ATP synthase) were analyzed in normal oral mucosa (n = 5), oral precursor lesions (simple hyperplasia, n = 11; squamous intraepithelial neoplasia, SIN I-III, n = 35), and OSCC specimen (n = 42) by immunohistochemistry and real-time polymerase chain reaction (qPCR) analysis in OSCC cell lines. Metabolism-related proteins were correlated with proliferation activity (Ki-67) and apoptotic properties (TUNEL assay) in OSCC. Specificity of antibodies was confirmed by western blotting in cancer cell lines. Expression of IGF-R1, glycolysis-related proteins (GLUT-1, HK 2, LDHA, TKTL1), and mitochondrial enzymes (SDHA, SDHB, ATP synthase) were significantly increased in the carcinogenesis of OSCC. Metabolic active regions of OSCC were strongly correlated with proliferating cancer (Ki-67+) cells without detection of apoptosis (TUNEL assay). This study provides the first evidence of the expression of IGF-R1, glycolysis-related proteins GLUT-1, HK 2, PFK-1, LDHA, and TKTL1, as well as mitochondrial enzymes SDHA, SDHB, and ATP synthase in the multi-step carcinogenesis of OSCC. Both, hypoxia-related glucose metabolism and mitochondrial oxidative phosphorylation characteristics are associated with the carcinogenesis of OSCC. Acidosis and OXPHOS may drive a metabolic shift towards the pentose phosphate pathway (PPP). Therefore, inhibition of the PPP, glycolysis, and targeted anti-mitochondrial therapies (ROS generation) by natural compounds or synthetic vitamin derivatives may act as sensitizer for apoptosis in cancer cells mediated by adjuvant therapies in OSCC.

  11. Circulating levels of fatty acid-binding protein family and metabolic phenotype in the general population.

    Directory of Open Access Journals (Sweden)

    Shutaro Ishimura

    Full Text Available OBJECTIVE: Fatty acid-binding proteins (FABPs are a family of 14-15-kDa proteins, and some FABPs have been to be used as biomarkers of tissue injury by leak from cells. However, recent studies have shown that FABPs can be secreted from cells into circulation. Here we examined determinants and roles of circulating FABPs in a general population. METHODS: From the database of the Tanno-Sobetsu Study, a study with a population-based cohort design, data in 2011 for 296 subjects on no medication were retrieved, and FABP1~5 in their serum samples were assayed. RESULTS: Level of FABP4, but not the other isoforms, showed a gender difference, being higher in females than in males. Levels of all FABPs were negatively correlated with estimated glomerular filtration rate (eGFR, but a distinct pattern of correlation with other clinical parameters was observed for each FABP isoform; significant correlates were alanine aminotransferase (ALT, blood pressure (BP, and brain natriuretic peptide (BNP for FABP1, none besides eGFR for FABP2, age, BP, and BNP for FABP3, age, waist circumference (WC, BP, BNP, lipid variables, high-sensitivity C-reactive protein (hsCRP, and HOMA-R for FABP4, and age, WC, BP, ALT, BNP, and HOMA-R for FABP5. FABP4 is the most strongly related to metabolic markers among FABPs. In a multivariate regression analysis, FABP4 level was an independent predictor of HOMA-R after adjustment of age, gender, WC, BP, HDL cholesterol, and hsCRP. CONCLUSIONS: Each FABP isoform level showed a distinct pattern of correlation with clinical parameters, although levels of all FABPs were negatively determined by renal function. Circulating FABP4 appears to be a useful biomarker for detecting pre-clinical stage of metabolic syndrome, especially insulin resistance, in the general population.

  12. Spectroscopic properties of the CP43 core antenna protein of photosystem II

    NARCIS (Netherlands)

    Groot, ML; Frese, RN; de Weerd, FL; Bromek, K.; Pettersson, A.; Peterman, EJG; van Stokkum, IHM; van Grondelle, R; Dekker, JP

    1999-01-01

    CP43 is a chlorophyll-protein complex that funnels excitation energy from the main light-harvesting system of photosystem II to the photochemical reaction center. We purified CP43 from spinach photosystem II membranes in the presence of the nonionic detergent n-dodecyl-β,d-maltoside and recorded its

  13. Role of Mitogen-Activated Protein Kinase Pathways in Multifactorial Adverse Cardiac Remodeling Associated with Metabolic Syndrome

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    Mohamed Asrih

    2013-01-01

    Full Text Available Metabolic syndrome has been widely associated with an increased risk for acute cardiovascular events. Emerging evidence supports metabolic syndrome as a condition favoring an adverse cardiac remodeling, which might evolve towards heart dysfunction and failure. This pathological remodeling has been described to result from the cardiac adaptive response to clinical mechanical conditions (such as hypertension, dyslipidemia, and hyperglycemia, soluble inflammatory molecules (such as cytokines and chemokines, as well as hormones (such as insulin, characterizing the pathophysiology of metabolic syndrome. Moreover, these cardiac processes (resulting in cardiac hypertrophy and fibrosis are also associated with the modulation of intracellular signalling pathways within cardiomyocytes. Amongst the different intracellular kinases, mitogen-activated protein kinases (MAPKs were shown to be involved in heart damage in metabolic syndrome. However, their role remains controversial. In this paper, we will discuss and update evidence on MAPK-mediated mechanisms underlying cardiac adverse remodeling associated with metabolic syndrome.

  14. Drosophila proteins involved in metabolism of uracil-DNA possess different types of nuclear localization signals.

    Science.gov (United States)

    Merényi, Gábor; Kónya, Emese; Vértessy, Beáta G

    2010-05-01

    Adequate transport of large proteins that function in the nucleus is indispensable for cognate molecular events within this organelle. Selective protein import into the nucleus requires nuclear localization signals (NLS) that are recognized by importin receptors in the cytoplasm. Here we investigated the sequence requirements for nuclear targeting of Drosophila proteins involved in the metabolism of uracil-substituted DNA: the recently identified uracil-DNA degrading factor, dUTPase, and the two uracil-DNA glycosylases present in Drosophila. For the uracil-DNA degrading factor, NLS prediction identified two putative NLS sequences [PEKRKQE(320-326) and PKRKKKR(347-353)]. Truncation and site-directed mutagenesis using YFP reporter constructs showed that only one of these basic stretches is critically required for efficient nuclear localization in insect cells. This segment corresponds to the well-known prototypic NLS of SV40 T-antigen. An almost identical NLS segment is also present in the Drosophila thymine-DNA glycosylase, but no NLS elements were predicted in the single-strand-specific monofunctional uracil-DNA glycosylase homolog protein. This latter protein has a molecular mass of 31 kDa, which may allow NLS-independent transport. For Drosophila dUTPase, two isoforms with distinct features regarding molecular mass and subcellular distribution were recently described. In this study, we characterized the basic PAAKKMKID(10-18) segment of dUTPase, which has been predicted to be a putative NLS by in silico analysis. Deletion studies, using YFP reporter constructs expressed in insect cells, revealed the importance of the PAA(10-12) tripeptide and the ID(17-18) dipeptide, as well as the role of the PAAK(10-13) segment in nuclear localization of dUTPase. We constructed a structural model that shows the molecular basis of such recognition in three dimensions.

  15. Advanced oxidation protein products are more related to metabolic syndrome components than biomarkers of lipid peroxidation.

    Science.gov (United States)

    Venturini, Danielle; Simão, Andréa Name Colado; Dichi, Isaias

    2015-09-01

    Although advanced oxidation protein products (AOPPs) have been reported as the most appropriate parameter for determination of oxidative stress in patients with metabolic syndrome (MetS), a direct comparison between protein and lipid peroxidation has not been performed yet. The aim of this study was to compare protein peroxidation with lipid peroxidation measured by 2 different methodologies (tert-butyl hydroperoxide-initiated chemiluminescence and ferrous oxidation-xylenol orange assay). The hypothesis of this study was that AOPPs would be more related to MetS than to oxidative markers of lipid peroxidation. This cross-sectional study evaluated 76 patients with MetS and 20 healthy subjects. Prooxidant-antioxidant index (PAI) assessed as AOPP/total radical-trapping antioxidant parameter ratio progressively increased (P peroxidation determined by AOPPs, and especially by PAI, is more related to MetS components than lipid peroxidation. In addition, PAI progressively increased with the number of MetS components. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Regulated transport into the nucleus of herpesviridae DNA replication core proteins.

    Science.gov (United States)

    Gualtiero, Alvisi; Jans, David A; Camozzi, Daria; Avanzi, Simone; Loregian, Arianna; Ripalti, Alessandro; Palù, Giorgio

    2013-09-16

    The Herpesvirdae family comprises several major human pathogens belonging to three distinct subfamilies. Their double stranded DNA genome is replicated in the nuclei of infected cells by a number of host and viral products. Among the latter the viral replication complex, whose activity is strictly required for viral replication, is composed of six different polypeptides, including a two-subunit DNA polymerase holoenzyme, a trimeric primase/helicase complex and a single stranded DNA binding protein. The study of herpesviral DNA replication machinery is extremely important, both because it provides an excellent model to understand processes related to eukaryotic DNA replication and it has important implications for the development of highly needed antiviral agents. Even though all known herpesviruses utilize very similar mechanisms for amplification of their genomes, the nuclear import of the replication complex components appears to be a heterogeneous and highly regulated process to ensure the correct spatiotemporal localization of each protein. The nuclear transport process of these enzymes is controlled by three mechanisms, typifying the main processes through which protein nuclear import is generally regulated in eukaryotic cells. These include cargo post-translational modification-based recognition by the intracellular transporters, piggy-back events allowing coordinated nuclear import of multimeric holoenzymes, and chaperone-assisted nuclear import of specific subunits. In this review we summarize these mechanisms and discuss potential implications for the development of antiviral compounds aimed at inhibiting the Herpesvirus life cycle by targeting nuclear import of the Herpesvirus DNA replicating enzymes.

  17. Specificity Protein 1 Regulates Gene Expression Related to Fatty Acid Metabolism in Goat Mammary Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Jiangjiang Zhu

    2015-01-01

    Full Text Available Specificity protein 1 (SP1 is a ubiquitous transcription factor that plays an important role in controlling gene expression. Although important in mediating the function of various hormones, the role of SP1 in regulating milk fat formation remains unknown. To investigate the sequence and expression information, as well as its role in modulating lipid metabolism, we cloned SP1 gene from mammary gland of Xinong Saanen dairy goat. The full-length cDNA of the SP1 gene is 4376 bp including 103 bp of 5'UTR, 2358 bp of ORF (HM_236311 and 1915 bp of 3'UTR, which is predicted to encode a 786 amino acids polypeptide. Phylogenetic tree analysis showed that goat SP1 has the closest relationship with sheep, followed by bovines (bos taurus, odobenus and ceratotherium, pig, primates (pongo, gorilla, macaca and papio and murine (rattus and mus, while the furthest relationship was with canis and otolemur. Expression was predominant in the lungs, small intestine, muscle, spleen, mammary gland and subcutaneous fat. There were no significant expression level differences between the mammary gland tissues collected at lactation and dry-off period. Overexpression of SP1 in goat mammary epithelial cells (GMECs led to higher mRNA expression level of peroxisome proliferator-activated receptor-γ (PPARγ and lower liver X receptor α (LXRα mRNA level, both of which were crucial in regulating fatty acid metabolism, and correspondingly altered the expression of their downstream genes in GMECs. These results were further enhanced by the silencing of SP1. These findings suggest that SP1 may play an important role in fatty acid metabolism.

  18. Novel Synthesis of Core-Shell Silica Nanoparticles for the Capture of Low Molecular Weight Proteins and Peptides

    Directory of Open Access Journals (Sweden)

    Sergio G. Hernandez-Leon

    2017-10-01

    Full Text Available Silica nanoparticles were functionalized with immobilized molecular bait, Cibacron Blue, and a porous polymeric bis-acrylamide shell. These nanoparticles represent a new alternative to capture low molecular weight (LMW proteins/peptides, that might be potential biomarkers. Functionalized core-shell silica nanoparticles (FCSNP presented a size distribution of 243.9 ± 11.6 nm and an estimated surface charge of −38.1 ± 0.9 mV. The successful attachment of compounds at every stage of synthesis was evidenced by ATR-FTIR. The capture of model peptides was determined by mass spectrometry, indicating that only the peptide with a long sequence of hydrophobic amino acids (alpha zein 34-mer interacted with the molecular bait. FCSNP excluded the high molecular weight protein (HMW, BSA, and captured LMW proteins (myoglobin and aprotinin, as evidenced by SDS-PAGE. Functionalization of nanoparticles with Cibacron Blue was crucial to capture these molecules. FCSNP were stable after twelve months of storage and maintained a capacity of 3.1–3.4 µg/mg.

  19. A sequence-specific core promoter-binding transcription factor recruits TRF2 to coordinately transcribe ribosomal protein genes.

    Science.gov (United States)

    Baumann, Douglas G; Gilmour, David S

    2017-10-13

    Ribosomal protein (RP) genes must be coordinately expressed for proper assembly of the ribosome yet the mechanisms that control expression of RP genes in metazoans are poorly understood. Recently, TATA-binding protein-related factor 2 (TRF2) rather than the TATA-binding protein (TBP) was found to function in transcription of RP genes in Drosophila. Unlike TBP, TRF2 lacks sequence-specific DNA binding activity, so the mechanism by which TRF2 is recruited to promoters is unclear. We show that the transcription factor M1BP, which associates with the core promoter region, activates transcription of RP genes. Moreover, M1BP directly interacts with TRF2 to recruit it to the RP gene promoter. High resolution ChIP-exo was used to analyze in vivo the association of M1BP, TRF2 and TFIID subunit, TAF1. Despite recent work suggesting that TFIID does not associate with RP genes in Drosophila, we find that TAF1 is present at RP gene promoters and that its interaction might also be directed by M1BP. Although M1BP associates with thousands of genes, its colocalization with TRF2 is largely restricted to RP genes, suggesting that this combination is key to coordinately regulating transcription of the majority of RP genes in Drosophila. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. Structure of the protein core of translation initiation factor 2 in apo, GTP-bound and GDP-bound forms

    Energy Technology Data Exchange (ETDEWEB)

    Simonetti, Angelita [IGBMC (Institute of Genetics and of Molecular and Cellular Biology), Centre National de la Recherche Scientifique (CNRS) UMR 7104/Institut National de la Santé de la Recherche Médicale - INSERM U964/Université de Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France); Marzi, Stefano [Architecture et Réactivité de l’ARN, UPR 9002 CNRS, IBMC (Institute of Molecular and Cellular Biology), 15 Rue R. Descartes, 67084 Strasbourg, France, Université de Strasbourg, 67000 Strasbourg (France); Fabbretti, Attilio [University of Camerino, 62032 Camerino (Monaco) (Italy); Hazemann, Isabelle; Jenner, Lasse [IGBMC (Institute of Genetics and of Molecular and Cellular Biology), Centre National de la Recherche Scientifique (CNRS) UMR 7104/Institut National de la Santé de la Recherche Médicale -INSERM U964/Université de Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France); Urzhumtsev, Alexandre [IGBMC (Institute of Genetics and of Molecular and Cellular Biology), Centre National de la Recherche Scientifique (CNRS) UMR 7104/Institut National de la Santé de la Recherche Médicale - INSERM U964/Université de Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France); Université de Lorraine, 54506 Vandoeuvre-lès-Nancy (France); Gualerzi, Claudio O. [University of Camerino, 62032 Camerino (Monaco) (Italy); Klaholz, Bruno P., E-mail: klaholz@igbmc.fr [IGBMC (Institute of Genetics and of Molecular and Cellular Biology), Centre National de la Recherche Scientifique (CNRS) UMR 7104/Institut National de la Santé de la Recherche Médicale - INSERM U964/Université de Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France)

    2013-06-01

    The crystal structures of the eubacterial translation initiation factor 2 in apo form and with bound GDP and GTP reveal conformational changes upon nucleotide binding and hydrolysis, notably of the catalytically important histidine in the switch II region. Translation initiation factor 2 (IF2) is involved in the early steps of bacterial protein synthesis. It promotes the stabilization of the initiator tRNA on the 30S initiation complex (IC) and triggers GTP hydrolysis upon ribosomal subunit joining. While the structure of an archaeal homologue (a/eIF5B) is known, there are significant sequence and functional differences in eubacterial IF2, while the trimeric eukaryotic IF2 is completely unrelated. Here, the crystal structure of the apo IF2 protein core from Thermus thermophilus has been determined by MAD phasing and the structures of GTP and GDP complexes were also obtained. The IF2–GTP complex was trapped by soaking with GTP in the cryoprotectant. The structures revealed conformational changes of the protein upon nucleotide binding, in particular in the P-loop region, which extend to the functionally relevant switch II region. The latter carries a catalytically important and conserved histidine residue which is observed in different conformations in the GTP and GDP complexes. Overall, this work provides the first crystal structure of a eubacterial IF2 and suggests that activation of GTP hydrolysis may occur by a conformational repositioning of the histidine residue.

  1. Differential regulation of hepatitis B virus core protein expression and genome replication by a small upstream open reading frame and naturally occurring mutations in the precore region.

    Science.gov (United States)

    Zong, Li; Qin, Yanli; Jia, Haodi; Ye, Lei; Wang, Yongxiang; Zhang, Jiming; Wands, Jack R; Tong, Shuping; Li, Jisu

    2017-05-01

    Hepatitis B virus (HBV) transcribes two subsets of 3.5-kb RNAs: precore RNA for hepatitis B e antigen (HBeAg) expression, and pregenomic RNA for core and P protein translation as well as genome replication. HBeAg expression could be prevented by mutations in the precore region, while an upstream open reading frame (uORF) has been proposed as a negative regulator of core protein translation. We employed replication competent HBV DNA constructs and transient transfection experiments in Huh7 cells to verify the uORF effect and to explore the alternative function of precore RNA. Optimized Kozak sequence for the uORF or extra ATG codons as present in some HBV genotypes reduced core protein expression. G1896A nonsense mutation promoted more efficient core protein expression than mutated precore ATG, while a +1 frameshift mutation was ineffective. In conclusion, various HBeAg-negative precore mutations and mutations affecting uORF differentially regulate core protein expression and genome replication. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Skin sensitization: Modeling based on skin metabolism simulation and formation of protein conjugates

    DEFF Research Database (Denmark)

    Dimitrov, Sabcho; Low, Lawrence; Patlewicz, Grace

    2005-01-01

    A quantitative structure-activity relationship (QSAR) system for estimating skin sensitization potency has been developed that incorporates skin metabolism and considers the potential of parent chemicals and/or their activated metabolites to react with skin proteins. A training set of diverse...... alerting groups, three-dimensional (3D)-QSARs were developed to describe the multiplicity of physicochemical, steric, and electronic parameters. These 3D-QSARs, so-called pattern recognition-type models, were applied each time a latent alerting group was identified in a parent chemical or its generated...... metabolite(s). The concept of the mutual influence amongst atoms in a molecule was used to define the structural domain of the skin sensitization model. The utility of the structural model domain and the predictability of the model were evaluated using sensitization potency data for 96 chemicals not used...

  3. The structure of membrane associated proteins in eicosanoid and glutathione metabolism as determined by electron crystallography.

    Science.gov (United States)

    Hebert, Hans; Jegerschöld, Caroline

    2007-08-01

    Membrane associated proteins in eicosanoid and glutathione metabolism (MAPEG) are involved in biosynthesis of arachidonic-derived mediators of pain, fever, and inflammation as well as in biotransformation and detoxification of electrophilic substances. Structure determination of microsomal glutathione transferase 1 using electron crystallography has provided the first atomic model of an MAPEG member. The homotrimer consists of three repeats of a four-helix transmembrane bundle with the largest extramembranous domain connecting the first and second helix and with a short proline rich loop on the same side between helices three and four. Residues of importance for intramolecular or intermolecular contacts as well as for stabilizing the active site have been identified and the results can be applied for interpreting structure-function relationship for similar MAPEG members.

  4. Hepatic mitogen-activated protein kinase phosphatase 1 selectively regulates glucose metabolism and energy homeostasis.

    Science.gov (United States)

    Lawan, Ahmed; Zhang, Lei; Gatzke, Florian; Min, Kisuk; Jurczak, Michael J; Al-Mutairi, Mashael; Richter, Patric; Camporez, Joao Paulo G; Couvillon, Anthony; Pesta, Dominik; Roth Flach, Rachel J; Shulman, Gerald I; Bennett, Anton M

    2015-01-01

    The liver plays a critical role in glucose metabolism and communicates with peripheral tissues to maintain energy homeostasis. Obesity and insulin resistance are highly associated with nonalcoholic fatty liver disease (NAFLD). However, the precise molecular details of NAFLD remain incomplete. The p38 mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK) regulate liver metabolism. However, the physiological contribution of MAPK phosphatase 1 (MKP-1) as a nuclear antagonist of both p38 MAPK and JNK in the liver is unknown. Here we show that hepatic MKP-1 becomes overexpressed following high-fat feeding. Liver-specific deletion of MKP-1 enhances gluconeogenesis and causes hepatic insulin resistance in chow-fed mice while selectively conferring protection from hepatosteatosis upon high-fat feeding. Further, hepatic MKP-1 regulates both interleukin-6 (IL-6) and fibroblast growth factor 21 (FGF21). Mice lacking hepatic MKP-1 exhibit reduced circulating IL-6 and FGF21 levels that were associated with impaired skeletal muscle mitochondrial oxidation and susceptibility to diet-induced obesity. Hence, hepatic MKP-1 serves as a selective regulator of MAPK-dependent signals that contributes to the maintenance of glucose homeostasis and peripheral tissue energy balance. These results also demonstrate that hepatic MKP-1 overexpression in obesity is causally linked to the promotion of hepatosteatosis. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  5. The role of Monosaccharide Transport Proteins in carbohydrate assimilation, distribution, metabolism and homeostasis

    Science.gov (United States)

    Cura, Anthony J.; Carruthers, Anthony

    2012-01-01

    The facilitated diffusion of glucose, galactose, fructose, urate, myoinositol and dehydroascorbic acid in mammals is catalyzed by a family of 14 monosaccharide transport proteins called GLUTs. These transporters may be divided into 3 classes according to sequence similarity and function/substrate specificity. GLUT1 appears to be highly expressed in glycolytically active cells and has been co-opted in vitamin C auxotrophs to maintain the redox state of the blood through transport of dehydroascorbate. Several GLUTs are definitive glucose/galactose transporters, GLUT2 and GLUT5 are physiologically important fructose transporters, GLUT9 appears to be a urate transporter while GLUT13 (HMIT1) is a proton/myoinositol co-transporter. The physiologic substrates of some GLUTs remain to be established. The GLUTs are expressed in a tissue specific manner where affinity, specificity and capacity for substrate transport are paramount for tissue function. Although great strides have been made in characterizing GLUT-catalyzed monosaccharide transport and mapping GLUT membrane topography and determinants of substrate specificity, a unifying model for GLUT structure and function remains elusive. The GLUTs play a major role in carbohydrate homeostasis and the redistribution of sugar-derived carbons among the various organ systems. This is accomplished through a multiplicity of GLUT-dependent glucose sensing and effector mechanisms that regulate monosaccharide ingestion, absorption, distribution, cellular transport and metabolism and recovery/retention. Glucose transport and metabolism have co-evolved in mammals to support cerebral glucose utilization. PMID:22943001

  6. C-reactive protein and metabolic syndrome in youth: a strong relationship?

    Science.gov (United States)

    Oliveira, Antônio C; Oliveira, Ana M; Adan, Luis F; Oliveira, Nelson F; Silva, Agnaluce M; Ladeia, Ana M

    2008-05-01

    Metabolic syndrome (MS) is on the rise in youth. As high-sensitivity C-reactive protein (hs-CRP) is associated with cardiovascular/metabolic disorders, we evaluated the association between MS and its components and hs-CRP in a sample of Brazilian overweight and obese youth. A total of 407 students (229 girls, 273 with excessive weight, 11.3+/-3.2 years) were evaluated. Measurement included BMI, waist circumference (WC), blood pressure, lipids, insulin, and hs-CRP. Excessive weight was defined using BMI z -score; MS by the modified National Cholesterol Education Program-Adult Treatment Panel III. Subjects were classified into two groups: with MS (n=72) and without (n=335). hs-CRP means and medians were higher in MS group (1.41 mg/l vs. 1.06 mg/l, Plow HDL-c (P=0.023) were significant; adjustment of hs-CRP for BMI z-score eliminated the previous association, except for the number of MS components (nMSc) (Pconfidence interval (CI), 4.7-13.4; P=0.000), hypertension (OR, 2.3; CI, 1.3-4.2; P=0.003), and hypertriglyceridemia (OR, 2.3; CI, 1.5-3.7; Pyouth, hs-CRP is strongly related with MS and its components, and is also determined by the body composition. This association indicates a precocious proinflammatory state.

  7. Selective processing and metabolism of disease-causing mutant prion proteins.

    Directory of Open Access Journals (Sweden)

    Aarthi Ashok

    2009-06-01

    Full Text Available Prion diseases are fatal neurodegenerative disorders caused by aberrant metabolism of the cellular prion protein (PrP(C. In genetic forms of these diseases, mutations in the globular C-terminal domain are hypothesized to favor the spontaneous generation of misfolded PrP conformers (including the transmissible PrP(Sc form that trigger downstream pathways leading to neuronal death. A mechanistic understanding of these diseases therefore requires knowledge of the quality control pathways that recognize and degrade aberrant PrPs. Here, we present comparative analyses of the biosynthesis, trafficking, and metabolism of a panel of genetic disease-causing prion protein mutants in the C-terminal domain. Using quantitative imaging and biochemistry, we identify a misfolded subpopulation of each mutant PrP characterized by relative detergent insolubility, inaccessibility to the cell surface, and incomplete glycan modifications. The misfolded populations of mutant PrPs were neither recognized by ER quality control pathways nor routed to ER-associated degradation despite demonstrable misfolding in the ER. Instead, mutant PrPs trafficked to the Golgi, from where the misfolded subpopulation was selectively trafficked for degradation in acidic compartments. Surprisingly, selective re-routing was dependent not only on a mutant globular domain, but on an additional lysine-based motif in the highly conserved unstructured N-terminus. These results define a specific trafficking and degradation pathway shared by many disease-causing PrP mutants. As the acidic lysosomal environment has been implicated in facilitating the conversion of PrP(C to PrP(Sc, our identification of a mutant-selective trafficking pathway to this compartment may provide a cell biological basis for spontaneous generation of PrP(Sc in familial prion disease.

  8. The identification of the heat-stable microsomal protein required for methoxyflurane metabolism as cytochrome b5.

    Science.gov (United States)

    Canova-Davis, E; Waskell, L

    1984-02-25

    Methoxyflurane is an anesthetic whose metabolism by cytochrome P-450LM2 has been shown to be dependent upon a heat-stable microsomal protein (Canova-Davis, E., and Waskell, L. A. (1982) Biochem. Biophys. Res. Commun. 108, 1264-1270). Treatment of this protein with diethylpyrocarbonate, which modifies selected amino acids, caused a dose-dependent loss in its ability to effect the metabolism of methoxyflurane by purified cytochrome P-450LM2. This protein factor has been identified as cytochrome b5 by demonstrating that cytochrome b5 and the heat-stable factor coelute during cytochrome b5 purification. Neither ferriheme nor apocytochrome b5 was able to substitute for the activating factor, while cytochrome b5 reconstituted from apocytochrome b5 and heme exhibited an activity similar to that of native b5. Examination of the cytochrome b5 molecule by computer graphics suggested that diethylpyrocarbonate did not inactivate b5 by reacting with the anionic surface of the cytochrome b5 molecule. Maximal rates of methoxyflurane metabolism were obtained at a ratio of 1:1:1 of the three proteins, cytochrome P-450LM2:reductase:cytochrome b5. In summary, it has been demonstrated that the heat-stable protein, cytochrome b5, is obligatory for the metabolism of methoxyflurane by cytochrome P-450LM2. These data also suggest that cytochrome b5 may be acting as an electron donor to P-450LM2 in the O-demethylation of methoxyflurane.

  9. Moderate physical training counterbalances harmful effects of low-protein diet on heart: metabolic, oxidative and morphological parameters

    Directory of Open Access Journals (Sweden)

    Déborah S. Carthagenes

    2017-12-01

    Full Text Available ABSTRACT Aims Maternal low-protein diet induces several impairments on cardiac system. Conversely, moderate exercise has been widely recommended to health improvement due to its effects on heart function. Thus, we investigated whether the moderate physical training is capable to offset the lasting injuries of a maternal protein restriction on the hearts of male adult rats. Methods Pregnant rats were divided into two groups: Control (C=17% casein and undernutrition (U=8% casein. Offspring from the undernutrition group, at 60 days of life, were subdivided into undernutrition (U and undernutrition+exercise (UT groups. Treadmill exercise was performed: (8 weeks, 5 days/week, 60 min/day at 70% of VO2máx. 48 hours after last exercise session, tissues were collected for morphological and biochemical analysis. Results Despite the deleterious effect induced by low-protein diet, physical training was able to restore morphological parameters to similar levels to the control group. Additionally, oxidative stress index was also improved in UT group, due to the increase in antioxidant enzymatic defense. In metabolic enzymes, maternal low-protein diet induced a change in metabolism, and moderate physical training improved oxidative metabolism. Conclusion We demonstrated that moderate physical training can offset the cardiac metabolism in adult rats that were exposed to a maternal low-protein diet.

  10. Effects of different protein and glycemic index diets on metabolic profiles and substrate partitioning in lean healthy males.

    Science.gov (United States)

    Munsters, Marjet J; Geraedts, Maartje C; Saris, Wim H

    2013-11-01

    Dietary glycemic index (GI) and protein affects postprandial insulin responses and consequently 24 h glucose metabolism and therefore substrate partitioning. This study investigated the mechanistic effects of different protein and GI diets on 24 h profiles of metabolic markers and substrate partitioning. After 3 days of diet and physical activity standardization, 10 healthy male subjects (BMI: 22.5 ± 0.6 kg/m(2)) stayed in a respiration chamber 4 times for 36 h each time to measure substrate partitioning. All subjects randomly received four isoenergetic diets: a normal (15En%) dairy protein and low GI (60 units) (NDP-HGI) diet. During the day, blood was sampled at fixed time points for the measurement of metabolic markers and satiety hormones. The HDP-LGI diet increased 24 h protein oxidation and sleeping metabolic rate (SMR) compared with the NDP-LGI diet (p carbohydrate and fat oxidation (day and night) were found between all intervention diets. Net incremental area under the curve (net iAUC) of 24 h plasma glucose decreased in the HDP-LGI diet compared with the NDP-LGI diet (p insulin levels. No difference in appetite profiles were observed between all intervention diets. The lower 24 h glycemic profile as a result of a high dairy protein diet did not lead to changes in 24 h substrate partitioning in lean healthy subjects with a normal insulin sensitivity.

  11. Role of Decorin Core Protein in Collagen Organisation in Congenital Stromal Corneal Dystrophy (CSCD)

    Science.gov (United States)

    Kamma-Lorger, Christina S.; Pinali, Christian; Martínez, Juan Carlos; Harris, Jon; Young, Robert D.; Bredrup, Cecilie; Crosas, Eva; Malfois, Marc; Rødahl, Eyvind

    2016-01-01

    The role of Decorin in organising the extracellular matrix was examined in normal human corneas and in corneas from patients with Congenital Stromal Corneal Dystrophy (CSCD). In CSCD, corneal clouding occurs due to a truncating mutation (c.967delT) in the decorin (DCN) gene. Normal human Decorin protein and the truncated one were reconstructed in silico using homology modelling techniques to explore structural changes in the diseased protein. Corneal CSCD specimens were also examined using 3-D electron tomography and Small Angle X-ray diffraction (SAXS), to image the collagen-proteoglycan arrangement and to quantify fibrillar diameters, respectively. Homology modelling showed that truncated Decorin had a different spatial geometry to the normal one, with the truncation removing a major part of the site that interacts with collagen, compromising its ability to bind effectively. Electron tomography showed regions of abnormal stroma, where collagen fibrils came together to form thicker fibrillar structures, showing that Decorin plays a key role in the maintenance of the order in the normal corneal extracellular matrix. Average diameter of individual fibrils throughout the thickness of the cornea however remained normal. PMID:26828927

  12. Role of Decorin Core Protein in Collagen Organisation in Congenital Stromal Corneal Dystrophy (CSCD.

    Directory of Open Access Journals (Sweden)

    Christina S Kamma-Lorger

    Full Text Available The role of Decorin in organising the extracellular matrix was examined in normal human corneas and in corneas from patients with Congenital Stromal Corneal Dystrophy (CSCD. In CSCD, corneal clouding occurs due to a truncating mutation (c.967delT in the decorin (DCN gene. Normal human Decorin protein and the truncated one were reconstructed in silico using homology modelling techniques to explore structural changes in the diseased protein. Corneal CSCD specimens were also examined using 3-D electron tomography and Small Angle X-ray diffraction (SAXS, to image the collagen-proteoglycan arrangement and to quantify fibrillar diameters, respectively. Homology modelling showed that truncated Decorin had a different spatial geometry to the normal one, with the truncation removing a major part of the site that interacts with collagen, compromising its ability to bind effectively. Electron tomography showed regions of abnormal stroma, where collagen fibrils came together to form thicker fibrillar structures, showing that Decorin plays a key role in the maintenance of the order in the normal corneal extracellular matrix. Average diameter of individual fibrils throughout the thickness of the cornea however remained normal.

  13. Conserved function of core clock proteins in the gymnosperm Norway spruce (Picea abies L. Karst).

    Science.gov (United States)

    Karlgren, Anna; Gyllenstrand, Niclas; Källman, Thomas; Lagercrantz, Ulf

    2013-01-01

    From studies of the circadian clock in the plant model species Arabidopsis (Arabidopsis thaliana), a number of important properties and components have emerged. These include the genes CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), GIGANTEA (GI), ZEITLUPE (ZTL) and TIMING OF CAB EXPRESSION 1 (TOC1 also known as PSEUDO-RESPONSE REGULATOR 1 (PRR1)) that via gene expression feedback loops participate in the circadian clock. Here, we present results from ectopic expression of four Norway spruce (Picea abies) putative homologs (PaCCA1, PaGI, PaZTL and PaPRR1) in Arabidopsis, their flowering time, circadian period length, red light response phenotypes and their effect on endogenous clock genes were assessed. For PaCCA1-ox and PaZTL-ox the results were consistent with Arabidopsis lines overexpressing the corresponding Arabidopsis genes. For PaGI consistent results were obtained when expressed in the gi2 mutant, while PaGI and PaPRR1 expressed in wild type did not display the expected phenotypes. These results suggest that protein function of PaCCA1, PaGI and PaZTL are at least partly conserved compared to Arabidopsis homologs, however further studies are needed to reveal the protein function of PaPRR1. Our data suggest that components of the three-loop network typical of the circadian clock in angiosperms were present before the split of gymnosperms and angiosperms.

  14. Conserved function of core clock proteins in the gymnosperm Norway spruce (Picea abies L. Karst.

    Directory of Open Access Journals (Sweden)

    Anna Karlgren

    Full Text Available From studies of the circadian clock in the plant model species Arabidopsis (Arabidopsis thaliana, a number of important properties and components have emerged. These include the genes CIRCADIAN CLOCK ASSOCIATED 1 (CCA1, GIGANTEA (GI, ZEITLUPE (ZTL and TIMING OF CAB EXPRESSION 1 (TOC1 also known as PSEUDO-RESPONSE REGULATOR 1 (PRR1 that via gene expression feedback loops participate in the circadian clock. Here, we present results from ectopic expression of four Norway spruce (Picea abies putative homologs (PaCCA1, PaGI, PaZTL and PaPRR1 in Arabidopsis, their flowering time, circadian period length, red light response phenotypes and their effect on endogenous clock genes were assessed. For PaCCA1-ox and PaZTL-ox the results were consistent with Arabidopsis lines overexpressing the corresponding Arabidopsis genes. For PaGI consistent results were obtained when expressed in the gi2 mutant, while PaGI and PaPRR1 expressed in wild type did not display the expected phenotypes. These results suggest that protein function of PaCCA1, PaGI and PaZTL are at least partly conserved compared to Arabidopsis homologs, however further studies are needed to reveal the protein function of PaPRR1. Our data suggest that components of the three-loop network typical of the circadian clock in angiosperms were present before the split of gymnosperms and angiosperms.

  15. Protein costs do not explain evolution of metabolic strategies and regulation of ribosomal content : does protein investment explain an anaerobic bacterial Crabtree effect?

    NARCIS (Netherlands)

    Goel, Anisha; Eckhardt, Thomas H.; Puri, Pranav; de Jong, Anne; Branco dos Santos, Filipe; Giera, Martin; Fusetti, Fabrizia; de Vos, Willem M.; Kok, Jan; Poolman, Bert; Molenaar, Douwe; Kuipers, Oscar P.; Teusink, Bas

    Protein investment costs are considered a major driver for the choice of alternative metabolic strategies. We tested this premise in Lactococcus lactis, a bacterium that exhibits a distinct, anaerobic version of the bacterial Crabtree/Warburg effect; with increasing growth rates it shifts from a

  16. Protein costs do not explain evolution of metabolic strategies and regulation of ribosomal content: does protein investment explain an anaerobic bacterial Crabtree effect?

    NARCIS (Netherlands)

    Goel, A.; Eckhardt, T.H.; Puri, P.; de Jong, A.; Branco dos Santos, F.; Giera, M.; Fusetti, F.; de Vos, W.M.; de Kok, J.; Poolman, B.; Molenaar, D.; Kuipers, O.P.; Teusink, B.

    2015-01-01

    Protein investment costs are considered a major driver for the choice of alternative metabolic strategies. We tested this premise in Lactococcus lactis, a bacterium that exhibits a distinct, anaerobic version of the bacterial Crabtree/Warburg effect; with increasing growth rates it shifts from a

  17. A Single Amino Acid Substitution in the Core Protein of West Nile Virus Increases Resistance to Acidotropic Compounds

    Science.gov (United States)

    Martín-Acebes, Miguel A.; Blázquez, Ana-Belén; de Oya, Nereida Jiménez; Escribano-Romero, Estela; Shi, Pei-Yong; Saiz, Juan-Carlos

    2013-01-01

    West Nile virus (WNV) is a worldwide distributed mosquito-borne flavivirus that naturally cycles between birds and mosquitoes, although it can infect multiple vertebrate hosts including horses and humans. This virus is responsible for recurrent epidemics of febrile illness and encephalitis, and has recently become a global concern. WNV requires to transit through intracellular acidic compartments at two different steps to complete its infectious cycle. These include fusion between the viral envelope and the membrane of endosomes during viral entry, and virus maturation in the trans-Golgi network. In this study, we followed a genetic approach to study the connections between viral components and acidic pH. A WNV mutant with increased resistance to the acidotropic compound NH4Cl, which blocks organelle acidification and inhibits WNV infection, was selected. Nucleotide sequencing revealed that this mutant displayed a single amino acid substitution (Lys 3 to Glu) on the highly basic internal capsid or core (C) protein. The functional role of this replacement was confirmed by its introduction into a WNV infectious clone. This single amino acid substitution also increased resistance to other acidification inhibitor (concanamycin A) and induced a reduction of the neurovirulence in mice. Interestingly, a naturally occurring accompanying mutation found on prM protein abolished the resistant phenotype, supporting the idea of a genetic crosstalk between the internal C protein and the external glycoproteins of the virion. The findings here reported unveil a non-previously assessed connection between the C viral protein and the acidic pH necessary for entry and proper exit of flaviviruses. PMID:23874963

  18. The dense core vesicle protein IA-2, but not IA-2β, is required for active avoidance learning.

    Science.gov (United States)

    Carmona, G N; Nishimura, T; Schindler, C W; Panlilio, L V; Notkins, A L

    2014-06-06

    The islet-antigens IA-2 and IA-2β are major autoantigens in type-1 diabetes and transmembrane proteins in dense core vesicles (DCV). Recently we showed that deletion of both IA-2 and IA-2β alters the secretion of hormones and neurotransmitters and impairs behavior and learning. The present study was designed to evaluate the contribution to learning of each of these genes by using single knockout (SKO) and double knockout (DKO) mice in an active avoidance test. After 5 days of training, wild-type (WT) mice showed 60-70% active avoidance responses, whereas the DKO mice showed only 10-15% active avoidance responses. The degree of active avoidance responses in the IA-2 SKO mice was similar to that of the DKO mice, but in contrast, the IA-2β SKO mice behaved like WT mice showing 60-70% active avoidance responses. Molecular studies revealed a marked decrease in the phosphorylation of the cAMP response element-binding protein (CREB) and Ca(2+)/calmodulin-dependent protein kinase II (CAMKII) in the striatum and hippocampus of the IA-2 SKO and DKO mice, but not in the IA-2β SKO mice. To evaluate the role of CREB and CAMKII in the SKO and DKO mice, GBR-12909, which selectively blocks the dopamine uptake transporter and increases CREB and CAMKII phosphorylation, was administered. GBR-12909 restored the phosphorylation of CREB and CAMKII and increased active avoidance learning in the DKO and IA-2 SKO to near the normal levels found in the WT and IA-2β SKO mice. We conclude that in the absence of the DCV protein IA-2, active avoidance learning is impaired. Published by Elsevier Ltd.

  19. The diverse functions of the hepatitis B core/capsid protein (HBc) in the viral life cycle: Implications for the development of HBc-targeting antivirals.

    Science.gov (United States)

    Diab, Ahmed; Foca, Adrien; Zoulim, Fabien; Durantel, David; Andrisani, Ourania

    2018-01-01

    Virally encoded proteins have evolved to perform multiple functions, and the core protein (HBc) of the hepatitis B virus (HBV) is a perfect example. While HBc is the structural component of the viral nucleocapsid, additional novel functions for the nucleus-localized HBc have recently been described. These results extend for HBc, beyond its structural role, a regulatory function in the viral life cycle and potentially a role in pathogenesis. In this article, we review the diverse roles of HBc in HBV replication and pathogenesis, emphasizing how the unique structure of this protein is key to its various functions. We focus in particular on recent advances in understanding the significance of HBc phosphorylations, its interaction with host proteins and the role of HBc in regulating the transcription of host genes. We also briefly allude to the emerging niche for new direct-acting antivirals targeting HBc, known as Core (protein) Allosteric Modulators (CAMs). Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Systematic functional comparative analysis of four single-stranded DNA-binding proteins and their affection on viral RNA metabolism.

    Directory of Open Access Journals (Sweden)

    Haiyan Shi

    Full Text Available The accumulation of single-stranded DNA-binding (SSB proteins is essential for organisms and has various applications. However, no study has simultaneously and systematically compared the characteristics of SSB proteins. In addition, SSB proteins may bind RNA and play an unknown biological role in RNA metabolism. Here, we expressed a novel species of SSB protein derived from Thermococcus kodakarensis KOD1 (KOD, as well as SSB proteins from Thermus thermophilus (TTH, Escherichia coli, and Sulfolobus Solfataricus P2 (SSOB, abbreviated kod, tth, bl21, and ssob, respectively. These SSB proteins could bind ssDNA and viral RNA. bl21 resisted heat treatment for more than 9 h, Ssob and kod could withstand 95°C for 10 h and retain its ssDNA- and RNA-binding ability. Four SSB proteins promoted the specificity of the DNA polymerase in PCR-based 5- and 9-kb genome fragment amplification. kod also increased the amplification of a 13-kb PCR product, and SSB protein-bound RNA resisted Benzonase digestion. The SSB proteins could also enter the host cell bound to RNA, which resulted in modulation of viral RNA metabolism, particularly ssob and bl21.

  1. The SNARE protein vti1a functions in dense-core vesicle biogenesis

    DEFF Research Database (Denmark)

    Walter, Alexander M; Kurps, Julia; de Wit, Heidi

    2014-01-01

    The SNARE protein vti1a is proposed to drive fusion of intracellular organelles, but recent data also implicated vti1a in exocytosis. Here we show that vti1a is absent from mature secretory vesicles in adrenal chromaffin cells, but localizes to a compartment near the trans-Golgi network, partially...... overlapping with syntaxin-6. Exocytosis is impaired in vti1a null cells, partly due to fewer Ca(2+)-channels at the plasma membrane, partly due to fewer vesicles of reduced size and synaptobrevin-2 content. In contrast, release kinetics and Ca(2+)-sensitivity remain unchanged, indicating that the final fusion...... reaction leading to transmitter release is unperturbed. Additional deletion of the closest related SNARE, vti1b, does not exacerbate the vti1a phenotype, and vti1b null cells show no secretion defects, indicating that vti1b does not participate in exocytosis. Long-term re-expression of vti1a (days...

  2. Destabilizing polymorphism in cervid prion protein hydrophobic core determines prion conformation and conversion efficiency.

    Directory of Open Access Journals (Sweden)

    Samia Hannaoui

    2017-08-01

    Full Text Available Prion diseases are infectious neurodegenerative disorders of humans and animals caused by misfolded forms of the cellular prion protein PrPC. Prions cause disease by converting PrPC into aggregation-prone PrPSc. Chronic wasting disease (CWD is the most contagious prion disease with substantial lateral transmission, affecting free-ranging and farmed cervids. Although the PrP primary structure is highly conserved among cervids, the disease phenotype can be modulated by species-specific polymorphisms in the prion protein gene. How the resulting amino-acid substitutions impact PrPC and PrPSc structure and propagation is poorly understood. We investigated the effects of the cervid 116A>G substitution, located in the most conserved PrP domain, on PrPC structure and conversion and on 116AG-prion conformation and infectivity. Molecular dynamics simulations revealed structural de-stabilization of 116G-PrP, which enhanced its in vitro conversion efficiency when used as recombinant PrP substrate in real-time quaking-induced conversion (RT-QuIC. We demonstrate that 116AG-prions are conformationally less stable, show lower activity as a seed in RT-QuIC and exhibit reduced infectivity in vitro and in vivo. Infectivity of 116AG-prions was significantly enhanced upon secondary passage in mice, yet conformational features were retained. These findings indicate that structurally de-stabilized PrPC is readily convertible by cervid prions of different genetic background and results in a prion conformation adaptable to cervid wild-type PrP. Conformation is an important criterion when assessing transmission barrier, and conformational variants can target a different host range. Therefore, a thorough analysis of CWD isolates and re-assessment of species-barriers is important in order to fully exclude a zoonotic potential of CWD.

  3. Destabilizing polymorphism in cervid prion protein hydrophobic core determines prion conformation and conversion efficiency

    Science.gov (United States)

    Hannaoui, Samia; Amidian, Sara; Cheng, Yo Ching; Duque Velásquez, Camilo; Law, Sampson; Telling, Glenn; Stepanova, Maria; McKenzie, Debbie

    2017-01-01

    Prion diseases are infectious neurodegenerative disorders of humans and animals caused by misfolded forms of the cellular prion protein PrPC. Prions cause disease by converting PrPC into aggregation-prone PrPSc. Chronic wasting disease (CWD) is the most contagious prion disease with substantial lateral transmission, affecting free-ranging and farmed cervids. Although the PrP primary structure is highly conserved among cervids, the disease phenotype can be modulated by species-specific polymorphisms in the prion protein gene. How the resulting amino-acid substitutions impact PrPC and PrPSc structure and propagation is poorly understood. We investigated the effects of the cervid 116A>G substitution, located in the most conserved PrP domain, on PrPC structure and conversion and on 116AG-prion conformation and infectivity. Molecular dynamics simulations revealed structural de-stabilization of 116G-PrP, which enhanced its in vitro conversion efficiency when used as recombinant PrP substrate in real-time quaking-induced conversion (RT-QuIC). We demonstrate that 116AG-prions are conformationally less stable, show lower activity as a seed in RT-QuIC and exhibit reduced infectivity in vitro and in vivo. Infectivity of 116AG-prions was significantly enhanced upon secondary passage in mice, yet conformational features were retained. These findings indicate that structurally de-stabilized PrPC is readily convertible by cervid prions of different genetic background and results in a prion conformation adaptable to cervid wild-type PrP. Conformation is an important criterion when assessing transmission barrier, and conformational variants can target a different host range. Therefore, a thorough analysis of CWD isolates and re-assessment of species-barriers is important in order to fully exclude a zoonotic potential of CWD. PMID:28800624

  4. Ptc7p Dephosphorylates Select Mitochondrial Proteins to Enhance Metabolic Function

    Directory of Open Access Journals (Sweden)

    Xiao Guo

    2017-01-01

    Full Text Available Proper maintenance of mitochondrial activity is essential for metabolic homeostasis. Widespread phosphorylation of mitochondrial proteins may be an important element of this process; yet, little is known about which enzymes control mitochondrial phosphorylation or which phosphosites have functional impact. We investigate these issues by disrupting Ptc7p, a conserved but largely uncharacterized mitochondrial matrix PP2C-type phosphatase. Loss of Ptc7p causes respiratory growth defects concomitant with elevated phosphorylation of select matrix proteins. Among these, Δptc7 yeast exhibit an increase in phosphorylation of Cit1p, the canonical citrate synthase of the tricarboxylic acid (TCA cycle, that diminishes its activity. We find that phosphorylation of S462 can eliminate Cit1p enzymatic activity likely by disrupting its proper dimerization, and that Ptc7p-driven dephosphorylation rescues Cit1p activity. Collectively, our work connects Ptc7p to an essential TCA cycle function and to additional phosphorylation events that may affect mitochondrial activity inadvertently or in a regulatory manner.

  5. Coupling Bioorthogonal Chemistries with Artificial Metabolism: Intracellular Biosynthesis of Azidohomoalanine and Its Incorporation into Recombinant Proteins

    Directory of Open Access Journals (Sweden)

    Ying Ma

    2014-01-01

    Full Text Available In this paper, we present a novel, “single experiment” methodology based on genetic engineering of metabolic pathways for direct intracellular production of non-canonical amino acids from simple precursors, coupled with expanded genetic code. In particular, we engineered the intracellular biosynthesis of L-azidohomoalanine from O-acetyl-L-homoserine and NaN3, and achieved its direct incorporation into recombinant target proteins by AUG codon reassignment in a methionine-auxotroph E. coli strain. In our system, the host’s methionine biosynthetic pathway was first diverted towards the production of the desired non-canonical amino acid by exploiting the broad reaction specificity of recombinant pyridoxal phosphate-dependent O-acetylhomoserine sulfhydrylase from Corynebacterium glutamicum. Then, the expression of the target protein barstar, accompanied with efficient L-azidohomoalanine incorporation in place of L-methionine, was accomplished. This work stands as proof-of-principle and paves the way for additional work towards intracellular production and site-specific incorporation of biotechnologically relevant non-canonical amino acids directly from common fermentable sources.

  6. Difference in fibril core stability between two tau four-repeat domain proteins: a hydrogen-deuterium exchange coupled to mass spectrometry study.

    Science.gov (United States)

    Ramachandran, Gayathri; Udgaonkar, Jayant B

    2013-12-10

    One of the signatures of Alzheimer's disease and tauopathies is fibrillization of the microtubule-associated protein tau. The purpose of this study was to compare the high-resolution structure of fibrils formed by two different tau four-repeat domain constructs, tau4RD and tauK18, using hydrogen-deuterium exchange coupled to mass spectrometry as a tool. While the two fibrils are found to be constructed on similar structural principles, the tauK18 fibril has a slightly more stable core. This difference in fibril core stability appears to be reflective of the mechanistic differences in the aggregation pathways of the two proteins.

  7. Critical analysis of protein signaling networks involved in the regulation of plant secondary metabolism: focus on anthocyanins.

    Science.gov (United States)

    Bulgakov, Victor P; Avramenko, Tatiana V; Tsitsiashvili, Gurami Sh

    2017-09-01

    Anthocyanin biosynthesis in Arabidopsis is a convenient and relatively simple model for investigating the basic principles of secondary metabolism regulation. In recent years, many publications have described links between anthocyanin biosynthesis and general defense reactions in plants as well as photomorphogenesis and hormonal signaling. These relationships are complex, and they cannot be understood intuitively. Upon observing the lacuna in the Arabidopsis interactome (an interaction map of the factors involved in the regulation of Arabidopsis secondary metabolism is not available), we attempted to connect various cellular processes that affect anthocyanin biosynthesis. In this review, we revealed the main signaling protein modules that regulate anthocyanin biosynthesis. To our knowledge, this is the first reconstruction of a network of proteins involved in plant secondary metabolism.

  8. A DNA Structural Alphabet Distinguishes Structural Features of DNA Bound to Regulatory Proteins and in the Nucleosome Core Particle.

    Science.gov (United States)

    Schneider, Bohdan; Božíková, Paulína; Čech, Petr; Svozil, Daniel; Černý, Jiří

    2017-10-18

    We analyzed the structural behavior of DNA complexed with regulatory proteins and the nucleosome core particle (NCP). The three-dimensional structures of almost 25 thousand dinucleotide steps from more than 500 sequentially non-redundant crystal structures were classified by using DNA structural alphabet CANA (Conformational Alphabet of Nucleic Acids) and associations between ten CANA letters and sixteen dinucleotide sequences were investigated. The associations showed features discriminating between specific and non-specific binding of DNA to proteins. Important is the specific role of two DNA structural forms, A-DNA, and BII-DNA, represented by the CANA letters AAA and BB2: AAA structures are avoided in non-specific NCP complexes, where the wrapping of the DNA duplex is explained by the periodic occurrence of BB2 every 10.3 steps. In both regulatory and NCP complexes, the extent of bending of the DNA local helical axis does not influence proportional representation of the CANA alphabet letters, namely the relative incidences of AAA and BB2 remain constant in bent and straight duplexes.

  9. Different intracellular distribution of avian reovirus core protein sigmaA in cells of avian and mammalian origin

    Energy Technology Data Exchange (ETDEWEB)

    Vazquez-Iglesias, Lorena; Lostale-Seijo, Irene; Martinez-Costas, Jose [Departamento de Bioquimica y Biologia Molecular, Facultad de Farmacia, y Centro Singular de Investigacion en Quimica Biologica y Materiales Moleculares (CIQUS), Universidad de Santiago de Compostela, 15782-Santiago de Compostela (Spain); Benavente, Javier, E-mail: franciscojavier.benavente@usc.es [Departamento de Bioquimica y Biologia Molecular, Facultad de Farmacia, y Centro Singular de Investigacion en Quimica Biologica y Materiales Moleculares (CIQUS), Universidad de Santiago de Compostela, 15782-Santiago de Compostela (Spain)

    2012-10-25

    A comparative analysis of the intracellular distribution of avian reovirus (ARV) core protein sigmaA in cells of avian and mammalian origin revealed that, whereas the viral protein accumulates in the cytoplasm and nucleolus of avian cells, most sigmaA concentrates in the nucleoplasm of mammalian cells in tight association with the insoluble nuclear matrix fraction. Our results further showed that sigmaA becomes arrested in the nucleoplasm of mammalian cells via association with mammalian cell-specific factors and that this association prevents nucleolar targeting. Inhibition of RNA polymerase II activity, but not of RNA polymerase I activity, in infected mammalian cells induces nucleus-to-cytoplasm sigmaA translocation through a CRM1- and RanGTP-dependent mechanism, yet a heterokaryon assay suggests that sigmaA does not shuttle between the nucleus and cytoplasm. The scarcity of sigmaA in cytoplasmic viral factories of infected mammalian cells could be one of the factors contributing to limited ARV replication in mammalian cells.

  10. Muscle protein metabolism in neonatal alloxan-administered rats: effects of continuous and intermittent swimming training.

    Science.gov (United States)

    Ribeiro, Carla; Cambri, Lucieli T; Dalia, Rodrigo A; Araújo, Michel B; Ghezzi, Ana C; Moura, Leandro P; Araújo, Gustavo G; Botezelli, Jose D; Mello, Maria Ar

    2012-02-06

    This study aimed to examine the effects of intermittent and continuous swimming training on muscle protein metabolism in neonatal alloxan-administered rats. Wistar rats were used and divided into six groups: sedentary alloxan (SA), sedentary control (SC), continuous trained alloxan (CA), intermittent trained alloxan (IA), continuous trained control (CC) and intermittent trained control (IC). Alloxan (250 mg/kg body weight) was injected into newborn rats at 6 days of age. The continuous training protocol consisted of 12 weeks of swimming training in individual cylinder tanks while supporting a load that was 5% of body weight; uninterrupted swimming for 1 h/day, five days a week. The intermittent training protocol consisted of 12 weeks of swimming training in individual cylinder tanks while supporting a load that was 15% of body weight; 30 s of activity interrupted by 30 s of rest for a total of 20 min/day, five days a week. At 28 days, the alloxan animals displayed higher glycemia after glucose overload than the control animals. No differences in insulinemia among the groups were detected. At 120 days, no differences in serum albumin and total protein among the groups were observed. Compared to the other groups, DNA concentrations were higher in the alloxan animals that were subjected to continuous training, whereas the DNA/protein ratio was higher in the alloxan animals that were subjected to intermittent training. It was concluded that continuous and intermittent training sessions were effective in altering muscle growth by hyperplasia and hypertrophy, respectively, in alloxan-administered animals.

  11. Muscle protein metabolism in neonatal alloxan-administered rats: effects of continuous and intermittent swimming training

    Directory of Open Access Journals (Sweden)

    Ribeiro Carla

    2012-02-01

    Full Text Available Abstract Background This study aimed to examine the effects of intermittent and continuous swimming training on muscle protein metabolism in neonatal alloxan-administered rats. Methods Wistar rats were used and divided into six groups: sedentary alloxan (SA, sedentary control (SC, continuous trained alloxan (CA, intermittent trained alloxan (IA, continuous trained control (CC and intermittent trained control (IC. Alloxan (250 mg/kg body weight was injected into newborn rats at 6 days of age. The continuous training protocol consisted of 12 weeks of swimming training in individual cylinder tanks while supporting a load that was 5% of body weight; uninterrupted swimming for 1 h/day, five days a week. The intermittent training protocol consisted of 12 weeks of swimming training in individual cylinder tanks while supporting a load that was 15% of body weight; 30 s of activity interrupted by 30 s of rest for a total of 20 min/day, five days a week. Results At 28 days, the alloxan animals displayed higher glycemia after glucose overload than the control animals. No differences in insulinemia among the groups were detected. At 120 days, no differences in serum albumin and total protein among the groups were observed. Compared to the other groups, DNA concentrations were higher in the alloxan animals that were subjected to continuous training, whereas the DNA/protein ratio was higher in the alloxan animals that were subjected to intermittent training. Conclusion It was concluded that continuous and intermittent training sessions were effective in altering muscle growth by hyperplasia and hypertrophy, respectively, in alloxan-administered animals.

  12. Coevolved Mutations Reveal Distinct Architectures for Two Core Proteins in the Bacterial Flagellar Motor.

    Directory of Open Access Journals (Sweden)

    Alessandro Pandini

    Full Text Available Switching of bacterial flagellar rotation is caused by large domain movements of the FliG protein triggered by binding of the signal protein CheY to FliM. FliG and FliM form adjacent multi-subunit arrays within the basal body C-ring. The movements alter the interaction of the FliG C-terminal (FliGC "torque" helix with the stator complexes. Atomic models based on the Salmonella entrovar C-ring electron microscopy reconstruction have implications for switching, but lack consensus on the relative locations of the FliG armadillo (ARM domains (amino-terminal (FliGN, middle (FliGM and FliGC as well as changes during chemotaxis. The generality of the Salmonella model is challenged by the variation in motor morphology and response between species. We studied coevolved residue mutations to determine the unifying elements of switch architecture. Residue interactions, measured by their coevolution, were formalized as a network, guided by structural data. Our measurements reveal a common design with dedicated switch and motor modules. The FliM middle domain (FliMM has extensive connectivity most simply explained by conserved intra and inter-subunit contacts. In contrast, FliG has patchy, complex architecture. Conserved structural motifs form interacting nodes in the coevolution network that wire FliMM to the FliGC C-terminal, four-helix motor module (C3-6. FliG C3-6 coevolution is organized around the torque helix, differently from other ARM domains. The nodes form separated, surface-proximal patches that are targeted by deleterious mutations as in other allosteric systems. The dominant node is formed by the EHPQ motif at the FliMMFliGM contact interface and adjacent helix residues at a central location within FliGM. The node interacts with nodes in the N-terminal FliGc α-helix triad (ARM-C and FliGN. ARM-C, separated from C3-6 by the MFVF motif, has poor intra-network connectivity consistent with its variable orientation revealed by structural data. ARM

  13. Impact of CHO Metabolism on Cell Growth and Protein Production: An Overview of Toxic and Inhibiting Metabolites and Nutrients

    DEFF Research Database (Denmark)

    Pereira, Sara; Kildegaard, Helene F.; Andersen, Mikael R.

    2018-01-01

    For over three decades, Chinese hamster ovary (CHO) cells have been the chosen expression platform for the production of therapeutic proteins with complex post-translational modifications. However, the metabolism of these cells is far from perfect and optimized, and requires substantial knowhow...

  14. Induced lung inflammation and dietary protein supply affect nitrogen retention and amino acid metabolism in growing pigs

    NARCIS (Netherlands)

    Kampman-van de Hoek, E.; Sakkas, P.; Gerrits, W.J.J.; Borne, van den J.J.G.C.; Peet-Schwering, van der C.M.C.; Jansman, A.J.M.

    2015-01-01

    It is hypothesised that during immune system activation, there is a competition for amino acids (AA) between body protein deposition and immune system functioning. The aim of the present study was to quantify the effect of immune system activation on N retention and AA metabolism in growing pigs,

  15. DASH-like diets high in protein or monounsaturated fats improve metabolic syndrome and calculated vascular risk.

    Science.gov (United States)

    Root, Martin M; Dawson, Hannah R

    2013-01-01

    Weight-loss diets with varying proportions of macronutrients have had varying effects on weight loss, and components of metabolic syndrome and risk factors for vascular diseases. However, little work has examined the effect of weight-neutral dietary changes in macronutrients on these factors. This is an investigation using the OMNI Heart datasets available from the NHLBI BioLINCC program. This study compared a DASH-like diet high in carbohydrates with similar diets high in protein and high in unsaturated fats. Measures of metabolic syndrome, except waist, and measures of risk factors for vascular diseases were taken at the end of each dietary period. All 3 diets significantly lowered the number of metabolic syndrome components (p ≤ 0.002) with a standardized measure of changes in metabolic syndrome components, suggesting that the high-protein, high-fat diet was most efficacious overall (p = 0.035). All 3 diets lowered a calculated 10-year risk of cardiovascular disease, with the high-protein and unsaturated fat diet being the most efficacious (p fat diet showed a slightly decreased calculated 9-year risk of diabetes (p = 0.11). Of the 3 weight-neutral diets, those high in protein and unsaturated fats appeared partially or wholly most beneficial.

  16. Repletion of branched chain amino acids reverses mTORC1 signaling but not improved metabolism during dietary protein dilution

    DEFF Research Database (Denmark)

    Maida, Adriano; Chan, Jessica S K; Sjøberg, Kim Anker

    2017-01-01

    OBJECTIVE: Dietary protein dilution (PD) has been associated with metabolic advantages such as improved glucose homeostasis and increased energy expenditure. This phenotype involves liver-induced release of FGF21 in response to amino acid insufficiency; however, it has remained unclear whether di...

  17. Multi-omic profiling of EPO producing Chinese hamster ovary cell panel reveals metabolic adaptation to heterologous protein production

    DEFF Research Database (Denmark)

    Ley, Daniel; Kazemi Seresht, Ali; Engmark, Mikael

    Heterologous protein production in CHO cells imposes a burden on the host cell metabolism and impact cellular physiology on a global scale. In this work, a multi-omics approach was applied to characterize the physiological impact of erythropoietin production, and discover production bottlenecks...

  18. Effect of water stress and foliar boron application on seed protein oil fatty acids and nitrogen metabolism in soybean

    Science.gov (United States)

    Effects of water stress and foliar boron (FB) application on soybean (Glycine max (L) Merr.) seed composition and nitrogen metabolism have not been well investigated. Therefore, the objective of this study was to investigate the effects of water stress and FB on seed protein, oil, fatty acids, nitra...

  19. Effect of Genomic Integration Location on Heterologous Protein Expression and Metabolic Engineering in E. coli.

    Science.gov (United States)

    Englaender, Jacob A; Jones, J Andrew; Cress, Brady F; Kuhlman, Thomas E; Linhardt, Robert J; Koffas, Mattheos A G

    2017-04-21

    Chromosomal integration offers a selection-free alternative to DNA plasmids for expression of foreign proteins and metabolic pathways. Episomal plasmid DNA is convenient but has drawbacks including increased metabolic burden and the requirement for selection in the form of antibiotics. E. coli has long been used for the expression of foreign proteins and for the production of valuable metabolites by expression of complete metabolic pathways. The gene encoding the fluorescent reporter protein mCherry was integrated into four genomic loci on the E. coli chromosome to measure protein expression at each site. Expression levels ranged from 25% to 500% compared to the gene expressed on a high-copy plasmid. Modular expression of DNA is one of the most commonly used methods for optimizing metabolite production by metabolic engineering. By combining a recently developed method for integration of large synthetic DNA constructs into the genome, we were able to integrate two foreign pathways into the same four genomic loci. We have demonstrated that only one of the genomic loci resulted in the production of violacein, and that all four loci produced trans-cinnamic acid from the TAL pathway.

  20. Abomasal protein infusion in postpartum transition dairy cows: effect on performance and mammary metabolism.

    Science.gov (United States)

    Larsen, M; Lapierre, H; Kristensen, N B

    2014-09-01

    The effect of increasing the postpartum metabolizable protein (MP) supply on performance and mammary metabolism was studied using 8 Holstein cows in a complete randomized design. At parturition, cows were assigned to abomasal infusion of water (CTRL) or casein (CAS). Arterial and epigastric venous blood samples were taken 14 d before expected parturition and at 4, 15, and 29 d in milk (DIM). To compensate previously estimated deficiency of essential AA and to avoid oversupply, casein protein infusion was graduated with 696±1, 490±9, and 212±10g/d at 4, 15 and 29 DIM, respectively. Dry matter intake was unaffected by CAS. Compared with CTRL, MP supply was greater at 4 DIM with CAS but did not differ by 29 DIM. Milk yield was greater with CAS (+7.2±1.3kg/d from 1 to 29 DIM). Milk protein yield was greater with CAS at 4 DIM and averaged 1,664±39g/d compared with 1,212±86g/d for CTRL, but did not differ at 29 DIM (1,383±48g/d). The ratio of MP total supply to requirement was numerically greater at 4 DIM for CAS compared with CTRL, indicating less postpartum protein deficiency. In contrast, a greater net energy deficiency tended to be induced with CAS, but the greater milk yield allowed a large part of mobilized fat to be secreted in milk. Arterial concentration of total essential AA increased sharply after parturition for CAS compared with slight decreases for CTRL. The patterns of arterial concentrations combined with arterial-mammary venous concentration differences indicated that Lys, Leu, and Tyr were the first-limiting AA at 4 DIM with CTRL. Mammary plasma flow was unaffected by treatment, indicating similar perfusion of mammary tissue. The greater milk yield with CAS was associated with greater mammary uptake of individual essential AA, tendencies to greater uptake of glucose, lactate, and β-hydroxybutyrate, whereas uptakes of volatile fatty acids were unaffected. Despite similar MP supply by 29 DIM, milk and lactose yields were greater with CAS

  1. Heterogeneous N-terminal acylation of retinal proteins results from the retina's unusual lipid metabolism.

    Science.gov (United States)

    Bereta, Grzegorz; Palczewski, Krzysztof

    2011-05-10

    Protein N-myristoylation occurs by a covalent attachment of a C14:0 fatty acid to the N-terminal Gly residue. This reaction is catalyzed by a N-myristoyltransferase that uses myristoyl-coenzyme A as substrate. But proteins in the retina also undergo heterogeneous N-acylation with C14:2, C14:1, and C12:0 fatty acids. The basis and the role of this retina-specific phenomenon are poorly understood. We studied guanylate cyclase-activating protein 1 (GCAP1) as an example of retina-specific heterogeneously N-acylated protein. The types and the abundance of fatty acids bound to bovine retinal GCAP1 were C14:2, 37.0%; C14:0, 32.4%; C14:1, 22.3%; and C12:0, 8.3% as quantified by liquid chromatography coupled mass spectrometry. We also devised a method for N-acylating proteins in vitro and used it to modify GCAP1 with acyl moieties of different lengths. Analysis of these GCAPs both confirmed that N-terminal acylation of GCAP1 is critical for its high activity and proper Ca(2+)-dependent response and revealed comparable functionality for GCAP1 with acyl moieties of various lengths. We also tested the hypothesis that retinal heterogeneous N-acylation results from retinal enrichment of unusual N-myristoyltransferase substrates. Thus, acyl-coenzyme A esters were purified from both bovine retina and brain and analyzed by liquid chromatography coupled mass spectrometry. Substantial differences in acyl-coenzyme A profiles between the retina and brain were detected. Importantly, the ratios of uncommon N-acylation substrates--C14:2- and C14:1-coenyzme A to C14:0-coenzyme A--were higher in the retina than in the brain. Thus, our results suggest that heterogeneous N-acylation, responsible for expansion of retinal proteome, reflects the unique character of retinal lipid metabolism. Additionally, we propose a new hypothesis explaining the physiological relevance of elevated retinal ratios of C14:2- and C14:1-coenzyme A to C14:0-coenzyme A.

  2. Identifying molecular effects of diet through systems biology: influence of herring diet on sterol metabolism and protein turnover in mice.

    Directory of Open Access Journals (Sweden)

    Intawat Nookaew

    Full Text Available BACKGROUND: Changes in lifestyle have resulted in an epidemic development of obesity-related diseases that challenge the healthcare systems worldwide. To develop strategies to tackle this problem the focus is on diet to prevent the development of obesity-associated diseases such as cardiovascular disease (CVD. This will require methods for linking nutrient intake with specific metabolic processes in different tissues. METHODOLOGY/PRINCIPAL FINDING: Low-density lipoprotein receptor-deficient (Ldlr -/- mice were fed a high fat/high sugar diet to mimic a westernized diet, being a major reason for development of obesity and atherosclerosis. The diets were supplemented with either beef or herring, and matched in macronutrient contents. Body composition, plasma lipids and aortic lesion areas were measured. Transcriptomes of metabolically important tissues, e.g. liver, muscle and adipose tissue were analyzed by an integrated approach with metabolic networks to directly map the metabolic effects of diet in these different tissues. Our analysis revealed a reduction in sterol metabolism and protein turnover at the transcriptional level in herring-fed mice. CONCLUSION: This study shows that an integrated analysis of transcriptome data using metabolic networks resulted in the identification of signature pathways. This could not have been achieved using standard clustering methods. In particular, this systems biology analysis could enrich the information content of biomedical or nutritional data where subtle changes in several tissues together affects body metabolism or disease progression. This could be applied to improve diets for subjects exposed to health risks associated with obesity.

  3. Identifying molecular effects of diet through systems biology: influence of herring diet on sterol metabolism and protein turnover in mice.

    Science.gov (United States)

    Nookaew, Intawat; Gabrielsson, Britt G; Holmäng, Agneta; Sandberg, Ann-Sofie; Nielsen, Jens

    2010-08-24

    Changes in lifestyle have resulted in an epidemic development of obesity-related diseases that challenge the healthcare systems worldwide. To develop strategies to tackle this problem the focus is on diet to prevent the development of obesity-associated diseases such as cardiovascular disease (CVD). This will require methods for linking nutrient intake with specific metabolic processes in different tissues. Low-density lipoprotein receptor-deficient (Ldlr -/-) mice were fed a high fat/high sugar diet to mimic a westernized diet, being a major reason for development of obesity and atherosclerosis. The diets were supplemented with either beef or herring, and matched in macronutrient contents. Body composition, plasma lipids and aortic lesion areas were measured. Transcriptomes of metabolically important tissues, e.g. liver, muscle and adipose tissue were analyzed by an integrated approach with metabolic networks to directly map the metabolic effects of diet in these different tissues. Our analysis revealed a reduction in sterol metabolism and protein turnover at the transcriptional level in herring-fed mice. This study shows that an integrated analysis of transcriptome data using metabolic networks resulted in the identification of signature pathways. This could not have been achieved using standard clustering methods. In particular, this systems biology analysis could enrich the information content of biomedical or nutritional data where subtle changes in several tissues together affects body metabolism or disease progression. This could be applied to improve diets for subjects exposed to health risks associated with obesity.

  4. Mitochondrial thiol modification by a targeted electrophile inhibits metabolism in breast adenocarcinoma cells by inhibiting enzyme activity and protein levels

    Directory of Open Access Journals (Sweden)

    M. Ryan Smith

    2016-08-01

    Full Text Available 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

  5. Identification of Genetic Variants Linking Protein C and Lipoprotein Metabolism: The ARIC Study (Atherosclerosis Risk in Communities).

    Science.gov (United States)

    Pankow, James S; Tang, Weihong; Pankratz, Nathan; Guan, Weihua; Weng, Lu-Chen; Cushman, Mary; Boerwinkle, Eric; Folsom, Aaron R

    2017-03-01

    Previous studies have identified common genetic variants in 4 chromosomal regions that together account for 14% to 15% of the variance in circulating levels of protein C. To further characterize the genetic architecture of protein C, we obtained denser coverage at some loci, extended investigation of protein C to low-frequency and rare variants, and searched for new associations in genes known to influence protein C. Genetic associations with protein C antigen level were evaluated in ≤10 778 European and 3190 black participants aged 45 to 64 years. Analyses included >26 million autosomal variants available after imputation to the 1000 Genomes reference panel along with additional low-frequency and rare variants directly genotyped using the Illumina ITMAT-Broad-CARe chip and Illumina HumanExome BeadChip. Genome-wide significant associations (PC level in both whites and blacks, reaching genome-wide significance in a meta-analysis combining results from both groups (P=1.4×10(-9)). To further investigate a possible link between lipid metabolism and protein C level, we conducted Mendelian randomization analyses using 185 lipid-related genetic variants as instrumental variables. The results indicated that triglycerides, and possibly low-density lipoprotein cholesterol, influence protein C levels. Discovery of variants influencing circulating protein C levels in the CELSR2-PSRC1-SORT1 region may indicate a novel genetic link between lipoprotein metabolism and hemostasis. © 2017 American Heart Association, Inc.

  6. Differential effects of cooked beans and cooked lentils on protein metabolism in intestine and muscle in growing rats.

    Science.gov (United States)

    Pirman, Tatjana; Combe, Etiennette; Ribeyre, Marie Claude; Prugnaud, Jacques; Stekar, Jasna; Patureau Mirand, Philippe

    2006-01-01

    The effect of diets based on cooked beans or lentils on protein metabolism in intestines and muscles was studied in rats. The cooked seeds were used as the unique protein source in balanced diets (containing 229 and 190 g of crude protein per kg dry matter) fed to young growing rats for 20 days. Their effects were compared with those of the control casein diet in pair-fed rats. Protein synthesis rates in small and large intestines and in gastrocnemius and soleus muscles were determined in vivo, in a fed state, by the flooding dose method, using 13C-valine. In the small and large intestine tissues of the legume fed groups, protein, RNA relative masses (mg.100 g BM(-1)) and protein synthesis rates (FSR and ASR) were higher than in the control rats (p lentils in the large intestine and in gastrocnemius muscle. Copyright 2006 S. Karger AG, Basel.

  7. Metabolically inert perfluorinated fatty acids directly activate uncoupling protein 1 in brown-fat mitochondria.

    Science.gov (United States)

    Shabalina, Irina G; Kalinovich, Anastasia V; Cannon, Barbara; Nedergaard, Jan

    2016-05-01

    The metabolically inert perfluorinated fatty acids perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) can display fatty acid-like activity in biological systems. The uncoupling protein 1 (UCP1) in brown adipose tissue is physiologically (re)activated by fatty acids, including octanoate. This leads to bioenergetically uncoupled energy dissipation (heat production, thermogenesis). We have examined here the possibility that PFOA/PFOS can directly (re)activate UCP1 in isolated mouse brown-fat mitochondria. In wild-type brown-fat mitochondria, PFOS and PFOA overcame GDP-inhibited thermogenesis, leading to increased oxygen consumption and dissipated membrane potential. The absence of this effect in brown-fat mitochondria from UCP1-ablated mice indicated that it occurred through activation of UCP1. A competitive type of inhibition by increased GDP concentrations indicated interaction with the same mechanistic site as that utilized by fatty acids. No effect was observed in heart mitochondria, i.e., in mitochondria without UCP1. The stimulatory effect of PFOA/PFOS was not secondary to non-specific mitochondrial membrane permeabilization or to ROS production. Thus, metabolic effects of perfluorinated fatty acids could include direct brown adipose tissue (UCP1) activation. The possibility that this may lead to unwarranted extra heat production and thus extra utilization of food resources, leading to decreased fitness in mammalian wildlife, is discussed, as well as possible negative effects in humans. However, a possibility to utilize PFOA-/PFOS-like substances for activating UCP1 therapeutically in obesity-prone humans may also be envisaged.

  8. Metabolic syndrome and C-reactive protein in patients with depressive disorder on antidepressive medication

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    Stanojević Albina

    2013-01-01

    Full Text Available Introduction. Recurrent depression is a psychiatric disorder of which etiology and pathogenesis might be related to immune response. Metabolic Syndrome (MetS and its components are also strongly associated with elevated inflammatory indicators, as so as the body mass index (BMI and total cholesterol levels. Objective. Objective of this study was to investigate if there was any difference in C-reactive protein (CRP levels in patients with recurrent depressive disorder, treated with antidepressants, compared to a healthy control group of subjects and if there was an association between increased CRP levels and the presence of MetS in these two groups. Methods. Sixty subjects entered the study; of these 35 patients with the diagnosis of recurrent depressive disorder, while the healthy control group included 25 subjects. MetS was defined according to the NCEP ATP III criteria. The cut-off point for CRP was set at >5 mg /L. Results. There was no statistically significant difference in the prevalence of MetS and CRP values between the studied groups. Waist circumference and total cholesterol levels were significantly higher in the experimental group. Patients that fulfilled the criteria for MetS showed significantly higher values of central obesity and arterial hypertension in the experimental group as well. The elevated CRP levels were associated with increased frequency of MetS in depressed patients. Conclusion. Both CRP levels and metabolic risk profile screening, according to the international criteria, may be beneficial in order to obtain better assessment for depressive long term medicated patients.

  9. Fatty acid transport protein 1 regulates retinoid metabolism and photoreceptor development in mouse retina

    Science.gov (United States)

    Cubizolle, Aurélie; Guillou, Laurent; Mollereau, Bertrand; Hamel, Christian P.

    2017-01-01

    In retinal pigment epithelium (RPE), RPE65 catalyzes the isomerization of all-trans-retinyl fatty acid esters to 11-cis-retinol in the visual cycle and controls the rhodopsin regeneration rate. However, the mechanisms by which these processes are regulated are still unclear. Fatty Acid Transport Protein 1 (FATP1) is involved in fatty acid uptake and lipid metabolism in a variety of cell types. FATP1 co-localizes with RPE65 in RPE and inhibits its isomerase activity in vitro. Here, we further investigated the role of FATP1 in the visual cycle using transgenic mice that overexpress human FATP1 specifically in the RPE (hFATP1TG mice). The mice displayed no delay in the kinetics of regeneration of the visual chromophore 11-cis-retinal after photobleaching and had no defects in light sensitivity. However, the total retinoid content was higher in the hFATP1TG mice than in wild type mice, and the transgenic mice also displayed an age-related accumulation (up to 40%) of all-trans-retinal and retinyl esters that was not observed in control mice. Consistent with these results, hFATP1TG mice were more susceptible to light-induced photoreceptor degeneration. hFATP1 overexpression also induced an ~3.5-fold increase in retinosome autofluorescence, as measured by two-photon microscopy. Interestingly, hFATP1TG retina contained ~25% more photoreceptor cells and ~35% longer outer segments than wild type mice, revealing a non-cell-autonomous effect of hFATP1 expressed in the RPE. These data are the first to show that FATP1-mediated fatty acid uptake in the RPE controls both retinoid metabolism in the outer retina and photoreceptor development. PMID:28672005

  10. HCV core protein-induced down-regulation of microRNA-152 promoted aberrant proliferation by regulating Wnt1 in HepG2 cells.

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    Shifeng Huang

    Full Text Available BACKGROUND: Hepatitis C virus (HCV has been reported to regulate cellular microRNAs (miRNAs. The HCV core protein is considered to be a potential oncoprotein in HCV-related hepatocellular carcinoma (HCV-HCC, but HCV core-regulated miRNAs are largely unknown. Our preliminary experiments revealed significant down-regulation of microRNA-152 (miR-152 by HCV core protein in HepG2 cells. Through target gene prediction softwares, Wnt1 was predicted to be a potential target of miR-152. The present study was initiated to investigate whether miR-152 is aberrantly regulated by the HCV core protein, and involved in the regulation of the aberrant proliferation of HCV-HCC cells. METHODS: MiR-152 levels were examined by stem-loop real-time RT-PCR (SLqRT-PCR. Cell proliferation was analyzed by MTT and colony formation assay. Cell cycle analysis was performed by flow cytometry. Luciferase reporter assay was conducted to confirm miRNA-target association. Wnt1 expression was determined by real-time qPCR and Western blotting. RESULTS: HCV core protein significantly suppressed miR-152 expression, and led to significant Wnt1 up-regulation with a concomitant aberrantly promoted proliferation. Moreover, we validated that miR-152 inhibition promoted, while miR-152 mimics inhibited cell proliferation. Using, qRT-PCR and western blot, Wnt1 was demonstrated to be regulated by miR-152. Luciferase activity assay showed that while miR-152 mimics significantly reduced the luciferase activity by 83.76% (P<0.0001, miR-152 inhibitor showed no effect on luciferase reporter. Most notably, salvage expression of miR-152 after Ad-HCV core infection for 24 h almost totally reversed the proliferation-promoting effect of the HCV core protein, and meanwhile, reduced the expression of both Wnt1 mRNA and protein to basal levels. CONCLUSION: These findings provide important evidence that the reduced miR-152 expression by HCV core protein can indirectly lose an inhibitory effect on Wnt1

  11. Quantitative Comparison of Dense-Core Amyloid Plaque Accumulation in Amyloid-β Precursor Protein Transgenic Mice

    Science.gov (United States)

    Liu, Peng; Reichl, John H.; Rao, Eshaan R.; McNellis, Brittany M.; Huang, Eric S.; Hemmy, Laura S.; Forster, Colleen L.; Kuskowski, Michael A.; Borchelt, David R.; Vassar, Robert; Ashe, Karen H.; Zahs, Kathleen R.

    2016-01-01

    There exist several dozen lines of transgenic mice that express human amyloid-β precursor protein (AβPP) with Alzheimer’s disease (AD)-linked mutations. AβPP transgenic mouse lines differ in the types and amounts of Aβ that they generate and in their spatiotemporal patterns of expression of Aβ assemblies, providing a toolkit to study Aβ amyloidosis and the influence of Aβ aggregation on brain function. More complete quantitative descriptions of the types of Aβ assemblies present in transgenic mice and in humans during disease progression should add to our understanding of how Aβ toxicity in mice relates to the pathogenesis of AD. Here, we provide a direct quantitative comparison of amyloid plaque burdens and plaque sizes in four lines of AβPP transgenic mice. We measured the fraction of cortex and hippocampus occupied by dense-core plaques, visualized by staining with Thioflavin S, in mice from young adulthood through advanced age. We found that the plaque burdens among the transgenic lines varied by an order of magnitude: at 15 months of age, the oldest age studied, the median cortical plaque burden in 5XFAD mice was already ~4.5 times that of 21-month Tg2576 mice and ~15 times that of 21–24-month rTg9191 mice. Plaque-size distributions changed across the lifespan in a line- and region-dependent manner. We also compared the dense-core plaque burdens in the mice to those measured in a set of pathologically-confirmed AD cases from the Nun Study. Cortical plaque burdens in Tg2576, APPSwePS1ΔE9, and 5XFAD mice eventually far exceeded those measured in the human cohort. PMID:28059792

  12. Combined inflammatory and metabolic defects reflected by reduced serum protein levels in patients with Buruli ulcer disease.

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    Richard O Phillips

    2014-04-01

    Full Text Available 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.

  13. Accumulation of p21 proteins at DNA damage sites independent of p53 and core NHEJ factors following irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Koike, Manabu, E-mail: m_koike@nirs.go.jp [DNA Repair Gene Res., National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Yutoku, Yasutomo [DNA Repair Gene Res., National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Graduate School of Science, Chiba University, Chiba 263-8522 (Japan); Koike, Aki [DNA Repair Gene Res., National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan)

    2011-08-19

    Highlights: {yields} p21 accumulated rapidly at laser-irradiated sites via its C-terminal region. {yields} p21 colocalized with the DSB marker {gamma}-H2AX and the DSB sensor Ku80. {yields} Accumulation of p21 is dependent on PCNA, but not p53 and the NHEJ core factors. {yields} Accumulation activity of p21 was conserved among human and animal cells. {yields} p21 is a useful tool as a detection marker of DNA damaged sites. -- Abstract: The cyclin-dependent kinase (CDK) inhibitor p21 plays key roles in p53-dependent DNA-damage responses, i.e., cell cycle checkpoints, senescence, or apoptosis. p21 might also play a role in DNA repair. p21 foci arise at heavy-ion-irradiated DNA-double-strand break (DSB) sites, which are mainly repaired by nonhomologous DNA-end-joining (NHEJ). However, no mechanisms of p21 accumulation at double-strand break (DSB) sites have been clarified in detail. Recent works indicate that Ku70 and Ku80 are essential for the accumulation of other NHEJ core factors, e.g., DNA-PKcs, XRCC4 and XLF, and other DNA damage response factors, e.g., BRCA1. Here, we show that p21 foci arise at laser-irradiated sites in cells from various tissues from various species. The accumulation of EGFP-p21 was detected in not only normal cells, but also transformed or cancer cells. Our results also showed that EGFP-p21 accumulated rapidly at irradiated sites, and colocalized with the DSB marker {gamma}-H2AX and with the DSB sensor protein Ku80. On the other hand, the accumulation occurred in Ku70-, Ku80-, or DNA-PKcs-deficient cell lines and in human papillomavirus 18-positive cells, whereas the p21 mutant without the PCNA-binding region (EGFP-p21(1-146)) failed to accumulate at the irradiated sites. These findings suggest that the accumulation of p21, but not functional p53 and the NHEJ core factors, is dependent on PCNA. These findings also suggest that the accumulation activity of p21 at DNA damaged sites is conserved among human and animal cells, and p21 is a useful

  14. Novel insights into the origin and diversification of photosynthesis based on analyses of conserved indels in the core reaction center proteins.

    Science.gov (United States)

    Khadka, Bijendra; Adeolu, Mobolaji; Blankenship, Robert E; Gupta, Radhey S

    2017-02-01

    The evolution and diversification of different types of photosynthetic reaction centers (RCs) remains an important unresolved problem. We report here novel sequence features of the core proteins from Type I RCs (RC-I) and Type II RCs (RC-II) whose analyses provide important insights into the evolution of the RCs. The sequence alignments of the RC-I core proteins contain two conserved inserts or deletions (indels), a 3 amino acid (aa) indel that is uniquely found in all RC-I homologs from Cyanobacteria (both PsaA and PsaB) and a 1 aa indel that is specifically shared by the Chlorobi and Acidobacteria homologs. Ancestral sequence reconstruction provides evidence that the RC-I core protein from Heliobacteriaceae (PshA), lacking these indels, is most closely related to the ancestral RC-I protein. Thus, the identified 3 aa and 1 aa indels in the RC-I protein sequences must have been deletions, which occurred, respectively, in an ancestor of the modern Cyanobacteria containing a homodimeric form of RC-I and in a common ancestor of the RC-I core protein from Chlorobi and Acidobacteria. We also report a conserved 1 aa indel in the RC-II protein sequences that is commonly shared by all homologs from Cyanobacteria but not found in the homologs from Chloroflexi, Proteobacteria and Gemmatimonadetes. Ancestral sequence reconstruction provides evidence that the RC-II subunits lacking this indel are more similar to the ancestral RC-II protein. The results of flexible structural alignments of the indel-containing region of the RC-II protein with the homologous region in the RC-I core protein, which shares structural similarity with the RC-II homologs, support the view that the 1 aa indel present in the RC-II homologs from Cyanobacteria is a deletion, which was not present in the ancestral form of the RC-II protein. Our analyses of the conserved indels found in the RC-I and RC-II proteins, thus, support the view that the earliest photosynthetic lineages with living descendants

  15. Effects of immobilization and aerobic training on proteins related to intramuscular substrate storage and metabolism in young and older men.

    Science.gov (United States)

    Vigelsø, Andreas; Gram, Martin; Wiuff, Caroline; Hansen, Christina Neigaard; Prats, Clara; Dela, Flemming; Helge, Jørn Wulff

    2016-03-01

    Aging and inactivity lead to skeletal muscle metabolic inflexibility, but the underlying molecular mechanisms are not entirely elucidated. Therefore, we investigated how muscle lipid and glycogen stores and major regulatory proteins were affected by short-term immobilization followed by aerobic training in young and older men. 17 young (23 ± 1 years, 24 ± 1 kg m(-2), and 20 ± 2% body fat) and 15 older men (68 ± 1 years; 27 ± 1 kg m(-2), and 29 ± 2% body fat) underwent 2 weeks' one leg immobilization followed by 6 weeks' cycle training. Biopsies were obtained from m. vastus lateralis just before immobilization (at inclusion), after immobilization, and the after 6 weeks' training. The biopsies were analyzed for muscle substrates; muscle perilipin protein (PLIN), glycogen synthase (GS), synaptosomal-associated protein of 23 kDa (SNAP23) protein content, and muscle 3-hydroxyacyl-CoA dehydrogenase (HAD) activity The older men had higher intramuscular triglyceride (IMTG) (73 %) and Glycogen (16%) levels compared to the young men, and IMTG tended to increase with immobilization. PLIN2 and 3 protein content increased with immobilization in the older men only. The young men had higher GS (74%) protein compared to the older men. Immobilization decreased and training restored HAD activity, GS and SNAP23 protein content in young and older men. Evidence of age-related metabolic inflexibility is presented, seen as body fat and IMTG accumulation. The question arises as to whether IMTG accumulation in the older men is caused by or leading to the increase in PLIN2 and 3 protein content. Training decreased body fat and IMTG levels in both young and older men; hence, training should be prioritized to reduce the detrimental effect of aging on metabolism.

  16. High-mobility group protein B1: a new biomarker of metabolic syndrome in obese children.

    Science.gov (United States)

    Arrigo, Teresa; Chirico, Valeria; Salpietro, Vincenzo; Munafò, Caterina; Ferraù, Valeria; Gitto, Eloisa; Lacquaniti, Antonio; Salpietro, Carmelo

    2013-04-01

    Obesity is associated with a chronic low-grade inflammation. High-mobility group box 1 protein (HMGB1) plays a key role in inflammation and immunostimulatory and chemotactic processes. The aim of the study was to assess the role of HMGB1 in obese children and to evaluate its diagnostic profile in identifying childhood obesity-related complications, such as the metabolic syndrome (MS). Sixty obese children were enrolled and compared with 40 healthy children (control). Homeostasis model assessment of insulin resistance (HOMA-IR), lipid profile, thyroid hormones, and pro- and anti-inflammatory peptides such as C-reactive protein (CRP), adiponectin, interleukin 6 (IL6), IL18, IL23, TNFα, resistin, and HMGB1 were evaluated. Receiver operating characteristics (ROC) analysis was employed to calculate the area under the curve (AUC) for HMGB1, IL6, and adiponectin to find the best cutoff values capable of identifying MS in obese children. HMGB1 levels were statistically higher in obese patients than in the control group (19.4±6.8 vs 3.7±1.2 ng/ml; Pobese patients, IL18, IL6, and resistin levels were significantly high, while adiponectin levels were low. At multivariate analysis, HMGB1 was found to be independently correlated with BMI, IL23, IL6, free triiodothyronine, HDL, and HOMA-IR. At ROC analysis, HMGB1 showed higher sensitivity and specificity (AUC, 0. 992; sensitivity, 94.7%; specificity, 97.5%) than IL6 and adiponectin in identifying MS in obese children. HMGB1 plays an important role in the inflammatory process associated with childhood obesity. This peptide may be an important diagnostic marker for obesity-related complications, such as MS.

  17. Hierarchical core/shell Fe3O4@SiO2@γ-AlOOH@Au micro/nanoflowers for protein immobilization.

    Science.gov (United States)

    Xuan, Shouhu; Wang, Feng; Gong, Xinglong; Kong, Siu-Kai; Yu, Jimmy C; Leung, Ken Cham-Fai

    2011-03-07

    A facile synthesis of monodispersed microparticles composed of superparamagnetic Fe(3)O(4) cores, SiO(2) shell, hierarchical γ-AlOOH periphery with Au nanoparticles is reported. These particles are found to be useful for protein immobilization and bear resemblance to daisy flowers, and are hereafter termed "nanoflowers".

  18. PACAP38 differentially effects genes and CRMP2 protein expression in ischemic core and penumbra regions of permanent middle cerebral artery occlusion model mice brain.

    Science.gov (United States)

    Hori, Motohide; Nakamachi, Tomoya; Shibato, Junko; Rakwal, Randeep; Tsuchida, Masachi; Shioda, Seiji; Numazawa, Satoshi

    2014-09-23

    Pituitary adenylate-cyclase activating polypeptide (PACAP) has neuroprotective and axonal guidance functions, but the mechanisms behind such actions remain unclear. Previously we examined effects of PACAP (PACAP38, 1 pmol) injection intracerebroventrically in a mouse model of permanent middle cerebral artery occlusion (PMCAO) along with control saline (0.9% NaCl) injection. Transcriptomic and proteomic approaches using ischemic (ipsilateral) brain hemisphere revealed differentially regulated genes and proteins by PACAP38 at 6 and 24 h post-treatment. However, as the ischemic hemisphere consisted of infarct core, penumbra, and non-ischemic regions, specificity of expression and localization of these identified molecular factors remained incomplete. This led us to devise a new experimental strategy wherein, ischemic core and penumbra were carefully sampled and compared to the corresponding contralateral (healthy) core and penumbra regions at 6 and 24 h post PACAP38 or saline injections. Both reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to examine targeted gene expressions and the collapsin response mediator protein 2 (CRMP2) protein profiles, respectively. Clear differences in expression of genes and CRMP2 protein abundance and degradation product/short isoform was observed between ischemic core and penumbra and also compared to the contralateral healthy tissues after PACAP38 or saline treatment. Results indicate the importance of region-specific analyses to further identify, localize and functionally analyse target molecular factors for clarifying the neuroprotective function of PACAP38.

  19. PACAP38 Differentially Effects Genes and CRMP2 Protein Expression in Ischemic Core and Penumbra Regions of Permanent Middle Cerebral Artery Occlusion Model Mice Brain

    Directory of Open Access Journals (Sweden)

    Motohide Hori

    2014-09-01

    Full Text Available Pituitary adenylate-cyclase activating polypeptide (PACAP has neuroprotective and axonal guidance functions, but the mechanisms behind such actions remain unclear. Previously we examined effects of PACAP (PACAP38, 1 pmol injection intracerebroventrically in a mouse model of permanent middle cerebral artery occlusion (PMCAO along with control saline (0.9% NaCl injection. Transcriptomic and proteomic approaches using ischemic (ipsilateral brain hemisphere revealed differentially regulated genes and proteins by PACAP38 at 6 and 24 h post-treatment. However, as the ischemic hemisphere consisted of infarct core, penumbra, and non-ischemic regions, specificity of expression and localization of these identified molecular factors remained incomplete. This led us to devise a new experimental strategy wherein, ischemic core and penumbra were carefully sampled and compared to the corresponding contralateral (healthy core and penumbra regions at 6 and 24 h post PACAP38 or saline injections. Both reverse transcription-polymerase chain reaction (RT-PCR and Western blotting were used to examine targeted gene expressions and the collapsin response mediator protein 2 (CRMP2 protein profiles, respectively. Clear differences in expression of genes and CRMP2 protein abundance and degradation product/short isoform was observed between ischemic core and penumbra and also compared to the contralateral healthy tissues after PACAP38 or saline treatment. Results indicate the importance of region-specific analyses to further identify, localize and functionally analyse target molecular factors for clarifying the neuroprotective function of PACAP38.

  20. Liver cancer-derived hepatitis C virus core proteins shift TGF-beta responses from tumor suppression to epithelial-mesenchymal transition.

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    Serena Battaglia

    Full Text Available BACKGROUND: Chronic hepatitis C virus (HCV infection and associated liver cirrhosis represent a major risk factor for hepatocellular carcinoma (HCC development. TGF-beta is an important driver of liver fibrogenesis and cancer; however, its actual impact in human cancer progression is still poorly known. The aim of this study was to investigate the role of HCC-derived HCV core natural variants on cancer progression through their impact on TGF-beta signaling. PRINCIPAL FINDINGS: We provide evidence that HCC-derived core protein expression in primary human or mouse hepatocyte alleviates TGF-beta responses in terms or growth inhibition or apoptosis. Instead, in these hepatocytes TGF-beta was still able to induce an epithelial to mesenchymal transition (EMT, a process that contributes to the promotion of cell invasion and metastasis. Moreover, we demonstrate that different thresholds of Smad3 activation dictate the TGF-beta responses in hepatic cells and that HCV core protein, by decreasing Smad3 activation, may switch TGF-beta growth inhibitory effects to tumor promoting responses. CONCLUSION/SIGNIFICANCE: Our data illustrate the capacity of hepatocytes to develop EMT and plasticity under TGF-beta, emphasize the role of HCV core protein in the dynamic of these effects and provide evidence for a paradigm whereby a viral protein implicated in oncogenesis is capable to shift TGF-beta responses from cytostatic effects to EMT development.

  1. Evidence for Posttranslational Protein Flavinylation in the Syphilis Spirochete Treponema pallidum: Structural and Biochemical Insights from the Catalytic Core of a Periplasmic Flavin-Trafficking Protein.

    Science.gov (United States)

    Deka, Ranjit K; Brautigam, Chad A; Liu, Wei Z; Tomchick, Diana R; Norgard, Michael V

    2015-05-05

    -trafficking protein (Ftp), not only exploits its catalytic center to mediate posttranslational flavinylation of proteins (to create flavoproteins) but also likely maintains the periplasmic flavin pool via its unique ability to hydrolyze FAD. This functional diversity within a single lipoprotein is quite remarkable and reflects the enzymatic versatility of the treponemal lipoproteins, as well as molecular parsimony in an organism with a limited genome. Ftp-mediated protein flavinylation in the periplasm also likely is a key aspect of a predicted flavin-dependent Rnf-based redox homeostasis system at the cytoplasmic membrane of T. pallidum. In addition to its importance in T. pallidum physiology, Ftp homologs exist in other bacteria, thereby expanding our understanding of the bacterial periplasm as a metabolically active subcellular compartment for flavoprotein biogenesis as well as flavin homeostasis. Copyright © 2015 Deka et al.

  2. Systems-Level Response to Point Mutations in a Core Metabolic Enzyme Modulates Genotype-Phenotype Relationship

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    Shimon Bershtein

    2015-04-01

    Full Text Available Linking the molecular effects of mutations to fitness is central to understanding evolutionary dynamics. Here, we establish a quantitative relation between the global effect of mutations on the E. coli proteome and bacterial fitness. We created E. coli strains with specific destabilizing mutations in the chromosomal folA gene encoding dihydrofolate reductase (DHFR and quantified the ensuing changes in the abundances of 2,000+ E. coli proteins in mutant strains using tandem mass tags with subsequent LC-MS/MS. mRNA abundances in the same E. coli strains were also quantified. The proteomic effects of mutations in DHFR are quantitatively linked to phenotype: the SDs of the distributions of logarithms of relative (to WT protein abundances anticorrelate with bacterial growth rates. Proteomes hierarchically cluster first by media conditions, and within each condition, by the severity of the perturbation to DHFR function. These results highlight the importance of a systems-level layer in the genotype-phenotype relationship.

  3. Metabolism and excretion of anacetrapib, a novel inhibitor of the cholesteryl ester transfer protein, in humans.

    Science.gov (United States)

    Kumar, Sanjeev; Tan, Eugene Y; Hartmann, Georgy; Biddle, Zachary; Bergman, Arthur J; Dru, James; Ho, Jonathan Z; Jones, Allen N; Staskiewicz, Steve J; Braun, Matthew P; Karanam, Bindhu; Dean, Dennis C; Gendrano, Isaias Noel; Graves, Mark W; Wagner, John A; Krishna, Rajesh

    2010-03-01

    Anacetrapib is a novel cholesteryl ester transfer protein inhibitor being developed for the treatment of primary hypercholesterolemia and mixed dyslipidemia. The absorption, distribution, metabolism, and excretion of anacetrapib were investigated in an open-label study in which six healthy male subjects received a single oral dose of 150 mg and 165 microCi of [(14)C]anacetrapib. Plasma, urine, and fecal samples were collected at predetermined times for up to 14 days postdose and were analyzed for total radioactivity, the parent compound, and metabolites. The majority of the administered radioactivity (87%) was eliminated by fecal excretion, with negligible amounts present in urine (0.1%). The peak level of radioactivity in plasma (approximately 2 microM equivalents of [(14)C]anacetrapib) was achieved approximately 4 h postdose. The parent compound was the major radioactive component (79-94% of total radioactivity) in both plasma and feces. Three oxidative metabolites, M1, M2, and M3, were detected in plasma and feces and were identified as the O-demethylated species (M1) and two secondary hydroxylated derivatives of M1 (M2 and M3). Each metabolite was detected at low levels, representing excretion of metabolites by the biliary-fecal route.

  4. Alterations in glucose and protein metabolism in animals subjected to simulated microgravity

    Science.gov (United States)

    Mondon, C. E.; Rodnick, K. J.; Azhar, S.; Reaven, G. M.; Dolkas, C. B.

    1992-01-01

    Reduction of physical activity due to disease or environmental restraints, such as total bed rest or exposure to spaceflight, leads to atrophy of skeletal muscle and is frequently accompanied by alterations in food intake and the concentration of metabolic regulatory hormones such as insulin. Hindlimb suspension of laboratory rats, as a model for microgravity, also shows marked atrophy of gravity-dependent muscles along with a reduced gain in body weight. Suspended rats exhibit enhanced sensitivity to insulin-induced glucose uptake when compared with normal control rats and resistance to insulin action when compared with control rats matched similarly for reduced body weight gain. These changes are accompanied by decreased insulin binding and tyrosine kinase activity in soleus but not plantaris muscle, unchanged glucose uptake by perfused hindlimb and decreased sensitivity but not responsiveness to insulin-induced suppression of net proteolysis in hindlimb skeletal muscle. These findings suggest that loss of insulin sensitivity during muscle atrophy is associated with decreased insulin binding and tyrosine kinase activity in atrophied soleus muscle along with decreased sensitivity to the effects of insulin on suppressing net protein breakdown but not on enhancing glucose uptake by perfused hindlimb.

  5. Alterations in glucose and protein metabolism in animals subjected to simulated microgravity

    Science.gov (United States)

    Mondon, C. E.; Rodnick, K. J.; Dolkas, C. B.; Azhar, S.; Reaven, G. M.

    1992-09-01

    Reduction of physical activity due to disease or environmental restraints, such as total bed rest or exposure to spaceflight, leads to atrophy of skeletal muscle and is frequently accompanied by alterations in food intake and the concentration of metabolic regulatory hormones such as insulin. Hindlimb suspension of laboratory rats, as a model for microgravity, also shows marked atrophy of gravity dependent muscles along with a reduced gain in body weight. Suspended rats exhibit enhanced sensitivity to insulin-induced glucose uptake when compared with normal control rats and resistance to insulin action when compared with control rats matched similarly for reduced body weight gain. These changes are accompanied by decreased insulin binding and tyrosine kinase activity in soleus but not plantaris muscle, unchanged glucose uptake by perfused hindlimb and decreased sensitivity but not responsiveness to insulin-induced suppression of net proteolysis in hindlimb skeletal muscle. These findings suggest that loss of insulin sensitivity during muscle atrophy is associated with decreased insulin binding and tyrosine kinase activity in atrophied soleus muscle along with decreased sensitivity to the effects of insulin on suppressing net protein breakdown but not on enhancing glucose uptake by perfused hindlimb.

  6. Multiplex iterative plasmid engineering for combinatorial optimization of metabolic pathways and diversification of protein coding sequences.

    Science.gov (United States)

    Li, Yifan; Gu, Qun; Lin, Zhenquan; Wang, Zhiwen; Chen, Tao; Zhao, Xueming

    2013-11-15

    Engineering complex biological systems typically requires combinatorial optimization to achieve the desired functionality. Here, we present Multiplex Iterative Plasmid Engineering (MIPE), which is a highly efficient and customized method for combinatorial diversification of plasmid sequences. MIPE exploits ssDNA mediated λ Red recombineering for the introduction of mutations, allowing it to target several sites simultaneously and generate libraries of up to 10(7) sequences in one reaction. We also describe "restriction digestion mediated co-selection (RD CoS)", which enables MIPE to produce enhanced recombineering efficiencies with greatly simplified coselection procedures. To demonstrate this approach, we applied MIPE to fine-tune gene expression level in the 5-gene riboflavin biosynthetic pathway and successfully isolated a clone with 2.67-fold improved production in less than a week. We further demonstrated the ability of MIPE for highly multiplexed diversification of protein coding sequence by simultaneously targeting 23 codons scattered along the 750 bp sequence. We anticipate this method to benefit the optimization of diverse biological systems in synthetic biology and metabolic engineering.

  7. Effects of immobilization and aerobic training on proteins related to intramuscular substrate storage and metabolism in young and older men

    DEFF Research Database (Denmark)

    Vigelsø Hansen, Andreas; Gram, Martin; Wiuff, Caroline

    2016-01-01

    PURPOSE: Aging and inactivity lead to skeletal muscle metabolic inflexibility, but the underlying molecular mechanisms are not entirely elucidated. Therefore, we investigated how muscle lipid and glycogen stores and major regulatory proteins were affected by short-term immobilization followed...... lateralis just before immobilization (at inclusion), after immobilization, and the after 6 weeks' training. The biopsies were analyzed for muscle substrates; muscle perilipin protein (PLIN), glycogen synthase (GS), synaptosomal-associated protein of 23 kDa (SNAP23) protein content, and muscle 3-hydroxyacyl......-CoA dehydrogenase (HAD) activity RESULTS: The older men had higher intramuscular triglyceride (IMTG) (73 %) and Glycogen (16%) levels compared to the young men, and IMTG tended to increase with immobilization. PLIN2 and 3 protein content increased with immobilization in the older men only. The young men had higher...

  8. Differential regulation of lipid and protein metabolism in obese vs. lean subjects before and after a 72-h fast

    DEFF Research Database (Denmark)

    Bak, Ann Mosegaard; Møller, Andreas Buch; Vendelbo, Mikkel Holm

    2016-01-01

    release in obese subjects under basal and fasting conditions. We therefore studied nine lean and nine obese subjects twice, after 12 and 72 h of fasting, using measurements of mRNA and protein expression and phosphorylation of lipolytic and protein metabolic signaling molecules in fat and muscle together...... with whole body and forearm tracer techniques. Obese subjects displayed increased whole body lipolysis, decreased urea production rates, and decreased forearm muscle protein breakdown per 100 ml of forearm tissue, differences that persisted after 72 h of fasting. Lipolysis per fat mass unit was reduced...... in obese subjects and, correspondingly, adipose tissue hormone-sensitive lipase (HSL) phosphorylation and mRNA and protein levels of the adipose triglyceride lipase (ATGL) coactivator CGI58 were decreased. Fasting resulted in higher HSL phosphorylations and lower protein levels of the ATGL inhibitor G0S2...

  9. Uncoupling of Metabolic Health from Longevity through Genetic Alteration of Adipose Tissue Lipid-Binding Proteins

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    Khanichi N. Charles

    2017-10-01

    Full Text Available Deterioration of metabolic health is a hallmark of aging and generally assumed to be detrimental to longevity. Exposure to a high-calorie diet impairs metabolism and accelerates aging; conversely, calorie restriction (CR prevents age-related metabolic diseases and extends lifespan. However, it is unclear whether preservation of metabolic health is sufficient to extend lifespan. We utilized a genetic mouse model lacking Fabp4/5 that confers protection against metabolic diseases and shares molecular and lipidomic features with CR to address this question. Fabp-deficient mice exhibit extended metabolic healthspan, with protection against insulin resistance and glucose intolerance, inflammation, deterioration of adipose tissue integrity, and fatty liver disease. Surprisingly, however, Fabp-deficient mice did not exhibit any extension of lifespan. These data indicate that extension of metabolic healthspan in the absence of CR can be uncoupled from lifespan, indicating the potential for independent drivers of these pathways, at least in laboratory mice.

  10. Systematization of the protein sequence diversity in enzymes related to secondary metabolic pathways in plants, in the context of big data biology inspired by the KNApSAcK motorcycle database.

    Science.gov (United States)

    Ikeda, Shun; Abe, Takashi; Nakamura, Yukiko; Kibinge, Nelson; Hirai Morita, Aki; Nakatani, Atsushi; Ono, Naoaki; Ikemura, Toshimichi; Nakamura, Kensuke; Altaf-Ul-Amin, Md; Kanaya, Shigehiko

    2013-05-01

    Biology is increasingly becoming a data-intensive science with the recent progress of the omics fields, e.g. genomics, transcriptomics, proteomics and metabolomics. The species-metabolite relationship database, KNApSAcK Core, has been widely utilized and cited in metabolomics research, and chronological analysis of that research work has helped to reveal recent trends in metabolomics research. To meet the needs of these trends, the KNApSAcK database has been extended by incorporating a secondary metabolic pathway database called Motorcycle DB. We examined the enzyme sequence diversity related to secondary metabolism by means of batch-learning self-organizing maps (BL-SOMs). Initially, we constructed a map by using a big data matrix consisting of the frequencies of all possible dipeptides in the protein sequence segments of plants and bacteria. The enzyme sequence diversity of the secondary metabolic pathways was examined by identifying clusters of segments associated with certain enzyme groups in the resulting map. The extent of diversity of 15 secondary metabolic enzyme groups is discussed. Data-intensive approaches such as BL-SOM applied to big data matrices are needed for systematizing protein sequences. Handling big data has become an inevitable part of biology.

  11. BET Bromodomain Proteins Brd2, Brd3 and Brd4 Selectively Regulate Metabolic Pathways in the Pancreatic β-Cell.

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    Jude T Deeney

    Full Text Available Displacement of Bromodomain and Extra-Terminal (BET proteins from chromatin has promise for cancer and inflammatory disease treatments, but roles of BET proteins in metabolic disease remain unexplored. Small molecule BET inhibitors, such as JQ1, block BET protein binding to acetylated lysines, but lack selectivity within the BET family (Brd2, Brd3, Brd4, Brdt, making it difficult to disentangle contributions of each family member to transcriptional and cellular outcomes. Here, we demonstrate multiple improvements in pancreatic β-cells upon BET inhibition with JQ1 or BET-specific siRNAs. JQ1 (50-400 nM increases insulin secretion from INS-1 cells in a concentration dependent manner. JQ1 increases insulin content in INS-1 cells, accounting for increased secretion, in both rat and human islets. Higher concentrations of JQ1 decrease intracellular triglyceride stores in INS-1 cells, a result of increased fatty acid oxidation. Specific inhibition of both Brd2 and Brd4 enhances insulin transcription, leading to increased insulin content. Inhibition of Brd2 alone increases fatty acid oxidation. Overlapping yet discrete roles for individual BET proteins in metabolic regulation suggest new isoform-selective BET inhibitors may be useful to treat insulin resistant/diabetic patients. Results imply that cancer and diseases of chronic inflammation or disordered metabolism are related through shared chromatin regulatory mechanisms.

  12. Niemann-Pick C2 protein expression regulates lithogenic diet-induced gallstone formation and dietary cholesterol metabolism in mice.

    Science.gov (United States)

    Balboa, Elisa; Morales, Gabriela; Aylwin, Paula; Carrasco, Gonzalo; Amigo, Ludwig; Castro, Juan; Rigotti, Attilio; Zanlungo, Silvana

    2012-01-01

    Niemann-Pick C2 protein (NPC2) is a lysosomal soluble protein that is highly expressed in the liver; it binds to cholesterol and is involved in intracellular cholesterol trafficking, allowing the exit of lysosomal cholesterol obtained via the lipoprotein endocytic pathway. Thus, this protein may play an important role in controlling hepatic cholesterol transport and metabolism. The aim of this work was to study the relevance of NPC2 protein expression in hepatic cholesterol metabolism, biliary lipid secretion and gallstone formation by comparing NPC2 hypomorph [NPC2 (h/h)] and wild-type mice fed control, 2% cholesterol, and lithogenic diets. NPC2 (h/h) mice exhibited resistance to a diet-induced increase in plasma cholesterol levels. When consuming the chow diet, we observed increased biliary cholesterol and phospholipid secretions in NPC2 (h/h) mice. When fed the 2% cholesterol diet, NPC2 (h/h) mice exhibited low and high gallbladder bile cholesterol and phospholipid concentrations, respectively. NPC2 (h/h) mice fed with the lithogenic diet showed reduced biliary cholesterol secretion, gallbladder bile cholesterol saturation, and cholesterol crystal and gallstone formation. This work indicates that hepatic NPC2 expression is an important factor in the regulation of diet-derived cholesterol metabolism and disposal as well as in diet-induced cholesterol gallstone formation in mice.

  13. Sex difference in the association of metabolic syndrome with high sensitivity C-reactive protein in a Taiwanese population

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    Lin Wen-Yuan

    2010-07-01

    Full Text Available Abstract Background Although sex differences have been reported for associations between components of metabolic syndrome and inflammation, the question of whether there is an effect modification by sex in the association between inflammation and metabolic syndrome has not been investigated in detail. Therefore, the aim of this study was to compare associations of high sensitivity C-creative protein (hs-CRP with metabolic syndrome and its components between men and women. Methods A total of 1,305 subjects aged 40 years and over were recruited in 2004 in a metropolitan city in Taiwan. The biochemical indices, such as hs-CRP, fasting glucose levels, lipid profiles, urinary albumin, urinary creatinine and anthropometric indices, were measured. Metabolic syndrome was defined using the American Heart Association and the National Heart, lung and Blood Institute (AHA/NHLBI definition. The relationship between metabolic syndrome and hs-CRP was examined using multivariate logistic regression analysis. Results After adjustment for age and lifestyle factors including smoking, and alcohol intake, elevated concentrations of hs-CRP showed a stronger association with metabolic syndrome in women (odds ratio comparing tertile extremes 4.80 [95% CI: 3.31-6.97] than in men (2.30 [1.65-3.21]. The p value for the sex interaction was 0.002. All components were more strongly associated with metabolic syndrome in women than in men, and all sex interactions were significant except for hypertension. Conclusions Our data suggest that inflammatory processes may be of particular importance in the pathogenesis of metabolic syndrome in women.

  14. Treatment of metabolic syndrome by combination of physical activity and diet needs an optimal protein intake: a randomized controlled trial.

    Science.gov (United States)

    Dutheil, Frédéric; Lac, Gérard; Courteix, Daniel; Doré, Eric; Chapier, Robert; Roszyk, Laurence; Sapin, Vincent; Lesourd, Bruno

    2012-09-17

    The recommended dietary allowance (RDA) for protein intake has been set at 1.0-1.3 g/kg/day for senior. To date, no consensus exists on the lower threshold intake (LTI = RDA/1.3) for the protein intake (PI) needed in senior patients ongoing both combined caloric restriction and physical activity treatment for metabolic syndrome. Considering that age, caloric restriction and exercise are three increasing factors of protein need, this study was dedicated to determine the minimal PI in this situation, through the determination of albuminemia that is the blood marker of protein homeostasis. Twenty eight subjects (19 M, 9 F, 61.8 ± 6.5 years, BMI 33.4 ± 4.1 kg/m²) with metabolic syndrome completed a three-week residential programme (Day 0 to Day 21) controlled for nutrition (energy balance of -500 kcal/day) and physical activity (3.5 hours/day). Patients were randomly assigned in two groups: Normal-PI (NPI: 1.0 g/kg/day) and High-PI (HPI: 1.2 g/kg/day). Then, patients returned home and were followed for six months. Albuminemia was measured at D0, D21, D90 and D180. At baseline, PI was spontaneously 1.0 g/kg/day for both groups. Albuminemia was 40.6 g/l for NPI and 40.8 g/l for HPI. A marginal protein under-nutrition appeared in NPI with a decreased albuminemia at D90 below 35 g/l (34.3 versus 41.5 g/l for HPI, p metabolic syndrome, the lower threshold intake for protein must be set at 1.2 g/kg/day to maintain blood protein homeostasis.

  15. Type and amount of dietary protein in the treatment of metabolic syndrome: a randomized controlled trial12

    Science.gov (United States)

    Hill, Alison M; Harris Jackson, Kristina A; Roussell, Michael A; West, Sheila G; Kris-Etherton, Penny M

    2015-01-01

    Background: Food-based dietary patterns emphasizing plant protein that were evaluated in the Dietary Approaches to Stop Hypertension (DASH) and OmniHeart trials are recommended for the treatment of metabolic syndrome (MetS). However, the contribution of plant protein to total protein in these diets is proportionally less than that of animal protein. Objective: This study compared 3 diets varying in type (animal compared with plant) and amount of protein on MetS criteria. Design: Sixty-two overweight adults with MetS consumed a healthy American diet for 2 wk before being randomly allocated to either a modified DASH diet rich in plant protein (18% protein, two-thirds plant sources, n = 9 males, 12 females), a modified DASH diet rich in animal protein (Beef in an Optimal Lean Diet: 18.4% protein, two-thirds animal sources, n = 9 males, 11 females), or a moderate-protein diet (Beef in an Optimal Lean Diet Plus Protein: 27% protein, two-thirds animal sources, n = 10 males, 11 females). Diets were compared across 3 phases of energy balance: 5 wk of controlled (all foods provided) weight maintenance (WM), 6 wk of controlled weight loss (minimum 500-kcal/d deficit) including exercise (WL), and 12 wk of prescribed, free-living weight loss (FL). The primary endpoint was change in MetS criteria. Results: All groups achieved ∼5% weight loss at the end of the WL phase and maintained it through FL, with no between-diet differences (WM compared with WL, FL, P protein source or amount. Our findings demonstrate that heart-healthy weight-loss dietary patterns that emphasize either animal or plant protein improve MetS criteria similarly. This study was registered at clinicaltrials.gov as NCT00937638. PMID:26354540

  16. The Effect of Vegan Protein-Based Diets on Metabolic Parameters, Expressions of Adiponectin and Its Receptors in Wistar Rats.

    Science.gov (United States)

    Chen, Jie-Hua; Song, Jia; Chen, Yan; Ding, Qiang; Peng, Anfang; Mao, Limei

    2016-10-18

    Vegan protein-based diet has attracted increasing interest in the prevention of metabolic syndrome (MetS). Meanwhile, adiponectin has become a highly potential molecular target in the prevention of MetS. Our study will identify a potential vegan protein diet for the prevention of MetS using rat models. Thirty-six Wistar rats were randomly assigned into three groups and given diets containing one of the following proteins for 12 weeks: casein (CAS, control diet), soy protein (SOY), and gluten-soy mixed protein (GSM). Changes in metabolic parameters as well as the expressions of adiponectin and its receptors were identified. Compared to CAS diet, both SOY and GSM diets led to decreases in blood total cholesterol and triglycerides, but only GSM diet led to an increase in HDL-cholesterol; no marked difference was observed in blood glucose in all three groups; HOMA-IR was found lower only in SOY group. Among groups, the order of serum adiponectin level was found as GSM > SOY > CAS. Similar order pattern was also observed in expression of adiponectin in adipose tissue and AdipoR1 mRNA in skeletal muscle. Our results suggested for the first time that, besides SOY diet, GSM diet could also be a possible substitute of animal protein to prevent MetS.

  17. Rice protein improves adiposity, body weight and reduces lipids level in rats through modification of triglyceride metabolism

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    Yang Lin

    2012-02-01

    Full Text Available Abstract Background To elucidate whether rice protein can possess a vital function in improving lipids level and adiposity, the effects of rice proteins extracted by alkaline (RP-A and α-amylase (RP-E on triglyceride metabolism were investigated in 7-week-old male Wistar rats fed cholesterol-enriched diets for 2 weeks, as compared with casein (CAS. Results Compared with CAS, plasma concentrations of glucose and lipids were significantly reduced by RP-feeding (P P P P P > 0.05. There was a significant positive correlation between protein digestibility and deposit fat (r = 0.8567, P P Conclusions The present study demonstrates that rice protein can modify triglyceride metabolism, leading to an improvement of body weight and adiposity. Results suggest that the triglyceride-lowering action as well as the potential of anti-adiposity induced by rice protein is attributed to upregulation of lipolysis and downregulation of lipogenesis, and the lower digestibility of rice protein may be the main modulator responsible for the lipid-lowering action.

  18. The Effect of Vegan Protein-Based Diets on Metabolic Parameters, Expressions of Adiponectin and Its Receptors in Wistar Rats

    Directory of Open Access Journals (Sweden)

    Jie-Hua Chen

    2016-10-01

    Full Text Available Vegan protein-based diet has attracted increasing interest in the prevention of metabolic syndrome (MetS. Meanwhile, adiponectin has become a highly potential molecular target in the prevention of MetS. Our study will identify a potential vegan protein diet for the prevention of MetS using rat models. Thirty-six Wistar rats were randomly assigned into three groups and given diets containing one of the following proteins for 12 weeks: casein (CAS, control diet, soy protein (SOY, and gluten-soy mixed protein (GSM. Changes in metabolic parameters as well as the expressions of adiponectin and its receptors were identified. Compared to CAS diet, both SOY and GSM diets led to decreases in blood total cholesterol and triglycerides, but only GSM diet led to an increase in HDL-cholesterol; no marked difference was observed in blood glucose in all three groups; HOMA-IR was found lower only in SOY group. Among groups, the order of serum adiponectin level was found as GSM > SOY > CAS. Similar order pattern was also observed in expression of adiponectin in adipose tissue and AdipoR1 mRNA in skeletal muscle. Our results suggested for the first time that, besides SOY diet, GSM diet could also be a possible substitute of animal protein to prevent MetS.

  19. The strawberry pathogenesis-related 10 (PR-10) Fra a proteins control flavonoid biosynthesis by binding to metabolic intermediates.

    Science.gov (United States)

    Casañal, Ana; Zander, Ulrich; Muñoz, Cristina; Dupeux, Florine; Luque, Irene; Botella, Miguel Angel; Schwab, Wilfried; Valpuesta, Victoriano; Marquez, José A

    2013-12-06

    Pathogenesis-related 10 (PR-10) proteins are involved in many aspects of plant biology but their molecular function is still unclear. They are related by sequence and structural homology to mammalian lipid transport and plant abscisic acid receptor proteins and are predicted to have cavities for ligand binding. Recently, three new members of the PR-10 family, the Fra a proteins, have been identified in strawberry, where they are required for the activity of the flavonoid biosynthesis pathway, which is essential for the development of color and flavor in fruits. Here, we show that Fra a proteins bind natural flavonoids with different selectivity and affinities in the low μm range. The structural analysis of Fra a 1 E and a Fra a 3-catechin complex indicates that loops L3, L5, and L7 surrounding the ligand-binding cavity show significant flexibility in the apo forms but close over the ligand in the Fra a 3-catechin complex. Our findings provide mechanistic insight on the function of Fra a proteins and suggest that PR-10 proteins, which are widespread in plants, may play a role in the control of secondary metabolic pathways by binding to metabolic intermediates.

  20. Quantitative Metabolomics and Instationary 13C-Metabolic Flux Analysis Reveals Impact of Recombinant Protein Production on Trehalose and Energy Metabolism in Pichia pastoris

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    Joel Jordà

    2014-05-01

    Full Text Available Pichia pastoris has been recognized as an effective host for recombinant protein production. In this work, we combine metabolomics and instationary 13C metabolic flux analysis (INST 13C-MFA using GC-MS and LC-MS/MS to evaluate the potential impact of the production of a Rhizopus oryzae lipase (Rol on P. pastoris central carbon metabolism. Higher oxygen uptake and CO2 production rates and slightly reduced biomass yield suggest an increased energy demand for the producing strain. This observation is further confirmed by 13C-based metabolic flux analysis. In particular, the flux through the methanol oxidation pathway and the TCA cycle was increased in the Rol-producing strain compared to the reference strain. Next to changes in the flux distribution, significant variations in intracellular metabolite concentrations were observed. Most notably, the pools of trehalose, which is related to cellular stress response, and xylose, which is linked to methanol assimilation, were significantly increased in the recombinant strain.

  1. In vivo versus in vitro protein abundance analysis of Shigella dysenteriae type 1 reveals changes in the expression of proteins involved in virulence, stress and energy metabolism

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    Donohue-Rolfe Arthur

    2011-06-01

    Full Text Available Abstract Background Shigella dysenteriae serotype 1 (SD1 causes the most severe form of epidemic bacillary dysentery. Quantitative proteome profiling of Shigella dysenteriae serotype 1 (SD1 in vitro (derived from LB cell cultures and in vivo (derived from gnotobiotic piglets was performed by 2D-LC-MS/MS and APEX, a label-free computationally modified spectral counting methodology. Results Overall, 1761 proteins were quantitated at a 5% FDR (false discovery rate, including 1480 and 1505 from in vitro and in vivo samples, respectively. Identification of 350 cytoplasmic membrane and outer membrane (OM proteins (38% of in silico predicted SD1 membrane proteome contributed to the most extensive survey of the Shigella membrane proteome reported so far. Differential protein abundance analysis using statistical tests revealed that SD1 cells switched to an anaerobic energy metabolism under in vivo conditions, resulting in an increase in fermentative, propanoate, butanoate and nitrate metabolism. Abundance increases of transcription activators FNR and Nar supported the notion of a switch from aerobic to anaerobic respiration in the host gut environment. High in vivo abundances of proteins involved in acid resistance (GadB, AdiA and mixed acid fermentation (PflA/PflB indicated bacterial survival responses to acid stress, while increased abundance of oxidative stress proteins (YfiD/YfiF/SodB implied that defense mechanisms against oxygen radicals were mobilized. Proteins involved in peptidoglycan turnover (MurB were increased, while β-barrel OM proteins (OmpA, OM lipoproteins (NlpD, chaperones involved in OM protein folding pathways (YraP, NlpB and lipopolysaccharide biosynthesis (Imp were decreased, suggesting unexpected modulations of the outer membrane/peptidoglycan layers in vivo. Several virulence proteins of the Mxi-Spa type III secretion system and invasion plasmid antigens (Ipa proteins required for invasion of colonic epithelial cells, and release

  2. In vivo versus in vitro protein abundance analysis of Shigella dysenteriae type 1 reveals changes in the expression of proteins involved in virulence, stress and energy metabolism.

    Science.gov (United States)

    Kuntumalla, Srilatha; Zhang, Quanshun; Braisted, John C; Fleischmann, Robert D; Peterson, Scott N; Donohue-Rolfe, Arthur; Tzipori, Saul; Pieper, Rembert

    2011-06-24

    Shigella dysenteriae serotype 1 (SD1) causes the most severe form of epidemic bacillary dysentery. Quantitative proteome profiling of Shigella dysenteriae serotype 1 (SD1) in vitro (derived from LB cell cultures) and in vivo (derived from gnotobiotic piglets) was performed by 2D-LC-MS/MS and APEX, a label-free computationally modified spectral counting methodology. Overall, 1761 proteins were quantitated at a 5% FDR (false discovery rate), including 1480 and 1505 from in vitro and in vivo samples, respectively. Identification of 350 cytoplasmic membrane and outer membrane (OM) proteins (38% of in silico predicted SD1 membrane proteome) contributed to the most extensive survey of the Shigella membrane proteome reported so far. Differential protein abundance analysis using statistical tests revealed that SD1 cells switched to an anaerobic energy metabolism under in vivo conditions, resulting in an increase in fermentative, propanoate, butanoate and nitrate metabolism. Abundance increases of transcription activators FNR and Nar supported the notion of a switch from aerobic to anaerobic respiration in the host gut environment. High in vivo abundances of proteins involved in acid resistance (GadB, AdiA) and mixed acid fermentation (PflA/PflB) indicated bacterial survival responses to acid stress, while increased abundance of oxidative stress proteins (YfiD/YfiF/SodB) implied that defense mechanisms against oxygen radicals were mobilized. Proteins involved in peptidoglycan turnover (MurB) were increased, while β-barrel OM proteins (OmpA), OM lipoproteins (NlpD), chaperones involved in OM protein folding pathways (YraP, NlpB) and lipopolysaccharide biosynthesis (Imp) were decreased, suggesting unexpected modulations of the outer membrane/peptidoglycan layers in vivo. Several virulence proteins of the Mxi-Spa type III secretion system and invasion plasmid antigens (Ipa proteins) required for invasion of colonic epithelial cells, and release of bacteria into the host cell

  3. Boesenbergia pandurata attenuates diet-induced obesity by activating AMP-activated protein kinase and regulating lipid metabolism.

    Science.gov (United States)

    Kim, Dae-Young; Kim, Myung-Suk; Sa, Bo-Kyung; Kim, Mi-Bo; Hwang, Jae-Kwan

    2012-01-01

    Obesity, a chronic metabolic disorder, is characterized by enlarged fat mass and dysregulation of lipid metabolism. The medicinal plant, Boesenbergia pandurata (Roxb.) Schltr., has been reported to possess anti-oxidative and anti-inflammatory properties; however, its anti-obesity activity is unexplored. The present study was conducted to determine whether B. pandurata extract (BPE), prepared from its rhizome parts, attenuated high-fat diet (HFD)-induced obesity in C57BL/6J mice. The molecular mechanism was investigated in 3T3-L1 adipocytes and HepG2 human hepatoma cells. BPE treatment decreased triglyceride accumulation in both 3T3-L1 adipocytes and HepG2 hepatocytes by activating AMP-activated protein kinase (AMPK) signaling and regulating the expression of lipid metabolism-related proteins. In the animal model, oral administration of BPE (200 mg/kg/day for 8 weeks) significantly reduced HFD-induced body weight gain without altering the amount of food intake. In addition, elevated serum levels of total cholesterol, low-density lipoprotein cholesterol, and triglycerides were suppressed by BPE administration. Fat pad masses were reduced in BPE-treated mice, as evidenced by reduced adipocyte size. Furthermore, BPE protected against the development of nonalcoholic fatty liver by decreasing hepatic triglyceride accumulation. BPE also activated AMPK signaling and altered the expression of lipid metabolism-related proteins in white adipose tissue and liver. Taken together, these findings indicate that BPE attenuates HFD-induced obesity by activating AMPK and regulating lipid metabolism, suggesting a potent anti-obesity agent.

  4. Road to the future of systems biotechnology: CRISPR-Cas-mediated metabolic engineering for recombinant protein production.

    Science.gov (United States)

    Roointan, Amir; Morowvat, Mohammad Hossein

    The rising potential for CRISPR-Cas-mediated genome editing has revolutionized our strategies in basic and practical bioengineering research. It provides a predictable and precise method for genome modification in a robust and reproducible fashion. Emergence of systems biotechnology and synthetic biology approaches coupled with CRISPR-Cas technology could change the future of cell factories to possess some new features which have not been found naturally. We have discussed the possibility and versatile potentials of CRISPR-Cas technology for metabolic engineering of a recombinant host for heterologous protein production. We describe the mechanisms involved in this metabolic engineering approach and present the diverse features of its application in biotechnology and protein production.

  5. Proteins involved in wine aroma compounds metabolism by a Saccharomyces cerevisiae flor-velum yeast strain grown in two conditions.

    Science.gov (United States)

    Moreno-García, Jaime; García-Martínez, Teresa; Millán, M Carmen; Mauricio, Juan Carlos; Moreno, Juan

    2015-10-01

    A proteomic and exometabolomic study was conducted on Saccharomyces cerevisiae flor yeast strain growing under biofilm formation condition (BFC) with ethanol and glycerol as carbon sources and results were compared with those obtained under no biofilm formation condition (NBFC) containing glucose as carbon source. By using modern techniques, OFFGEL fractionator and LTQ-Orbitrap for proteome and SBSE-TD-GC-MS for metabolite analysis, we quantified 84 proteins including 33 directly involved in the metabolism of glycerol, ethanol and 17 aroma compounds. Contents in acetaldehyde, acetic acid, decanoic acid, 1,1-diethoxyethane, benzaldehyde and 2-phenethyl acetate, changed above their odor thresholds under BFC, and those of decanoic acid, ethyl octanoate, ethyl decanoate and isoamyl acetate under NBFC. Of the twenty proteins involved in the metabolism of ethanol, acetaldehyde, acetoin, 2,3-butanediol, 1,1-diethoxyethane, benzaldehyde, organic acids and ethyl esters, only Adh2p, Ald4p, Cys4p, Fas3p, Met2p and Plb1p were detected under BFC and as many Acs2p, Ald3p, Cem1p, Ilv2p, Ilv6p and Pox1p, only under NBFC. Of the eight proteins involved in glycerol metabolism, Gut2p was detected only under BFC while Pgs1p and Rhr2p were under NBFC. Finally, of the five proteins involved in the metabolism of higher alcohols, Thi3p was present under BFC, and Aro8p and Bat2p were under NBFC. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. iTRAQ-based protein profiling provides insights into the central metabolism changes driving grape berry development and ripening.

    Science.gov (United States)

    Martínez-Esteso, María José; Vilella-Antón, María Teresa; Pedreño, María Ángeles; Valero, María Luz; Bru-Martínez, Roque

    2013-10-24

    Grapevine (Vitis vinifera L.) is an economically important fruit crop. Quality-determining grape components such as sugars, acids, flavors, anthocyanins, tannins, etc., accumulate in the different grape berry development stages. Thus, correlating the proteomic profiles with the biochemical and physiological changes occurring in grape is of paramount importance to advance in our understanding of berry development and ripening processes. We report the developmental analysis of Vitis vinifera cv. Muscat Hamburg berries at the protein level from fruit set to full ripening. An iTRAQ-based bottom-up proteomic approach followed by tandem mass spectrometry led to the identification and quantitation of 411 and 630 proteins in the green and ripening phases, respectively. Two key points in development relating to changes in protein level were detected: end of the first growth period (7 mm-to-15 mm) and onset of ripening (15 mm-to-V100, V100-to-110). A functional analysis was performed using the Blast2GO software based on the enrichment of GO terms during berry growth. The study of the proteome contributes to decipher the biological processes and metabolic pathways involved in the development and quality traits of fruit and its derived products. These findings lie mainly in metabolism and storage of sugars and malate, energy-related pathways such as respiration, photosynthesis and fermentation, and the synthesis of polyphenolics as major secondary metabolites in grape berry. In addition, some key steps in carbohydrate and malate metabolism have been identified in this study, i.e., PFP-PFK or SuSy-INV switches among others, which may influence the final sugar and acid balance in ripe fruit. In conclusion, some proteins not reported to date have been detected to be deregulated in specific tissues and developmental stages, leading to formulate new hypotheses on the metabolic processes underlying grape berry development. These results open up new lines to decipher the

  7. Multi-omic profiling of EPO-producing Chinese hamster ovary cell panel reveals metabolic adaptation to heterologous protein production

    DEFF Research Database (Denmark)

    Ley, Daniel; Kazemi Seresht, Ali; Engmark, Mikael

    2015-01-01

    Chinese hamster ovary (CHO) cells are the preferred production host for many therapeutic proteins. The production of heterologous proteins in CHO cells imposes a burden on the host cell metabolism and impact cellular physiology on a global scale. In this work, a multi-omics approach was applied...... to 5 pg/cell/day. Time-course analysis of high- and low-producing clones in chemostat culture revealed rapid adaptation of transcription levels of amino acid catabolic genes in favor of EPO production within nine generations. Interestingly, the adaptation was followed by an increase in specific EPO...

  8. Metabolic syndrome: prevalence, associated factors, and C-reactive protein: the MADRIC (MADrid RIesgo Cardiovascular) Study.

    Science.gov (United States)

    Martínez, Maria A; Puig, Juan G; Mora, Marta; Aragón, Rosa; O'Dogherty, Pascual; Antón, José L; Sánchez-Villares, Teresa; Rubio, José M; Rosado, Javier; Torres, Rosa; Marcos, Joaquín; Pallardo, Luis F; Banegas, José R

    2008-09-01

    The metabolic syndrome (MS) is defined by the clustering of a number of cardiovascular risk factors. The aims of the present study were to estimate the prevalence of MS in Madrid (Spain) by 2 definitions and to investigate its relationship with several sociodemographic factors and C-reactive protein (CRP) levels. This was a cross-sectional population study, and participants were 1344 subjects aged 31 to 70 years. Clinical evaluation included data on sociodemographic and cardiovascular background, physical examination, fasting glucose, triglycerides, and high-density lipoprotein cholesterol. The CRP levels were determined in a subgroup of 843 subjects. The diagnosis of MS was made according to the 2005 Adult Treatment Panel III (ATP III) and International Diabetes Federation (IDF) definitions. The age- and sex-adjusted prevalence of MS was 24.6% (95% confidence interval [CI], 22.3%-26.9%) using the ATP III definition and 30.9% (95% CI, 28.4%-33.3%) using the International Diabetes Federation definition. The overall agreement rate was 91.5% (kappa = 0.80; 95% CI, 0.76-0.83). Prevalence figures by both definitions were higher in men than in women and increased with age. Male sex, older age, low educational level, and physical inactivity were all determinants of ATP III-defined MS. The presence of MS or any of its components was associated with high CRP levels. In a logistic regression analysis, low educational level and waist circumference were the best predictors for high CRP level. The prevalence of MS in the Madrid region is one of the highest in Europe and confirms the strong Spanish regional variability in this syndrome frequency. Some sociodemographic and lifestyle factors, particularly educational level, are predictors for MS and high CRP levels.

  9. Muscle metabolism during exercise with carbohydrate or protein-carbohydrate ingestion.

    Science.gov (United States)

    Cermak, Naomi M; Solheim, Amy S; Gardner, Melanie S; Tarnopolsky, Mark A; Gibala, Martin J

    2009-12-01

    Ingesting protein (PRO) with CHO during prolonged exercise is purported to improve performance compared with CHO alone by altering the regulation of skeletal muscle energy provision. However, no study has directly investigated this issue. We tested the hypothesis that compared with CHO alone, coingestion of PRO would alter markers of metabolic control, including the magnitude of glycogen use and the net expansion of the tricarboxylic acid cycle intermediate pool, which has been linked to the capacity for oxidative energy delivery. Eight trained men (mean +/- SE: age = 29 +/- 2 yr; VO2peak = 55 +/- 2 mL x kg(-1) x min(-1)) cycled at 69% +/- 1% VO2peak for 90 min on two occasions, and biopsy samples (vastus lateralis) were obtained before and after exercise. In a randomized, double-blind manner, subjects ingested one of two drinks during exercise that contained either 6% CHO or 6% CHO + 2% PRO (CHO + PRO) at a rate of 1 L x h(-1) to deliver 60 g x h(-1) CHO +/- 20 g x h(-1) PRO. CHO + PRO ingestion increased the plasma concentration of branched chain (561 +/- 46 vs 301 +/- 32 micromol x L(-1)) and essential amino acids (1071 +/- 98 vs 670 +/- 71 micromol x L(-1)) after exercise versus CHO (both P values exercise bout were not different between treatments. When trained men ingest CHO at a rate on the upper end of the range generally recommended to improve endurance performance, coingestion of PRO does not alter specific markers proposed to reflect an enhanced capacity for skeletal muscle energy delivery.

  10. High sensitivity C-reactive protein in airline pilots with metabolic syndrome.

    Science.gov (United States)

    Alonso-Rodríguez, César; Medina-Font, Juan

    2012-05-01

    Airline pilots belong to a relatively high-income, healthy population, with sedentary behavior during their flight activity, who often eat unsuitable meals. We assessed the prevalence of metabolic syndrome (MS) and the levels of high sensitivity C-reactive protein (hs-CRP) in a population of airline pilot in order to study a possible relationship between the hs-CRP and MS. MS was established according to the National Cholesterol Education Program, Adult Treatment Panel III. hs-CRP was classified into three categories: Low 3 mg x L(-1). The prevalence of MS was 14.8%. The hs-CRP level in the population studied was 1.68 +/- 1.79 mg x L(-1). hs-CRP significantly increased with age. The pilots with MS presented significantly higher hs-CRP levels (median = 1.9 with an interquartile range (IQR) = 2.5 mg x L(-1)) than the pilots without MS (median = 0.9 and IQR = 1.275 mg x L(-1)). MS significantly increased in the groups with high hs-CRP in comparison with pilots with intermediate hs-CRP levels and with those with low hs-CRP levels. A similar association was found between the levels of hs-CRP and the prevalence of MS in the three age groups. The levels of hs-CRP increased in pilots as they presented greater numbers of MS diagnostic criteria. hs-CRP rises significantly in pilots of increasing age, in pilots with MS as compared to those without the syndrome, and in pilots as they present greater numbers of MS diagnostic criteria. The prevalence of MS increased among the groups with higher levels of hs-CRP.

  11. Marked over expression of uncoupling protein-2 in beta cells exerts minor effects on mitochondrial metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Hals, Ingrid K., E-mail: ingrid.hals@ntnu.no [Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology (NTNU), Trondheim (Norway); Ogata, Hirotaka; Pettersen, Elin [Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology (NTNU), Trondheim (Norway); Ma, Zuheng; Bjoerklund, Anneli [Department of Molecular Medicine and Surgery, Karolinska Institute, Stockholm (Sweden); Skorpen, Frank [Department of Laboratory Medicine, NTNU, Trondheim (Norway); Egeberg, Kjartan Wollo [Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology (NTNU), Trondheim (Norway); Grill, Valdemar [Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology (NTNU), Trondheim (Norway); Department of Molecular Medicine and Surgery, Karolinska Institute, Stockholm (Sweden)

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer The impact of UCP-2 over expression on mitochondrial function is controversial. Black-Right-Pointing-Pointer We tested mitochondrial functions at defined levels of overexpression. Black-Right-Pointing-Pointer We find minor increases of fatty acid oxidation and uncoupling. Black-Right-Pointing-Pointer Effects were seen only at high level (fourfold) of over expression. Black-Right-Pointing-Pointer Hence it is doubtful whether these effects are of importance in diabetes. -- Abstract: Evidence is conflicting as to the impact of elevated levels of uncoupling protein-2 (UCP-2) on insulin-producing beta cells. Here we investigated effects of a fourfold induction of UCP-2 protein primarily on mitochondrial parameters and tested for replication of positive findings at a lower level of induction. We transfected INS-1 cells to obtain a tet-on inducible cell line. A 48 h exposure to 1 {mu}g/ml of doxycycline (dox) induced UCP-2 fourfold (424 {+-} 113%, mean {+-} SEM) and 0.1 {mu}g/ml twofold (178 {+-} 29%, n = 3). Fourfold induced cells displayed normal viability (MTT, apoptosis), normal cellular insulin contents and, glucose-induced insulin secretion (+27 {+-} 11%) as well as D-[U-{sup 14}C]-glucose oxidation (+5 {+-} 9% at 11 mM glucose). Oxidation of [1-{sup 14}C]-oleate was increased from 4088 to 5797 fmol/{mu}g prot/2 h at 3.3 mM glucose, p < 0.03. Oxidation of L-[{sup 14}C(U)]-glutamine was unaffected. Induction of UCP-2 did not significantly affect measures of mitochondrial membrane potential (Rhodamine 123) or mitochondrial mass (Mitotracker Green) and did not affect ATP levels. Oligomycin-inhibited oxygen consumption (a measure of mitochondrial uncoupling) was marginally increased, the effect being significant in comparison with dox-only treated cells, p < 0.05. Oxygen radicals, assessed by dichlorofluorescin diacetate, were decreased by 30%, p < 0.025. Testing for the lower level of UCP-2 induction did not reproduce any of the

  12. C-Terminal Binding Protein: A Molecular Link between Metabolic Imbalance and Epigenetic Regulation in Breast Cancer

    Directory of Open Access Journals (Sweden)

    Jung S. Byun

    2013-01-01

    Full Text Available The prevalence of obesity has given rise to significant global concerns as numerous population-based studies demonstrate an incontrovertible association between obesity and breast cancer. Mechanisms proposed to account for this linkage include exaggerated levels of carbohydrate substrates, elevated levels of circulating mitogenic hormones, and inflammatory cytokines that impinge on epithelial programming in many tissues. Moreover, recently many scientists have rediscovered the observation, first described by Otto Warburg nearly a century ago, that most cancer cells undergo a dramatic metabolic shift in energy utilization and expenditure that fuels and supports the cellular expansion associated with malignant proliferation. This shift in substrate oxidation comes at the cost of sharp changes in the levels of the high energy intermediate, nicotinamide adenine dinucleotide (NADH. In this review, we discuss a novel example of how shifts in the concentration and flux of substrates metabolized and generated during carbohydrate metabolism represent components of a signaling network that can influence epigenetic regulatory events in the nucleus. We refer to this regulatory process as “metabolic transduction” and describe how the C-terminal binding protein (CtBP family of NADH-dependent nuclear regulators represents a primary example of how cellular metabolic status can influence epigenetic control of cellular function and fate.

  13. Exercise Training and Work Task Induced Metabolic and Stress-Related mRNA and Protein Responses in Myalgic Muscles

    Directory of Open Access Journals (Sweden)

    Gisela Sjøgaard

    2013-01-01

    Full Text Available The aim was to assess mRNA and/or protein levels of heat shock proteins, cytokines, growth regulating, and metabolic proteins in myalgic muscle at rest and in response to work tasks and prolonged exercise training. A randomized controlled trial included 28 females with trapezius myalgia and 16 healthy controls. Those with myalgia performed ~7 hrs repetitive stressful work and were subsequently randomized to 10 weeks of specific strength training, general fitness training, or reference intervention. Muscles biopsies were taken from the trapezius muscle at baseline, after work and after 10 weeks intervention. The main findings are that the capacity of carbohydrate oxidation was reduced in myalgic compared with healthy muscle. Repetitive stressful work increased mRNA content for heat shock proteins and decreased levels of key regulators for growth and oxidative metabolism. In contrast, prolonged general fitness as well as specific strength training decreased mRNA content of heat shock protein while the capacity of carbohydrate oxidation was increased only after specific strength training.

  14. Effect of 21-day head down bed rest on urine proteins related to endothelium: Correlations with changes in carbohydrate metabolism

    Science.gov (United States)

    Kashirina, D.; Pastushkova, L.; Custaud, M. A.; Dobrokhotov, I.; Brzhozovsky, A.; Navasiolava, N.; Nosovsky, A.; Kononikhin, A.; Nikolaev, E.; Larina, I.

    2017-08-01

    We performed liquid chromatography-mass spectrometric study of the urine proteome in 8 healthy volunteers aged between 20 and 44 y.o. who have completed 21-day head-down bed rest. ANDSystem software which builds associative networks was used to identify the urinary proteins functionally related to the endothelium. We identified 7 endothelium-related biological processes, directly linked to 13 urine proteins. We performed manual annotation of the proteins which were the most important in terms of endothelial functions. Analysis of the correlations with biochemical variables revealed a positive correlation between fasting blood glucose and the following urine proteins: albumin, CD44 antigen, endothelial protein C receptor, mucin-1, osteopontin, receptor tyrosine kinase. As well, we found a positive correlation between HOMA-insulin resistance index and the following urine proteins: endothelial protein C receptor and syndecan-4. These results might suggest the involvement of above-mentioned proteins in glucose metabolism and their participation in the response to changes in blood glucose level.

  15. Aroclor 1254, a developmental neurotoxicant, alters energy metabolism- and intracellular signaling-associated protein networks in rat cerebellum and hippocampus

    Energy Technology Data Exchange (ETDEWEB)

    Kodavanti, Prasada Rao S., E-mail: kodavanti.prasada@epa.gov [Neurotoxicology Branch, NHEERL, ORD, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina (United States); Osorio, Cristina [Systems Proteomics Center, University of North Carolina at Chapel Hill, North Carolina (United States); Program on Molecular Biology and Biotechnology, University of North Carolina at Chapel Hill, North Carolina (United States); Royland, Joyce E.; Ramabhadran, Ram [Genetic and Cellular Toxicology Branch, NHEERL, ORD, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina (United States); Alzate, Oscar [Department of Cellular and Developmental Biology, University of North Carolina at Chapel Hill, North Carolina (United States); Systems Proteomics Center, University of North Carolina at Chapel Hill, North Carolina (United States); Program on Molecular Biology and Biotechnology, University of North Carolina at Chapel Hill, North Carolina (United States)

    2011-11-15

    The vast literature on the mode of action of polychlorinated biphenyls (PCBs) indicates that PCBs are a unique model for understanding the mechanisms of toxicity of environmental mixtures of persistent chemicals. PCBs have been shown to adversely affect psychomotor function and learning and memory in humans. Although the molecular mechanisms for PCB effects are unclear, several studies indicate that the disruption of Ca{sup 2+}-mediated signal transduction plays significant roles in PCB-induced developmental neurotoxicity. Culminating events in signal transduction pathways include the regulation of gene and protein expression, which affects the growth and function of the nervous system. Our previous studies showed changes in gene expression related to signal transduction and neuronal growth. In this study, protein expression following developmental exposure to PCB is examined. Pregnant rats (Long Evans) were dosed with 0.0 or 6.0 mg/kg/day of Aroclor-1254 from gestation day 6 through postnatal day (PND) 21, and the cerebellum and hippocampus from PND14 animals were analyzed to determine Aroclor 1254-induced differential protein expression. Two proteins were found to be differentially expressed in the cerebellum following PCB exposure while 18 proteins were differentially expressed in the hippocampus. These proteins are related to energy metabolism in mitochondria (ATP synthase, sub unit {beta} (ATP5B), creatine kinase, and malate dehydrogenase), calcium signaling (voltage-dependent anion-selective channel protein 1 (VDAC1) and ryanodine receptor type II (RyR2)), and growth of the nervous system (dihydropyrimidinase-related protein 4 (DPYSL4), valosin-containing protein (VCP)). Results suggest that Aroclor 1254-like persistent chemicals may alter energy metabolism and intracellular signaling, which might result in developmental neurotoxicity. -- Highlights: Black-Right-Pointing-Pointer We performed brain proteomic analysis of rats exposed to the neurotoxicant

  16. The effects of wet cupping on serum high-sensitivity C-reactive protein and heat shock protein 27 antibody titers in patients with metabolic syndrome.

    Science.gov (United States)

    Farahmand, Seyed Kazem; Gang, Li Zhi; Saghebi, Seyed Ahmad; Mohammadi, Maryam; Mohammadi, Shabnam; Mohammadi, Ghazaleh; Ferns, Gordan A; Ghanbarzadeh, Majid; Razmgah, Gholamreza Ghayour; Ramazani, Zahra; Ghayour-Mobarhan, Majid; Esmaily, Habibollah; Bahrami Taghanaki, Hamidreza; Azizi, Hoda

    2014-08-01

    It has previously been reported that increased level of serum heat shock proteins (Hsps) antibody in patients with metabolic syndrome. It is possible that the expression of Hsp and inflammatory markers can be affected by cupping and traditional Chinese medicine. There is a little data investigating the effects of cupping on markers of inflammation and Hsp proteins, hence, the objective of this study was evaluation of the effects of wet cupping on serum high-sensitivity C-reactive protein (hs-CRP) and Hsp27 antibody titers in patients with metabolic syndrome. Serum Hs-CRP and Hsp27 antibody titers were assessed in samples from 126 patients with metabolic syndrome (18-65 years of age) at baseline, and after 6 and 12 weeks after treatment. One hundred and twenty-six patients were randomly divided into the experimental group treated with wet cupping combined with dietary advice, and the control group treated with dietary advice alone using a random number table. Eight patients in case group and five subjects in control groups were excluded from the study. Data were analyzed using SPSS 15.0 software and a repeated measure ANCOVA. Serum hs-CRP titers did not change significantly between groups (p>0.05) and times (p=0.27). The same result was found for Hsp27 titers (p>0.05). Wet-cupping on the interscapular region has no effect on serum hs-CRP and Hsp27 patients with metabolic syndrome. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Interactions between vertebrate hemoglobins and red cell proteins: Possible roles in regulating cellular metabolism and rheology

    DEFF Research Database (Denmark)

    Weber, Roy E.

    2007-01-01

    Red blood cells (RBCs) play a vital role in vertebrate metabolism. Tissue O2 delivery depends on their O2 transporting properties and rheology, an integral determinant of tissue perfusion. The mechanical characteristics and key metabolic characteristics of RBCs (such as glycolysis rate, pentose...

  18. Hepatitis C virus core protein activates autophagy through EIF2AK3 and ATF6 UPR pathway-mediated MAP1LC3B and ATG12 expression

    Science.gov (United States)

    Wang, Ji; Kang, Rongyan; Huang, He; Xi, Xueyan; Wang, Bei; Wang, Jianwei; Zhao, Zhendong

    2014-01-01

    HCV infection induces autophagy, but how this occurs is unclear. Here, we report the induction of autophagy by the structural HCV core protein and subsequent endoplasmic reticular (ER) stress in Huh7 hepatoma cells. During ER stress, both the EIF2AK3 and ATF6 pathways of the unfolded protein response (UPR) were activated by HCV core protein. Then, these pathways upregulated transcription factors ATF4 and DDIT3. The ERN1-XBP1 pathway was not activated. Through ATF4 in the EIF2AK3 pathway, the autophagy gene ATG12 was upregulated. DDIT3 upregulated the transcription of autophagy gene MAP1LC3B (LC3B) by directly binding to the –253 to –99 base region of the LC3B promoter, contributing to the development of autophagy. Collectively, these data suggest not only a novel role for the HCV core protein in autophagy but also offer new insight into detailed molecular mechanisms with respect to HCV-induced autophagy, specifically how downstream UPR molecules regulate key autophagic gene expression. PMID:24589849

  19. Ex Vivo Transepithelial Collagen Cross-linking in Porcine and Human Corneas Using Human Decorin Core Protein.

    Science.gov (United States)

    Metzler, Kimberly M; Roberts, Cynthia J; Mahmoud, Ashraf M; Agarwal, Gunjan; Liu, Jun

    2016-06-01

    To investigate changes in corneal biomechanics after cross-linking with human decorin core protein (decoron), which is a small, naturally occurring proteoglycan that bridges collagen fibrils, organizing and stabilizing lamellar collagen architecture. Five human donor pairs (10 eyes) and 5 porcine pairs (10 eyes) had one random eye treated transepithelially with decoron, with the untreated fellow eye serving as control. Pretreatment (45 to 60 seconds) and penetration enhancer (45 to 60 seconds) preceded instillation of decoron (45 to 60 seconds). Total treatment time was less than 4 minutes per eye. Human donor eyes were evaluated using the CorVis ST (Oculus Optikgeräte GmbH, Wetzlar, Germany) at 15, 20, 30, 40, and 50 mm Hg of intraocular pressure. Elastic modulus was calculated for human corneas, using parameters derived from Scheimpflug images. Analysis of variance was performed. Porcine corneas underwent uniaxial tensile testing with a Rheometrics Systems Analyzer (RSA III; TA Instruments, New Castle, DE). Secant modulus was calculated and paired t tests were performed between treated and control groups. One human eye pair was excluded based on initial corneal thickness greater than 850 µm. Analysis of variance of the included four pairs demonstrated a significant treatment effect (P cross-linking. Elastic modulus demonstrated a significant treatment effect with a higher elastic modulus in the treatment group. In porcine eye pairs, the secant modulus was significantly higher in the treated than the untreated corneas at 4%, 5%, and 6% strain (P biomechanical behavior and higher elastic modulus in both human and porcine corneas in this preliminary ex vivo study. Further studies are needed to evaluate clinical safety, efficacy, and long-term stability. [J Refract Surg. 2016;32(6):410-417.]. Copyright 2016, SLACK Incorporated.

  20. C-Terminal Substitution of HBV Core Proteins with Those from DHBV Reveals That Arginine-Rich 167RRRSQSPRR175 Domain Is Critical for HBV Replication

    Science.gov (United States)

    Kim, Taeyeung; Shin, Bo-Hye; Park, Gil-Soon; Park, Sun; Chwae, Yong-Joon; Shin, Ho-Joon; Kim, Kyongmin

    2012-01-01

    To investigate the contributions of carboxyl-terminal nucleic acid binding domain of HBV core (C) protein for hepatitis B virus (HBV) replication, chimeric HBV C proteins were generated by substituting varying lengths of the carboxyl-terminus of duck hepatitis B virus (DHBV) C protein for the corresponding regions of HBV C protein. All chimeric C proteins formed core particles. A chimeric C protein with 221–262 amino acids of DHBV C protein, in place of 146–185 amino acids of the HBV C protein, supported HBV pregenomic RNA (pgRNA) encapsidation and DNA synthesis: 40% amino acid sequence identity or 45% homology in the nucleic-acid binding domain of HBV C protein was sufficient for pgRNA encapsidation and DNA synthesis, although we predominantly detected spliced DNA. A chimeric C protein with 221–241 and 251–262 amino acids of DHBV C, in place of HBV C 146–166 and 176–185 amino acids, respectively, could rescue full-length DNA synthesis. However, a reciprocal C chimera with 242–250 of DHBV C (242RAGSPLPRS250) introduced in place of 167–175 of HBV C (167RRRSQSPRR175) significantly decreased pgRNA encapsidation and DNA synthesis, and full-length DNA was not detected, demonstrating that the arginine-rich 167RRRSQSPRR175 domain may be critical for efficient viral replication. Five amino acids differing between viral species (underlined above) were tested for replication rescue; R169 and R175 were found to be important. PMID:22911745

  1. Interaction of E1 and E3 components with the core proteins of the human pyruvate dehydrogenase complex.

    Science.gov (United States)

    Patel, Mulchand S; Korotchkina, Lioubov G; Sidhu, Sukhdeep

    2009-11-01

    The human (h) pyruvate dehydrogenase complex (hPDC) consists of multiple copies of several components: pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), dihydrolipoamide dehydrogenase (E3), E3-binding protein (BP), and specific kinases and phosphatases. Mammalian PDC has a well organized structure with an icosahedral symmetry of the central E2/BP core to which the other component proteins bind non-covalently. Both hE2 and hBP consist of three well defined domains, namely the lipoyl domain, the subunit-binding domain and the inner domain, connected with flexible linkers. hE1 (alpha(2)beta(2)) binds to the subunit-binding domain of hE2; whereas hE3 binds to the E3-binding domain of hBP. Among several residues of the C-terminal surface of the hE1beta E1betaD289 was found to interact with hE2K276. The C-terminal residue I329 of the hE1beta did not participate in binding to hE2. This latter finding shows specificity in the interaction between E1beta and E2 in hPDC. The selective binding between hE3 and the E3-binding domain of hBP was investigated using specific mutants. E3R460G and E3340K showed significant reductions in affinity for hBP as determined by surface plasmon resonance. Both residues are involved in the structural organization of the binding site on hE3. Substitution of I157, N137 and R155 of hBP resulted in variable increases in the K(D) for binding with wild-type hE3, suggesting that the binding results from several weak electrostatic bonds and hydrophobic interactions among residues of hBP with residues at the interface of dimeric hE3. These results provide insight in the mono-specificity of binding of E1 to E2 and E3 to BP in hPDC and showed the differences in the binding of peripheral components (E1 and E3) in human and bacterial PDCs.

  2. Recent advances in the application of core-shell structured magnetic materials for the separation and enrichment of proteins and peptides.

    Science.gov (United States)

    Zhao, Man; Xie, Yiqin; Deng, Chunhui; Zhang, Xiangmin

    2014-08-29

    Many endogenous proteins/peptides and proteins/peptides with post-translational modifications (PTMs) are presented at extremely low abundance, and they usually suffer strong interference with highly abundant proteins/peptides as well as other contaminants, resulting in low ionization efficiency in MS analysis. Therefore, the separation and enrichment of proteins/peptides from complex mixtures is of great importance to the successful identification of them. Core-shell structured magnetic microspheres have been widely used in the enrichment and isolation of proteins/peptides, thanks to unique properties such as strong magnetic responsiveness, outstanding binding capacity, excellent biocompatibility, robust mechanical strength and admirable recoverability. The aim of this review is to update the advances in the application of core-shell structured magnetic materials for proteomics analysis, including the separation and enrichment of low-concentration proteins/peptides, the selective enrichment of phosphoproteins and the selective enrichment of glycoproteins, and to compare the enrichment performance of magnetic microspheres with different kinds of functionalization. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Intrauterine growth retarded progeny of pregnant sows fed high protein:low carbohydrate diet is related to metabolic energy deficit.

    Directory of Open Access Journals (Sweden)

    Cornelia C Metges

    Full Text Available High and low protein diets fed to pregnant adolescent sows led to intrauterine growth retardation (IUGR. To explore underlying mechanisms, sow plasma metabolite and hormone concentrations were analyzed during different pregnancy stages and correlated with litter weight (LW at birth, sow body weight and back fat thickness. Sows were fed diets with low (6.5%, LP, adequate (12.1%, AP, and high (30%, HP protein levels, made isoenergetic by adjusted carbohydrate content. At -5, 24, 66, and 108 days post coitum (dpc fasted blood was collected. At 92 dpc, diurnal metabolic profiles were determined. Fasted serum urea and plasma glucagon were higher due to the HP diet. High density lipoprotein cholesterol (HDLC, %HDLC and cortisol were reduced in HP compared with AP sows. Lowest concentrations were observed for serum urea and protein, plasma insulin-like growth factor-I, low density lipoprotein cholesterol, and progesterone in LP compared with AP and HP sows. Fasted plasma glucose, insulin and leptin concentrations were unchanged. Diurnal metabolic profiles showed lower glucose in HP sows whereas non-esterified fatty acids (NEFA concentrations were higher in HP compared with AP and LP sows. In HP and LP sows, urea concentrations were 300% and 60% of AP sows, respectively. Plasma total cholesterol was higher in LP than in AP and HP sows. In AP sows, LW correlated positively with insulin and insulin/glucose and negatively with glucagon/insulin at 66 dpc, whereas in HP sows LW associated positively with NEFA. In conclusion, IUGR in sows fed high protein:low carbohydrate diet was probably due to glucose and energy deficit whereas in sows with low protein:high carbohydrate diet it was possibly a response to a deficit of indispensable amino acids which impaired lipoprotein metabolism and favored maternal lipid disposal.

  4. A proteomic approach to determine changes in proteins involved in the myocardial metabolism in left ventricles of spontaneously hypertensive rats.

    Science.gov (United States)

    Zamorano-León, Jose J; Modrego, Javier; Mateos-Cáceres, Petra J; Macaya, Carlos; Martín-Fernández, Beatriz; Miana, María; de las Heras, Natalia; Cachofeiro, Victoria; Lahera, Vicente; López-Farré, Antonio J

    2010-01-01

    Different works have suggested that in the hypertrophied heart the energy metabolic pathway shifts to glycolysis. Our aim was to evaluate using proteomics the expression of proteins associated with different energetic metabolic pathways in hypertrophied left ventricles of spontaneously hypertensive rats (SHR). 24-weeks-old SHR with stable hypertension and established left ventricle hypertrophy were used. Normotensive Wistar Kyoto rats were used as control. Proteins from left ventricles were analyzed by 2-dimensional electrophoresis and identified by comparison with a virtual rat heart proteomic map and mass spectrometry. Enoyl-CoA hydratase expression, an enzyme involved in fatty acid beta-oxidation, was reduced whereas the expression of other beta-oxidation enzymes, 3-ketoacyl-CoA thiolase and the mitochondrial precursor of acyl-CoA thioester hydrolase, was increased in the hypertrophied left ventricles. The expression of two enzymes involved in the first steps of glycolysis, fructose bisphosphate aldolase and triosephosphate isomerase, was reduced in the left ventricle of SHR. Pyruvate dehydrogenase expression, enzyme involved in glucose oxidation, was enhanced in the hypertrophied ventricles whereas proteins of the tricarboxylic acid cycle were not modified. Proteins involved in the mitochondrial oxidative phosphorylation were overexpressed whereas the alpha-subunit of the mitochondrial precursor of ATP synthase was downexpressed. Several proteins involved in the main energy metabolic pathways were up and downexpressed. Moreover, our results seem to suggest that probably neither fatty acid beta-oxidation nor glycolysis are the only sources for energy in the hypertrophied left ventricle. Copyright 2010 S. Karger AG, Basel.

  5. Increased ratio of dietary carbohydrate to protein shifts the focus of metabolic signaling from skeletal muscle to adipose

    Directory of Open Access Journals (Sweden)

    Devkota Suzanne

    2011-03-01

    Full Text Available Abstract Background The Dietary Reference Intakes (DRI established acceptable macronutrient distribution ranges (AMDR for carbohydrates and protein, however little is known about differences in glycemic regulations and metabolic signaling across this range. This study examined metabolic outcomes associated with intake of two diets differing in carbohydrate:protein ratios representing the upper and lower ends of the AMDR. Methods Adult, male rats were fed either a high carbohydrate (CHO diet (60% of energy from carbohydrates, 12% protein, 28% fat; n = 30 or a high protein (PRO diet (35% carbohydrate, 35% protein, 30% fat; n = 30. Rats were meal-fed 3x/d the respective diets for 10 d and then terminated after overnight food deprivation or 30, 60, 90, 120 min post-prandial (PP. Plasma was collected at each of these points to provide a time course for glucose, insulin and C-peptide. Skeletal muscle and adipose tissues were collected at 0, 30 and 90 min for measurements of basal, early and delayed activation of Akt, p70S6K and Erk 1/2. Data were analyzed by two-way ANOVA. Results The CHO group produced a consistently elevated response in plasma glucose, insulin and C-peptide following the meal through the 120 min time course. In addition, Akt and Erk 1/2 activation in adipose was much higher than in skeletal muscle. Conversely, the PRO group PP glucose response was minimal and insulin maintained a response similar to a biphasic pattern. Tissue responses for the PRO group were greater for Akt and p70S6K signaling in skeletal muscle compared with adipose. Conclusion Taken together these data suggest that altering CHO:PRO ratios within the AMDR produce different glycemic response patterns accompanied by differential metabolic signaling in skeletal muscle and adipose.

  6. Sterol regulatory element-binding protein-1c orchestrates metabolic remodeling of white adipose tissue by caloric restriction.

    Science.gov (United States)

    Fujii, Namiki; Narita, Takumi; Okita, Naoyuki; Kobayashi, Masaki; Furuta, Yurika; Chujo, Yoshikazu; Sakai, Masahiro; Yamada, Atsushi; Takeda, Kanae; Konishi, Tomokazu; Sudo, Yuka; Shimokawa, Isao; Higami, Yoshikazu

    2017-06-01

    Caloric restriction (CR) can delay onset of several age-related pathophysiologies and extend lifespan in various species, including rodents. CR also induces metabolic remodeling involved in activation of lipid metabolism, enhancement of mitochondrial biogenesis, and reduction of oxidative stress in white adipose tissue (WAT). In studies using genetically modified mice with extended lifespans, WAT characteristics influenced mammalian lifespans. However, molecular mechanisms underlying CR-associated metabolic remodeling of WAT remain unclear. Sterol regulatory element-binding protein-1c (Srebp-1c), a master transcription factor of fatty acid (FA) biosynthesis, is responsible for the pathogenesis of fatty liver (steatosis). Our study showed that, under CR conditions, Srebp-1c enhanced mitochondrial biogenesis via increased expression of peroxisome proliferator-activated receptor gamma coactivator-1α (Pgc-1α) and upregulated expression of proteins involved in FA biosynthesis within WAT. However, via Srebp-1c, most of these CR-associated metabolic alterations were not observed in other tissues, including the liver. Moreover, our data indicated that Srebp-1c may be an important factor both for CR-associated suppression of oxidative stress, through increased synthesis of glutathione in WAT, and for the prolongevity action of CR. Our results strongly suggested that Srebp-1c, the primary FA biosynthesis-promoting transcriptional factor implicated in fatty liver disease, is also the food shortage-responsive factor in WAT. This indicated that Srebp-1c is a key regulator of metabolic remodeling leading to the beneficial effects of CR. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  7. Glucokinase regulatory protein genetic variant interacts with omega-3 PUFA to influence insulin resistance and inflamma