The Not4 E3 Ligase and CCR4 Deadenylase Play Distinct Roles in Protein Quality Control

PubMed Central

Halter, David; Collart, Martine A.; Panasenko, Olesya O.

2014-01-01

Eukaryotic cells control their proteome by regulating protein production and protein clearance. Protein production is determined to a large extent by mRNA levels, whereas protein degradation depends mostly upon the proteasome. Dysfunction of the proteasome leads to the accumulation of non-functional proteins that can aggregate, be toxic for the cell, and, in extreme cases, lead to cell death. mRNA levels are controlled by their rates of synthesis and degradation. Recent evidence indicates that these rates have oppositely co-evolved to ensure appropriate mRNA levels. This opposite co-evolution has been correlated with the mutations in the Ccr4-Not complex. Consistently, the deadenylation enzymes responsible for the rate-limiting step in eukaryotic mRNA degradation, Caf1 and Ccr4, are subunits of the Ccr4-Not complex. Another subunit of this complex is a RING E3 ligase, Not4. It is essential for cellular protein solubility and has been proposed to be involved in co-translational quality control. An open question has been whether this role of Not4 resides strictly in the regulation of the deadenylation module of the Ccr4-Not complex. However, Not4 is important for proper assembly of the proteasome, and the Ccr4-Not complex may have multiple functional modules that participate in protein quality control in different ways. In this work we studied how the functions of the Caf1/Ccr4 and Not4 modules are connected. We concluded that Not4 plays a role in protein quality control independently of the Ccr4 deadenylase, and that it is involved in clearance of aberrant proteins at least in part via the proteasome. PMID:24465968

Distinct Mechanisms of Pathogenic DJ-1 Mutations in Mitochondrial Quality Control

PubMed Central

Strobbe, Daniela; Robinson, Alexis A.; Harvey, Kirsten; Rossi, Lara; Ferraina, Caterina; de Biase, Valerio; Rodolfo, Carlo; Harvey, Robert J.; Campanella, Michelangelo

2018-01-01

The deglycase and chaperone protein DJ-1 is pivotal for cellular oxidative stress responses and mitochondrial quality control. Mutations in PARK7, encoding DJ-1, are associated with early-onset familial Parkinson’s disease and lead to pathological oxidative stress and/or disrupted protein degradation by the proteasome. The aim of this study was to gain insights into the pathogenic mechanisms of selected DJ-1 missense mutations, by characterizing protein–protein interactions, core parameters of mitochondrial function, quality control regulation via autophagy, and cellular death following dopamine accumulation. We report that the DJ-1M26I mutant influences DJ-1 interactions with SUMO-1, in turn enhancing removal of mitochondria and conferring increased cellular susceptibility to dopamine toxicity. By contrast, the DJ-1D149A mutant does not influence mitophagy, but instead impairs Ca2+ dynamics and free radical homeostasis by disrupting DJ-1 interactions with a mitochondrial accessory protein known as DJ-1-binding protein (DJBP/EFCAB6). Thus, individual DJ-1 mutations have different effects on mitochondrial function and quality control, implying mutation-specific pathomechanisms converging on impaired mitochondrial homeostasis. PMID:29599708

Yeast prions are useful for studying protein chaperones and protein quality control.

PubMed

Masison, Daniel C; Reidy, Michael

2015-01-01

Protein chaperones help proteins adopt and maintain native conformations and play vital roles in cellular processes where proteins are partially folded. They comprise a major part of the cellular protein quality control system that protects the integrity of the proteome. Many disorders are caused when proteins misfold despite this protection. Yeast prions are fibrous amyloid aggregates of misfolded proteins. The normal action of chaperones on yeast prions breaks the fibers into pieces, which results in prion replication. Because this process is necessary for propagation of yeast prions, even small differences in activity of many chaperones noticeably affect prion phenotypes. Several other factors involved in protein processing also influence formation, propagation or elimination of prions in yeast. Thus, in much the same way that the dependency of viruses on cellular functions has allowed us to learn much about cell biology, the dependency of yeast prions on chaperones presents a unique and sensitive way to monitor the functions and interactions of many components of the cell's protein quality control system. Our recent work illustrates the utility of this system for identifying and defining chaperone machinery interactions.

Controlling Vapor Pressure In Hanging-Drop Crystallization

NASA Technical Reports Server (NTRS)

Carter, Daniel C.; Smith, Robbie

1988-01-01

Rate of evaporation adjusted to produce larger crystals. Device helps to control vapor pressure of water and other solvents in vicinity of hanging drop of solution containing dissolved enzyme protein. Well of porous frit (sintered glass) holds solution in proximity to drop of solution containing protein or enzyme. Vapor from solution in frit controls evaporation of solvent from drop to control precipitation of protein or enzyme. With device, rate of nucleation limited to decrease number and increase size (and perhaps quality) of crystals - large crystals of higher quality needed for x-ray diffraction studies of macromolecules.

MALDI-TOF MS for quality control of high protein content sport supplements.

PubMed

De Ceglie, Cristina; Calvano, Cosima D; Zambonin, Carlo G

2015-06-01

High protein content sport nutritional supplements are found as powder products containing, as ingredients, amino acids and proteins with important nutritional values as milk, soy and egg proteins. An EU Food Supplements Directive (2002) requires that supplements should be safe, both in dosages and in purity. It is important, then, to develop rapid and sensitive methods to be employed for the quality control of these substances. In this work, we apply, for the first time, matrix-assisted laser desorption ionization-mass spectrometry as a fast, reproducible and sensitive method for the quality control of sport nutritional supplements based on proteins. To this aim, several commercial egg- and/or milk-based powder products have been processed by in gel or in solution digestion and analyzed in comparison to pure standard products. This strategy allowed to assess the reliability of the indications on proteins (as caseins, whey proteins and ovalbumin) declared in the label of several sport nutritional supplements. Copyright © 2014 Elsevier Ltd. All rights reserved.

Quality control in the secretory assembly line.

PubMed Central

Helenius, A

2001-01-01

As a rule, only proteins that have reached a native, folded and assembled structure are transported to their target organelles and compartments within the cell. In the secretory pathway of eukaryotic cells, this type of sorting is particularly important. A variety of molecular mechanisms are involved that distinguish between folded and unfolded proteins, modulate their intracellular transport, and induce degradation if they fail to fold. This phenomenon, called quality control, occurs at several levels and involves different types of folding sensors. The quality control system provides a stringent and versatile molecular sorting system that guaranties fidelity of protein expression in the secretory pathway. PMID:11260794

A Decade of Boon or Burden: What Has the CHIP Ever Done for Cellular Protein Quality Control Mechanism Implicated in Neurodegeneration and Aging?

PubMed Central

Joshi, Vibhuti; Amanullah, Ayeman; Upadhyay, Arun; Mishra, Ribhav; Kumar, Amit; Mishra, Amit

2016-01-01

Cells regularly synthesize new proteins to replace old and abnormal proteins for normal cellular functions. Two significant protein quality control pathways inside the cellular milieu are ubiquitin proteasome system (UPS) and autophagy. Autophagy is known for bulk clearance of cytoplasmic aggregated proteins, whereas the specificity of protein degradation by UPS comes from E3 ubiquitin ligases. Few E3 ubiquitin ligases, like C-terminus of Hsc70-interacting protein (CHIP) not only take part in protein quality control pathways, but also plays a key regulatory role in other cellular processes like signaling, development, DNA damage repair, immunity and aging. CHIP targets misfolded proteins for their degradation through proteasome, as well as autophagy; simultaneously, with the help of chaperones, it also regulates folding attempts for misfolded proteins. The broad range of CHIP substrates and their associations with multiple pathologies make it a key molecule to work upon and focus for future therapeutic interventions. E3 ubiquitin ligase CHIP interacts and degrades many protein inclusions formed in neurodegenerative diseases. The presence of CHIP at various nodes of cellular protein-protein interaction network presents this molecule as a potential candidate for further research. In this review, we have explored a wide range of functionality of CHIP inside cells by a detailed presentation of its co-chaperone, E3 and E4 enzyme like functions, with central focus on its protein quality control roles in neurodegenerative diseases. We have also raised many unexplored but expected fundamental questions regarding CHIP functions, which generate hopes for its future applications in research, as well as drug discovery. PMID:27757073

Structural Elements Regulating AAA+ Protein Quality Control Machines.

PubMed

Chang, Chiung-Wen; Lee, Sukyeong; Tsai, Francis T F

2017-01-01

Members of the ATPases Associated with various cellular Activities (AAA+) superfamily participate in essential and diverse cellular pathways in all kingdoms of life by harnessing the energy of ATP binding and hydrolysis to drive their biological functions. Although most AAA+ proteins share a ring-shaped architecture, AAA+ proteins have evolved distinct structural elements that are fine-tuned to their specific functions. A central question in the field is how ATP binding and hydrolysis are coupled to substrate translocation through the central channel of ring-forming AAA+ proteins. In this mini-review, we will discuss structural elements present in AAA+ proteins involved in protein quality control, drawing similarities to their known role in substrate interaction by AAA+ proteins involved in DNA translocation. Elements to be discussed include the pore loop-1, the Inter-Subunit Signaling (ISS) motif, and the Pre-Sensor I insert (PS-I) motif. Lastly, we will summarize our current understanding on the inter-relationship of those structural elements and propose a model how ATP binding and hydrolysis might be coupled to polypeptide translocation in protein quality control machines.

Prefoldin Promotes Proteasomal Degradation of Cytosolic Proteins with Missense Mutations by Maintaining Substrate Solubility

PubMed Central

Young, Barry P.; Loewen, Christopher J.; Mayor, Thibault

2016-01-01

Misfolded proteins challenge the ability of cells to maintain protein homeostasis and can accumulate into toxic protein aggregates. As a consequence, cells have adopted a number of protein quality control pathways to prevent protein aggregation, promote protein folding, and target terminally misfolded proteins for degradation. In this study, we employed a thermosensitive allele of the yeast Guk1 guanylate kinase as a model misfolded protein to investigate degradative protein quality control pathways. We performed a flow cytometry based screen to identify factors that promote proteasomal degradation of proteins misfolded as the result of missense mutations. In addition to the E3 ubiquitin ligase Ubr1, we identified the prefoldin chaperone subunit Gim3 as an important quality control factor. Whereas the absence of GIM3 did not impair proteasomal function or the ubiquitination of the model substrate, it led to the accumulation of the poorly soluble model substrate in cellular inclusions that was accompanied by delayed degradation. We found that Gim3 interacted with the Guk1 mutant allele and propose that prefoldin promotes the degradation of the unstable model substrate by maintaining the solubility of the misfolded protein. We also demonstrated that in addition to the Guk1 mutant, prefoldin can stabilize other misfolded cytosolic proteins containing missense mutations. PMID:27448207

A pH-Regulated Quality Control Cycle for Surveillance of Secretory Protein Assembly

PubMed Central

Vavassori, Stefano; Cortini, Margherita; Masui, Shoji; Sannino, Sara; Anelli, Tiziana; Caserta, Imma R.; Fagioli, Claudio; Mossuto, Maria F.; Fornili, Arianna; van Anken, Eelco; Degano, Massimo; Inaba, Kenji; Sitia, Roberto

2013-01-01

Summary To warrant the quality of the secretory proteome, stringent control systems operate at the endoplasmic reticulum (ER)-Golgi interface, preventing the release of nonnative products. Incompletely assembled oligomeric proteins that are deemed correctly folded must rely on additional quality control mechanisms dedicated to proper assembly. Here we unveil how ERp44 cycles between cisGolgi and ER in a pH-regulated manner, patrolling assembly of disulfide-linked oligomers such as IgM and adiponectin. At neutral, ER-equivalent pH, the ERp44 carboxy-terminal tail occludes the substrate-binding site. At the lower pH of the cisGolgi, conformational rearrangements of this peptide, likely involving protonation of ERp44’s active cysteine, simultaneously unmask the substrate binding site and −RDEL motif, allowing capture of orphan secretory protein subunits and ER retrieval via KDEL receptors. The ERp44 assembly control cycle couples secretion fidelity and efficiency downstream of the calnexin/calreticulin and BiP-dependent quality control cycles. PMID:23685074

NFκB is a central regulator of protein quality control in response to protein aggregation stresses via autophagy modulation

PubMed Central

Nivon, Mathieu; Fort, Loïc; Muller, Pascale; Richet, Emma; Simon, Stéphanie; Guey, Baptiste; Fournier, Maëlenn; Arrigo, André-Patrick; Hetz, Claudio; Atkin, Julie D.; Kretz-Remy, Carole

2016-01-01

During cell life, proteins often misfold, depending on particular mutations or environmental changes, which may lead to protein aggregates that are toxic for the cell. Such protein aggregates are the root cause of numerous diseases called “protein conformational diseases,” such as myofibrillar myopathy and familial amyotrophic lateral sclerosis. To fight against aggregates, cells are equipped with protein quality control mechanisms. Here we report that NFκB transcription factor is activated by misincorporation of amino acid analogues into proteins, inhibition of proteasomal activity, expression of the R120G mutated form of HspB5 (associated with myofibrillar myopathy), or expression of the G985R and G93A mutated forms of superoxide dismutase 1 (linked to familial amyotrophic lateral sclerosis). This noncanonical stimulation of NFκB triggers the up-regulation of BAG3 and HspB8 expression, two activators of selective autophagy, which relocalize to protein aggregates. Then NFκB-dependent autophagy allows the clearance of protein aggregates. Thus NFκB appears as a central and major regulator of protein aggregate clearance by modulating autophagic activity. In this context, the pharmacological stimulation of this quality control pathway might represent a valuable strategy for therapies against protein conformational diseases. PMID:27075172

The human immunodeficiency virus antigen Nef forms protein bodies in leaves of transgenic tobacco when fused to zeolin

PubMed Central

de Virgilio, Maddalena; Bellucci, Michele; Mainieri, Davide; Rossi, Marika; Benvenuto, Eugenio; Arcioni, Sergio; Vitale, Alessandro

2008-01-01

Protein bodies (PB) are stable polymers naturally formed by certain seed storage proteins within the endoplasmic reticulum (ER). The human immunodeficiency virus negative factor (Nef) protein, a potential antigen for the development of an anti-viral vaccine, is highly unstable when introduced into the plant secretory pathway, probably because of folding defects in the ER environment. The aim of this study was to promote the formation of Nef-containing PB in tobacco (Nicotiana tabacum) leaves by fusing the Nef sequence to the N-terminal domains of the maize storage protein γ-zein or to the chimeric protein zeolin (which efficiently forms PB and is composed of the vacuolar storage protein phaseolin fused to the N-terminal domains of γ-zein). Protein blots and pulse–chase indicate that fusions between Nef and the same γ-zein domains present in zeolin are degraded by ER quality control. Consistently, a mutated zeolin, in which wild-type phaseolin was substituted with a defective version known to be degraded by ER quality control, is unstable in plant cells. Fusion of Nef to the entire zeolin sequence instead allows the formation of PB detectable by electron microscopy and subcellular fractionation, leading to zeolin–Nef accumulation higher than 1% of total soluble protein, consistently reproduced in independent transgenic plants. It is concluded that zeolin, but not its γ-zein portion, has a positive dominant effect over ER quality control degradation. These results provide insights into the requirements for PB formation and avoidance of quality-control degradation, and indicate a strategy for enhancing foreign protein accumulation in plants. PMID:18540021

Periplasmic quality control in biogenesis of outer membrane proteins.

PubMed

Lyu, Zhi Xin; Zhao, Xin Sheng

2015-04-01

The β-barrel outer membrane proteins (OMPs) are integral membrane proteins that reside in the outer membrane of Gram-negative bacteria and perform a diverse range of biological functions. Synthesized in the cytoplasm, OMPs must be transported across the inner membrane and through the periplasmic space before they are assembled in the outer membrane. In Escherichia coli, Skp, SurA and DegP are the most prominent factors identified to guide OMPs across the periplasm and to play the role of quality control. Although extensive genetic and biochemical analyses have revealed many basic functions of these periplasmic proteins, the mechanism of their collaboration in assisting the folding and insertion of OMPs is much less understood. Recently, biophysical approaches have shed light on the identification of the intricate network. In the present review, we summarize recent advances in the characterization of these key factors, with a special emphasis on the multifunctional protein DegP. In addition, we present our proposed model on the periplasmic quality control in biogenesis of OMPs.

Guidelines to reach high-quality purified recombinant proteins.

PubMed

Oliveira, Carla; Domingues, Lucília

2018-01-01

The final goal in recombinant protein production is to obtain high-quality pure protein samples. Indeed, the successful downstream application of a recombinant protein depends on its quality. Besides production, which is conditioned by the host, the quality of a recombinant protein product relies mainly on the purification procedure. Thus, the purification strategy must be carefully designed from the molecular level. On the other hand, the quality control of a protein sample must be performed to ensure its purity, homogeneity and structural conformity, in order to validate the recombinant production and purification process. Therefore, this review aims at providing succinct information on the rational purification design of recombinant proteins produced in Escherichia coli, specifically the tagging purification, as well as on accessible tools for evaluating and optimizing protein quality. The classical techniques for structural protein characterization-denaturing protein gel electrophoresis (SDS-PAGE), size exclusion chromatography (SEC), dynamic light scattering (DLS) and circular dichroism (CD)-are revisited with focus on the protein and their main advantages and disadvantages. Furthermore, methods for determining protein concentration and protein storage are also presented. The guidelines compiled herein will aid preparing pure, soluble and homogeneous functional recombinant proteins from the very beginning of the molecular cloning design.

Biological Regulation of Bone Quality

PubMed Central

Alliston, Tamara

2014-01-01

The ability of bone to resist fracture is determined by the combination of bone mass and bone quality. Like bone mass, bone quality is carefully regulated. Of the many aspects of bone quality, this review focuses on biological mechanisms that control the material quality of the bone extracellular matrix (ECM). Bone ECM quality depends upon ECM composition and organization. Proteins and signaling pathways that affect the mineral or organic constituents of bone ECM impact bone ECM material properties, such as elastic modulus and hardness. These properties are also sensitive to pathways that regulate bone remodeling by osteoblasts, osteoclasts, and osteocytes. Several extracellular proteins, signaling pathways, intracellular effectors, and transcription regulatory networks have been implicated in the control of bone ECM quality. A molecular understanding of these mechanisms will elucidate the biological control of bone quality and suggest new targets for the development of therapies to prevent bone fragility. PMID:24894149

Analysis and quality control of carbohydrates in therapeutic proteins with fluorescence HPLC

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

Liu, Kun; Huang, Jian; Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu 610054

Conbercept is an Fc fusion protein with very complicated carbohydrate profiles which must be carefully monitored through manufacturing process. Here, we introduce an optimized fluorescence derivatization high-performance liquid chromatographic method for glycan mapping in conbercept. Compared with conventional glycan analysis method, this method has much better resolution and higher reproducibility making it excellent for product quality control.

PIGN prevents protein aggregation in the endoplasmic reticulum independently of its function in the GPI synthesis.

PubMed

Ihara, Shinji; Nakayama, Sohei; Murakami, Yoshiko; Suzuki, Emiko; Asakawa, Masayo; Kinoshita, Taroh; Sawa, Hitoshi

2017-02-01

Quality control of proteins in the endoplasmic reticulum (ER) is essential for ensuring the integrity of secretory proteins before their release into the extracellular space. Secretory proteins that fail to pass quality control form aggregates. Here we show the PIGN-1/PIGN is required for quality control in Caenorhabditis elegans and in mammalian cells. In C. elegans pign-1 mutants, several proteins fail to be secreted and instead form abnormal aggregation. PIGN-knockout HEK293 cells also showed similar protein aggregation. Although PIGN-1/PIGN is responsible for glycosylphosphatidylinositol (GPI)-anchor biosynthesis in the ER, certain mutations in C. elegans pign-1 caused protein aggregation in the ER without affecting GPI-anchor biosynthesis. These results show that PIGN-1/PIGN has a conserved and non-canonical function to prevent deleterious protein aggregation in the ER independently of the GPI-anchor biosynthesis. PIGN is a causative gene for some human diseases including multiple congenital seizure-related syndrome (MCAHS1). Two pign-1 mutations created by CRISPR/Cas9 that correspond to MCAHS1 also cause protein aggregation in the ER, implying that the dysfunction of the PIGN non-canonical function might affect symptoms of MCAHS1 and potentially those of other diseases. © 2017. Published by The Company of Biologists Ltd.

The Endoplasmic Reticulum-Associated Degradation Pathways of Budding Yeast

PubMed Central

Thibault, Guillaume; Ng, Davis T.W.

2012-01-01

Protein misfolding is a common cellular event that can produce intrinsically harmful products. To reduce the risk, quality control mechanisms are deployed to detect and eliminate misfolded, aggregated, and unassembled proteins. In the secretory pathway, it is mainly the endoplasmic reticulum-associated degradation (ERAD) pathways that perform this role. Here, specialized factors are organized to monitor and process the folded states of nascent polypeptides. Despite the complex structures, topologies, and posttranslational modifications of client molecules, the ER mechanisms are the best understood among all protein quality-control systems. This is the result of convergent and sometimes serendipitous discoveries by researchers from diverse fields. Although major advances in ER quality control and ERAD came from all model organisms, this review will focus on the discoveries culminating from the simple budding yeast. PMID:23209158

Meal-based enhancement of protein quality and quantity during weight loss in obese older adults with mobility limitations: rationale and design for the MEASUR-UP trial.

PubMed

McDonald, Shelley R; Porter Starr, Kathryn N; Mauceri, Luisa; Orenduff, Melissa; Granville, Esther; Ocampo, Christine; Payne, Martha E; Pieper, Carl F; Bales, Connie W

2015-01-01

Obese older adults with even modest functional limitations are at a disadvantage for maintaining their independence into late life. However, there is no established intervention for obesity in older individuals. The Measuring Eating, Activity, and Strength: Understanding the Response - Using Protein (MEASUR-UP) trial is a randomized controlled pilot study of obese women and men aged ≥60 years with mild to moderate functional impairments. Changes in body composition (lean and fat mass) and function (Short Physical Performance Battery) in an enhanced protein weight reduction (Protein) arm will be compared to those in a traditional weight loss (Control) arm. The Protein intervention is based on evidence that older adults achieve optimal rates of muscle protein synthesis when consuming about 25-30 g of high quality protein per meal; these participants will consume ~30 g of animal protein at each meal via a combination of provided protein (beef) servings and diet counseling. This trial will provide information on the feasibility and efficacy of enhancing protein quantity and quality in the context of a weight reduction regimen and determine the impact of this intervention on body weight, functional status, and lean muscle mass. We hypothesize that the enhancement of protein quantity and quality in the Protein arm will result in better outcomes for function and/or lean muscle mass than in the Control arm. Ultimately, we hope our findings will help identify a safe weight loss approach that can delay or prevent late life disability by changing the trajectory of age-associated functional impairment associated with obesity. Copyright © 2014 Elsevier Inc. All rights reserved.

Heat shock proteins as potential targets for protective strategies in neurodegeneration.

PubMed

Kampinga, Harm H; Bergink, Steven

2016-06-01

Protein aggregates are hallmarks of nearly all age-related neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and several polyglutamine diseases such as Huntington's disease and different forms of spinocerebellar ataxias (SCA; SCA1-3, SCA6, and SCA7). The collapse of cellular protein homoeostasis can be both a cause and a consequence of this protein aggregation. Boosting components of the cellular protein quality control system has been widely investigated as a strategy to counteract protein aggregates or their toxic consequences. Heat shock proteins (HSPs) play a central part in regulating protein quality control and contribute to protein aggregation and disaggregation. Therefore, HSPs are viable targets for the development of drugs aimed at reducing pathogenic protein aggregates that are thought to contribute to the development of so many neurodegenerative disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

Control of Protein Crystal Nucleation and Growth Using Stirring Solution

NASA Astrophysics Data System (ADS)

Niino, Ai; Adachi, Hiroaki; Takano, Kazufumi; Matsumura, Hiroyoshi; Kinoshita, Takayoshi; Warizaya, Masaichi; Inoue, Tsuyoshi; Mori, Yusuke; Sasaki, Takatomo

2004-11-01

We have previously developed a protein crystallization technique using a stirring protein solution and revealed that (i) continuous stirring prevents excess spontaneous nucleation and accelerates the growth of protein crystals and (ii) prestirring (solution stirring in advance) promotes the crystal nucleation of hen egg-white lysozyme. In bovine adenosine deaminase (ADA) crystallization, continuous stirring improves the crystal quality but elongates the nucleation time. In this paper, in order to control both the crystal nucleation and growth of ADA using a Micro-Stirring technique, we carried out five different stirring patterns such as (i) no stirring, (ii) continuous stirring, (iii) prestirring, (iv) poststirring (stirring late in the growth period) and (v) restirring (combined pre- and poststirring). The results showed that high-quality well-shaped crystals were obtained under the continuous stirring and restirring conditions and the nucleation time under the prestirring and restirring conditions was shorter than that under the continuous stirring and poststirring conditions. Consequently, high-quality crystals were promptly obtained under the restirring condition. These results suggest that we are able to control both the nucleation and growth of protein crystals with the stirring techniques.

Anomalous Diffusion Reports on the Interaction of Misfolded Proteins with the Quality Control Machinery in the Endoplasmic Reticulum

PubMed Central

Malchus, Nina; Weiss, Matthias

2010-01-01

A multitude of transmembrane proteins enters the endoplasmic reticulum (ER) as unfolded polypeptide chains. During their folding process, they interact repetitively with the ER's quality control machinery. Here, we have used fluorescence correlation spectroscopy to probe these interactions for a prototypical transmembrane protein, VSVG ts045, in vivo. While both folded and unfolded VSVG ts045 showed anomalous diffusion, the unfolded protein had a significantly stronger anomaly. This difference subsided when unfolded VSVG ts045 was in a complex with its chaperone calnexin, or when a mutant form of VSVG ts045 with only one glycan was used. Our experimental data and accompanying simulations suggest that the folding sensor of the quality control (UGT1) oligomerizes unfolded VSVG ts045, leading to a more anomalous/obstructed diffusion. In contrast, calnexin dissolves the oligomers, rendering unfolded VSVG ts045 more mobile, and hence prevents poisoning of the ER. PMID:20713018

Therapeutic intervention at cellular quality control systems in Alzheimer's and Parkinson's diseases.

PubMed

Arduino, Daniela M; Esteves, A Raquel; Silva, Diana F F; Martins-Branco, Diogo; Santos, Daniel; Pimentel, Diana F Gomes; Cardoso, Sandra M

2011-01-01

Cellular homeostasis relies on quality control systems so that damaged biologic structures are either repaired or degraded and entirely replaced by newly formed proteins or even organelles. The clearance of dysfunctional cellular structures in long-lived postmitotic cells, like neurons, is essential to eliminate, per example, defective mitochondria, lipofuscin-loaded lysosomes and oxidized proteins. Short-lived proteins are degraded mainly by proteases and proteasomes whether most long-lived proteins and all organelles are digested by autophagy in the lysosomes. Recently, it an interplay was established between the ubiquitin-proteasome system and macroautophagy, so that both degradative mechanisms compensate for each other. In this article we describe each of these clearance systems and their contribution to neuronal quality control. We will highlight some of the findings that provide evidence for the dysfunction of these systems in Alzheimer's and Parkinson's diseases. Ultimately, we provide an outline on potential therapeutic interventions based on the modulation of cellular degradative systems.

Protein Kinase A Regulates Molecular Chaperone Transcription and Protein Aggregation

PubMed Central

Prince, Thomas; Calderwood, Stuart K.

2011-01-01

Heat shock factor 1 (HSF1) regulates one of the major pathways of protein quality control and is essential for deterrence of protein-folding disorders, particularly in neuronal cells. However, HSF1 activity declines with age, a change that may open the door to progression of neurodegenerative disorders such as Huntington's disease. We have investigated mechanisms of HSF1 regulation that may become compromised with age. HSF1 binds stably to the catalytic domain of protein kinase A (PKAcα) and becomes phosphorylated on at least one regulatory serine residue (S320). We show here that PKA is essential for effective transcription of HSP genes by HSF1. PKA triggers a cascade involving HSF1 binding to the histone acetylase p300 and positive translation elongation factor 1 (p-TEFb) and phosphorylation of the c-terminal domain of RNA polymerase II, a key mechanism in the downstream steps of HSF1-mediated transcription. This cascade appears to play a key role in protein quality control in neuronal cells expressing aggregation-prone proteins with long poly-glutamine (poly-Q) tracts. Such proteins formed inclusion bodies that could be resolved by HSF1 activation during heat shock. Resolution of the inclusions was inhibited by knockdown of HSF1, PKAcα, or the pTEFb component CDK9, indicating a key role for the HSF1-PKA cascade in protein quality control. PMID:22216146

Quality control of mRNP biogenesis: networking at the transcription site.

PubMed

Eberle, Andrea B; Visa, Neus

2014-08-01

Eukaryotic cells carry out quality control (QC) over the processes of RNA biogenesis to inactivate or eliminate defective transcripts, and to avoid their production. In the case of protein-coding transcripts, the quality controls can sense defects in the assembly of mRNA-protein complexes, in the processing of the precursor mRNAs, and in the sequence of open reading frames. Different types of defect are monitored by different specialized mechanisms. Some of them involve dedicated factors whose function is to identify faulty molecules and target them for degradation. Others are the result of a more subtle balance in the kinetics of opposing activities in the mRNA biogenesis pathway. One way or another, all such mechanisms hinder the expression of the defective mRNAs through processes as diverse as rapid degradation, nuclear retention and transcriptional silencing. Three major degradation systems are responsible for the destruction of the defective transcripts: the exosome, the 5'-3' exoribonucleases, and the nonsense-mediated mRNA decay (NMD) machinery. This review summarizes recent findings on the cotranscriptional quality control of mRNA biogenesis, and speculates that a protein-protein interaction network integrates multiple mRNA degradation systems with the transcription machinery. Copyright © 2014 Elsevier Ltd. All rights reserved.

Protein Aggregation and Its Impact on Product Quality

PubMed Central

Roberts, Christopher J.

2014-01-01

Protein pharmaceutical products are typically active as folded monomers that are composed of one or more protein chains, such as the heavy and light chains in monoclonal antibodies that are a mainstay of current drug pipelines. There are numerous possible aggregated states for a given protein, some of which are potentially useful, while most of which are considered deleterious from the perspective of pharmaceutical product quality and performance. This review provides an overview of how and why different aggregated states of proteins occur, how this potentially impacts product quality and performance, fundamental approaches to control aggregate formation, and the practical approaches that are currently used in the pharmaceutical industry. PMID:25173826

Thermal Optimization of Growth and Quality in Protein Crystals

NASA Technical Reports Server (NTRS)

Wiencek, John M.

1996-01-01

Experimental evidence suggests that larger and higher quality crystals can be attained in the microgravity of space; however, the effect of growth rate on protein crystal quality is not well documented. This research is the first step towards providing strategies to grow crystals under constant rates of growth. Controlling growth rates at a constant value allows for direct one-to-one comparison of results obtained in microgravity and on earth. The overall goal of the project was to control supersaturation at a constant value during protein crystal growth by varying temperature in a predetermined manner. Applying appropriate theory requires knowledge of specific physicochemical properties of the protein solution including the effect of supersaturation on growth rates and the effect of temperature on protein solubility. Such measurements typically require gram quantities of protein and many months of data acquisition. A second goal of the project applied microcalorimetry for the rapid determination of these physicochemical properties using a minimum amount of protein. These two goals were successfully implemented on hen egg-white lysozyme. Results of these studies are described in the attached reprints.

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

PubMed

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

2017-10-25

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

Life-span extension by a metacaspase in the yeast Saccharomyces cerevisiae.

PubMed

Hill, Sandra Malmgren; Hao, Xinxin; Liu, Beidong; Nyström, Thomas

2014-06-20

Single-cell species harbor ancestral structural homologs of caspase proteases, although the evolutionary benefit of such apoptosis-related proteins in unicellular organisms is unclear. Here, we found that the yeast metacaspase Mca1 is recruited to the insoluble protein deposit (IPOD) and juxtanuclear quality-control compartment (JUNQ) during aging and proteostatic stress. Elevating MCA1 expression counteracted accumulation of unfolded proteins and aggregates and extended life span in a heat shock protein Hsp104 disaggregase- and proteasome-dependent manner. Consistent with a role in protein quality control, genetic interaction analysis revealed that MCA1 buffers against deficiencies in the Hsp40 chaperone YDJ1 in a caspase cysteine-dependent manner. Life-span extension and aggregate management by Mca1 was only partly dependent on its conserved catalytic cysteine, which suggests that Mca1 harbors both caspase-dependent and independent functions related to life-span control. Copyright © 2014, American Association for the Advancement of Science.

The Exosome Associates Cotranscriptionally with the Nascent Pre-mRNP through Interactions with Heterogeneous Nuclear Ribonucleoproteins

PubMed Central

Hessle, Viktoria; Björk, Petra; Sokolowski, Marcus; de Valdivia, Ernesto González; Silverstein, Rebecca; Artemenko, Konstantin; Tyagi, Anu; Maddalo, Gianluca; Ilag, Leopold; Helbig, Roger; Zubarev, Roman A.

2009-01-01

Eukaryotic cells have evolved quality control mechanisms to degrade aberrant mRNA molecules and prevent the synthesis of defective proteins that could be deleterious for the cell. The exosome, a protein complex with ribonuclease activity, is a key player in quality control. An early quality checkpoint takes place cotranscriptionally but little is known about the molecular mechanisms by which the exosome is recruited to the transcribed genes. Here we study the core exosome subunit Rrp4 in two insect model systems, Chironomus and Drosophila. We show that a significant fraction of Rrp4 is associated with the nascent pre-mRNPs and that a specific mRNA-binding protein, Hrp59/hnRNP M, interacts in vivo with multiple exosome subunits. Depletion of Hrp59 by RNA interference reduces the levels of Rrp4 at transcription sites, which suggests that Hrp59 is needed for the exosome to stably interact with nascent pre-mRNPs. Our results lead to a revised mechanistic model for cotranscriptional quality control in which the exosome is constantly recruited to newly synthesized RNAs through direct interactions with specific hnRNP proteins. PMID:19494042

The influence of additives and drying methods on quality attributes of fish protein powder made from saithe (Pollachius virens).

PubMed

Shaviklo, Gholam Reza; Thorkelsson, Gudjon; Arason, Sigurjon; Kristinsson, Hordur G; Sveinsdottir, Kolbrun

2010-09-01

Fish protein powder (FPP) is used in the food industry for developing formulated food products. This study investigates the feasibility of increasing the value of saithe (Pollachius virens) by producing a functional FPP. Quality attributes of spray and freeze-dried saithe surimi containing lyoprotectants were studied. A freeze-dried saithe surimi without lyoprotectants was also prepared as a control sample. The amount of protein, moisture, fat and carbohydrate in the FPPs were 745-928, 39-58, 21-32 and 10-151 g kg(-1). Quality attributes of FPPs were influenced by the two drying methods and lyoprotectants. The highest level of lipid oxidation was found in the control and the second highest in the spray-dried FPP. The spray-dried fish protein had the lowest viscosity among all FPPs. Gel-forming ability of samples with lyoprotectants was higher than that of the control. Water-binding capacity, emulsion properties and solubility of the freeze-dried fish protein containing lyoprotectants were significantly higher than spray-dried and control samples. However, functional properties of spray-dried FPP were higher than the control sample. It is feasible to develop value-added FPP from saithe surimi using spray- and freeze-drying processes, but freeze-dried FPP containing lyoprotectant had superior functional properties and stability compared with spray-dried sample. Both products might be used as functional protein ingredients in various food systems. Copyright 2010 Society of Chemical Industry.

Analysis of Quality-Related Parameters in Mature Kernels of Polygalacturonase Inhibiting Protein (PGIP) Transgenic Bread Wheat Infected with Fusarium graminearum.

PubMed

Masci, Stefania; Laino, Paolo; Janni, Michela; Botticella, Ermelinda; Di Carli, Mariasole; Benvenuto, Eugenio; Danieli, Pier Paolo; Lilley, Kathryn S; Lafiandra, Domenico; D'Ovidio, Renato

2015-04-22

Fusarium head blight, caused by the fungus Fusarium graminearum, has a detrimental effect on both productivity and qualitative properties of wheat. To evaluate its impact on wheat flour, we compared its effect on quality-related parameters between a transgenic bread wheat line expressing a bean polygalacturonase inhibiting protein (PGIP) and its control line. We have compared metabolic proteins, the amounts of gluten proteins and their relative ratios, starch content, yield, extent of pathogen contamination, and deoxynivalenol (DON) accumulation. These comparisons showed that Fusarium significantly decreases the amount of starch in infected control plants, but not in infected PGIP plants. The flour of PGIP plants contained also a lower amount of pathogen biomass and DON accumulation. Conversely, both gluten and metabolic proteins were not significantly influenced either by the transgene or by fungal infection. These results indicate that the transgenic PGIP expression reduces the level of infection, without changing significantly the wheat seed proteome and other quality-related parameters.

The loss of SMG1 causes defects in quality control pathways in Physcomitrella patens

PubMed Central

Lang, Daniel; Zimmer, Andreas D; Causier, Barry

2018-01-01

Abstract Nonsense-mediated mRNA decay (NMD) is important for RNA quality control and gene regulation in eukaryotes. NMD targets aberrant transcripts for decay and also directly influences the abundance of non-aberrant transcripts. In animals, the SMG1 kinase plays an essential role in NMD by phosphorylating the core NMD factor UPF1. Despite SMG1 being ubiquitous throughout the plant kingdom, little is known about its function, probably because SMG1 is atypically absent from the genome of the model plant, Arabidopsis thaliana. By combining our previously established SMG1 knockout in moss with transcriptome-wide analysis, we reveal the range of processes involving SMG1 in plants. Machine learning assisted analysis suggests that 32% of multi-isoform genes produce NMD-targeted transcripts and that splice junctions downstream of a stop codon act as the major determinant of NMD targeting. Furthermore, we suggest that SMG1 is involved in other quality control pathways, affecting DNA repair and the unfolded protein response, in addition to its role in mRNA quality control. Consistent with this, smg1 plants have increased susceptibility to DNA damage, but increased tolerance to unfolded protein inducing agents. The potential involvement of SMG1 in RNA, DNA and protein quality control has major implications for the study of these processes in plants. PMID:29596649

Quality control methodology for high-throughput protein-protein interaction screening.

PubMed

Vazquez, Alexei; Rual, Jean-François; Venkatesan, Kavitha

2011-01-01

Protein-protein interactions are key to many aspects of the cell, including its cytoskeletal structure, the signaling processes in which it is involved, or its metabolism. Failure to form protein complexes or signaling cascades may sometimes translate into pathologic conditions such as cancer or neurodegenerative diseases. The set of all protein interactions between the proteins encoded by an organism constitutes its protein interaction network, representing a scaffold for biological function. Knowing the protein interaction network of an organism, combined with other sources of biological information, can unravel fundamental biological circuits and may help better understand the molecular basics of human diseases. The protein interaction network of an organism can be mapped by combining data obtained from both low-throughput screens, i.e., "one gene at a time" experiments and high-throughput screens, i.e., screens designed to interrogate large sets of proteins at once. In either case, quality controls are required to deal with the inherent imperfect nature of experimental assays. In this chapter, we discuss experimental and statistical methodologies to quantify error rates in high-throughput protein-protein interactions screens.

Chemical composition, starch digestibility and antioxidant capacity of tortilla made with a blend of quality protein maize and black bean.

PubMed

Grajales-García, Eva M; Osorio-Díaz, Perla; Goñi, Isabel; Hervert-Hernández, Deisy; Guzmán-Maldonado, Salvador H; Bello-Pérez, Luis A

2012-01-01

Tortilla and beans are the basic components in the diet of people in the urban and rural areas of Mexico. Quality protein maize is suggested for tortilla preparation because it presents an increase in lysine and tryptophan levels. Beans contain important amounts of dietary fiber. The objective of this study was to prepare tortilla with bean and assesses the chemical composition, starch digestibility and antioxidant capacity using a quality protein maize variety. Tortilla with bean had higher protein, ash, dietary fiber and resistant starch content, and lower digestible starch than control tortilla. The hydrolysis rate (60 to 50%) and the predicted glycemic index (88 to 80) of tortilla decreased with the addition of bean in the blend. Extractable polyphenols and proanthocyanidins were higher in the tortilla with bean than control tortilla. This pattern produced higher antioxidant capacity of tortilla with bean (17.6 μmol Trolox eq/g) than control tortilla (7.8 μmol Trolox eq/g). The addition of bean to tortilla modified the starch digestibility and antioxidant characteristics of tortilla, obtaining a product with nutraceutical characteristics.

[Biological evaluation of a protein mixture intended for enteral nutrition].

PubMed

Meneses, J Olza; Foulquie, J Porres; Valero, G Urbano; de Victoria, E Martínez; Hernández, A Gil

2008-01-01

Enteral nutrition is the best way to feed or supplement the diet when gastrointestinal tract functions of patients are partially or totally preserved. Whenever total enteral nutrition is needed, it represents the only source of nutrients for patients. Thus, it is mandatory to ensure that high biological value proteins are included in enteral formulae. To assess the biological quality of a protein blend constituted by 50% potassium caseinate, 25% whey protein and 25% pea protein intended to be used in enteral nutrition products. Forty Wistar rats (20 male and 20 female), with initial body weight of 51 g, where divided into four groups and feed for 10 days with: casein (Control), experimental protein blend (Experimental), liophylized normo- and hyperproteic enteral nutrition formulae adapted to the animal nutritional requirements (Normoproteic and Hyperproteic). Protein efficiency ratio (PER), apparent digestibility coefficient (ADC), relationship between retained and absorbed nitrogen (R/A) and relationship between retained and consumed nitrogen (R/I) where calculated. Experimental and control groups had similar values for all analysed indices (PER, ADC, R/A and R/I). These indices where also similar between normo and hyperproteic groups, but lower than experimental and control groups, except in PER, where normoproteic group was either similar to control and hiperproteic group. The quality of the protein blend used in this study is high. It is a good protein source to be used in the development of new enteral nutritional products.

Structural Basis for Modulation of Quality Control Fate in a Marginally Stable Protein.

PubMed

Brock, Kelly P; Abraham, Ayelet-chen; Amen, Triana; Kaganovich, Daniel; England, Jeremy L

2015-07-07

The human von Hippel-Lindau (VHL) tumor suppressor is a marginally stable protein previously used as a model substrate of eukaryotic refolding and degradation pathways. When expressed in the absence of its cofactors, VHL cannot fold and is quickly degraded by the quality control machinery of the cell. We combined computational methods with in vivo experiments to examine the basis of the misfolding propensity of VHL. By expressing a set of randomly mutated VHL sequences in yeast, we discovered a more stable mutant form. Subsequent modeling suggested the mutation had caused a conformational change affecting cofactor and chaperone interaction, and this hypothesis was then confirmed by additional knockout and overexpression experiments targeting a yeast cofactor homolog. These findings offer a detailed structural basis for the modulation of quality control fate in a model misfolded protein and highlight burial mode modeling as a rapid means to detect functionally important conformational changes in marginally stable globular domains. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Interaction between bacterial outer membrane proteins and periplasmic quality control factors: a kinetic partitioning mechanism.

PubMed

Wu, Si; Ge, Xi; Lv, Zhixin; Zhi, Zeyong; Chang, Zengyi; Zhao, Xin Sheng

2011-09-15

The OMPs (outer membrane proteins) of Gram-negative bacteria have to be translocated through the periplasmic space before reaching their final destination. The aqueous environment of the periplasmic space and high permeability of the outer membrane engender such a translocation process inevitably challenging. In Escherichia coli, although SurA, Skp and DegP have been identified to function in translocating OMPs across the periplasm, their precise roles and their relationship remain to be elucidated. In the present paper, by using fluorescence resonance energy transfer and single-molecule detection, we have studied the interaction between the OMP OmpC and these periplasmic quality control factors. The results of the present study reveal that the binding rate of OmpC to SurA or Skp is much faster than that to DegP, which may lead to sequential interaction between OMPs and different quality control factors. Such a kinetic partitioning mechanism for the chaperone-substrate interaction may be essential for the quality control of the biogenesis of OMPs.

Mitochondrial quality control and communications with the nucleus are important in maintaining mitochondrial function and cell health☆☆☆

PubMed Central

Kotiadis, Vassilios N.; Duchen, Michael R.; Osellame, Laura D.

2014-01-01

Background The maintenance of cell metabolism and homeostasis is a fundamental characteristic of living organisms. In eukaryotes, mitochondria are the cornerstone of these life supporting processes, playing leading roles in a host of core cellular functions, including energy transduction, metabolic and calcium signalling, and supporting roles in a number of biosynthetic pathways. The possession of a discrete mitochondrial genome dictates that the maintenance of mitochondrial ‘fitness’ requires quality control mechanisms which involve close communication with the nucleus. Scope of review This review explores the synergistic mechanisms that control mitochondrial quality and function and ensure cellular bioenergetic homeostasis. These include antioxidant defence mechanisms that protect against oxidative damage caused by reactive oxygen species, while regulating signals transduced through such free radicals. Protein homeostasis controls import, folding, and degradation of proteins underpinned by mechanisms that regulate bioenergetic capacity through the mitochondrial unfolded protein response. Autophagic machinery is recruited for mitochondrial turnover through the process of mitophagy. Mitochondria also communicate with the nucleus to exact specific transcriptional responses through retrograde signalling pathways. Major conclusions The outcome of mitochondrial quality control is not only reliant on the efficient operation of the core homeostatic mechanisms but also in the effective interaction of mitochondria with other cellular components, namely the nucleus. General significance Understanding mitochondrial quality control and the interactions between the organelle and the nucleus will be crucial in developing therapies for the plethora of diseases in which the pathophysiology is determined by mitochondrial dysfunction. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research. PMID:24211250

Unraveling the CHIP:Hsp70 complex as an information processor for protein quality control.

PubMed

VanPelt, Jamie; Page, Richard C

2017-02-01

The CHIP:Hsp70 complex stands at the crossroads of the cellular protein quality control system. Hsp70 facilitates active refolding of misfolded client proteins, while CHIP directs ubiquitination of misfolded client proteins bound to Hsp70. The direct competition between CHIP and Hsp70 for the fate of misfolded proteins leads to the question: how does the CHIP:Hsp70 complex execute triage decisions that direct misfolded proteins for either refolding or degradation? The current body of literature points toward action of the CHIP:Hsp70 complex as an information processor that takes inputs in the form of client folding state, dynamics, and posttranslational modifications, then outputs either refolded or ubiquitinated client proteins. Herein we examine the CHIP:Hsp70 complex beginning with the structure and function of CHIP and Hsp70, followed by an examination of recent studies of the interactions and dynamics of the CHIP:Hsp70 complex. Copyright © 2016 Elsevier B.V. All rights reserved.

Structural approaches to the study of oligosaccharides in glycoprotein quality control.

PubMed

Ito, Yukishige; Hagihara, Shinya; Matsuo, Ichiro; Totani, Kiichiro

2005-10-01

High-mannose-type oligosaccharides have been shown to play important roles in protein quality control. Several intracellular proteins, such as lectins, chaperones and glycan-processing enzymes, are involved in this process. These include calnexin/calreticulin, UDP-glucose:glycoprotein glucosyltransferase (UGGT), cargo receptors (such as VIP36 and ERGIC-53), mannosidase-like proteins (e.g. EDEM and Htm1p) and ubiquitin ligase (Fbs). They are thought to recognize high-mannose-type glycans with subtly different structures, although the precise specificities are yet to be clarified. In order to gain a clear understanding of these protein-carbohydrate interactions, comprehensive synthesis of high-mannose-type glycans was conducted. In addition, two approaches to the synthesis of artificial glycoproteins with homogeneous oligosaccharides were investigated. Furthermore, a novel substrate of UGGT was discovered.

Proteomic Response and Quality Maintenance in Postharvest Fruit of Strawberry (Fragaria × ananassa) to Exogenous Cytokinin.

PubMed

Li, Li; Li, Dongdong; Luo, Zisheng; Huang, Xinhong; Li, Xihong

2016-06-01

The limitations in current understanding of the molecular mechanisms underlying fruit response to the application of plant growth regulators have increasingly become major challenges in improvement of crop quality. This study aimed to evaluate the response of strawberry to the preharvest application of exogenous cytokinin known as forchlorfenuron (CPPU). Postharvest internal and physiological quality attributes were characterized following storage under different conditions. Hierarchical clustering analysis via a label-free proteomic quantitative approach identified a total of 124 proteins in strawberries across all treatments. The expression profiles of both proteins and genes spanned the ranged role of cytokinin involved in primary and secondary metabolism, stress response, and so on. Eighty-eight proteins and fifty-six proteins were significantly regulated immediately at harvest and after storage, respectively. In general, the glycolysis in strawberry was only regulated by CPPU before storage; in addition to the accelerated photosynthesis and acid metabolism, CPPU application maintained higher capacity of resistance in strawberry to stress stimuli after storage, in comparison to control. Nevertheless, the volatile biosynthesis in strawberry has been suppressed by exogenous CPPU. Novel cytokinin response proteins and processes were identified in addition to the main transcriptomic expression to gain insights into the phytohormone control of fruit postharvest quality.

Chemical Composition, Starch Digestibility and Antioxidant Capacity of Tortilla Made with a Blend of Quality Protein Maize and Black Bean

PubMed Central

Grajales-García, Eva M.; Osorio-Díaz, Perla; Goñi, Isabel; Hervert-Hernández, Deisy; Guzmán-Maldonado, Salvador H.; Bello-Pérez, Luis A.

2012-01-01

Tortilla and beans are the basic components in the diet of people in the urban and rural areas of Mexico. Quality protein maize is suggested for tortilla preparation because it presents an increase in lysine and tryptophan levels. Beans contain important amounts of dietary fiber. The objective of this study was to prepare tortilla with bean and assesses the chemical composition, starch digestibility and antioxidant capacity using a quality protein maize variety. Tortilla with bean had higher protein, ash, dietary fiber and resistant starch content, and lower digestible starch than control tortilla. The hydrolysis rate (60 to 50%) and the predicted glycemic index (88 to 80) of tortilla decreased with the addition of bean in the blend. Extractable polyphenols and proanthocyanidins were higher in the tortilla with bean than control tortilla. This pattern produced higher antioxidant capacity of tortilla with bean (17.6 μmol Trolox eq/g) than control tortilla (7.8 μmol Trolox eq/g). The addition of bean to tortilla modified the starch digestibility and antioxidant characteristics of tortilla, obtaining a product with nutraceutical characteristics. PMID:22312252

Effect of Replacing Animal Protein with Plant Protein on Glycemic Control in Diabetes: A Systematic Review and Meta-Analysis of Randomized Controlled Trials

PubMed Central

Viguiliouk, Effie; Stewart, Sarah E.; Jayalath, Viranda H.; Ng, Alena Praneet; Mirrahimi, Arash; de Souza, Russell J.; Hanley, Anthony J.; Bazinet, Richard P.; Blanco Mejia, Sonia; Leiter, Lawrence A.; Josse, Robert G.; Kendall, Cyril W.C.; Jenkins, David J.A.; Sievenpiper, John L.

2015-01-01

Previous research on the effect of replacing sources of animal protein with plant protein on glycemic control has been inconsistent. We therefore conducted a systematic review and meta-analysis of randomized controlled trials (RCTs) to assess the effect of this replacement on glycemic control in individuals with diabetes. We searched MEDLINE, EMBASE, and Cochrane databases through 26 August 2015. We included RCTs ≥ 3-weeks comparing the effect of replacing animal with plant protein on HbA1c, fasting glucose (FG), and fasting insulin (FI). Two independent reviewers extracted relevant data, assessed study quality and risk of bias. Data were pooled by the generic inverse variance method and expressed as mean differences (MD) with 95% confidence intervals (CIs). Heterogeneity was assessed (Cochran Q-statistic) and quantified (I2-statistic). Thirteen RCTs (n = 280) met the eligibility criteria. Diets emphasizing a replacement of animal with plant protein at a median level of ~35% of total protein per day significantly lowered HbA1c (MD = −0.15%; 95%-CI: −0.26, −0.05%), FG (MD = −0.53 mmol/L; 95%-CI: −0.92, −0.13 mmol/L) and FI (MD = −10.09 pmol/L; 95%-CI: −17.31, −2.86 pmol/L) compared with control arms. Overall, the results indicate that replacing sources of animal with plant protein leads to modest improvements in glycemic control in individuals with diabetes. Owing to uncertainties in our analyses there is a need for larger, longer, higher quality trials. Trial Registration: ClinicalTrials.gov registration number: NCT02037321. PMID:26633472

Aerobic exercise training rescues cardiac protein quality control and blunts endoplasmic reticulum stress in heart failure rats.

PubMed

Bozi, Luiz H M; Jannig, Paulo R; Rolim, Natale; Voltarelli, Vanessa A; Dourado, Paulo M M; Wisløff, Ulrik; Brum, Patricia C

2016-11-01

Cardiac endoplasmic reticulum (ER) stress through accumulation of misfolded proteins plays a pivotal role in cardiovascular diseases. In an attempt to reestablish ER homoeostasis, the unfolded protein response (UPR) is activated. However, if ER stress persists, sustained UPR activation leads to apoptosis. There is no available therapy for ER stress relief. Considering that aerobic exercise training (AET) attenuates oxidative stress, mitochondrial dysfunction and calcium imbalance, it may be a potential strategy to reestablish cardiac ER homoeostasis. We test the hypothesis that AET would attenuate impaired cardiac ER stress after myocardial infarction (MI). Wistar rats underwent to either MI or sham surgeries. Four weeks later, rats underwent to 8 weeks of moderate-intensity AET. Myocardial infarction rats displayed cardiac dysfunction and lung oedema, suggesting heart failure. Cardiac dysfunction in MI rats was paralleled by increased protein levels of UPR markers (GRP78, DERLIN-1 and CHOP), accumulation of misfolded and polyubiquitinated proteins, and reduced chymotrypsin-like proteasome activity. These results suggest an impaired cardiac protein quality control. Aerobic exercise training improved exercise capacity and cardiac function of MI animals. Interestingly, AET blunted MI-induced ER stress by reducing protein levels of UPR markers, and accumulation of both misfolded and polyubiquinated proteins, which was associated with restored proteasome activity. Taken together, our study provide evidence for AET attenuation of ER stress through the reestablishment of cardiac protein quality control, which contributes to better cardiac function in post-MI heart failure rats. These results reinforce the importance of AET as primary non-pharmacological therapy to cardiovascular disease. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Peroxisomal Proteostasis Involves a Lon Family Protein That Functions as Protease and Chaperone*

PubMed Central

Bartoszewska, Magdalena; Williams, Chris; Kikhney, Alexey; Opaliński, Łukasz; van Roermund, Carlo W. T.; de Boer, Rinse; Veenhuis, Marten; van der Klei, Ida J.

2012-01-01

Proteins are subject to continuous quality control for optimal proteostasis. The knowledge of peroxisome quality control systems is still in its infancy. Here we show that peroxisomes contain a member of the Lon family of proteases (Pln). We show that Pln is a heptameric protein and acts as an ATP-fueled protease and chaperone. Hence, Pln is the first chaperone identified in fungal peroxisomes. In cells of a PLN deletion strain peroxisomes contain protein aggregates, a major component of which is catalase-peroxidase. We show that this enzyme is sensitive to oxidative damage. The oxidatively damaged, but not the native protein, is a substrate of the Pln protease. Cells of the pln strain contain enhanced levels of catalase-peroxidase protein but reduced catalase-peroxidase enzyme activities. Together with the observation that Pln has chaperone activity in vitro, our data suggest that catalase-peroxidase aggregates accumulate in peroxisomes of pln cells due to the combined absence of Pln protease and chaperone activities. PMID:22733816

Wine and grape tannin interactions with salivary proteins and their impact on astringency: a review of current research.

PubMed

McRae, Jacqui M; Kennedy, James A

2011-03-11

Astringency is an important characteristic of red wine quality. The sensation is generally thought to be produced by the interaction of wine tannins with salivary proteins and the subsequent aggregation and precipitation of protein-tannin complexes. The importance of wine astringency for marketability has led to a wealth of research on the causes of astringency and how tannins impact the quality of the sensation, particularly with respect to tannin structure. Ultimately, the understanding of how tannin structure impacts astringency will allow the controlled manipulation of tannins via such methods as micro-oxygenation or fining to improve the quality of wines.

Protein quality and oil digestibility of Lupinus mutabilis: metabolic studies in children.

PubMed

Lopez de Romaãna, G; Graham, G G; Morales, E; Massa, E; MacLean, W C

1983-04-01

The nutritional quality of lupins (Lupinus mutabilis) for infants and children was evaluated in two sets of balance studies. In the first the digestibility and protein quality of diets based on lupin flour, with and without methionine supplementation, were compared with those of a control diet consisting of casein, sucrose and vegetable oil. Apparent nitrogen absorption from lupin flour (81.8 and 84.3% of intake) was slightly but significantly less than that during casein control periods (87.2 and 86.8% of intake, P less than 0.05 and less than 0.001). Apparent nitrogen retention from unsupplemented lupin (15.6 +/- 5.8% of intake) was significantly less than that from casein in the corresponding control periods (29.8 +/- 4.9%, P less than 0.001); a small but significant (P less than 0.05) increase in nitrogen retention was observed during the control period following the lupin diet when compared with that preceding it. Methionine supplementation of lupin produced a marked improvement in apparent nitrogen retention (to 22.2 +/- 6.9%, P less than 0.05). In the second set of studies the digestibility of lupin oil was compared with that of a blend of soybean and cottonseed oils (50:50). Excretion of fecal fat (9.8 +/- 3.0% of intake) and fecal energy (6.7 +/- 1.2% of intake) with the diet containing lupin oil were similar to those observed with the control diet. Both the protein quality and oil digestibility of Lupinus mutabilis are very similar to those from soybeans processed in a similar manner. For certain countries the lupin could be a valuable source of protein and edible oil for human consumption.

High-Protein and High-Dietary Fiber Breakfasts Result in Equal Feelings of Fullness and Better Diet Quality in Low-Income Preschoolers Compared with Their Usual Breakfast.

PubMed

Kranz, Sibylle; Brauchla, Mary; Campbell, Wayne W; Mattes, Rickard D; Schwichtenberg, Amy J

2017-03-01

Background: In the United States, 17% of children are currently obese. Increasing feelings of fullness may prevent excessive energy intake, lead to better diet quality, and promote long-term maintenance of healthy weight. Objective: The purpose of this study was to develop a fullness-rating tool (aim 1) and to determine whether a high-protein (HP), high-fiber (HF), and combined HP and HF (HPHF) breakfast increases preschoolers' feelings of fullness before (pre) and after (post) breakfast and pre-lunch, as well as their diet quality, as measured by using a composite diet quality assessment tool, the Revised Children's Diet Quality Index (aim 2). Methods: Children aged 4 and 5 y ( n = 41; 22 girls and 19 boys) from local Head Start centers participated in this randomized intervention trial. Sixteen percent of boys and 32% of girls were overweight or obese. After the baseline week, children rotated through four 1-wk periods of consuming ad libitum HP (19-20 g protein), HF (10-11 g fiber), HPHF (19-21 g protein, 10-12 g fiber), or usual (control) breakfasts. Food intake at breakfast was estimated daily, and for breakfast, lunch, and snack on day 3 of each study week Student's t tests and ANOVA were used to determine statistical differences. Results: Children's post-breakfast and pre-lunch fullness ratings were ≥1 point higher than those of pre-breakfast (aim 1). Although children consumed, on average, 65 kcal less energy during the intervention breakfasts ( P < 0.007) than during the control breakfast, fullness ratings did not differ ( P = 0.76). Relative to the control breakfast, improved diet quality (12%) was calculated for the HP and HF breakfasts ( P < 0.027) but not for the HPHF breakfast (aim 2). Conclusions: Post-breakfast fullness ratings were not affected by the intervention breakfasts relative to the control breakfast. HP and HF breakfasts resulted in higher diet quality. Serving HP or HF breakfasts may be valuable in improving diet quality without lowering feelings of satiation or satiety. This trial was registered at clinicaltrials.gov as NCT02122224. © 2017 American Society for Nutrition.

mRNA quality control is bypassed for immediate export of stress-responsive transcripts.

PubMed

Zander, Gesa; Hackmann, Alexandra; Bender, Lysann; Becker, Daniel; Lingner, Thomas; Salinas, Gabriela; Krebber, Heike

2016-12-12

Cells grow well only in a narrow range of physiological conditions. Surviving extreme conditions requires the instantaneous expression of chaperones that help to overcome stressful situations. To ensure the preferential synthesis of these heat-shock proteins, cells inhibit transcription, pre-mRNA processing and nuclear export of non-heat-shock transcripts, while stress-specific mRNAs are exclusively exported and translated. How cells manage the selective retention of regular transcripts and the simultaneous rapid export of heat-shock mRNAs is largely unknown. In Saccharomyces cerevisiae, the shuttling RNA adaptor proteins Npl3, Gbp2, Hrb1 and Nab2 are loaded co-transcriptionally onto growing pre-mRNAs. For nuclear export, they recruit the export-receptor heterodimer Mex67-Mtr2 (TAP-p15 in humans). Here we show that cellular stress induces the dissociation of Mex67 and its adaptor proteins from regular mRNAs to prevent general mRNA export. At the same time, heat-shock mRNAs are rapidly exported in association with Mex67, without the need for adapters. The immediate co-transcriptional loading of Mex67 onto heat-shock mRNAs involves Hsf1, a heat-shock transcription factor that binds to heat-shock-promoter elements in stress-responsive genes. An important difference between the export modes is that adaptor-protein-bound mRNAs undergo quality control, whereas stress-specific transcripts do not. In fact, regular mRNAs are converted into uncontrolled stress-responsive transcripts if expressed under the control of a heat-shock promoter, suggesting that whether an mRNA undergoes quality control is encrypted therein. Under normal conditions, Mex67 adaptor proteins are recruited for RNA surveillance, with only quality-controlled mRNAs allowed to associate with Mex67 and leave the nucleus. Thus, at the cost of error-free mRNA formation, heat-shock mRNAs are exported and translated without delay, allowing cells to survive extreme situations.

Misfolded Polyglutamine, Polyalanine, and Superoxide Dismutase 1 Aggregate via Distinct Pathways in the Cell*

PubMed Central

Polling, Saskia; Mok, Yee-Foong; Ramdzan, Yasmin M.; Turner, Bradley J.; Yerbury, Justin J.; Hill, Andrew F.; Hatters, Danny M.

2014-01-01

Protein aggregation into intracellular inclusions is a key feature of many neurodegenerative disorders. A common theme has emerged that inappropriate self-aggregation of misfolded or mutant polypeptide sequences is detrimental to cell health. Yet protein quality control mechanisms may also deliberately cluster them together into distinct inclusion subtypes, including the insoluble protein deposit (IPOD) and the juxtanuclear quality control (JUNQ). Here we investigated how the intrinsic oligomeric state of three model systems of disease-relevant mutant protein and peptide sequences relates to the IPOD and JUNQ patterns of aggregation using sedimentation velocity analysis. Two of the models (polyalanine (37A) and superoxide dismutase 1 (SOD1) mutants A4V and G85R) accumulated into the same JUNQ-like inclusion whereas the other, polyglutamine (72Q), formed spatially distinct IPOD-like inclusions. Using flow cytometry pulse shape analysis (PulSA) to separate cells with inclusions from those without revealed the SOD1 mutants and 37A to have abruptly altered oligomeric states with respect to the nonaggregating forms, regardless of whether cells had inclusions or not, whereas 72Q was almost exclusively monomeric until inclusions formed. We propose that mutations leading to JUNQ inclusions induce a constitutively “misfolded” state exposing hydrophobic side chains that attract and ultimately overextend protein quality capacity, which leads to aggregation into JUNQ inclusions. Poly(Q) is not misfolded in this same sense due to universal polar side chains, but is highly prone to forming amyloid fibrils that we propose invoke a different engagement mechanism with quality control. PMID:24425868

Misfolded polyglutamine, polyalanine, and superoxide dismutase 1 aggregate via distinct pathways in the cell.

PubMed

Polling, Saskia; Mok, Yee-Foong; Ramdzan, Yasmin M; Turner, Bradley J; Yerbury, Justin J; Hill, Andrew F; Hatters, Danny M

2014-03-07

Protein aggregation into intracellular inclusions is a key feature of many neurodegenerative disorders. A common theme has emerged that inappropriate self-aggregation of misfolded or mutant polypeptide sequences is detrimental to cell health. Yet protein quality control mechanisms may also deliberately cluster them together into distinct inclusion subtypes, including the insoluble protein deposit (IPOD) and the juxtanuclear quality control (JUNQ). Here we investigated how the intrinsic oligomeric state of three model systems of disease-relevant mutant protein and peptide sequences relates to the IPOD and JUNQ patterns of aggregation using sedimentation velocity analysis. Two of the models (polyalanine (37A) and superoxide dismutase 1 (SOD1) mutants A4V and G85R) accumulated into the same JUNQ-like inclusion whereas the other, polyglutamine (72Q), formed spatially distinct IPOD-like inclusions. Using flow cytometry pulse shape analysis (PulSA) to separate cells with inclusions from those without revealed the SOD1 mutants and 37A to have abruptly altered oligomeric states with respect to the nonaggregating forms, regardless of whether cells had inclusions or not, whereas 72Q was almost exclusively monomeric until inclusions formed. We propose that mutations leading to JUNQ inclusions induce a constitutively "misfolded" state exposing hydrophobic side chains that attract and ultimately overextend protein quality capacity, which leads to aggregation into JUNQ inclusions. Poly(Q) is not misfolded in this same sense due to universal polar side chains, but is highly prone to forming amyloid fibrils that we propose invoke a different engagement mechanism with quality control.

Cyclosporine A-Sensitive, Cyclophilin B-Dependent Endoplasmic Reticulum-Associated Degradation

PubMed Central

Luban, Jeremy; Molinari, Maurizio

2010-01-01

Peptidyl-prolyl cis/trans isomerases (PPIs) catalyze cis/trans isomerization of peptide bonds preceding proline residues. The involvement of PPI family members in protein refolding has been established in test tube experiments. Surprisingly, however, no data is available on the involvement of endoplasmic reticulum (ER)-resident members of the PPI family in protein folding, quality control or disposal in the living cell. Here we report that the immunosuppressive drug cyclosporine A (CsA) selectively inhibits the degradation of a subset of misfolded proteins generated in the ER. We identify cyclophilin B (CyPB) as the ER-resident target of CsA that catalytically enhances disposal from the ER of ERAD-LS substrates containing cis proline residues. Our manuscript presents the first evidence for enzymatic involvement of a PPI in protein quality control in the ER of living cells. PMID:20927389

Transferrin receptor-like proteins control the degradation of a yeast metal transporter

PubMed Central

Stimpson, Helen E M; Lewis, Michael J; Pelham, Hugh R B

2006-01-01

Plasma membrane transporters are often downregulated by their substrates. The yeast manganese transporter Smf1 is subject to two levels of regulation: heavy metals induce its sequestration within the cell, and also its ubiquitination and degradation in the vacuole. Degradation requires Bsd2, a membrane protein with a PPxY motif that recruits the ubiquitin ligase Rsp5, and which has a role in the quality control of membrane proteins, that expose hydrophilic residues to the lipid bilayer. We show that degradation of Smf1 requires in addition one of a pair of related yeast proteins, Tre1 and Tre2, that also contain PPxY motifs. Tre1 can partially inhibit manganese uptake without Bsd2, but requires Bsd2 to induce Smf1 degradation. It has a relatively hydrophilic transmembrane domain and binds to Bsd2. We propose that the Tre proteins specifically link Smf1 to the Bsd2-dependent quality control system. Their luminal domains are related to the transferrin receptor, but these are dispensable for Smf1 regulation. Tre proteins and the transferrin receptors appear to have evolved independently from the same family of membrane-associated proteases. PMID:16456538

Crucial HSP70 co–chaperone complex unlocks metazoan protein disaggregation

PubMed Central

Nillegoda, Nadinath B.; Kirstein, Janine; Szlachcic, Anna; Berynskyy, Mykhaylo; Stank, Antonia; Stengel, Florian; Arnsburg, Kristin; Gao, Xuechao; Scior, Annika; Aebersold, Ruedi; Guilbride, D. Lys; Wade, Rebecca C.; Morimoto, Richard I.; Mayer, Matthias P.; Bukau, Bernd

2016-01-01

Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathophysiological states1,2. Healthy metazoan cells effectively eliminate intracellular protein aggregates3,4, indicating that efficient disaggregation and/or degradation mechanisms exist. However, metazoans lack the key heat-shock protein disaggregase HSP100 of non-metazoan HSP70-dependent protein disaggregation systems5,6, and the human HSP70 system alone, even with the crucial HSP110 nucleotide exchange factor, has poor disaggregation activity in vitro4,7. This unresolved conundrum is central to protein quality control biology. Here we show that synergic cooperation between complexed J-protein co-chaperones of classes A and B unleashes highly efficient protein disaggregation activity in human and nematode HSP70 systems. Metazoan mixed-class J-protein complexes are transient, involve complementary charged regions conserved in the J-domains and carboxy-terminal domains of each J-protein class, and are flexible with respect to subunit composition. Complex formation allows J-proteins to initiate transient higher order chaperone structures involving HSP70 and interacting nucleotide exchange factors. A network of cooperative class A and B J-protein interactions therefore provides the metazoan HSP70 machinery with powerful, flexible, and finely regulatable disaggregase activity and a further level of regulation crucial for cellular protein quality control. PMID:26245380

DDX3 DEAD-box RNA helicase plays a central role in mitochondrial protein quality control in Leishmania

PubMed Central

Padmanabhan, Prasad Kottayil; Zghidi-Abouzid, Ouafa; Samant, Mukesh; Dumas, Carole; Aguiar, Bruno Guedes; Estaquier, Jerome; Papadopoulou, Barbara

2016-01-01

DDX3 is a highly conserved member of ATP-dependent DEAD-box RNA helicases with multiple functions in RNA metabolism and cellular signaling. Here, we describe a novel function for DDX3 in regulating the mitochondrial stress response in the parasitic protozoan Leishmania. We show that genetic inactivation of DDX3 leads to the accumulation of mitochondrial reactive oxygen species (ROS) associated with a defect in hydrogen peroxide detoxification. Upon stress, ROS production is greatly enhanced, causing mitochondrial membrane potential loss, mitochondrial fragmentation, and cell death. Importantly, this phenotype is exacerbated upon oxidative stress in parasites forced to use the mitochondrial oxidative respiratory machinery. Furthermore, we show that in the absence of DDX3, levels of major components of the unfolded protein response as well as of polyubiquitinated proteins increase in the parasite, particularly in the mitochondrion, as an indicator of mitochondrial protein damage. Consistent with these findings, immunoprecipitation and mass-spectrometry studies revealed potential interactions of DDX3 with key components of the cellular stress response, particularly the antioxidant response, the unfolded protein response, and the AAA-ATPase p97/VCP/Cdc48, which is essential in mitochondrial protein quality control by driving proteosomal degradation of polyubiquitinated proteins. Complementation studies using DDX3 deletion mutants lacking conserved motifs within the helicase core support that binding of DDX3 to ATP is essential for DDX3's function in mitochondrial proteostasis. As a result of the inability of DDX3-depleted Leishmania to recover from ROS damage and to survive various stresses in the host macrophage, parasite intracellular development was impaired. Collectively, these observations support a central role for the Leishmania DDX3 homolog in preventing ROS-mediated damage and in maintaining mitochondrial protein quality control. PMID:27735940

A proteomics performance standard to support measurement quality in proteomics.

PubMed

Beasley-Green, Ashley; Bunk, David; Rudnick, Paul; Kilpatrick, Lisa; Phinney, Karen

2012-04-01

The emergence of MS-based proteomic platforms as a prominent technology utilized in biochemical and biomedical research has increased the need for high-quality MS measurements. To address this need, National Institute of Standards and Technology (NIST) reference material (RM) 8323 yeast protein extract is introduced as a proteomics quality control material for benchmarking the preanalytical and analytical performance of proteomics-based experimental workflows. RM 8323 yeast protein extract is based upon the well-characterized eukaryote Saccharomyces cerevisiae and can be utilized in the design and optimization of proteomics-based methodologies from sample preparation to data analysis. To demonstrate its utility as a proteomics quality control material, we coupled LC-MS/MS measurements of RM 8323 with the NIST MS Quality Control (MSQC) performance metrics to quantitatively assess the LC-MS/MS instrumentation parameters that influence measurement accuracy, repeatability, and reproducibility. Due to the complexity of the yeast proteome, we also demonstrate how NIST RM 8323, along with the NIST MSQC performance metrics, can be used in the evaluation and optimization of proteomics-based sample preparation methods. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The effect of G protein-coupled receptor kinase 2 (GRK2) on lactation and on proliferation of mammary epithelial cells from dairy cows.

PubMed

Hou, Xiaoming; Hu, Hongliu; Lin, Ye; Qu, Bo; Gao, Xuejun; Li, Qingzhang

2016-07-01

Milk protein is an important component of milk and a nutritional source for human consumption. To better understand the molecular events underlying synthesis of milk proteins, the global gene expression patterns in mammary glands of dairy cow with high-quality milk (>3% milk protein; >3.5% milk fat) and low-quality milk (<3% milk protein; <3.5% milk fat) were examined via digital gene expression study. A total of 139 upregulated and 66 downregulated genes were detected in the mammary tissues of lactating cows with high-quality milk compared with the tissues of cows with low-quality milk. A pathway enrichment study of these genes revealed that the top 5 pathways that were differentially affected in the tissues of cows with high- versus low-quality milk involved metabolic pathways, cancer, cytokine-cytokine receptor interactions, regulation of the actin cytoskeleton, and insulin signaling. We also found that the G protein-coupled receptor kinase 2 (GRK2) was one of the most highly upregulated genes in lactating mammary tissue with low-quality milk compared with tissue with high-quality milk. The knockdown of GRK2 in cultured bovine mammary epithelial cells enhanced CSN2 expression and activated signaling molecules related to translation, including protein kinase B, mammalian target of rapamycin, and p70 ribosomal protein S6 kinase 1 (S6K1), whereas overexpression of GRK2 had the opposite effects. However, expression of genes involved in the mitogen-activated protein kinase pathway was positively regulated by GRK2. Therefore, GRK2 seems to act as a negative mediator of milk-protein synthesis via the protein kinase B-mammalian target of rapamycin signaling axis. Furthermore, GRK2 may negatively control milk-protein synthesis by activating the mitogen-activated protein kinase pathway in dairy cow mammary epithelial cells. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Repurposing a bacterial quality control mechanism to enhance enzyme production in living cells

USDA-ARS?s Scientific Manuscript database

Heterologous expression of many proteins in bacteria, yeasts, and plants is often limited by low titers of functional protein. To address this problem, we have created a two-tiered directed evolution strategy in Escherichia coli that enables optimization of protein production while maintaining high ...

Emulsion stability, thermo-rheology and quality characteristics of ground pork patties prepared with soy protein isolate and carrageenan.

PubMed

Gao, Xue-qin; Zhang, Wan-gang; Zhou, Guang-hong

2015-11-01

Plant proteins and polysaccharides are often utilised in ground meat products as meat binders, gelling agents, texture stabilisers or fat substitutes. The aim of this study was to investigate the effects of the incorporation of 57 g kg(-1) soy protein isolate (SPI), 7 g kg(-1) carrageenan (CAR) and SPI/CAR mixture on the quality of ground pork patties. Ground pork patties with individual SPI and CAR or SPI/CAR mixture showed either retained or improved emulsion stability, physicochemical properties and dynamic rheology compared with control samples. Although there were no significant colour differences among treatments, samples with SPI/CAR mixture presented higher texture profile values for hardness and chewiness compared with other treatments (P < 0.05). Patties with additives showed significantly lower cooking loss and better thermal emulsion stability than control samples owing to a lower release rate of water and fat (P < 0.05). Compared with control samples or those with individual SPI and CAR, patties with SPI/CAR mixture formed a smoother and more continuous structure with a more compact protein matrix. The results indicate that a mixture of SPI and CAR can be effectively used as a functional ingredient to improve the quality of ground pork patties. © 2014 Society of Chemical Industry.

Optimization of mold wheat bread fortified with soy flour, pea flour and whey protein concentrate.

PubMed

Erben, Melina; Osella, Carlos A

2017-07-01

The objective of this work was to study the effect of replacing a selected wheat flour for defatted soy flour, pea flour and whey protein concentrate on both dough rheological characteristics and the performance and nutritional quality of bread. A mixture design was used to analyze the combination of the ingredients. The optimization process suggested that a mixture containing 88.8% of wheat flour, 8.2% of defatted soy flour, 0.0% of pea flour and 3.0% of whey protein concentrate could be a good combination to achieve the best fortified-bread nutritional quality. The fortified bread resulted in high protein concentration, with an increase in dietary fiber content and higher calcium levels compared with those of control (wheat flour 100%). Regarding protein quality, available lysine content was significantly higher, thus contributing with the essential amino acid requirement.

Protein Quality Control Acts on Folding Intermediates to Shape the Effects of Mutations on Organismal Fitness

PubMed Central

Bershtein, Shimon; Mu, Wanmeng; Serohijos, Adrian W. R.; Zhou, Jingwen; Shakhnovich, Eugene I.

2012-01-01

Summary What are the molecular properties of proteins that fall on the radar of protein quality control (PQC)? Here we mutate the E. coli’s gene encoding dihydrofolate reductase (DHFR), and replace it with bacterial orthologous genes to determine how components of PQC modulate fitness effects of these genetic changes. We find that chaperonins GroEL/ES and protease Lon compete for binding to molten globule intermediate of DHFR, resulting in a peculiar symmetry in their action: Over-expression of GroEL/ES and deletion of Lon both restore growth of deleterious DHFR mutants and most of the slow-growing orthologous DHFR strains. Kinetic steady-state modeling predicts and experimentation verifies that mutations affect fitness by shifting the flux balance in cellular milieu between protein production, folding and degradation orchestrated by PQC through the interaction with folding intermediates. PMID:23219534

Proteasomal and lysosomal protein degradation and heart disease.

PubMed

Wang, Xuejun; Robbins, Jeffrey

2014-06-01

In the cell, the proteasome and lysosomes represent the most important proteolytic machineries, responsible for the protein degradation in the ubiquitin-proteasome system (UPS) and autophagy, respectively. Both the UPS and autophagy are essential to protein quality and quantity control. Alterations in cardiac proteasomal and lysosomal degradation are remarkably associated with most heart disease in humans and are implicated in the pathogenesis of congestive heart failure. Studies carried out in animal models and in cell culture have begun to establish both sufficiency and, in some cases, the necessity of proteasomal functional insufficiency or lysosomal insufficiency as a major pathogenic factor in the heart. This review article highlights some recent advances in the research into proteasome and lysosome protein degradation in relation to cardiac pathology and examines the emerging evidence for enhancing degradative capacities of the proteasome and/or lysosome as a new therapeutic strategy for heart disease. This article is part of a Special Issue entitled "Protein Quality Control, the Ubiquitin Proteasome System, and Autophagy". Copyright © 2013 Elsevier Ltd. All rights reserved.

Cmr1/WDR76 defines a nuclear genotoxic stress body linking genome integrity and protein quality control.

PubMed

Gallina, Irene; Colding, Camilla; Henriksen, Peter; Beli, Petra; Nakamura, Kyosuke; Offman, Judith; Mathiasen, David P; Silva, Sonia; Hoffmann, Eva; Groth, Anja; Choudhary, Chunaram; Lisby, Michael

2015-03-30

DNA replication stress is a source of genomic instability. Here we identify changed mutation rate 1 (Cmr1) as a factor involved in the response to DNA replication stress in Saccharomyces cerevisiae and show that Cmr1--together with Mrc1/Claspin, Pph3, the chaperonin containing TCP1 (CCT) and 25 other proteins--define a novel intranuclear quality control compartment (INQ) that sequesters misfolded, ubiquitylated and sumoylated proteins in response to genotoxic stress. The diversity of proteins that localize to INQ indicates that other biological processes such as cell cycle progression, chromatin and mitotic spindle organization may also be regulated through INQ. Similar to Cmr1, its human orthologue WDR76 responds to proteasome inhibition and DNA damage by relocalizing to nuclear foci and physically associating with CCT, suggesting an evolutionarily conserved biological function. We propose that Cmr1/WDR76 plays a role in the recovery from genotoxic stress through regulation of the turnover of sumoylated and phosphorylated proteins.

Ubiquitin and Parkinson's disease through the looking glass of genetics.

PubMed

Walden, Helen; Muqit, Miratul M K

2017-04-13

Biochemical alterations found in the brains of Parkinson's disease (PD) patients indicate that cellular stress is a major driver of dopaminergic neuronal loss. Oxidative stress, mitochondrial dysfunction, and ER stress lead to impairment of the homeostatic regulation of protein quality control pathways with a consequent increase in protein misfolding and aggregation and failure of the protein degradation machinery. Ubiquitin signalling plays a central role in protein quality control; however, prior to genetic advances, the detailed mechanisms of how impairment in the ubiquitin system was linked to PD remained mysterious. The discovery of mutations in the α-synuclein gene, which encodes the main protein misfolded in PD aggregates, together with mutations in genes encoding ubiquitin regulatory molecules, including PTEN-induced kinase 1 (PINK1), Parkin, and FBX07, has provided an opportunity to dissect out the molecular basis of ubiquitin signalling disruption in PD, and this knowledge will be critical for developing novel therapeutic strategies in PD that target the ubiquitin system. © 2017 The Author(s).

Controlled dehydration improves the diffraction quality of two RNA crystals.

PubMed

Park, HaJeung; Tran, Tuan; Lee, Jun Hyuck; Park, Hyun; Disney, Matthew D

2016-11-03

Post-crystallization dehydration methods, applying either vapor diffusion or humidity control devices, have been widely used to improve the diffraction quality of protein crystals. Despite the fact that RNA crystals tend to diffract poorly, there is a dearth of reports on the application of dehydration methods to improve the diffraction quality of RNA crystals. We use dehydration techniques with a Free Mounting System (FMS, a humidity control device) to recover the poor diffraction quality of RNA crystals. These approaches were applied to RNA constructs that model various RNA-mediated repeat expansion disorders. The method we describe herein could serve as a general tool to improve diffraction quality of RNA crystals to facilitate structure determinations.

Postproduction Handling and Administration of Protein Pharmaceuticals and Potential Instability Issues.

PubMed

Nejadnik, M Reza; Randolph, Theodore W; Volkin, David B; Schöneich, Christian; Carpenter, John F; Crommelin, Daan J A; Jiskoot, Wim

2018-04-14

The safety and efficacy of protein pharmaceuticals depend not only on biological activity but also on purity levels. Impurities may be process related because of limitations in manufacturing or product related because of protein degradation occurring throughout the life history of a product. Although the pharmaceutical biotechnology industry has made great progress in improving bulk and drug product manufacturing as well as company-controlled storage and transportation conditions to minimize the level of degradation, there is less control over the many factors that may subsequently affect product quality after the protein pharmaceuticals are released and shipped by the manufacturer. Routine handling or unintentional mishandling of therapeutic protein products may cause protein degradation that remains unnoticed but can potentially compromise the clinical safety and efficacy of the product. In this commentary, we address some potential risks associated with (mis)handling of protein pharmaceuticals after release by the manufacturer. We summarize the environmental stress factors that have been shown to cause protein degradation and that may be encountered during typical handling procedures of protein pharmaceuticals in a hospital setting or during self-administration by patients. Moreover, we provide recommendations for improvements in product handling to help ensure the quality of protein pharmaceuticals during use. Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Molecular chaperones and photoreceptor function

PubMed Central

Kosmaoglou, Maria; Schwarz, Nele; Bett, John S.; Cheetham, Michael E.

2008-01-01

Molecular chaperones facilitate and regulate protein conformational change within cells. This encompasses many fundamental cellular processes: including the correct folding of nascent chains; protein transport and translocation; signal transduction and protein quality control. Chaperones are, therefore, important in several forms of human disease, including neurodegeneration. Within the retina, the highly specialized photoreceptor cell presents a fascinating paradigm to investigate the specialization of molecular chaperone function and reveals unique chaperone requirements essential to photoreceptor function. Mutations in several photoreceptor proteins lead to protein misfolding mediated neurodegeneration. The best characterized of these are mutations in the molecular light sensor, rhodopsin, which cause autosomal dominant retinitis pigmentosa. Rhodopsin biogenesis is likely to require chaperones, while rhodopsin misfolding involves molecular chaperones in quality control and the cellular response to protein aggregation. Furthermore, the specialization of components of the chaperone machinery to photoreceptor specific roles has been revealed by the identification of mutations in molecular chaperones that cause inherited retinal dysfunction and degeneration. These chaperones are involved in several important cellular pathways and further illuminate the essential and diverse roles of molecular chaperones. PMID:18490186

Cholesterol regulates the endoplasmic reticulum exit of the major membrane protein P0 required for peripheral myelin compaction.

PubMed

Saher, Gesine; Quintes, Susanne; Möbius, Wiebke; Wehr, Michael C; Krämer-Albers, Eva-Maria; Brügger, Britta; Nave, Klaus-Armin

2009-05-13

Rapid impulse conduction requires electrical insulation of axons by myelin, a cholesterol-rich extension of the glial cell membrane with a characteristic composition of proteins and lipids. Mutations in several myelin protein genes cause endoplasmic reticulum (ER) retention and disease, presumably attributable to failure of misfolded proteins to pass the ER quality control. Because many myelin proteins partition into cholesterol-rich membrane rafts, their interaction with cholesterol could potentially be part of the ER quality control system. Here, we provide in vitro and in vivo evidence that the major peripheral myelin protein P0 requires cholesterol for exiting the ER and reaching the myelin compartment. Cholesterol dependency of P0 trafficking in heterologous cells is mediated by a cholesterol recognition/interaction amino acid consensus (CRAC) motif. Mutant mice lacking cholesterol biosynthesis in Schwann cells suffer from severe hypomyelination with numerous uncompacted myelin stretches. This demonstrates that high-level cholesterol coordinates P0 export with myelin membrane synthesis, which is required for the correct stoichiometry of myelin components and for myelin compaction.

A Small-scale Model to Assess the Risk of Leachables from Single-use Bioprocess Containers through Protein Quality Characterization.

PubMed

Xiao, Nina J; Medley, Colin D; Shieh, Ian C; Downing, Gregory; Pizarro, Shelly; Liu, Jun; Patel, Ankit R

Leachables from single-use bioprocess containers (BPCs) are a source of process-related impurities that have the potential to alter product quality of biotherapeutics and affect patient health. Leachables often exist at very low concentrations, making it difficult to detect their presence and challenging to assess their impact on protein quality. A small-scale stress model based on assessing protein stability was developed to evaluate the potential risks associated with storing biotherapeutics in disposable bags caused by the presence of leachables. Small-scale BPCs were filled with protein solution at high surface area-to-volume ratios (≥3× the surface area-to-volume ratio of manufacturing-scale BPCs) and incubated at stress temperatures (e.g., 25 °C or 30 °C for up to 12 weeks) along with an appropriate storage vessel (e.g., glass vial or stainless steel) as a control for side-by-side comparison. Changes in protein size variants measured by size exclusion chromatography, capillary electrophoresis, and particle formation for two monoclonal antibodies using both the small-scale stress model and a control revealed a detrimental effect of gamma-irradiated BPCs on protein aggregation and significant BPC difference between earlier and later batches. It was found that preincubation of the empty BPCs prior to protein storage improved protein stability, suggesting the presence of volatile or heat-sensitive leachables (heat-labile or thermally degraded). In addition, increasing the polysorbate 20 concentration lowered, but did not completely mitigate, the leachable-protein interactions, indicating the presence of a hydrophobic leachable. Overall, this model can inform the risk of BPC leachables on biotherapeutics during routine manufacturing and assist in making decisions on the selection of a suitable BPC for the manufacturing process by assessing changes in product quality. Leachables from single-use systems often exist in small quantities and are difficult to detect with existing analytical methods. The presence of relevant detrimental leachables from single-use bioprocess containers (BPCs) can be indirectly detected by studying the stability of monoclonal antibodies via changes by size exclusion chromatography, capillary electrophoresis sodium dodecyl sulfate, and visible/sub-visible particles using a small-scale stress model containing high surface area-to-volume ratio at elevated temperature alongside with an appropriate control (e.g., glass vials or stainless steel containers). These changes in protein quality attributes allowed the evaluation of potential risks associated with adopting single-use bioprocess containers for storage as well as bag quality and bag differences between earlier and later batches. These leachables appear to be generated during the bag sterilization process by gamma irradiation. Improvements in protein stability after storage in "preheated" bags indicated that these leachables may be thermally unstable or volatile. The effect of surfactant levels, storage temperatures, surface area-to-volume ratios, filtration, and buffer exchange on leachables and protein stability were also assessed. © PDA, Inc. 2016.

Silencing of omega-5 gliadins in transgenic wheat eliminates a major source of environmental variability and improves dough mixing properties of flour.

PubMed

Altenbach, Susan B; Tanaka, Charlene K; Seabourn, Bradford W

2014-12-24

The end-use quality of wheat flour varies as a result of the growth conditions of the plant. Among the wheat gluten proteins, the omega-5 gliadins have been identified as a major source of environmental variability, increasing in proportion in grain from plants that receive fertilizer or are subjected to high temperatures during grain development. The omega-5 gliadins also have been associated with the food allergy wheat-dependent exercise-induced anaphylaxis (WDEIA). Recently, transgenic lines with reduced levels of omega-5 gliadins were developed using RNA interference (RNAi). These lines make it possible to determine whether changes in the levels of omega-5 gliadins in response to environmental conditions and agronomic inputs may be responsible for changes in flour end-use quality. Two transgenic wheat lines and a non-transgenic control were grown under a controlled temperature regimen with or without post-anthesis fertilizer and the protein composition of the resulting flour was analyzed by quantitative two-dimensional gel electrophoresis (2-DE). In one transgenic line, all 2-DE spots identified as omega-5 gliadins were substantially reduced without effects on other proteins. In the other transgenic line, the omega-5 gliadins were absent and there was a partial reduction in the levels of the omega-1,2 gliadins and the omega-1,2 chain-terminating gliadins as well as small changes in several other proteins. With the exception of the omega gliadins, the non-transgenic control and the transgenic plants showed similar responses to the fertilizer treatment. Protein contents of flour were determined by the fertilizer regimen and were similar in control and transgenic samples produced under each regimen while both mixing time and mixing tolerance were improved in flour from transgenic lines when plants received post-anthesis fertilizer. The data indicate that omega-5 gliadins have a negative effect on flour quality and suggest that changes in quality with the growth environment may be due in part to alterations in the levels of the omega gliadins. Because a known food allergen and one of the major sources of environmentally-induced variation in wheat flour protein composition has been eliminated, the transgenic lines may yield flour with both improved end-use quality and more consistent functionality when grown in different locations.

Controlling protein adsorption on graphene for cryo-EM using low-energy hydrogen plasmas

PubMed Central

Russo, Christopher J.; Passmore, Lori A.

2014-01-01

Despite its many favorable properties as a sample support for biological electron microscopy, graphene is not widely used because its hydrophobicity precludes reliable protein deposition. We describe a method to modify graphene using a low-energy hydrogen plasma, which reduces hydrophobicity without degrading the graphene lattice. We show that the use of plasma-treated graphene enables better control of protein distribution in ice for electron cryo-microscopy and improved image quality by reducing radiation-induced sample motion. PMID:24747813

A microcrystal selection technique in protein crystallization

NASA Astrophysics Data System (ADS)

Han, Qing; Lin, Sheng-Xiang

1996-10-01

The goal of protein crystallization is to obtain high quality single crystals for X-ray diffraction analysis. A new and easy technique was employed to control the number and quality of crystals by eliminating poor microcrystals after the spontaneous nucleation. The process was carried out with two samples: human 17β-hydroxysteroid dehydrogenase (17β-HSD) and hen egg white lysozyme. The present study suggests a useful method for the successful crystal growth of biomacromolecules.

Functional specialization in regulation and quality control in thermal adaptive evolution.

PubMed

Yama, Kazuma; Matsumoto, Yuki; Murakami, Yoshie; Seno, Shigeto; Matsuda, Hideo; Gotoh, Kazuyoshi; Motooka, Daisuke; Nakamura, Shota; Ying, Bei-Wen; Yomo, Tetsuya

2015-11-01

Distinctive survival strategies, specialized in regulation and in quality control, were observed in thermal adaptive evolution with a laboratory Escherichia coli strain. The two specialists carried a single mutation either within rpoH or upstream of groESL, which led to the activated global regulation by sigma factor 32 or an increased amount of GroEL/ES chaperonins, respectively. Although both specialists succeeded in thermal adaptation, the common winner of the evolution was the specialist in quality control, that is, the strategy of chaperonin-mediated protein folding. To understand this evolutionary consequence, multilevel analyses of cellular status, for example, transcriptome, protein and growth fitness, were carried out. The specialist in quality control showed less change in transcriptional reorganization responding to temperature increase, which was consistent with the finding of that the two specialists showed the biased expression of molecular chaperones. Such repressed changes in gene expression seemed to be advantageous for long-term sustainability because a specific increase in chaperonins not only facilitated the folding of essential gene products but also saved cost in gene expression compared with the overall transcriptional increase induced by rpoH regulation. Functional specialization offered two strategies for successful thermal adaptation, whereas the evolutionary advantageous was more at the points of cost-saving in gene expression and the essentiality in protein folding. © 2015 The Authors. Genes to Cells published by Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

High-Protein and High–Dietary Fiber Breakfasts Result in Equal Feelings of Fullness and Better Diet Quality in Low-Income Preschoolers Compared with Their Usual Breakfast123

PubMed Central

Kranz, Sibylle; Brauchla, Mary; Campbell, Wayne W; Mattes, Rickard D

2017-01-01

Background: In the United States, 17% of children are currently obese. Increasing feelings of fullness may prevent excessive energy intake, lead to better diet quality, and promote long-term maintenance of healthy weight. Objective: The purpose of this study was to develop a fullness-rating tool (aim 1) and to determine whether a high-protein (HP), high-fiber (HF), and combined HP and HF (HPHF) breakfast increases preschoolers’ feelings of fullness before (pre) and after (post) breakfast and pre-lunch, as well as their diet quality, as measured by using a composite diet quality assessment tool, the Revised Children’s Diet Quality Index (aim 2). Methods: Children aged 4 and 5 y (n = 41; 22 girls and 19 boys) from local Head Start centers participated in this randomized intervention trial. Sixteen percent of boys and 32% of girls were overweight or obese. After the baseline week, children rotated through four 1-wk periods of consuming ad libitum HP (19–20 g protein), HF (10–11 g fiber), HPHF (19–21 g protein, 10–12 g fiber), or usual (control) breakfasts. Food intake at breakfast was estimated daily, and for breakfast, lunch, and snack on day 3 of each study week Student’s t tests and ANOVA were used to determine statistical differences. Results: Children’s post-breakfast and pre-lunch fullness ratings were ≥1 point higher than those of pre-breakfast (aim 1). Although children consumed, on average, 65 kcal less energy during the intervention breakfasts (P < 0.007) than during the control breakfast, fullness ratings did not differ (P = 0.76). Relative to the control breakfast, improved diet quality (12%) was calculated for the HP and HF breakfasts (P < 0.027) but not for the HPHF breakfast (aim 2). Conclusions: Post-breakfast fullness ratings were not affected by the intervention breakfasts relative to the control breakfast. HP and HF breakfasts resulted in higher diet quality. Serving HP or HF breakfasts may be valuable in improving diet quality without lowering feelings of satiation or satiety. This trial was registered at clinicaltrials.gov as NCT02122224. PMID:28077732

Growth performance and carcase quality in broiler chickens fed on bacterial protein grown on natural gas.

PubMed

Øverland, M; Schøyen, H F; Skrede, A

2010-10-01

1. The effects of increasing concentrations (0, 40, 80 or 120 g/kg) of bacterial protein meal (BPM) and bacterial protein autolysate (BPA) grown on natural gas on growth performance and carcase quality in broiler chickens were examined. 2. Adding BPM to diets reduced feed intake and improved gain: feed from 0 to 21 d and overall to 35 d, but did not significantly affect weight gain compared to the soybean meal based control diet. 3. Increasing concentrations of BPA significantly reduced growth rate, feed intake, gain: feed, carcase weight and dressing percentage, but significantly increased carcase dry matter, fat and energy content. 4. Adding BPM to diets had no effect on viscosity of diets and jejunal digesta, and minor effects on litter quality, whereas BPA increased the viscosity of diets and jejunal digesta, improved litter quality at 21 d, but decreased litter quality at 32 d. 5. To conclude, broiler chickens performed better on a BPM product with intact proteins than on an autolysate with ruptured cell walls and a high content of free amino acids and low molecular-weight peptides.

Regulatory mechanisms of RNA function: emerging roles of DNA repair enzymes.

PubMed

Jobert, Laure; Nilsen, Hilde

2014-07-01

The acquisition of an appropriate set of chemical modifications is required in order to establish correct structure of RNA molecules, and essential for their function. Modification of RNA bases affects RNA maturation, RNA processing, RNA quality control, and protein translation. Some RNA modifications are directly involved in the regulation of these processes. RNA epigenetics is emerging as a mechanism to achieve dynamic regulation of RNA function. Other modifications may prevent or be a signal for degradation. All types of RNA species are subject to processing or degradation, and numerous cellular mechanisms are involved. Unexpectedly, several studies during the last decade have established a connection between DNA and RNA surveillance mechanisms in eukaryotes. Several proteins that respond to DNA damage, either to process or to signal the presence of damaged DNA, have been shown to participate in RNA quality control, turnover or processing. Some enzymes that repair DNA damage may also process modified RNA substrates. In this review, we give an overview of the DNA repair proteins that function in RNA metabolism. We also discuss the roles of two base excision repair enzymes, SMUG1 and APE1, in RNA quality control.

Proteostasis: bad news and good news from the endoplasmic reticulum.

PubMed

Noack, Julia; Brambilla Pisoni, Giorgia; Molinari, Maurizio

2014-01-01

The endoplasmic reticulum (ER) is an intracellular compartment dedicated to the synthesis and maturation of secretory and membrane proteins, totalling about 30% of the total eukaryotic cells proteome. The capacity to produce correctly folded polypeptides and to transport them to their correct intra- or extracellular destinations relies on proteostasis networks that regulate and balance the activity of protein folding, quality control, transport and degradation machineries. Nutrient and environmental changes, pathogen infection aging and, more relevant for the topics discussed in this review, mutations that impair attainment of the correct 3D structure of nascent polypeptide chains may compromise the activity of the proteostasis networks with devastating consequences on cells, organs and organisms' homeostasis. Here we present a review of mechanisms regulating folding and quality control of proteins expressed in the ER, and we describe the protein degradation and the ER stress pathways activated by the expression of misfolded proteins in the ER lumen. Finally, we highlight select examples of proteopathies (also known as conformational disorders or protein misfolding diseases) caused by protein misfolding in the ER and/or affecting cellular proteostasis and therapeutic interventions that might alleviate or cure the disease symptoms.

Mitochondrial enzymes are protected from stress-induced aggregation by mitochondrial chaperones and the Pim1/LON protease

PubMed Central

Bender, Tom; Lewrenz, Ilka; Franken, Sebastian; Baitzel, Catherina; Voos, Wolfgang

2011-01-01

Proteins in a natural environment are constantly challenged by stress conditions, causing their destabilization, unfolding, and, ultimately, aggregation. Protein aggregation has been associated with a wide variety of pathological conditions, especially neurodegenerative disorders, stressing the importance of adequate cellular protein quality control measures to counteract aggregate formation. To secure protein homeostasis, mitochondria contain an elaborate protein quality control system, consisting of chaperones and ATP-dependent proteases. To determine the effects of protein aggregation on the functional integrity of mitochondria, we set out to identify aggregation-prone endogenous mitochondrial proteins. We could show that major metabolic pathways in mitochondria were affected by the aggregation of key enzyme components, which were largely inactivated after heat stress. Furthermore, treatment with elevated levels of reactive oxygen species strongly influenced the aggregation behavior, in particular in combination with elevated temperatures. Using specific chaperone mutant strains, we showed a protective effect of the mitochondrial Hsp70 and Hsp60 chaperone systems. Moreover, accumulation of aggregated polypeptides was strongly decreased by the AAA-protease Pim1/LON. We therefore propose that the proteolytic breakdown of aggregation-prone polypeptides represents a major protective strategy to prevent the in vivo formation of aggregates in mitochondria. PMID:21209324

Evaluation of the lime-cooking and tortilla making properties of quality protein maize hybrids grown in Mexico.

PubMed

Serna-Saldivar, Sergio O; Amaya Guerra, Carlos A; Herrera Macias, Pedro; Melesio Cuellar, Jose L; Preciado Ortiz, Ricardo E; Terron Ibarra, Arturo D; Vazquez Carrillo, Gricelda

2008-09-01

Eleven experimental and three commercial white quality protein maize (QPM) hybrids and two regular endosperm controls were planted at Celaya, Guanajuato, Mexico with the aim of comparing grain physical characteristics, protein quality, lime-cooking and tortilla making properties. All genotypes were planted under irrigation using a density of 80,000 plants/ha and fertilized with 250 kg N-60 P-60 K per hectare. When compared with the controls these QPM genotypes had lower test (77.4 vs. 76.5 kg/hL) and 1,000 kernel weights (327 vs. 307 g), softer endosperm texture (2.5 vs. 1.8 where 1 = soft, 2 intermediate and 3 hard endosperm), lower protein (10.0 vs. 8.0%), higher nixtamal water uptake after 30 min lime-cooking (50.0 vs. 53.1% moisture) and lower pericarp removal scores. The lower thousand-kernel weight and softer endosperm texture observed in the QPM genotypes lowered the optimum lime-cooking time as estimated with regression equations. Most QPM genotypes had higher amounts of lysine, tryptophan and albumins/globulins when compared with the controls. QPMs HEC 424973, HEC 774986 and HEC 734286 had the best grain traits for nixtamalization and therefore the best potential for industrial utilization. The commercial use of these QPM hybrids should benefit Mexicans who depend on tortillas as the main staple.

Secreted Proteins Defy the Expression Level–Evolutionary Rate Anticorrelation

PubMed Central

Feyertag, Felix; Berninsone, Patricia M.; Alvarez-Ponce, David

2017-01-01

The rates of evolution of the proteins of any organism vary across orders of magnitude. A primary factor influencing rates of protein evolution is expression. A strong negative correlation between expression levels and evolutionary rates (the so-called E–R anticorrelation) has been observed in virtually all studied organisms. This effect is currently attributed to the abundance-dependent fitness costs of misfolding and unspecific protein–protein interactions, among other factors. Secreted proteins are folded in the endoplasmic reticulum, a compartment where chaperones, folding catalysts, and stringent quality control mechanisms promote their correct folding and may reduce the fitness costs of misfolding. In addition, confinement of secreted proteins to the extracellular space may reduce misinteractions and their deleterious effects. We hypothesize that each of these factors (the secretory pathway quality control and extracellular location) may reduce the strength of the E–R anticorrelation. Indeed, here we show that among human proteins that are secreted to the extracellular space, rates of evolution do not correlate with protein abundances. This trend is robust to controlling for several potentially confounding factors and is also observed when analyzing protein abundance data for 6 human tissues. In addition, analysis of mRNA abundance data for 32 human tissues shows that the E–R correlation is always less negative, and sometimes nonsignificant, in secreted proteins. Similar observations were made in Caenorhabditis elegans and in Escherichia coli, and to a lesser extent in Drosophila melanogaster, Saccharomyces cerevisiae and Arabidopsis thaliana. Our observations contribute to understand the causes of the E–R anticorrelation. PMID:28007979

Identification of quantitative trait loci (QTL) controlling protein, oil, and five major fatty acids’ contents in soybean

USDA-ARS?s Scientific Manuscript database

Improved seed composition in soybean (Glycine max L. Merr.) for protein and oil quality is one of the major goals of soybean breeders. A group of genes that act as quantitative traits with their effects can alter protein, oil, palmitic, stearic, oleic, linoleic, and linolenic acids percentage in soy...

Study of Fluid Flow Control In Protein Crystallization Using Strong Magnetic Fields

NASA Technical Reports Server (NTRS)

Ramachandran, N.; Leslie, F.; Ciszak, E.; Curreri, Peter A. (Technical Monitor)

2002-01-01

An important component in biotechnology, particularly in the area of protein engineering and rational drug design is the knowledge of the precise three-dimensional molecular structure of proteins. The quality of structural information obtained from X-ray diffraction methods is directly dependent on the degree of perfection of the protein crystals. As a consequence, the growth of high quality macromolecular crystals for diffraction analyses has been the central focus for biochemists, biologists, and bioengineers. Macromolecular crystals are obtained from solutions that contain the crystallizing species in equilibrium with higher aggregates, ions, precipitants, other possible phases of the protein, foreign particles, the walls of the container, and a likely host of other impurities. By changing transport modes in general, i.e., reduction of convection and sedimentation, as is achieved in 'microgravity', researchers have been able to dramatically affect the movement and distribution of macromolecules in the fluid, and thus their transport, formation of crystal nuclei, and adsorption to the crystal surface. While a limited number of high quality crystals from space flights have been obtained, as the recent National Research Council (NRC) review of the NASA microgravity crystallization program pointed out, the scientific approach and research in crystallization of proteins has been mainly empirical yielding inconclusive results. We postulate that we can reduce convection in ground-based experiments and we can understand the different aspects of convection control through the use of strong magnetic fields and field gradients. Whether this limited convection in a magnetic field will provide the environment for the growth of high quality crystals is still a matter of conjecture that our research will address. The approach exploits the variation of fluid magnetic susceptibility with concentration for this purpose and the convective damping is realized by appropriately positioning the crystal growth cell so that the magnetic susceptibility force counteracts terrestrial gravity. The general objective is to test the hypothesis of convective control using a strong magnetic field and magnetic field gradient and to understand the nature of the various forces that come into play. Specifically we aim to delineate causative factors and to quantify them through experiments, analysis and numerical modeling. Once the basic understanding is obtained, the study will focus on testing the hypothesis on proteins of pyruvate dehydrogenase complex (PDC), proteins E1 and E3. Obtaining high crystal quality of these proteins is of great importance to structural biologists since their structures need to be determined.

Screening for the anti-inflammation quality markers of Xiaojin Pills based on HPLC-MS/MS method, COX-2 inhibition test and protein interaction network.

PubMed

Xiong, Xi; He, Ya-Nan; Feng, Bi; Pan, Yuan; Zhang, Hai-Zhu; Ke, Xiu-Mei; Zhang, Yi; Yang, Ming; Han, Li; Zhang, Ding-Kun

2018-05-10

Nowadays, breast disorders seriously affect women's health in an increasing number. In China, Xiaojin Pills are commonly used in the treatment of breast diseases. Doctors have concluded that the combined use of Xiaojin Pills with conventional therapy can significantly improve the efficacy with fewer side effects. However, the prescription of Xiaojin Pills is complicated and their quality control methods cannot completely ensure the quality of Xiaojin Pills. On the basis of its mechanism, our study combined chemical evaluation and biological evaluation to identify the anti-inflammatory markers of Xiaojin Pills. In this manuscript, 13 compounds in Xiaojin Pills were quantified. At the same time, the cyclooxygenase-2 inhibition rates of different Xiaojin Pills were measured and the possible markers were screened by spectrum-effect relationship. Further, anti-inflammatory activities of markers were verified and protein interaction network was analyzed, identifying the components of Protocatechuate, Beta-Boswellic acid and Levistilide A as the anti-inflammatory quality markers of Xiaojin Pills. We hope our studies can provide a scientific theoretical basis for accurately quality control of Xiaojin Pills and reasonable suggestions for pharmaceutical companies and new ideas for the quality control of other medicines.

Effect of incorporation of soy flour to wheat flour on nutritional and sensory quality of biscuits fortified with mushroom

PubMed Central

Farzana, Tasnim; Mohajan, Suman

2015-01-01

The research study was conducted to evaluate the quality characteristics of soy-mushroom-enriched biscuits which could be used as a protein supplemented cereal snack food. In this study, wheat flour was replaced with soy flour at different levels that is 20% (T3), 15% (T2), and 10% (T1) and without soy flour was kept as control (To). Mushroom was added in both biscuits. Biscuits were analyzed for chemical and sensory parameters. Protein content of soy flour-supplemented biscuits increased from 11.07% to 17.86% as compared to control along with a significant increased in fat (17.36–20.89%), fiber (0.48–0.92%), iron (1.56–1.99 mg/100 g), and energy value (463–485 Kcal/g). Ash content also increased but not significantly. Results from chemical analyses and organoleptic evaluation indicate that good quality biscuits can be prepared by substituting wheat flour with 15% soy flour and addition of mushroom powders may affect the backing quality. Protein Energy Malnutrition (PEM) of the Bangladeshi population can be reduced through the development of biscuits in this way. PMID:26405522

Effect of incorporation of soy flour to wheat flour on nutritional and sensory quality of biscuits fortified with mushroom.

PubMed

Farzana, Tasnim; Mohajan, Suman

2015-09-01

The research study was conducted to evaluate the quality characteristics of soy-mushroom-enriched biscuits which could be used as a protein supplemented cereal snack food. In this study, wheat flour was replaced with soy flour at different levels that is 20% (T3), 15% (T2), and 10% (T1) and without soy flour was kept as control (To). Mushroom was added in both biscuits. Biscuits were analyzed for chemical and sensory parameters. Protein content of soy flour-supplemented biscuits increased from 11.07% to 17.86% as compared to control along with a significant increased in fat (17.36-20.89%), fiber (0.48-0.92%), iron (1.56-1.99 mg/100 g), and energy value (463-485 Kcal/g). Ash content also increased but not significantly. Results from chemical analyses and organoleptic evaluation indicate that good quality biscuits can be prepared by substituting wheat flour with 15% soy flour and addition of mushroom powders may affect the backing quality. Protein Energy Malnutrition (PEM) of the Bangladeshi population can be reduced through the development of biscuits in this way.

Cellular protein quality control and the evolution of aggregates in spinocerebellar ataxia type 3 (SCA3).

PubMed

Seidel, K; Meister, M; Dugbartey, G J; Zijlstra, M P; Vinet, J; Brunt, E R P; van Leeuwen, F W; Rüb, U; Kampinga, H H; den Dunnen, W F A

2012-10-01

A characteristic of polyglutamine diseases is the increased propensity of disease proteins to aggregate, which is thought to be a major contributing factor to the underlying neurodegeneration. Healthy cells contain mechanisms for handling protein damage, the protein quality control, which must be impaired or inefficient to permit proteotoxicity under pathological conditions. We used a quantitative analysis of immunohistochemistry of the pons of eight patients with the polyglutamine disorder spinocerebellar ataxia type 3. We employed the anti-polyglutamine antibody 1C2, antibodies against p62 that is involved in delivering ubiquitinated protein aggregates to autophagosomes, antibodies against the chaperones HSPA1A and DNAJB1 and the proteasomal stress marker UBB⁺¹. The 1C2 antibody stained neuronal nuclear inclusions (NNIs), diffuse nuclear staining (DNS), granular cytoplasmic staining (GCS) and combinations, with reproducible distribution. P62 always co-localized with 1C2 in NNI. DNS and GCS co-stained with a lower frequency. UBB⁺¹ was present in a subset of neurones with NNI. A subset of UBB⁺¹-containing neurones displayed increased levels of HSPA1A, while DNAJB1 was sequestered into the NNI. Based on our results, we propose a model for the aggregation-associated pathology of spinocerebellar ataxia type 3: GCS and DNS aggregation likely represents early stages of pathology, which progresses towards formation of p62-positive NNI. A fraction of NNI exhibits UBB⁺¹ staining, implying proteasomal overload at a later stage. Subsequently, the stress-inducible HSPA1A is elevated while DNAJB1 is recruited into NNIs. This indicates that the stress response is only induced late when all endogenous protein quality control systems have failed. © 2011 The Authors. Neuropathology and Applied Neurobiology © 2011 British Neuropathological Society.

Split Nitrogen Application Improves Wheat Baking Quality by Influencing Protein Composition Rather Than Concentration.

PubMed

Xue, Cheng; Auf'm Erley, Gunda Schulte; Rossmann, Anne; Schuster, Ramona; Koehler, Peter; Mühling, Karl-Hermann

2016-01-01

The use of late nitrogen (N) fertilization (N application at late growth stages of wheat, e.g., booting, heading or anthesis) to improve baking quality of wheat has been questioned. Although it increases protein concentration, the beneficial effect on baking quality (bread loaf volume) needs to be clearly understood. Two pot experiments were conducted aiming to evaluate whether late N is effective under controlled conditions and if these effects result from increased N rate or N splitting. Late N fertilizers were applied either as additional N or split from the basal N at late boot stage or heading in the form of nitrate-N or urea. Results showed that late N fertilization improved loaf volume of wheat flour by increasing grain protein concentration and altering its composition. Increasing N rate mainly enhanced grain protein quantitatively. However, N splitting changed grain protein composition by enhancing the percentages of gliadins and glutenins as well as certain high molecular weight glutenin subunits (HMW-GS), which led to an improved baking quality of wheat flour. The late N effects were greater when applied as nitrate-N than urea. The proportions of glutenin and x-type HMW-GS were more important than the overall protein concentration in determining baking quality. N splitting is more effective in improving wheat quality than the increase in the N rate by late N, which offers the potential to cut down N fertilization rates in wheat production systems.

Split Nitrogen Application Improves Wheat Baking Quality by Influencing Protein Composition Rather Than Concentration

PubMed Central

Xue, Cheng; auf’m Erley, Gunda Schulte; Rossmann, Anne; Schuster, Ramona; Koehler, Peter; Mühling, Karl-Hermann

2016-01-01

The use of late nitrogen (N) fertilization (N application at late growth stages of wheat, e.g., booting, heading or anthesis) to improve baking quality of wheat has been questioned. Although it increases protein concentration, the beneficial effect on baking quality (bread loaf volume) needs to be clearly understood. Two pot experiments were conducted aiming to evaluate whether late N is effective under controlled conditions and if these effects result from increased N rate or N splitting. Late N fertilizers were applied either as additional N or split from the basal N at late boot stage or heading in the form of nitrate-N or urea. Results showed that late N fertilization improved loaf volume of wheat flour by increasing grain protein concentration and altering its composition. Increasing N rate mainly enhanced grain protein quantitatively. However, N splitting changed grain protein composition by enhancing the percentages of gliadins and glutenins as well as certain high molecular weight glutenin subunits (HMW-GS), which led to an improved baking quality of wheat flour. The late N effects were greater when applied as nitrate-N than urea. The proportions of glutenin and x-type HMW-GS were more important than the overall protein concentration in determining baking quality. N splitting is more effective in improving wheat quality than the increase in the N rate by late N, which offers the potential to cut down N fertilization rates in wheat production systems. PMID:27313585

Cmr1/WDR76 defines a nuclear genotoxic stress body linking genome integrity and protein quality control

PubMed Central

Gallina, Irene; Colding, Camilla; Henriksen, Peter; Beli, Petra; Nakamura, Kyosuke; Offman, Judith; Mathiasen, David P.; Silva, Sonia; Hoffmann, Eva; Groth, Anja; Choudhary, Chunaram; Lisby, Michael

2015-01-01

DNA replication stress is a source of genomic instability. Here we identify changed mutation rate 1 (Cmr1) as a factor involved in the response to DNA replication stress in Saccharomyces cerevisiae and show that Cmr1—together with Mrc1/Claspin, Pph3, the chaperonin containing TCP1 (CCT) and 25 other proteins—define a novel intranuclear quality control compartment (INQ) that sequesters misfolded, ubiquitylated and sumoylated proteins in response to genotoxic stress. The diversity of proteins that localize to INQ indicates that other biological processes such as cell cycle progression, chromatin and mitotic spindle organization may also be regulated through INQ. Similar to Cmr1, its human orthologue WDR76 responds to proteasome inhibition and DNA damage by relocalizing to nuclear foci and physically associating with CCT, suggesting an evolutionarily conserved biological function. We propose that Cmr1/WDR76 plays a role in the recovery from genotoxic stress through regulation of the turnover of sumoylated and phosphorylated proteins. PMID:25817432

A Community Standard Format for the Representation of Protein Affinity Reagents*

PubMed Central

Gloriam, David E.; Orchard, Sandra; Bertinetti, Daniela; Björling, Erik; Bongcam-Rudloff, Erik; Borrebaeck, Carl A. K.; Bourbeillon, Julie; Bradbury, Andrew R. M.; de Daruvar, Antoine; Dübel, Stefan; Frank, Ronald; Gibson, Toby J.; Gold, Larry; Haslam, Niall; Herberg, Friedrich W.; Hiltke, Tara; Hoheisel, Jörg D.; Kerrien, Samuel; Koegl, Manfred; Konthur, Zoltán; Korn, Bernhard; Landegren, Ulf; Montecchi-Palazzi, Luisa; Palcy, Sandrine; Rodriguez, Henry; Schweinsberg, Sonja; Sievert, Volker; Stoevesandt, Oda; Taussig, Michael J.; Ueffing, Marius; Uhlén, Mathias; van der Maarel, Silvère; Wingren, Christer; Woollard, Peter; Sherman, David J.; Hermjakob, Henning

2010-01-01

Protein affinity reagents (PARs), most commonly antibodies, are essential reagents for protein characterization in basic research, biotechnology, and diagnostics as well as the fastest growing class of therapeutics. Large numbers of PARs are available commercially; however, their quality is often uncertain. In addition, currently available PARs cover only a fraction of the human proteome, and their cost is prohibitive for proteome scale applications. This situation has triggered several initiatives involving large scale generation and validation of antibodies, for example the Swedish Human Protein Atlas and the German Antibody Factory. Antibodies targeting specific subproteomes are being pursued by members of Human Proteome Organisation (plasma and liver proteome projects) and the United States National Cancer Institute (cancer-associated antigens). ProteomeBinders, a European consortium, aims to set up a resource of consistently quality-controlled protein-binding reagents for the whole human proteome. An ultimate PAR database resource would allow consumers to visit one on-line warehouse and find all available affinity reagents from different providers together with documentation that facilitates easy comparison of their cost and quality. However, in contrast to, for example, nucleotide databases among which data are synchronized between the major data providers, current PAR producers, quality control centers, and commercial companies all use incompatible formats, hindering data exchange. Here we propose Proteomics Standards Initiative (PSI)-PAR as a global community standard format for the representation and exchange of protein affinity reagent data. The PSI-PAR format is maintained by the Human Proteome Organisation PSI and was developed within the context of ProteomeBinders by building on a mature proteomics standard format, PSI-molecular interaction, which is a widely accepted and established community standard for molecular interaction data. Further information and documentation are available on the PSI-PAR web site. PMID:19674966

Secreted Proteins Defy the Expression Level-Evolutionary Rate Anticorrelation.

PubMed

Feyertag, Felix; Berninsone, Patricia M; Alvarez-Ponce, David

2017-03-01

The rates of evolution of the proteins of any organism vary across orders of magnitude. A primary factor influencing rates of protein evolution is expression. A strong negative correlation between expression levels and evolutionary rates (the so-called E-R anticorrelation) has been observed in virtually all studied organisms. This effect is currently attributed to the abundance-dependent fitness costs of misfolding and unspecific protein-protein interactions, among other factors. Secreted proteins are folded in the endoplasmic reticulum, a compartment where chaperones, folding catalysts, and stringent quality control mechanisms promote their correct folding and may reduce the fitness costs of misfolding. In addition, confinement of secreted proteins to the extracellular space may reduce misinteractions and their deleterious effects. We hypothesize that each of these factors (the secretory pathway quality control and extracellular location) may reduce the strength of the E-R anticorrelation. Indeed, here we show that among human proteins that are secreted to the extracellular space, rates of evolution do not correlate with protein abundances. This trend is robust to controlling for several potentially confounding factors and is also observed when analyzing protein abundance data for 6 human tissues. In addition, analysis of mRNA abundance data for 32 human tissues shows that the E-R correlation is always less negative, and sometimes nonsignificant, in secreted proteins. Similar observations were made in Caenorhabditis elegans and in Escherichia coli, and to a lesser extent in Drosophila melanogaster, Saccharomyces cerevisiae and Arabidopsis thaliana. Our observations contribute to understand the causes of the E-R anticorrelation. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Advanced control of dissolved oxygen concentration in fed batch cultures during recombinant protein production.

PubMed

Kuprijanov, A; Gnoth, S; Simutis, R; Lübbert, A

2009-02-01

Design and experimental validation of advanced pO(2) controllers for fermentation processes operated in the fed-batch mode are described. In most situations, the presented controllers are able to keep the pO(2) in fermentations for recombinant protein productions exactly on the desired value. The controllers are based on the gain-scheduling approach to parameter-adaptive proportional-integral controllers. In order to cope with the most often appearing distortions, the basic gain-scheduling feedback controller was complemented with a feedforward control component. This feedforward/feedback controller significantly improved pO(2) control. By means of numerical simulations, the controller behavior was tested and its parameters were determined. Validation runs were performed with three Escherichia coli strains producing different recombinant proteins. It is finally shown that the new controller leads to significant improvements in the signal-to-noise ratio of other key process variables and, thus, to a higher process quality.

Label-free quantitative proteomics to investigate strawberry fruit proteome changes under controlled atmosphere and low temperature storage.

PubMed

Li, Li; Luo, Zisheng; Huang, Xinhong; Zhang, Lu; Zhao, Pengyu; Ma, Hongyuan; Li, Xihong; Ban, Zhaojun; Liu, Xia

2015-04-29

To elucidate the mechanisms contributing to fruit responses to senescence and stressful environmental stimuli under low temperature (LT) and controlled atmosphere (CA) storage, a label-free quantitative proteomic investigation was conducted in strawberry (Fragaria ananassa, Duch. cv. 'Akihime'). Postharvest physiological quality traits including firmness, total soluble solids, total acidity, ascorbic acid and volatile production were characterized following storage under different conditions. The observed post-storage protein expression profiles may be associated with delayed senescence features in strawberry. A total of 454 proteins were identified in differentially treated strawberry fruits. Quantitative analysis, using normalized spectral counts, revealed 73 proteins common to all treatments, which formed three clusters in a hierarchical clustering analysis. The proteins spanned a range of functions in various metabolic pathways and networks involved in carbohydrate and energy metabolism, volatile biosynthesis, phenylpropanoid activity, stress response and protein synthesis, degradation and folding. After CA and LT storage, 16 (13) and 11 (17) proteins, respectively, were significantly increased (decreased) in abundance, while expression profile of 12 proteins was significantly changed by both CA and LT. To summarize, the differential variability of abundance in strawberry proteome, working in a cooperative manner, provided an overview of the biological processes that occurred during CA and LT storage. Controlled atmosphere storage at an optimal temperature is regarded to be an effective postharvest technology to delay fruit senescence and maintain fruit quality during shelf life. Nonetheless, little information on fruit proteomic changes under controlled atmosphere and/or low temperature storage is available. The significance of this paper is that it is the first study employing a label-free approach in the investigation of strawberry fruit response to controlled atmosphere and cold storage. Changes in postharvest physiological quality traits including volatile production, firmness, ascorbic acid, soluble solids and total acidity were also characterized. Significant biological changes associated with senescence were revealed and differentially abundant proteins under various storage conditions were identified. Proteomic profiles were linked to physiological aspects of strawberry fruit senescence in order to provide new insights into possible regulation mechanisms. Findings from this study not only provide proteomic information on fruit regulation, but also pave the way for further quantitative studies at the transcriptomic and metabolomic levels. Copyright © 2015 Elsevier B.V. All rights reserved.

External Quality Control for Dried Blood Spot Based C-reactive Protein Assay: Experience from the Indonesia Family Life Survey and the Longitudinal Aging Study in India

PubMed Central

Hu, Peifeng; Herningtyas, Elizabeth H.; Kale, Varsha; Crimmins, Eileen M.; Risbud, Arun R.; McCreath, Heather; Lee, Jinkook; Strauss, John; O’Brien, Jennifer C.; Bloom, David E.; Seeman, Teresa E.

2015-01-01

Measurement of C-reactive protein, a marker of inflammation, in dried blood spots has been increasingly incorporated in community-based social surveys internationally. Although the dried blood spot based CRP assay protocol has been validated in the United States, it remains unclear whether laboratories in other less developed countries can generate C-reactive protein results of similar quality. We therefore conducted external quality monitoring for dried blood spot based C-reactive protein measurement for the Indonesia Family Life Survey and the Longitudinal Aging Study in India. Our results show that dried blood spot based C-reactive protein results in these two countries have excellent and consistent correlations with serum-based values and dried blood spot based results from the reference laboratory in the United States. Even though the results from duplicate samples may have fluctuations in absolute values over time, the relative order of C-reactive protein levels remains similar and the estimates are reasonably precise for population-based studies that investigate the association between socioeconomic factors and health. PMID:25879265

Rewiring protein synthesis: From natural to synthetic amino acids.

PubMed

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

2017-11-01

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

Proteomic Biomarkers for Acute Interstitial Lung Disease in Gefitinib-Treated Japanese Lung Cancer Patients

PubMed Central

Kawakami, Takao; Nagasaka, Keiko; Takami, Sachiko; Wada, Kazuya; Tu, Hsiao-Kun; Otsuji, Makiko; Kyono, Yutaka; Dobashi, Tae; Komatsu, Yasuhiko; Kihara, Makoto; Akimoto, Shingo; Peers, Ian S.; South, Marie C.; Higenbottam, Tim; Fukuoka, Masahiro; Nakata, Koichiro; Ohe, Yuichiro; Kudoh, Shoji; Clausen, Ib Groth; Nishimura, Toshihide; Marko-Varga, György; Kato, Harubumi

2011-01-01

Interstitial lung disease (ILD) events have been reported in Japanese non-small-cell lung cancer (NSCLC) patients receiving EGFR tyrosine kinase inhibitors. We investigated proteomic biomarkers for mechanistic insights and improved prediction of ILD. Blood plasma was collected from 43 gefitinib-treated NSCLC patients developing acute ILD (confirmed by blinded diagnostic review) and 123 randomly selected controls in a nested case-control study within a pharmacoepidemiological cohort study in Japan. We generated ∼7 million tandem mass spectrometry (MS/MS) measurements with extensive quality control and validation, producing one of the largest proteomic lung cancer datasets to date, incorporating rigorous study design, phenotype definition, and evaluation of sample processing. After alignment, scaling, and measurement batch adjustment, we identified 41 peptide peaks representing 29 proteins best predicting ILD. Multivariate peptide, protein, and pathway modeling achieved ILD prediction comparable to previously identified clinical variables; combining the two provided some improvement. The acute phase response pathway was strongly represented (17 of 29 proteins, p = 1.0×10−25), suggesting a key role with potential utility as a marker for increased risk of acute ILD events. Validation by Western blotting showed correlation for identified proteins, confirming that robust results can be generated from an MS/MS platform implementing strict quality control. PMID:21799770

Protein crystal growth in low gravity

NASA Technical Reports Server (NTRS)

Feigelson, Robert S.

1991-01-01

The objective of this research is to study the effect of low gravity on the growth of protein crystals and those parameters which will affect growth and crystal quality. The application of graphoepitaxy (artificial epitaxy) to proteins is detailed. The development of a method for the control of nucleation is discussed. The factor affecting the morphology of isocitrate lyase crystals is presented.

Deficiency of Suppressor Enhancer Lin12 1 Like (SEL1L) in Mice Leads to Systemic Endoplasmic Reticulum Stress and Embryonic Lethality*

PubMed Central

Francisco, Adam B.; Singh, Rajni; Li, Shuai; Vani, Anish K.; Yang, Liu; Munroe, Robert J.; Diaferia, Giuseppe; Cardano, Marina; Biunno, Ida; Qi, Ling; Schimenti, John C.; Long, Qiaoming

2010-01-01

Stress in the endoplasmic reticulum (ER) plays an important causal role in the pathogenesis of several chronic diseases such as Alzheimer, Parkinson, and diabetes mellitus. Insight into the genetic determinants responsible for ER homeostasis will greatly facilitate the development of therapeutic strategies for the treatment of these debilitating diseases. Suppressor enhancer Lin12 1 like (SEL1L) is an ER membrane protein and was thought to be involved in the quality control of secreted proteins. Here we show that the mice homozygous mutant for SEL1L were embryonic lethal. Electron microscopy studies revealed a severely dilated ER in the fetal liver of mutant embryos, indicative of alteration in ER homeostasis. Consistent with this, several ER stress responsive genes were significantly up-regulated in the mutant embryos. Mouse embryonic fibroblast cells deficient in SEL1L exhibited activated unfolded protein response at the basal state, impaired ER-associated protein degradation, and reduced protein secretion. Furthermore, markedly increased apoptosis was observed in the forebrain and dorsal root ganglions of mutant embryos. Taken together, our results demonstrate an essential role for SEL1L in protein quality control during mouse embryonic development. PMID:20197277

Deficiency of suppressor enhancer Lin12 1 like (SEL1L) in mice leads to systemic endoplasmic reticulum stress and embryonic lethality.

PubMed

Francisco, Adam B; Singh, Rajni; Li, Shuai; Vani, Anish K; Yang, Liu; Munroe, Robert J; Diaferia, Giuseppe; Cardano, Marina; Biunno, Ida; Qi, Ling; Schimenti, John C; Long, Qiaoming

2010-04-30

Stress in the endoplasmic reticulum (ER) plays an important causal role in the pathogenesis of several chronic diseases such as Alzheimer, Parkinson, and diabetes mellitus. Insight into the genetic determinants responsible for ER homeostasis will greatly facilitate the development of therapeutic strategies for the treatment of these debilitating diseases. Suppressor enhancer Lin12 1 like (SEL1L) is an ER membrane protein and was thought to be involved in the quality control of secreted proteins. Here we show that the mice homozygous mutant for SEL1L were embryonic lethal. Electron microscopy studies revealed a severely dilated ER in the fetal liver of mutant embryos, indicative of alteration in ER homeostasis. Consistent with this, several ER stress responsive genes were significantly up-regulated in the mutant embryos. Mouse embryonic fibroblast cells deficient in SEL1L exhibited activated unfolded protein response at the basal state, impaired ER-associated protein degradation, and reduced protein secretion. Furthermore, markedly increased apoptosis was observed in the forebrain and dorsal root ganglions of mutant embryos. Taken together, our results demonstrate an essential role for SEL1L in protein quality control during mouse embryonic development.

Altered mitochondrial quality control signaling in muscle of old gastric cancer patients with cachexia.

PubMed

Marzetti, Emanuele; Lorenzi, Maria; Landi, Francesco; Picca, Anna; Rosa, Fausto; Tanganelli, Fabiana; Galli, Marco; Doglietto, Giovanni Battista; Pacelli, Fabio; Cesari, Matteo; Bernabei, Roberto; Calvani, Riccardo; Bossola, Maurizio

2017-01-01

Mitochondrial dysfunction is involved in the loss of muscle featuring both aging and cancer cachexia (CC). Whether mitochondrial quality control (MQC) is altered in skeletal myocytes of old patients with CC is unclear. The present investigation therefore sought to preliminarily characterize MQC pathways in muscle of old gastric cancer patients with cachexia. The study followed a case-control cross-sectional design. Intraoperative biopsies of the rectus abdominis muscle were obtained from 18 patients with gastric adenocarcinoma (nine with CC and nine non-cachectic) and nine controls, and assayed for the expression of a set of MQC mediators. The mitofusin 2 expression was reduced in cancer patients compared with controls, independent of CC. Fission protein 1 was instead up-regulated in CC patients relative to the other groups. The mitophagy regulators PTEN-induced putative kinase 1 and Parkin were both down-regulated in cancer patients compared with controls. The ratio between the protein content of the lipidated and non-lipidated forms of microtubule-associated protein 1 light chain 3B was lower in CC patients relative to controls and non-cachectic cancer patients. Finally, the expression of autophagy-associated protein 7, lysosome-associated membrane protein 2, peroxisome proliferator-activated receptor-γ coactivator-1α, and mitochondrial transcription factor A was unvarying among groups. Collectively, our findings indicate that, in old patients with gastric cancer, cachexia is associated with derangements of the muscular MQC axis at several checkpoints: mitochondrial dynamics, mitochondrial tagging for disposal, and mitophagy signaling. Further investigations are needed to corroborate these preliminary findings and determine whether MQC pathways may become target for future interventions. Copyright © 2016 Elsevier Inc. All rights reserved.

Ubiquitin-proteasome pathway and cellular responses to oxidative stress

USDA-ARS?s Scientific Manuscript database

The ubiquitin-proteasome pathway (UPP) is the primary cytosolic proteolytic machinery for the selective degradation of various forms of damaged proteins. Thus, the UPP is an important protein quality control mechanism. In the canonical UPP, both ubiquitin and the 26S proteasome are involved. Subs...

Studies on effect of additives on protein profile, microstructure and quality characteristics of pasta.

PubMed

Purnima, C; Ramasarma, P R; Prabhasankar, P

2012-02-01

Wheat storage proteins play a vital role in pasta making quality. In the present study, SDS-PAGE, Gel filtration chromatography and Scanning electron microscopy techniques were employed to understand the changes in the wheat protein fractions and their interactions with additives namely Sodium Steroyl Lactate (SSL), Glycerol Monostearate (GMS) and Hydroxy Propyl Methyl Cellulose (HPMC) during processing of pasta. SDS-PAGE studies indicated changes in High Molecular Weight Glutenin (HMW) fractions during drying stages of pasta preparation and in cooked pasta samples. In uncooked pasta, gel filtration patterns showed four peaks corresponding to different storage proteins whereas in the case of cooked pasta, these peaks were merged into three peaks. Pasta quality characteristics studies indicated that pasta with HPMC was found to have minimum percentage of cooking loss (5.6%), increased cooked weight (82 g), firmness (2.97 N) and high overall quality score (27) than GMS, SSL and control. Microstructure studies confirm the beneficial effect of HPMC. The present study indicated that HPMC is better additive for pasta manufacture followed by GMS. This could be due to interaction of HPMC with starch and protein matrix is different from that of GMS and SSL.

The effect of transportation of broilers during summer on the expression of heat shock protein 70, postmortem metabolism and meat quality.

PubMed

Xing, T; Xu, X L; Zhou, G H; Wang, P; Jiang, N N

2015-01-01

The objective of this study was to determine the effects of different transport times on broilers during summer on stress, meat quality, and early postmortem muscle metabolites. Arbor Acres broiler chickens (n = 105) were randomly categorized into 5 treatments: unstressed control, 0.5 h, 1 h, 2 h, and 4 h transport. Each treatment consisted of 3 replicates with 7 birds each. All birds (except the control group) were transported according to a designed protocol. With the extension of transport time, the activities of plasma creatine kinase (CK) and lactate dehydrogenase (LDH) gradually increased. The content of heat shock protein 70 (Hsp70) did not change significantly during 0.5 h transport compared to the control group, but was significantly higher (P < 0.05) at 1 h or more of transport time. Also, transport times of 2 h or more resulted in a death rate of 20%-33% of broilers. We found that the breast meat in the 0.5 h transport group had significantly (P < 0.05) higher L* values, drip loss, cooking loss, AMP/ATP ratio, and phosphorylation of AMP-activated protein kinase (p-AMPK). In addition, pH24h was lower compared to the control group, increasing the likelihood of pale, soft, and exudative (PSE)-like meat. However, no significant variations were found in meat color, drip loss, or cooking loss in other transport groups compared to the control group under the condition of this study. Muscle glycogen content decreased with time of transportation. There were significant correlations among p-AMPK and meat quality (P < 0.05). These results indicate that preslaughter transport during summer may cause severe physiological and biochemical changes of broilers. Further investigations studying the deeper relationship between biological indicators and meat quality according to the similar transport conditions would provide a better understanding of the effect of transport duration on meat quality.

Storage study and quality evaluation of coconut protein powder.

PubMed

Naik, Aduja; Prakash, Maya; R, Ravi; Raghavarao, Ksms

2013-11-01

Coconut skim milk and insoluble protein are 2 major byproducts in the production of virgin coconut oil. Coconut skim milk was homogenized along with insoluble protein and spray dried to obtain a value-added product, namely, coconut protein powder (CPP). This study deals with the storage study of CPP under different conditions (refrigerated [control], ambient and accelerated). CPP samples were withdrawn periodically at designated intervals of 15 d for accelerated and control, and 30 d for ambient condition. CPP stored at different conditions exhibited marginal moisture uptake (by 0.74 % w/w for control, 0.76 % w/w for ambient, and 1.26 % w/w for accelerated condition) and as a result, had very little effect on the functional properties of the powder. Withdrawn CPP was tested for sensory quality aspects and subjected to instrumental analysis as well. Withdrawn CPP was incorporated as a milk substitute in dessert (Kheer). Quantitative descriptive analysis of the powder and product (Kheer) showed no significant difference in attributes of CPP during the storage period of 2 mo. Electronic nose analysis revealed that CPP samples were not much different with respect to aroma pattern matching, respectively. © 2013 CSIR-Central Food Technological Research Institute.

Regulation of protein turnover by heat shock proteins.

PubMed

Bozaykut, Perinur; Ozer, Nesrin Kartal; Karademir, Betul

2014-12-01

Protein turnover reflects the balance between synthesis and degradation of proteins, and it is a crucial process for the maintenance of the cellular protein pool. The folding of proteins, refolding of misfolded proteins, and also degradation of misfolded and damaged proteins are involved in the protein quality control (PQC) system. Correct protein folding and degradation are controlled by many different factors, one of the most important of which is the heat shock protein family. Heat shock proteins (HSPs) are in the class of molecular chaperones, which may prevent the inappropriate interaction of proteins and induce correct folding. On the other hand, these proteins play significant roles in the degradation pathways, including endoplasmic reticulum-associated degradation (ERAD), the ubiquitin-proteasome system, and autophagy. This review focuses on the emerging role of HSPs in the regulation of protein turnover; the effects of HSPs on the degradation machineries ERAD, autophagy, and proteasome; as well as the role of posttranslational modifications in the PQC system. Copyright © 2014 Elsevier Inc. All rights reserved.

Degradation of misfolded proteins in neurodegenerative diseases: therapeutic targets and strategies.

PubMed

Ciechanover, Aaron; Kwon, Yong Tae

2015-03-13

Mammalian cells remove misfolded proteins using various proteolytic systems, including the ubiquitin (Ub)-proteasome system (UPS), chaperone mediated autophagy (CMA) and macroautophagy. The majority of misfolded proteins are degraded by the UPS, in which Ub-conjugated substrates are deubiquitinated, unfolded and cleaved into small peptides when passing through the narrow chamber of the proteasome. The substrates that expose a specific degradation signal, the KFERQ sequence motif, can be delivered to and degraded in lysosomes via the CMA. Aggregation-prone substrates resistant to both the UPS and the CMA can be degraded by macroautophagy, in which cargoes are segregated into autophagosomes before degradation by lysosomal hydrolases. Although most misfolded and aggregated proteins in the human proteome can be degraded by cellular protein quality control, some native and mutant proteins prone to aggregation into β-sheet-enriched oligomers are resistant to all known proteolytic pathways and can thus grow into inclusion bodies or extracellular plaques. The accumulation of protease-resistant misfolded and aggregated proteins is a common mechanism underlying protein misfolding disorders, including neurodegenerative diseases such as Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD), prion diseases and Amyotrophic Lateral Sclerosis (ALS). In this review, we provide an overview of the proteolytic pathways in neurons, with an emphasis on the UPS, CMA and macroautophagy, and discuss the role of protein quality control in the degradation of pathogenic proteins in neurodegenerative diseases. Additionally, we examine existing putative therapeutic strategies to efficiently remove cytotoxic proteins from degenerating neurons.

Protein aggregation, cardiovascular diseases, and exercise training: Where do we stand?

PubMed

Gouveia, Marisol; Xia, Ke; Colón, Wilfredo; Vieira, Sandra I; Ribeiro, Fernando

2017-11-01

Cells ensure their protein quality control through the proteostasis network. Aging and age-related diseases, such as neurodegenerative and cardiovascular diseases, have been associated to the reduction of proteostasis network efficiency and, consequently, to the accumulation of protein misfolded aggregates. The decline in protein homeostasis has been associated with the development and progression of atherosclerotic cardiovascular disease, cardiac hypertrophy, cardiomyopathies, and heart failure. Exercise training is a key component of the management of patients with cardiovascular disease, consistently improving quality of life and prognosis. In this review, we give an overview on age-related protein aggregation, the role of the increase of misfolded protein aggregates on cardiovascular pathophysiology, and describe the beneficial or deleterious effects of the proteostasis network on the development of cardiovascular disease. We subsequently discuss how exercise training, a key lifestyle intervention in those with cardiovascular disease, could restore proteostasis and improve disease status. Copyright © 2017 Elsevier B.V. All rights reserved.

Accuracy of near infrared spectroscopy for prediction of chemical composition, salt content and free amino acids in dry-cured ham.

PubMed

Prevolnik, Maja; Škrlep, Martin; Janeš, Lucija; Velikonja-Bolta, Spela; Škorjanc, Dejan; Čandek-Potokar, Marjeta

2011-06-01

The capability of near infrared (NIR) spectroscopy was examined for the purposes of quality control of the traditional Slovenian dry-cured ham "Kraški pršut." Predictive models were developed for moisture, salt, protein, non-protein nitrogen, intramuscular fat and free amino acids in biceps femoris muscle (n = 135). The models' quality was assessed using statistical parameters: coefficient of determination (R(2)) and standard error (se) of cross-validation (CV) and external validation (EV). Residual predictive deviation (RPD) was also assessed. Best results were obtained for salt content and salt percentage in moisture/dry matter (R(CV)(2)>0.90, RPD>3.0), it was satisfactory for moisture, non-protein nitrogen, intramuscular fat and total free amino acids (R(CV)(2) = 0.75-0.90, RPD = 2.0-3.0), while not so for protein content and proteolysis index (R(CV)(2) = 0.65-0.75, RPD<2.0). Calibrations for individual free amino acids yielded R(CV)(2) from 0.40 to 0.90 and RPD from 1.3 to 2.9. Additional external validation of models on independent samples yielded comparable results. Based on the results, NIR spectroscopy can replace chemical methods in quality control of dry-cured ham. Copyright © 2011 Elsevier Ltd. All rights reserved.

Protein Biosynthesis and Maturation in the ER.

PubMed

Pedrazzini, Emanuela; Vitale, Alessandro

2018-01-01

The endoplasmic reticulum takes care of the folding, assembly, and quality control of thousands of proteins destined to the different compartments of the endomembrane system, or to be secreted in the apoplast. Here we describe how these early events in the life of all these proteins can be followed biochemically by using velocity or isopycnic ultracentrifugation, metabolic labeling with radioactive amino acids, and immunoprecipitation in various conditions.

Influence of source and quantity of protein on the development of immunity and resistance to African trypanosomiasis.

PubMed Central

Norton, J D; Yang, S P; Diffley, P

1986-01-01

Although it is well documented that severe protein deprivation inhibits the development of the immune response and exacerbates certain infections, little has been done to study the effects of native diets on endemic diseases or immunity. Therefore, protein-restricted diets were formulated for mice to mimic the sources and amounts measured in human diets of the Batouri region of Cameroon, endemic for African trypanosomiasis. Weanling C57BL/6 female mice were fed a diet that contained 73% of the recommended daily allowance (RDA) of protein. The sources of protein were all plant (cornmeal), all animal (casein), or a ratio that reflected the native diet (2.2 parts plant to 1 part animal protein). Diets were isocaloric on a weight basis, equal in lipids, and adequate in vitamins and minerals. Control mice were fed laboratory chow or two times the RDA of animal protein (casein). Mice fed only cornmeal or the native diets consumed as much food but did not gain as much weight as mice fed only animal protein, indicating the poorer quality of protein in their diets. Upon infection with Trypanosoma brucei gambiense, however, significantly higher numbers of these mice controlled the first peak of parasitemia and survived the infection as compared with mice fed the other three diets. Since all mice developed patent infections and the parasite growth rate was unaffected by diet, innate immune factors were ruled out as the cause for the higher level of resistance to the parasite. To determine whether diet affected the development of the immune system, weanling mice were maintained on diets for 30 days before immunization with sheep erythrocytes or trinitrophenylated Ficoll. Mice fed only plant protein or native diets elicited higher direct plaque-forming-cell responses to both the T-cell-dependent and T-cell-independent antigens. Since variant-specific immunity which controls levels of African trypanosomes in the blood is a T-cell-independent humoral immunoglobulin M response, this suggests that cornmeal, a protein of poor quality, was adequate for the development of humoral immunity and resistance to African trypanosomiasis while casein, an animal protein of high quality, was not. This provides more evidence that diet plays an important role in infection and immunity. PMID:3484725

The Role of the Multifunctional BAG3 Protein in Cellular Protein Quality Control and in Disease.

PubMed

Stürner, Elisabeth; Behl, Christian

2017-01-01

In neurons, but also in all other cells the complex proteostasis network is monitored and tightly regulated by the cellular protein quality control (PQC) system. Beyond folding of newly synthesized polypeptides and their refolding upon misfolding the PQC also manages the disposal of aberrant proteins either by the ubiquitin-proteasome machinery or by the autophagic-lysosomal system. Aggregated proteins are primarily degraded by a process termed selective macroautophagy (or aggrephagy). One such recently discovered selective macroautophagy pathway is mediated by the multifunctional HSP70 co-chaperone BAG3 ( BCL-2-associated athanogene 3 ). Under acute stress and during cellular aging, BAG3 in concert with the molecular chaperones HSP70 and HSPB8 as well as the ubiquitin receptor p62/SQSTM1 specifically targets aggregation-prone proteins to autophagic degradation. Thereby, BAG3-mediated selective macroautophagy represents a pivotal adaptive safeguarding and emergency system of the PQC which is activated under pathophysiological conditions to ensure cellular proteostasis. Interestingly, BAG3-mediated selective macroautophagy is also involved in the clearance of aggregated proteins associated with age-related neurodegenerative disorders, like Alzheimer's disease (tau-protein), Huntington's disease (mutated huntingtin/polyQ proteins), and amyotrophic lateral sclerosis (mutated SOD1). In addition, based on its initial description BAG3 is an anti-apoptotic protein that plays a decisive role in other widespread diseases, including cancer and myopathies. Therefore, in the search for novel therapeutic intervention avenues in neurodegeneration, myopathies and cancer BAG3 is a promising candidate.

Protein quality control at the inner nuclear membrane

PubMed Central

Khmelinskii, Anton; Blaszczak, Ewa; Pantazopoulou, Marina; Fischer, Bernd; Omnus, Deike J.; Le Dez, Gaëlle; Brossard, Audrey; Gunnarsson, Alexander; Barry, Joseph D.; Meurer, Matthias; Kirrmaier, Daniel; Boone, Charles; Huber, Wolfgang; Rabut, Gwenaël; Ljungdahl, Per O.; Knop, Michael

2015-01-01

The nuclear envelope is a double membrane that separates the nucleus from the cytoplasm. The inner nuclear membrane (INM) functions in essential nuclear processes including chromatin organization and regulation of gene expression1. The outer nuclear membrane is continuous with the endoplasmic reticulum (ER) and is the site of membrane protein synthesis. Protein homeostasis in this compartment is ensured by ER-associated protein degradation (ERAD) pathways that in yeast involve the integral membrane E3 ubiquitin ligases Hrd1 and Doa10 operating with the E2 ubiquitin-conjugating enzymes Ubc6 and Ubc72,3. However, little is known regarding protein quality control at the INM. Here we describe a protein degradation pathway at the INM mediated by the Asi complex consisting of the RING domain proteins Asi1 and Asi34. We report that the As complex functions together with the ubiquitin conjugating enzymes Ubc6andUbc7to degrade soluble and integral membrane proteins. Genetic evidence suggest that the Asi ubiquitin ligase defines a pathway distinct from but complementary to ERAD. Using unbiased screening with a novel genome-wide yeast library based on a tandem fluorescent protein timer (tFT)5, we identify more than 50 substrates of the Asi, Hrd1 and Doa10 E3 ubiquity ligases. We show that the Asi ubiquitin ligase is involved in degradation of mislocalised integral membrane proteins, thus acting to maintain and safeguard the identity of the INM. PMID:25519137

Differential proteomic analysis to identify proteins associated with quality traits of frozen mud shrimp (Solenocera melantho) using an iTRAQ-based strategy.

PubMed

Shi, Jing; Zhang, Longteng; Lei, Yutian; Shen, Huixing; Yu, Xunpei; Luo, Yongkang

2018-06-15

An iTRAQ-based strategy was applied to investigate proteome changes in mud shrimp during long-term frozen storage under different conditions. A total of 226 proteins was identified as differential abundance proteins (DAPs) in mud shrimp from two frozen treatment groups (-20 °C and -40 °C) compared with the fresh control group. The proteome changes in mud shrimp muscle stored under -20 °C was much greater than that under -40 °C. Correlation analysis between DAPs and quality traits of mud shrimp muscle showed that 12 proteins were correlated closely with color (L ∗ , a ∗ , and b ∗ value) and texture (hardness, elasticity, and chewiness). Bioinformatic analysis revealed that most of these proteins were involved in protein structure, metabolic enzymes, and protein turnover. Among them, several proteins might be potential protein markers for color, and some proteins are good candidate predictors for textural properties of mud shrimp muscle. Copyright © 2018 Elsevier Ltd. All rights reserved.

Macromolecular crystallization in microgravity generated by a superconducting magnet.

PubMed

Wakayama, N I; Yin, D C; Harata, K; Kiyoshi, T; Fujiwara, M; Tanimoto, Y

2006-09-01

About 30% of the protein crystals grown in space yield better X-ray diffraction data than the best crystals grown on the earth. The microgravity environments provided by the application of an upward magnetic force constitute excellent candidates for simulating the microgravity conditions in space. Here, we describe a method to control effective gravity and formation of protein crystals in various levels of effective gravity. Since 2002, the stable and long-time durable microgravity generated by a convenient type of superconducting magnet has been available for protein crystal growth. For the first time, protein crystals, orthorhombic lysozyme, were grown at microgravity on the earth, and it was proved that this microgravity improved the crystal quality effectively and reproducibly. The present method always accompanies a strong magnetic field, and the magnetic field itself seems to improve crystal quality. Microgravity is not always effective for improving crystal quality. When we applied this microgravity to the formation of cubic porcine insulin and tetragonal lysozyme crystals, we observed no dependence of effective gravity on crystal quality. Thus, this kind of test will be useful for selecting promising proteins prior to the space experiments. Finally, the microgravity generated by the magnet is compared with that in space, considering the cost, the quality of microgravity, experimental convenience, etc., and the future use of this microgravity for macromolecular crystal growth is discussed.

Influence of transport conditions and pre-slaughter water shower spray during summer on protein characteristics and water distribution of broiler breast meat.

PubMed

Xing, Tong; Li, Yun Han; Li, Ming; Jiang, Nan Nan; Xu, Xing Lian; Zhou, Guang Hong

2016-11-01

This study investigated the effects of pre-slaughter transport during summer and subsequent water shower spray on broiler meat quality and protein characteristics. Arbor Acres broiler chickens (n = 126, 42 days old, mixed sex, 2.5-3 kg) were randomly categorized into three treatments: (i) control group without transport (C); (ii) 30 min transport (T); and (iii) 30 min transport followed by 10 min water shower spray and 20 min lairage (T/W). Each treatment consisted of six replicates with seven birds each. Ambient temperature was 32-35°C during transportation. Results indicated that transport during high ambient temperature denatured myosin and sarcoplasmic proteins, led to decreased protein solubility and resulted in glycogen phosphorylase precipitated to the myofibrillar fraction. Furthermore, meat quality in the transport group showed a pale, soft and exudative (PSE)-like syndrome. Water shower spray during lairage after transport reduced the degree of protein denaturation and lessened the deterioration of meat quality. © 2016 Japanese Society of Animal Science.

Mechanisms and kinetics of cellulose fermentation for protein production

NASA Technical Reports Server (NTRS)

Dunlap, C. A.

1971-01-01

The development of a process (and ancillary processing and analytical techniques) to produce bacterial single-cell protein of good nutritional quality from waste cellulose is discussed. A fermentation pilot plant and laboratory were developed and have been in operation for about two years. Single-cell protein (SCP) can be produced from sugarcane bagasse--a typical agricultural cellulosic waste. The optimization and understanding of this process and its controlling variables are examined. Both batch and continuous fermentation runs have been made under controlled conditions in the 535 liter pilot plant vessel and in the laboratory 14-liter fermenters.

Assembly and Function of Heterotypic Ubiquitin Chains in Cell-Cycle and Protein Quality Control.

PubMed

Yau, Richard G; Doerner, Kerstin; Castellanos, Erick R; Haakonsen, Diane L; Werner, Achim; Wang, Nan; Yang, X William; Martinez-Martin, Nadia; Matsumoto, Marissa L; Dixit, Vishva M; Rape, Michael

2017-11-02

Posttranslational modification with ubiquitin chains controls cell fate in all eukaryotes. Depending on the connectivity between subunits, different ubiquitin chain types trigger distinct outputs, as seen with K48- and K63-linked conjugates that drive protein degradation or complex assembly, respectively. Recent biochemical analyses also suggested roles for mixed or branched ubiquitin chains, yet without a method to monitor endogenous conjugates, the physiological significance of heterotypic polymers remained poorly understood. Here, we engineered a bispecific antibody to detect K11/K48-linked chains and identified mitotic regulators, misfolded nascent polypeptides, and pathological Huntingtin variants as their endogenous substrates. We show that K11/K48-linked chains are synthesized and processed by essential ubiquitin ligases and effectors that are mutated across neurodegenerative diseases; accordingly, these conjugates promote rapid proteasomal clearance of aggregation-prone proteins. By revealing key roles of K11/K48-linked chains in cell-cycle and quality control, we establish heterotypic ubiquitin conjugates as important carriers of biological information. Copyright © 2017 Elsevier Inc. All rights reserved.

Non-destructive monitoring of mouse embryo development and its qualitative evaluation at the molecular level using Raman spectroscopy

NASA Astrophysics Data System (ADS)

Ishigaki, Mika; Hashimoto, Kosuke; Sato, Hidetoshi; Ozaki, Yukihiro

2017-03-01

Current research focuses on embryonic development and quality not only by considering fundamental biology, but also by aiming to improve assisted reproduction technologies, such as in vitro fertilization. In this study, we explored the development of mouse embryo and its quality based on molecular information, obtained nondestructively using Raman spectroscopy. The detailed analysis of Raman spectra measured in situ during embryonic development revealed a temporary increase in protein content after fertilization. Proteins with a β-sheet structure—present in the early stages of embryonic development—are derived from maternal oocytes, while α-helical proteins are additionally generated by switching on a gene after fertilization. The transition from maternal to embryonic control during development can be non-destructively profiled, thus facilitating the in situ assessment of structural changes and component variation in proteins generated by metabolic activity. Furthermore, it was indicated that embryos with low-grade morphology had high concentrations of lipids and hydroxyapatite. This technique could be used for embryo quality testing in the future.

Regulation of RNA-binding proteins affinity to export receptors enables the nuclear basket proteins to distinguish and retain aberrant mRNAs

PubMed Central

Soheilypour, M.; Mofrad, M. R. K.

2016-01-01

Export of messenger ribonucleic acids (mRNAs) into the cytoplasm is a fundamental step in gene regulation processes, which is meticulously quality controlled by highly efficient mechanisms in eukaryotic cells. Yet, it remains unclear how the aberrant mRNAs are recognized and retained inside the nucleus. Using a new modelling approach for complex systems, namely the agent-based modelling (ABM) approach, we develop a minimal model of the mRNA quality control (QC) mechanism. Our results demonstrate that regulation of the affinity of RNA-binding proteins (RBPs) to export receptors along with the weak interaction between the nuclear basket protein (Mlp1 or Tpr) and RBPs are the minimum requirements to distinguish and retain aberrant mRNAs. Our results show that the affinity between Tpr and RBPs is optimized to maximize the retention of aberrant mRNAs. In addition, we demonstrate how the length of mRNA affects the QC process. Since longer mRNAs spend more time in the nuclear basket to form a compact conformation and initiate their export, nuclear basket proteins could more easily capture and retain them inside the nucleus. PMID:27805000

Protein Crystal Movements and Fluid Flows During Microgravity Growth

NASA Technical Reports Server (NTRS)

Boggon, Titus J.; Chayen, Naomi E.; Snell, Edward H.; Dong, Jun; Lautenschlager, Peter; Potthast, Lothar; Siddons, D. Peter; Stojanoff, Vivian; Gordon, Elspeth; Thompson, Andrew W.;

Regulation of RNA-binding proteins affinity to export receptors enables the nuclear basket proteins to distinguish and retain aberrant mRNAs.

PubMed

Soheilypour, M; Mofrad, M R K

2016-11-02

Export of messenger ribonucleic acids (mRNAs) into the cytoplasm is a fundamental step in gene regulation processes, which is meticulously quality controlled by highly efficient mechanisms in eukaryotic cells. Yet, it remains unclear how the aberrant mRNAs are recognized and retained inside the nucleus. Using a new modelling approach for complex systems, namely the agent-based modelling (ABM) approach, we develop a minimal model of the mRNA quality control (QC) mechanism. Our results demonstrate that regulation of the affinity of RNA-binding proteins (RBPs) to export receptors along with the weak interaction between the nuclear basket protein (Mlp1 or Tpr) and RBPs are the minimum requirements to distinguish and retain aberrant mRNAs. Our results show that the affinity between Tpr and RBPs is optimized to maximize the retention of aberrant mRNAs. In addition, we demonstrate how the length of mRNA affects the QC process. Since longer mRNAs spend more time in the nuclear basket to form a compact conformation and initiate their export, nuclear basket proteins could more easily capture and retain them inside the nucleus.

The E3 ubiquitin ligase and RNA-binding protein ZNF598 orchestrates ribosome quality control of premature polyadenylated mRNAs

PubMed Central

Garzia, Aitor; Jafarnejad, Seyed Mehdi; Meyer, Cindy; Chapat, Clément; Gogakos, Tasos; Morozov, Pavel; Amiri, Mehdi; Shapiro, Maayan; Molina, Henrik; Tuschl, Thomas; Sonenberg, Nahum

2017-01-01

Cryptic polyadenylation within coding sequences (CDS) triggers ribosome-associated quality control (RQC), followed by degradation of the aberrant mRNA and polypeptide, ribosome disassembly and recycling. Although ribosomal subunit dissociation and nascent peptide degradation are well-understood, the molecular sensors of aberrant mRNAs and their mechanism of action remain unknown. We studied the Zinc Finger Protein 598 (ZNF598) using PAR-CLIP and revealed that it cross-links to tRNAs, mRNAs and rRNAs, thereby placing the protein on translating ribosomes. Cross-linked reads originating from AAA-decoding tRNALys(UUU) were 10-fold enriched over its cellular abundance, and poly-lysine encoded by poly(AAA) induced RQC in a ZNF598-dependent manner. Encounter with translated polyA segments by ZNF598 triggered ubiquitination of several ribosomal proteins, requiring the E2 ubiquitin ligase UBE2D3 to initiate RQC. Considering that human CDS are devoid of >4 consecutive AAA codons, sensing of prematurely placed polyA tails by a specialized RNA-binding protein is a novel nucleic-acid-based surveillance mechanism of RQC. PMID:28685749

The E3 ubiquitin ligase and RNA-binding protein ZNF598 orchestrates ribosome quality control of premature polyadenylated mRNAs.

PubMed

Garzia, Aitor; Jafarnejad, Seyed Mehdi; Meyer, Cindy; Chapat, Clément; Gogakos, Tasos; Morozov, Pavel; Amiri, Mehdi; Shapiro, Maayan; Molina, Henrik; Tuschl, Thomas; Sonenberg, Nahum

2017-07-07

Cryptic polyadenylation within coding sequences (CDS) triggers ribosome-associated quality control (RQC), followed by degradation of the aberrant mRNA and polypeptide, ribosome disassembly and recycling. Although ribosomal subunit dissociation and nascent peptide degradation are well-understood, the molecular sensors of aberrant mRNAs and their mechanism of action remain unknown. We studied the Zinc Finger Protein 598 (ZNF598) using PAR-CLIP and revealed that it cross-links to tRNAs, mRNAs and rRNAs, thereby placing the protein on translating ribosomes. Cross-linked reads originating from AAA-decoding tRNA Lys (UUU) were 10-fold enriched over its cellular abundance, and poly-lysine encoded by poly(AAA) induced RQC in a ZNF598-dependent manner. Encounter with translated polyA segments by ZNF598 triggered ubiquitination of several ribosomal proteins, requiring the E2 ubiquitin ligase UBE2D3 to initiate RQC. Considering that human CDS are devoid of >4 consecutive AAA codons, sensing of prematurely placed polyA tails by a specialized RNA-binding protein is a novel nucleic-acid-based surveillance mechanism of RQC.

Molecular weight distribution of flour proteins in intermediate wheatgrass (Thinopyrum intermedium): Impact on end-use quality parameters

USDA-ARS?s Scientific Manuscript database

Thinopyrum intermedium, commonly known as intermediate wheatgrass (IWG) is a perennial crop shown to have both environmental and nutritional benefits. We have previously shown that in comparison to wheat controls, IWG lines had higher protein and dietary fiber contents. However, a deficiency in hi...

Quality and storage stability of extruded puffed corn-fish snacks during 6-month storage at ambient temperature.

PubMed

Shaviklo, Gholam Reza; Thorkelsson, Gudjon; Rafipour, Fereidon; Sigurgisladottir, Sjofn

2011-03-30

Cereal-based snacks are usually low in protein and other nutrients. Increased health awareness of consumers has led the food industry to develop fortified snacks with functional ingredients. Three types of extruded corn-fish snacks, containing 150 g kg(-1) carp mince and 150 g kg(-1) trout mince, 30 g kg(-1) freeze-dried saithe protein and a regular corn snack (control). were produced to study quality changes and storage stability of the products during 6-month storage at 27±2 °C. All products had the same level of water activity and proximate composition except for protein. Fortified snacks had a protein content of 93-98 g kg(-1) , compared with 65 g kg(-1) in the control. A significant increase was observed for peroxide value during storage (0.0 to 2.8 meq kg(-1)). Scores for attributes describing oxidation and off odors and flavors increased after 5-6 months' storage but attributes describing puffed corn snack odor and flavor did not change during storage of any of the products. Extrusion of corn grits with fish flesh/fish protein can be used to produce high-protein products that would be an option to provide nutrient snacks for consumers and to increase fish consumption. Copyright © 2011 Society of Chemical Industry.

Reviving the protein quality control system: therapeutic target for cardiac disease in the elderly.

PubMed

Meijering, Roelien A M; Henning, Robert H; Brundel, Bianca J J M

2015-04-01

It has been firmly established that ageing constitutes a principal risk factor for cardiac disease. Currently, the underlying mechanisms of ageing that contribute to the initiation or acceleration of cardiac disease are essentially unresolved. Prevailing theories of ageing center on the loss of cellular protein homeostasis, by either design (genetically) or "wear and tear" (environmentally). Either or both ways, the normal protein homeostasis in the cell is affected, resulting in aberrant and misfolded proteins. Should such misfolded proteins escape the protein quality control (PQC) system, they become proteotoxic and accelerate the loss of cellular integrity. Impairment of PQC plays a prominent role in the pathophysiology of ageing-related neurodegenerative disorders such as Parkinson's, Huntington׳s, and Alzheimer׳s disease. The concept of an impaired PQC driving ageing-related diseases has recently been expanded to cardiac diseases, including atrial fibrillation, cardiac hypertrophy, and cardiomyopathy. In this review, we provide a brief overview of the PQC system in relation to ageing and discuss the emerging concept of the loss of PQC in cardiomyocytes as a trigger for cardiac disease. Finally, we discuss the potential of boosting the PQC system as an innovative therapeutic target to treat cardiac disease in the elderly. Copyright © 2015 Elsevier Inc. All rights reserved.

Comparison of the effects of dietary supplementation of flavonoids on laying hen performance, egg quality and egg nutrient profile.

PubMed

Iskender, H; Yenice, G; Dokumacioglu, E; Kaynar, O; Hayirli, A; Kaya, A

2017-10-01

1. The aim of this experiment was to compare the effects of dietary supplementation of hesperidin, naringin and quercetin on laying hen performance, egg quality and egg yolk lipid and protein profiles. 2. A total of 96 Lohmann White laying hens weighing an average of 1500 g at 28 weeks of age were randomly assigned to a basal diet and the basal diet supplemented (0.5 g/kg) with either hesperidin, naringin or quercetin. Each treatment was replicated in 6 cages in an 8-week experimental period. Data were analysed using one-way analysis of variance. 3. None of the dietary flavonoids affected laying performance and eggshell quality. Hesperidin and quercetin supplementations decreased albumen and yolk indexes. 4. As compared to the control group, egg yolk cholesterol content decreased and egg yolk protein content increased in response to dietary hesperidin and quercetin supplementation. The mean egg yolk cholesterol (mg/g) and protein (g/100 g) contents were 10.08/14.28, 16.12/14.08, 14.75/15.04 and 15.15/14.85 for the control group and groups supplemented with naringin, hesperidin and quercetin, respectively. 5. Egg yolk lipid and protein profiles were variable. 6. In conclusion, dietary supplementation of hesperidin or quercetin could be used in the diets during the early laying period to reduce egg yolk cholesterol and increase egg yolk protein, which may be attractive to consumers.

Lysosome and endoplasmic reticulum quality control pathways in Niemann-Pick type C disease

PubMed Central

Schultz, Mark L.; Krus, Kelsey L.; Lieberman, Andrew P.

2017-01-01

Lysosomal storage diseases result from inherited deficiencies of lysosomal hydrolytic activities or lipid transport. Collectively, these disorders are a common cause of morbidity in the pediatric population and are often associated with severe neurodegeneration. Among this group of diseases is Niemann-Pick type C, an autosomal recessive disorder of lipid trafficking that causes cognitive impairment, ataxia and death, most often in childhood. Here, we review the current knowledge of disease pathogenesis, with particular focus on insights gleaned from genetics and the study of model systems. Critical advances in understanding mechanisms that regulate intracellular cholesterol trafficking have emerged from this work and are highlighted. We review effects of disease-causing mutations on quality control pathways involving the lysosome and endoplasmic reticulum, and discuss how they function to clear the most common mutant protein found in Niemann-Pick type C patients, NPC1-I1061T. Finally, we summarize insights into the mechanisms that degrade misfolded transmembrane proteins in the endoplasmic reticulum and how manipulating these quality control pathways may lead to the identification of novel targets for disease-modifying therapies. PMID:27026653

Regulation of SOBIR1 accumulation and activation of defense responses in bir1-1 by specific components of ER quality control.

PubMed

Sun, Tongjun; Zhang, Qian; Gao, Minghui; Zhang, Yuelin

2014-03-01

Receptor-like kinases play diverse roles in plant biology. Arabidopsis BAK1-INTERACTING RECEPTOR-LIKE KINASE 1 (BIR1) functions as a negative regulator of plant immunity. bir1-1 mutant plants display spontaneous cell death and constitutive defense responses that are dependent on SUPPRESSOR OF BIR1,1 (SOBIR1) and PHYTOALEXIN DEFICIENT4 (PAD4). Here we report that mutations in three components of ER quality control, CALRETICULIN3 (CRT3), ER-LOCALIZED DnaJ-LIKE PROTEIN 3b (ERdj3b) and STROMAL-DERIVED FACTOR-2 (SDF2), also suppress the spontaneous cell death and constitutive defense responses in bir1-1. Further analysis revealed that accumulation of the SOBIR1 protein is reduced in crt3-1 and erdj3b-1 mutant plants. These data suggest that ER quality control plays important roles in the biogenesis of SOBIR1, and is required for cell death and defense responses in bir1-1. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

The genetic control of milling yield, dough rheology and baking quality of wheat.

PubMed

Kuchel, H; Langridge, P; Mosionek, L; Williams, K; Jefferies, S P

2006-05-01

Improving the end-use quality of wheat is a key target for many breeding programmes. With the exception of the relationship between glutenin alleles and some dough rheological characters, knowledge concerning the genetic control of wheat quality traits is somewhat limited. A doubled haploid population produced from a cross between two Australian cultivars 'Trident' and 'Molineux' has been used to construct a linkage map based largely on microsatellite molecular makers. 'Molineux' is superior to 'Trident' for a number of milling, dough rheology and baking quality characteristics, although by international standards 'Trident' would still be regarded as possessing moderately good end-use quality. This population was therefore deemed useful for investigation of wheat end-use quality. A number of significant QTL identified for dough rheological traits mapped to HMW and LMW glutenin loci on chromosomes 1A and 1B. However, QTL associated with dough strength and loaf volume were also identified on chromosome 2A and a significant QTL associated with loaf volume and crumb quality was identified on chromosome 3A. A QTL for flour protein content and milling yield was identified on chromosome 6A and a QTL associated with flour colour reported previously on chromosome 7B was confirmed in this population. The detection of loci affecting dough strength, loaf volume and flour protein content may provide fresh opportunities for the application of marker-assisted selection to improve bread-making quality.

Recent Advances in Understanding the Control of Secretory Proteins by the Unfolded Protein Response in Plants

PubMed Central

Hayashi, Shimpei; Wakasa, Yuhya; Takaiwa, Fumio

2013-01-01

The membrane transport system is built on the proper functioning of the endoplasmic reticulum (ER). The accumulation of unfolded proteins in the ER lumen (ER stress) disrupts ER homeostasis and disturbs the transport system. In response to ER stress, eukaryotic cells activate intracellular signaling (named the unfolded protein response, UPR), which contributes to the quality control of secretory proteins. On the other hand, the deleterious effects of UPR on plant health and growth characteristics have frequently been overlooked, due to limited information on this mechanism. However, recent studies have shed light on the molecular mechanism of plant UPR, and a number of its unique characteristics have been elucidated. This study briefly reviews the progress of understanding what is happening in plants under ER stress conditions. PMID:23629671

Cln6 mutants associated with neuronal ceroid lipofuscinosis are degraded in a proteasome-dependent manner.

PubMed

Oresic, Kristina; Mueller, Britta; Tortorella, Domenico

2009-06-01

NCLs (neuronal ceroid lipofuscinoses), a group of inherited neurodegenerative lysosomal storage diseases that predominantly affect children, are the result of autosomal recessive mutations within one of the nine cln genes. The wild-type cln gene products are composed of membrane and soluble proteins that localize to the lysosome or the ER (endoplasmic reticulum). However, the destiny of the Cln variants has not been fully characterized. To explore a possible link between ER quality control and processing of Cln mutants, we investigated the fate of two NCL-related Cln6 mutants found in patient samples (Cln6(G123D) and Cln6(M241T)) in neuronal-derived human cells. The point mutations are predicted to be in the putative transmembrane domains and most probably generate misfolded membrane proteins that are subjected to ER quality control. Consistent with this paradigm, both mutants underwent rapid proteasome-mediated degradation and complexed with components of the ER extraction apparatus, Derlin-1 and p97. In addition, knockdown of SEL1L [sel-1 suppressor of lin-12-like (Caenorhabditis elegans)], a member of an E3 ubiquitin ligase complex involved in ER protein extraction, rescued significant amounts of Cln6(G123D) and Cln6(M241T) polypeptides. The results implicate ER quality control in the instability of the Cln variants that probably contributes to the development of NCL.

Nonsense-Mediated Decay in Genetic Disease: Friend or Foe?

PubMed Central

Miller, Jake N.; Pearce, David A.

2014-01-01

Eukaryotic cells utilize various RNA quality control mechanisms to ensure high fidelity of gene expression, thus protecting against the accumulation of nonfunctional RNA and the subsequent production of abnormal peptides. Messenger RNAs (mRNAs) are largely responsible for protein production, and mRNA quality control is particularly important for protecting the cell against the downstream effects of genetic mutations. Nonsense-mediated decay (NMD) is an evolutionarily conserved mRNA quality control system in all eukaryotes that degrades transcripts containing premature termination codons (PTCs). By degrading these aberrant transcripts, NMD acts to prevent the production of truncated proteins that could otherwise harm the cell through various insults, such as dominant negative effects or the ER stress response. Although NMD functions to protect the cell against the deleterious effects of aberrant mRNA, there is a growing body of evidence that mutation-, codon-, gene-, cell-, and tissue-specific differences in NMD efficiency can alter the underlying pathology of genetic disease. In addition, the protective role that NMD plays in genetic disease can undermine current therapeutic strategies aimed at increasing the production of full-length functional protein from genes harboring nonsense mutations. Here, we review the normal function of this RNA surveillance pathway and how it is regulated, provide current evidence for the role that it plays in modulating genetic disease phenotypes, and how NMD can be used as a therapeutic target. PMID:25485595

Effects of different source additives and wilt conditions on the pH value, aerobic stability, and carbohydrate and protein fractions of alfalfa silage.

PubMed

Tao, Lian; Zhou, He; Zhang, Naifeng; Si, Bingwen; Tu, Yan; Ma, Tao; Diao, Qiyu

2017-01-01

To improve the silage quality and reduce the silage additive cost, the present experiment was designed to evaluate the potential of applying the fermented juice of epiphytic lactic acid bacteria (FJLB) as an additive in alfalfa silage. The effects of FJLB on the fermentation quality, carbohydrate and protein fractions, and aerobic stability of alfalfa silage wilted under five different conditions were investigated and compared with commercial lactic acid bacteria (CLAB) and the control. The FJLB application decreased the pH value, the volatile fatty acids and non-protein nitrogen content, and the loss of sugar by 9.9%, 22.9%, 19.6% and 9.6%, respectively; it increased the lactic acid concentration by 29.5% and the aerobic stability by 17 h in comparison to the control. The FJLB application also decreased the pH value (4.44 vs. 4.66) and volatile fatty acid content (38.32 vs. 44.82) and increased the lactic acid concentration (68.99 vs. 63.29) in comparison to the CLAB-treated silage. However, the FJLB treatment had lower aerobic stability (254 h vs. 274 h) than the CLAB treatment. The FJLB application improved silage quality in comparison to the control; in addition, its effect as a fermentation stimulant may be comparable to or even better than CLAB. © 2016 Japanese Society of Animal Science.

Co-production of GroELS discriminates between intrinsic and thermally-induced recombinant protein aggregation during substrate quality control

PubMed Central

2011-01-01

Background The effects and effectiveness of the chaperone pair GroELS on the yield and quality of recombinant polypeptides produced in Escherichia coli are matter of controversy, as the reported activities of this complex are not always consistent and eventually indicate undesired side effects. The divergence in the reported data could be due, at least partially, to different experimental conditions in independent research approaches. Results We have then selected two structurally different model proteins (namely GFP and E. coli β-galactosidase) and two derived aggregation-prone fusions to explore, in a systematic way, the eventual effects of GroELS co-production on yield, solubility and conformational quality. Host cells were cultured at two alternative temperatures below the threshold at which thermal stress is expected to be triggered, to minimize the involvement of independent stress factors. Conclusions From the analysis of protein yield, solubility and biological activity of the four model proteins produced alone or along the chaperones, we conclude that GroELS impacts on yield and quality of aggregation-prone proteins with intrinsic determinants but not on thermally induced protein aggregation. No effective modifications of protein solubility have been observed, but significant stabilization of small (encapsulable) substrates and moderate chaperone-induced degradation of larger (excluded) polypeptides. These findings indicate that the activities of this chaperone pair in the context of actively producing recombinant bacteria discriminate between intrinsic and thermally-induced protein aggregation, and that the side effects of GroELS overproduction might be determined by substrate size. PMID:21992454

Multicapillary SDS-gel electrophoresis for the analysis of fluorescently labeled mAb preparations: a high throughput quality control process for the production of QuantiPlasma and PlasmaScan mAb libraries.

PubMed

Székely, Andrea; Szekrényes, Akos; Kerékgyártó, Márta; Balogh, Attila; Kádas, János; Lázár, József; Guttman, András; Kurucz, István; Takács, László

2014-08-01

Molecular heterogeneity of mAb preparations is the result of various co- and post-translational modifications and to contaminants related to the production process. Changes in molecular composition results in alterations of functional performance, therefore quality control and validation of therapeutic or diagnostic protein products is essential. A special case is the consistent production of mAb libraries (QuantiPlasma™ and PlasmaScan™) for proteome profiling, quality control of which represents a challenge because of high number of mAbs (>1000). Here, we devise a generally applicable multicapillary SDS-gel electrophoresis process for the analysis of fluorescently labeled mAb preparations for the high throughput quality control of mAbs of the QuantiPlasma™ and PlasmaScan™ libraries. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Nutritional evaluation of phosphorylated pumpkin seed (Cucurbita moschata) protein concentrate in silver catfish Rhamdia quelen (Quoy and Gaimard, 1824).

PubMed

Lovatto, Naglezi de Menezes; Goulart, Fernanda Rodrigues; de Freitas, Silvandro Tonetto; Mombach, Patricia Inês; Loureiro, Bruno Bianch; Bender, Ana Betine Beutinger; Boligon, Aline Augusti; Radünz Neto, João; da Silva, Leila Picolli

2015-12-01

An 8-week feeding trial was conducted to evaluate the effect of replacing fish meal with pumpkin seed meal (PSM) or phosphorylated protein concentrate of pumpkin seed meal (PPCPS) on growth and metabolic responses of silver catfish. Five isonitrogenous and isocaloric diets were formulated. Control diet contained fish meal as the main protein source. The treatment groups contained 25 and 50% of either PSM or PPCPS protein replaced the fishmeal protein. A total of 400 silver catfish, with initial mean weight of 24 ± 0.46 g, were distributed into 20 tanks. For data four orthogonal contrasts were applied: control diet versus PSM diets; control diets versus PPCPS diets; control versus other diets; PSM diets versus PPCPS diets. The results indicated that the fish fed PSM diets had lower weight gain when compared to either control diet or PPCPS. The PPCPS do not affect growth and protein efficiency ratio. Lower albumin contents were found for the control diet fish for the contrasts control diet versus PPCPS diet and control diet versus other diets. The hepatic ALAT enzyme activity was higher in the fish fed the control diet (P < 0.05). The hepatic ALP was most active in fish that received the PPCPS diets, when comparing control diet versus PPCPS diets and control diet versus other diets. The hepatosomatic index was higher for fish fed the PPCPS. Our results indicated that PPCPS presents relevant nutritional quality for fish and can replace the fish meal protein up to 50% without affecting growth, PER and intermediate metabolites in silver catfish.

Major quality trait analysis and QTL detection in hexaploid wheat in humid rain-fed agriculture.

PubMed

Li, H M; Tang, Z X; Zhang, H Q; Yan, B J; Ren, Z L

2013-05-21

Humid rain-fed agriculture is a special environment for wheat (Triticum aestivum) culture that tends to negatively affect wheat yield and quality. To identify quality characters of wheat in a humid environment, we conducted quality analysis and quantitative trait loci (QTL) detection in a recombinant inbred line whose parent had a high level of quality for several years. We found that high-quality wheat had less gluten content and lower protein content. Apparently, wheat quality and associated quantity traits were in a dynamic state of equilibrium. We detected 83 QTL for 10 wheat quality traits in this recombinant inbred line population. Nine QTL were detected in both evaluation years; Q.DT.scau-2A, linked to Xwmc522-2A, was detected at the same genetic location in both years. Other QTL for different traits were detected simultaneously in more than one location. Consequently, there appeared to be pleiotropic genes that control wheat quality. Based on previous studies and our research on QTL analysis of grain protein content, we conclude that there must be one or more genes for grain protein content on chromosome 6B, whose expression was little affected by environment. We constructed a consensus map and projected the QTL on it. It was useful for choosing optimal markers for marker-assisted breeding and map-based cloning.

Thermal stability and gel quality of myofibrillar protein as affected by soy protein isolates subjected to an acidic pH and mild heating.

PubMed

Niu, Haili; Xia, Xiufang; Wang, Chao; Kong, Baohua; Liu, Qian

2018-03-01

Thermal stability and gel quality of myofibrillar protein were evaluated with regard to the addition of native soy protein isolates (SPI) and SPI subjected to acidic pH and mild heating (modified SPI). Compared with the control, the addition of modified SPI increased the compression force of the protein gel and decreased water loss (P<0.05). Differential scanning calorimetry results showed that an addition of 0.75% native SPI decreased the first transition temperature (P<0.05), and addition of 0.5% and 0.75% modified SPI exhibited no appreciable changes on it (P>0.05), indicating that a higher concentration of modified SPI would not damage the protein thermal stability. Moreover, the addition of modified SPI enhanced hydrogen bonding and disulphide linkages. Atomic force microscopy analysis revealed that the addition of modified SPI decreased the roughness of the mixed myofibrillar protein gels. Overall, modified SPI has the potential to improve myofibrillar protein gel texture and water holding capacity. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Role of the Multifunctional BAG3 Protein in Cellular Protein Quality Control and in Disease

PubMed Central

Stürner, Elisabeth; Behl, Christian

2017-01-01

In neurons, but also in all other cells the complex proteostasis network is monitored and tightly regulated by the cellular protein quality control (PQC) system. Beyond folding of newly synthesized polypeptides and their refolding upon misfolding the PQC also manages the disposal of aberrant proteins either by the ubiquitin-proteasome machinery or by the autophagic-lysosomal system. Aggregated proteins are primarily degraded by a process termed selective macroautophagy (or aggrephagy). One such recently discovered selective macroautophagy pathway is mediated by the multifunctional HSP70 co-chaperone BAG3 (BCL-2-associated athanogene 3). Under acute stress and during cellular aging, BAG3 in concert with the molecular chaperones HSP70 and HSPB8 as well as the ubiquitin receptor p62/SQSTM1 specifically targets aggregation-prone proteins to autophagic degradation. Thereby, BAG3-mediated selective macroautophagy represents a pivotal adaptive safeguarding and emergency system of the PQC which is activated under pathophysiological conditions to ensure cellular proteostasis. Interestingly, BAG3-mediated selective macroautophagy is also involved in the clearance of aggregated proteins associated with age-related neurodegenerative disorders, like Alzheimer’s disease (tau-protein), Huntington’s disease (mutated huntingtin/polyQ proteins), and amyotrophic lateral sclerosis (mutated SOD1). In addition, based on its initial description BAG3 is an anti-apoptotic protein that plays a decisive role in other widespread diseases, including cancer and myopathies. Therefore, in the search for novel therapeutic intervention avenues in neurodegeneration, myopathies and cancer BAG3 is a promising candidate. PMID:28680391

Distinct transcriptional responses elicited by unfolded nuclear or cytoplasmic protein in mammalian cells

PubMed Central

Miyazaki, Yusuke; Chen, Ling-chun; Chu, Bernard W; Swigut, Tomek; Wandless, Thomas J

2015-01-01

Eukaryotic cells possess a variety of signaling pathways that prevent accumulation of unfolded and misfolded proteins. Chief among these is the heat shock response (HSR), which is assumed to respond to unfolded proteins in the cytosol and nucleus alike. In this study, we probe this axiom further using engineered proteins called ‘destabilizing domains’, whose folding state we control with a small molecule. The sudden appearance of unfolded protein in mammalian cells elicits a robust transcriptional response, which is distinct from the HSR and other known pathways that respond to unfolded proteins. The cellular response to unfolded protein is strikingly different in the nucleus and the cytosol, although unfolded protein in either compartment engages the p53 network. This response provides cross-protection during subsequent proteotoxic stress, suggesting that it is a central component of protein quality control networks, and like the HSR, is likely to influence the initiation and progression of human pathologies. DOI: http://dx.doi.org/10.7554/eLife.07687.001 PMID:26314864

NIR spectroscopy for the quality control of Moringa oleifera (Lam.) leaf powders: Prediction of minerals, protein and moisture contents.

PubMed

Rébufa, Catherine; Pany, Inès; Bombarda, Isabelle

2018-09-30

A rapid methodology was developed to simultaneously predict water content and activity values (a w ) of Moringa oleifera leaf powders (MOLP) using near infrared (NIR) signatures and experimental sorption isotherms. NIR spectra of MOLP samples (n = 181) were recorded. A Partial Least Square Regression model (PLS2) was obtained with low standard errors of prediction (SEP of 1.8% and 0.07 for water content and a w respectively). Experimental sorption isotherms obtained at 20, 30 and 40 °C showed similar profiles. This result is particularly important to use MOLP in food industry. In fact, a temperature variation of the drying process will not affect their available water content (self-life). Nutrient contents based on protein and selected minerals (Ca, Fe, K) were also predicted from PLS1 models. Protein contents were well predicted (SEP of 2.3%). This methodology allowed for an improvement in MOLP safety, quality control and traceability. Published by Elsevier Ltd.

CHIP protects against cardiac pressure overload through regulation of AMPK

PubMed Central

Schisler, Jonathan C.; Rubel, Carrie E.; Zhang, Chunlian; Lockyer, Pamela; Cyr, Douglas M.; Patterson, Cam

2013-01-01

Protein quality control and metabolic homeostasis are integral to maintaining cardiac function during stress; however, little is known about if or how these systems interact. Here we demonstrate that C terminus of HSC70-interacting protein (CHIP), a regulator of protein quality control, influences the metabolic response to pressure overload by direct regulation of the catalytic α subunit of AMPK. Induction of cardiac pressure overload in Chip–/– mice resulted in robust hypertrophy and decreased cardiac function and energy generation stemming from a failure to activate AMPK. Mechanistically, CHIP promoted LKB1-mediated phosphorylation of AMPK, increased the specific activity of AMPK, and was necessary and sufficient for stress-dependent activation of AMPK. CHIP-dependent effects on AMPK activity were accompanied by conformational changes specific to the α subunit, both in vitro and in vivo, identifying AMPK as the first physiological substrate for CHIP chaperone activity and establishing a link between cardiac proteolytic and metabolic pathways. PMID:23863712

Roles for the ubiquitin-proteasome pathway in protein quality control and signaling in the retina: implications in the pathogenesis of age-related macular degeneration

USDA-ARS?s Scientific Manuscript database

The accumulation of damaged or postsynthetically modified proteins and dysregulation of inflammatory responses and angiogenesis in the retina/RPE are thought be etiologically related to formation of drusen and choroidal neovascularization (CNV), hallmarks of age-related macular degeneration (AMD). T...

Early Response of Protein Quality Control in Gills Is Associated with Survival of Hypertonic Shock in Mozambique tilapia

PubMed Central

Tang, Cheng-Hao; Lee, Tsung-Han

2013-01-01

The protein quality control (PQC) mechanism is essential for cell function and viability. PQC with proper biological function depends on molecular chaperones and proteases. The hypertonicity-induced protein damage and responses of PQC mechanism in aquatic organisms, however, are poorly understood. In this study, we examine the short-term effects of different hypertonic shocks on the levels of heat shock proteins (HSPs, e.g., HSP70 and HSP90), ubiquitin-conjugated proteins and protein aggregation in gills of the Mozambique tilapia (Oreochromis mossambicus). Following transfer from fresh water (FW) to 20‰ hypertonicity, all examined individuals survived to the end of experiment. Moreover, the levels of branchial HSPs and ubiquitin-conjugated proteins significantly increased at 3 and 24 h post-transfer, respectively. Up-regulation of HSPs and ubiquitin-conjugated proteins was sufficient to prevent the accumulation of aggregated proteins. However, the survival rate of tilapia dramatically declined at 5 h and all fish died within 7 h after direct transfer to 30‰ hypertonicity. We presumed that this result was due to the failed activation of gill PQC system, which resulted in elevating the levels of aggregated proteins at 3 and 4 h. Furthermore, in aggregated protein fractions, the amounts of gill Na+/K+-ATPase (NKA) remained relatively low when fish were transferred to 20‰ hypertonicity, whereas abundant NKA was found at 4 h post-transfer to 30‰ hypertonicity. This study demonstrated that the response of PQC in gills is earlier than observable changes in localization of ion-secreting transport proteins upon hypertonic challenge. To our knowledge, this is the first study to investigate the regulation of PQC mechanism in fish and characterize its important role in euryhaline teleost survival in response to hypertonic stress. PMID:23690986

Current opinion in Microbiology Roles of adaptor proteins in regulation of bacterial proteolysis

PubMed Central

Battesti, Aurelia; Gottesman, Susan

2013-01-01

Elimination of non-functional or unwanted proteins is critical for cell growth and regulation. In bacteria, ATP-dependent proteases target cytoplasmic proteins for degradation, contributing to both protein quality control and regulation of specific proteins, thus playing roles parallel to that of the proteasome in eukaryotic cells. Adaptor proteins provide a way to modulate the substrate specificity of the proteases and allow regulated proteolysis. Advances over the past few years have provided new insight into how adaptor proteins interact with both substrates and proteases and how adaptor functions are regulated. An important advance has come with the recognition of the critical roles of anti-adaptor proteins in regulating adaptor availability. PMID:23375660

Using Strong Magnetic Fields to Control Solutal Convection

NASA Technical Reports Server (NTRS)

Ramachandran, N.; Leslie, F. W.

2003-01-01

An important component in biotechnology, particularly in the area of protein engineering and rational drug design is the knowledge of the precise three-dimensional molecular structure of proteins. The quality of structural information obtained from X-ray diffraction methods is directly dependent on the degree of perfection of the protein crystals. As a consequence, the growth of high quality macromolecular crystals for diffraction analyses has been the central focus for biochemists, biologists, and bioengineers. Macromolecular crystals are obtained from solutions that contain the crystallizing species in equilibrium with higher aggregates, ions, precipitants, other possible phases of the protein, foreign particles, the walls of the container, and a likely host of other impurities. By changing transport modes in general, i.e., reduction of convection and sedimentation, as is achieved in microgravity , we have been able to dramatically affect the movement and distribution of macromolecules in the fluid, and thus their transport, formation of crystal nuclei, and adsorption to the crystal surface. While a limited number of high quality crystals from space flights have been obtained, as the recent National Research Council (NRC) review of the NASA microgravity crystallization program pointed out, the scientific approach and research in crystallization of proteins has been mainly empirical yielding inconclusive results. We postulate that we can reduce convection in ground-based experiments and we can understand the different aspects of convection control through the use of strong magnetic fields and field gradients. We postulate that limited convection in a magnetic field will provide the environment for the growth of high quality crystals. The approach exploits the variation of fluid magnetic susceptibility with concentration for this purpose and the convective damping is realized by appropriately positioning the crystal growth cell so that the magnetic susceptibility force counteracts terrestrial gravity. The general objective is to test the hypothesis of convective control using a strong magnetic field and magnetic field gradient and to understand the nature of the various forces that come into play. Specifically we aim to delineate causative factors and to quantify them through experiments, analysis and numerical modeling. The paper will report on the experimental results using paramagnetic salts and solutions in magnetic fields and compare them to analytical predictions.

Genome sequence of the thermotolerant foodborne pathogen Salmonella enterica serovar Senftenberg ATCC 43845 and phylogenetic analysis of Loci encoding increased protein quality control mechanisms

USDA-ARS?s Scientific Manuscript database

Salmonella enterica subsp. enterica bacteria are important foodborne pathogens with major economic impact. Some isolates exhibit increased heat tolerance, a concern for food safety. Analysis of a finished-quality genome sequence of an isolate commonly used in heat resistance studies, S. enterica sub...

The sigmaR regulon of Streptomyces coelicolor A32 reveals a key role in protein quality control during disulphide stress.

PubMed

Kallifidas, Dimitris; Thomas, Derek; Doughty, Phillip; Paget, Mark S B

2010-06-01

Diamide is an artificial disulphide-generating electrophile that mimics an oxidative shift in the cellular thiol-disulphide redox state (disulphide stress). The Gram-positive bacterium Streptomyces coelicolor senses and responds to disulphide stress through the sigma(R)-RsrA system, which comprises an extracytoplasmic function (ECF) sigma factor and a redox-active anti-sigma factor. Known targets that aid in the protection and recovery from disulphide stress include the thioredoxin system and genes involved in producing the major thiol buffer mycothiol. Here we determine the global response to diamide in wild-type and sigR mutant backgrounds to understand the role of sigma(R) in this response and to reveal additional regulatory pathways that allow cells to cope with disulphide stress. In addition to thiol oxidation, diamide was found to cause protein misfolding and aggregation, which elicited the induction of the HspR heat-shock regulon. Although this response is sigma(R)-independent, sigma(R) does directly control Clp and Lon ATP-dependent AAA(+) proteases, which may partly explain the reduced ability of a sigR mutant to resolubilize protein aggregates. sigma(R) also controls msrA and msrB methionine sulphoxide reductase genes, implying that sigma(R)-RsrA is responsible for the maintenance of both cysteine and methionine residues during oxidative stress. This work shows that the sigma(R)-RsrA system plays a more significant role in protein quality control than previously realized, and emphasizes the importance of controlling the cellular thiol-disulphide redox balance.

Rescue of sarcoglycan mutations by inhibition of endoplasmic reticulum quality control is associated with minimal structural modifications.

PubMed

Soheili, Tayebeh; Gicquel, Evelyne; Poupiot, Jérôme; N'Guyen, Luu; Le Roy, Florence; Bartoli, Marc; Richard, Isabelle

2012-02-01

Sarcoglycanopathies (SGP) are a group of autosomal recessive muscle disorders caused by primary mutations in one of the four sarcoglycan genes. The sarcoglycans (α-, β-, γ-, and δ-sarcoglycan) form a tetrameric complex at the muscle membrane that is part of the dystrophin-glycoprotein complex and plays an essential role for membrane integrity during muscle contractions. We previously showed that the most frequent missense mutation in α-sarcoglycan (p.R77C) leads to the absence of the protein at the cell membrane due to its blockade by the endoplasmic reticulum (ER) quality control. Moreover, we demonstrated that inhibition of the ER α-mannosidase I activity using kifunensine could rescue the mutant protein localization at the cell membrane. Here, we investigate 25 additional disease-causing missense mutations in the sarcoglycan genes with respect to intracellular fate and localization rescue of the mutated proteins by kifunensine. Our studies demonstrate that, similarly to p.R77C, 22 of 25 of the selected mutations lead to defective intracellular trafficking of the SGs proteins. Six of these were saved from ER retention upon kifunensine treatment. The trafficking of SGs mutants rescued by kifunensine was associated with mutations that have moderate structural impact on the protein. © 2011 Wiley Periodicals, Inc.

Dynamic JUNQ inclusion bodies are asymmetrically inherited in mammalian cell lines through the asymmetric partitioning of vimentin.

PubMed

Ogrodnik, Mikołaj; Salmonowicz, Hanna; Brown, Rachel; Turkowska, Joanna; Średniawa, Władysław; Pattabiraman, Sundararaghavan; Amen, Triana; Abraham, Ayelet-chen; Eichler, Noam; Lyakhovetsky, Roman; Kaganovich, Daniel

2014-06-03

Aging is associated with the accumulation of several types of damage: in particular, damage to the proteome. Recent work points to a conserved replicative rejuvenation mechanism that works by preventing the inheritance of damaged and misfolded proteins by specific cells during division. Asymmetric inheritance of misfolded and aggregated proteins has been shown in bacteria and yeast, but relatively little evidence exists for a similar mechanism in mammalian cells. Here, we demonstrate, using long-term 4D imaging, that the vimentin intermediate filament establishes mitotic polarity in mammalian cell lines and mediates the asymmetric partitioning of damaged proteins. We show that mammalian JUNQ inclusion bodies containing soluble misfolded proteins are inherited asymmetrically, similarly to JUNQ quality-control inclusions observed in yeast. Mammalian IPOD-like inclusion bodies, meanwhile, are not always inherited by the same cell as the JUNQ. Our study suggests that the mammalian cytoskeleton and intermediate filaments provide the physical scaffold for asymmetric inheritance of dynamic quality-control JUNQ inclusions. Mammalian IPOD inclusions containing amyloidogenic proteins are not partitioned as effectively during mitosis as their counterparts in yeast. These findings provide a valuable mechanistic basis for studying the process of asymmetric inheritance in mammalian cells, including cells potentially undergoing polar divisions, such as differentiating stem cells and cancer cells.

Protein Oxidation and Sensory Quality of Brine-Injected Pork Loins Added Ascorbate or Extracts of Green Tea or Maté during Chill-Storage in High-Oxygen Modified Atmosphere.

PubMed

Jongberg, Sisse; Tørngren, Mari Ann; Skibsted, Leif H

2018-01-15

Background: Ascorbate is often applied to enhance stability and robustness of brine-injected pork chops sold for retail, but may affect protein oxidation, while plant extracts are potential substitutes. Methods: Brine-injected pork chops (weight-gain ~12%, NaCl ~0.9%) prepared with ascorbate (225 ppm), green tea extract (25 ppm gallic acid equivalents (GAE)), or maté extract (25 ppm GAE) stored (5 °C, seven days) in high-oxygen atmosphere packaging (MAP: 80% O₂ and 20% CO₂) were analyzed for color changes, sensory quality, and protein oxidation compared to a control without antioxidant. Results: No significant differences were observed for green tea and maté extracts as compared to ascorbate when evaluated based on lipid oxidation derived off-flavors, except for stale flavor, which maté significantly reduced. All treatments increased the level of the protein oxidation product, α-aminoadipic semialdehyde as compared to the control, and ascorbate was further found to increase thiol loss and protein cross-linking, with a concomitant decrease in the sensory perceived tenderness. Conclusions: Green tea and maté were found to equally protect against lipid oxidation derived off-flavors, and maté showed less prooxidative activity towards proteins as compared to ascorbate, resulting in more tender meat. Maté is a valuable substitute for ascorbate in brine-injected pork chops.

Trehalose supplementation reduces hepatic endoplasmic reticulum stress and inflammatory signaling in old mice.

PubMed

Pagliassotti, Michael J; Estrada, Andrea L; Hudson, William M; Wei, Yuren; Wang, Dong; Seals, Douglas R; Zigler, Melanie L; LaRocca, Thomas J

2017-07-01

The accumulation of damaged proteins can perturb cellular homeostasis and provoke aging and cellular damage. Quality control systems, such as the unfolded protein response (UPR), inflammatory signaling and protein degradation, mitigate the residence time of damaged proteins. In the present study, we have examined the UPR and inflammatory signaling in the liver of young (~6 months) and old (~28 months) mice (n=8/group), and the ability of trehalose, a compound linked to increased protein stability and autophagy, to counteract age-induced effects on these systems. When used, trehalose was provided for 4 weeks in the drinking water immediately prior to sacrifice (n=7/group). Livers from old mice were characterized by activation of the UPR, increased inflammatory signaling and indices of liver injury. Trehalose treatment reduced the activation of the UPR and inflammatory signaling, and reduced liver injury. Reductions in proteins involved in autophagy and proteasome activity observed in old mice were restored following trehalose treatment. The autophagy marker, LC3B-II, was increased in old mice treated with trehalose. Metabolomics analyses demonstrated that reductions in hexosamine biosynthetic pathway metabolites and nicotinamide in old mice were restored following trehalose treatment. Trehalose appears to be an effective intervention to reduce age-associated liver injury and mitigate the need for activation of quality control systems that respond to disruption of proteostasis. Copyright © 2017 Elsevier Inc. All rights reserved.

A View on the Importance of "Multi-Attribute Method" for Measuring Purity of Biopharmaceuticals and Improving Overall Control Strategy.

PubMed

Rogers, Richard S; Abernathy, Michael; Richardson, Douglas D; Rouse, Jason C; Sperry, Justin B; Swann, Patrick; Wypych, Jette; Yu, Christopher; Zang, Li; Deshpande, Rohini

2017-11-30

Today, we are experiencing unprecedented growth and innovation within the pharmaceutical industry. Established protein therapeutic modalities, such as recombinant human proteins, monoclonal antibodies (mAbs), and fusion proteins, are being used to treat previously unmet medical needs. Novel therapies such as bispecific T cell engagers (BiTEs), chimeric antigen T cell receptors (CARTs), siRNA, and gene therapies are paving the path towards increasingly personalized medicine. This advancement of new indications and therapeutic modalities is paralleled by development of new analytical technologies and methods that provide enhanced information content in a more efficient manner. Recently, a liquid chromatography-mass spectrometry (LC-MS) multi-attribute method (MAM) has been developed and designed for improved simultaneous detection, identification, quantitation, and quality control (monitoring) of molecular attributes (Rogers et al. MAbs 7(5):881-90, 2015). Based on peptide mapping principles, this powerful tool represents a true advancement in testing methodology that can be utilized not only during product characterization, formulation development, stability testing, and development of the manufacturing process, but also as a platform quality control method in dispositioning clinical materials for both innovative biotherapeutics and biosimilars.

Magnetic Control of Convection during Protein Crystallization

NASA Technical Reports Server (NTRS)

Ramachandran, N.; Leslie, F. W.

2004-01-01

An important component in biotechnology, particularly in the area of protein engineering and rational drug design is the knowledge of the precise three-dimensional molecular structure of proteins. The quality of structural information obtained from X-ray diffraction methods is directly dependent on the degree of perfection of the protein crystals. As a consequence, the growth of high quality macromolecular Crystals for diffraction analyses has been the central focus for bio-chemists, biologists, and bioengineers. Macromolecular crystals are obtained from solutions that contain the crystallizing species in equilibrium with higher aggregates, ions, precipitants, other possible phases of the protein, foreign particles, the walls of container, and a likely host of other impurities. By changing transport modes in general, i.e., reduction of convection and Sedimentation as is achieved in "microgravity", we have been able to dramatically affect the movement and distribution of macromolecules in the fluid, and thus their transport, f o d o n of crystal nuclei, and adsorption to the crystal surface. While a limited number of high quality crystals from space flights have been obtained, as the recent National Research Council (NRC) review of the NASA microgravity crystallization program pointed out, the scientific approach and research in crystallization of proteins has been mainly empirical yielding inconclusive results. We postulate that we can reduce convection in ground-based experiments and we can understand the different aspects of convection control through the use of strong magnetic fields and field gradients. We postulate that limited convection in a magnetic field will provide the environment for the growth of high quality crystals. The approach exploits the variation of fluid magnetic susceptibility with counteracts on for this purpose and the convective damping is realized by appropriately positioning the crystal growth cell so that the magnetic susceptibility force counteract terrestrial gravity. The genera1 objective is to test the hypothesis of convective control using a strong magnetic field and magnetic field gradient and to understand the nature of the various forces that come into play. Specifically we aim to delineate causative factors and to quantify them through experiments, analysis and numerical modeling. The paper will report on the experimental results using paramagentic salts and solutions in magnetic fields and compare them to analyticalprctions.

Identification of chromosome regions controlling seed storage proteins of narrow-leafed lupin (Lupinus angustifolius).

PubMed

Li, Xin; Islam, Shahidul; Yang, Huaan; Ma, Wujun; Yan, Guijun

2013-05-01

Narrow-leafed lupin (Lupinus angustifolius L.) is a valuable legume crop for animal feed and human health food because of its high proteins content. However, the genetics of seed storage proteins is unclear, limiting further improvement of protein quantity and quality. In this study, matrix-assisted laser desorption/ionization time of flight mass spectrometry was used for the first time to analyze lupin seed storage proteins and the spectra generated was treated as markers to investigate the chromosome locations controlling seed storage proteins in the narrow-leafed lupin. In a recombinant inbred line population of 89 individuals, 48 polymorphic protein peaks were identified and seven of which were successfully mapped onto four existing linkage groups: two on NLL-04, three on NLL-05, one on NLL-07 and one on NLL-14, with LOD values ranging from 2.6 to 7.7 confirming a significant linkage. Most protein-based markers showed distorted segregation and were failed to be integrated into the reference map. Among them, 31 were grouped into six clusters and the other ten were totally unlinked. This study provides a significant clue to study the comparative genomics/proteomics among legumes as well as for protein marker-assisted breeding. The distribution pattern of genes controlling seed storage protein revealed in this study probably exists universally among legumes or even all plants and animals. Whether genes controlling seed storage protein share the same gene expression pattern controlling other enzymes and what is the mechanism behind it are the questions which remain to be answered in the future.

Effects of Liuwei Dihuang Granule ([symbols; see text]) on the outcomes of in vitro fertilization pre-embryo transfer in infertility women with Kidney-yin deficiency syndrome and the proteome expressions in the follicular fluid.

PubMed

Lian, Fang; Wu, Hai-cui; Sun, Zhen-gao; Guo, Ying; Shi, Lei; Xue, Ming-yue

2014-07-01

To observe the effects of Liuwei Dihuang Granule ([symbols; see text], LDG) for tonifying Kidney (Shen) on the outcomes of in vitro fertilization pre-embryo transfer (IVF-ET) of infertility women with Kidney-yin deficiency syndrome and to explore its mechanism by detecting the proteome expression in the follicular fluid. Sixty-six infertility patients of Kidney-yin deficiency syndrome who would undergo IVF-ET, were randomly assigned to a treatment group and a control group according to a random number table, 33 cases in each group. Another 33 cases of non-Kidney-yin deficiency syndrome was taken as a syndrome-control group. Besides Western routine therapy, LDG was given 3 menstrual cycles before IVF to the treatment group, and a placebo granule to the control and syndrome-control groups. The scores of Kidney-yin deficiency symptoms (sore waist and knees, dry vagina, dysphoria with feverish sensation in the chest, palms and soles, etc.) were assessed, the number of retrieved oocytes, rates of high quality oocytes and embryos, fertility rate and clinical pregnancy rate were recorded, and the follicular fluid was collected on the day when the ovum was picked up, the differential protein expression was detected using two-dimensional gel electrophoresis, and then, matrix assisted laser desorption ionization time-of flight mass spectrometry (MALDI-TOF-MS) was applied to identify the proteins. The syndrome score in the treatment group decreased significantly from 16.09±2.58 to 8.67±2.13, while it changed insignificantly in the control group, with a significant difference in the lowering score between the two groups (P<0.05); the high quality rates of oocytes and embryos and clinical pregnancy rate were all superior in the treatment group to the control group (82.29% vs 78.08%, 76.76% vs 68.79%, 63.64% vs 36.36%, all P<0.05). The protein expression map from the follicular fluid showed that compared with the control group, 33 differential protein expressions were found in the syndrome-control group, among which 18 were down-regulated, and 15 up-regulated; in the treatment group 28 differential protein expressions were found, among which 15 were down-regulated, and 13 up-regulated. Through MALDI-TOF-MS, 14 proteins were identified (P<0.05). For the infertility patients undergoing IVF, LDG could alleviate clinical symptoms, improve rates of high quality oocytes and embryos, so as to raise clinical pregnancy rate. The mechanism may be through regulating proteome expression in the follicular fluid to improve the developmental microenvironment for oocytes which would lead to a successful embryo implantation.

[Inclusion body myopathy with Paget's disease of bone and frontotemporal dementia].

PubMed

Hayashi, Yukiko

2013-01-01

Inclusion body myopathy with Paget's disease of bone and frontotemporal dementia (IBMPFD) is an autosomal dominant disease caused by mutations in the VCP gene. VCP encodes a well-conserved multifunctional protein, valosin containing protein (VCP), which has important roles in protein quality control via proteasome and autophagy, protein aggregation, quality control of mitochondria, cell proliferation, and so on. Clinically, muscle weakness is the most common symptom of which disease onset is around 40 years. Affected muscles are variable, and the patients are sometimes diagnosed as limb girdle muscular dystrophy or GNE myopathy. Muscle pathology shows characteristic features including cytoplasmic/nuclear inclusions, rimmed vacuoles, and disorganized myofibrills, together with neurogenic changes. Paget's disease of bone is reported to be observed in a half of the patients around the age of 40 years, but less common in Japanese patients. Frontotemporal dementia is seen around one third of the patients which appears nearly 10 years later than muscle or bone disease. In addition to cognitive dysfunctions, motor neuron involvement and cerebellar signs were also seen in our series. IBMPFD is not so rare disease as previously thought, but complicate clinical findings may make its diagnosis difficult.

Compartmentalization of superoxide dismutase 1 (SOD1G93A) aggregates determines their toxicity

PubMed Central

Weisberg, Sarah J.; Lyakhovetsky, Roman; Werdiger, Ayelet-chen; Gitler, Aaron D.; Soen, Yoav; Kaganovich, Daniel

2012-01-01

Neurodegenerative diseases constitute a class of illnesses marked by pathological protein aggregation in the brains of affected individuals. Although these disorders are invariably characterized by the degeneration of highly specific subpopulations of neurons, protein aggregation occurs in all cells, which indicates that toxicity arises only in particular cell biological contexts. Aggregation-associated disorders are unified by a common cell biological feature: the deposition of the culprit proteins in inclusion bodies. The precise function of these inclusions remains unclear. The starting point for uncovering the origins of disease pathology must therefore be a thorough understanding of the general cell biological function of inclusions and their potential role in modulating the consequences of aggregation. Here, we show that in human cells certain aggregate inclusions are active compartments. We find that toxic aggregates localize to one of these compartments, the juxtanuclear quality control compartment (JUNQ), and interfere with its quality control function. The accumulation of SOD1G93A aggregates sequesters Hsp70, preventing the delivery of misfolded proteins to the proteasome. Preventing the accumulation of SOD1G93A in the JUNQ by enhancing its sequestration in an insoluble inclusion reduces the harmful effects of aggregation on cell viability. PMID:22967507

Supplementation of Pork Patties with Bovine Plasma Protein Hydrolysates Augments Antioxidant Properties and Improves Quality.

PubMed

Seo, Hyun-Woo; Seo, Jin-Kyu; Yang, Han-Sul

2016-01-01

This study investigated the effects of bovine plasma protein (PP) hydrolysates on the antioxidant and quality properties of pork patties during storage. Pork patties were divided into 4 groups: without butylated hydroxytoluene (BHT) and PP hydrolysates (control), 0.02% BHT (T1), 1% PP hydrolysates (T2), and 2% PP hydrolysates (T3). Pork patty supplemented with PP hydrolysates had higher pH values and lower weight loss during cooking than the control patties. Results showed that lightness and hardness both decreased upon the addition of PP hydrolysates. All samples containing BHT and PP hydrolysates had reduced TBARS and peroxide values during storage. In particular, 2% PP hydrolysates were more effective in delaying lipid oxidation than were the other treatments. It was concluded that treatment with 2% PP hydrolysates can enhance the acceptance of pork patty.

A Quadrupole Dalton-based multi-attribute method for product characterization, process development, and quality control of therapeutic proteins.

PubMed

Xu, Weichen; Jimenez, Rod Brian; Mowery, Rachel; Luo, Haibin; Cao, Mingyan; Agarwal, Nitin; Ramos, Irina; Wang, Xiangyang; Wang, Jihong

2017-10-01

During manufacturing and storage process, therapeutic proteins are subject to various post-translational modifications (PTMs), such as isomerization, deamidation, oxidation, disulfide bond modifications and glycosylation. Certain PTMs may affect bioactivity, stability or pharmacokinetics and pharmacodynamics profile and are therefore classified as potential critical quality attributes (pCQAs). Identifying, monitoring and controlling these PTMs are usually key elements of the Quality by Design (QbD) approach. Traditionally, multiple analytical methods are utilized for these purposes, which is time consuming and costly. In recent years, multi-attribute monitoring methods have been developed in the biopharmaceutical industry. However, these methods combine high-end mass spectrometry with complicated data analysis software, which could pose difficulty when implementing in a quality control (QC) environment. Here we report a multi-attribute method (MAM) using a Quadrupole Dalton (QDa) mass detector to selectively monitor and quantitate PTMs in a therapeutic monoclonal antibody. The result output from the QDa-based MAM is straightforward and automatic. Evaluation results indicate this method provides comparable results to the traditional assays. To ensure future application in the QC environment, this method was qualified according to the International Conference on Harmonization (ICH) guideline and applied in the characterization of drug substance and stability samples. The QDa-based MAM is shown to be an extremely useful tool for product and process characterization studies that facilitates facile understanding of process impact on multiple quality attributes, while being QC friendly and cost-effective.

Evaluation of capillary zone electrophoresis for the quality control of complex biologic samples: Application to snake venoms.

PubMed

Kpaibe, André P S; Ben-Ameur, Randa; Coussot, Gaëlle; Ladner, Yoann; Montels, Jérôme; Ake, Michèle; Perrin, Catherine

2017-08-01

Snake venoms constitute a very promising resource for the development of new medicines. They are mainly composed of very complex peptide and protein mixtures, which composition may vary significantly from batch to batch. This latter consideration is a challenge for routine quality control (QC) in the pharmaceutical industry. In this paper, we report the use of capillary zone electrophoresis for the development of an analytical fingerprint methodology to assess the quality of snake venoms. The analytical fingerprint concept is being widely used for the QC of herbal drugs but rarely for venoms QC so far. CZE was chosen for its intrinsic efficiency in the separation of protein and peptide mixtures. The analytical fingerprint methodology was first developed and evaluated for a particular snake venom, Lachesis muta. Optimal analysis conditions required the use of PDADMAC capillary coating to avoid protein and peptide adsorption. Same analytical conditions were then applied to other snake venom species. Different electrophoretic profiles were obtained for each venom. Excellent repeatability and intermediate precision was observed for each batch. Analysis of different batches of the same species revealed inherent qualitative and quantitative composition variations of the venoms between individuals. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Variation in cooking and eating quality traits in Japanese rice germplasm accessions

PubMed Central

Hori, Kiyosumi; Suzuki, Keitaro; Iijima, Ken; Ebana, Kaworu

2016-01-01

The eating quality of cooked rice is important and determines its market price and consumer acceptance. To comprehensively describe the variation of eating quality in 183 rice germplasm accessions, we evaluated 33 eating-quality traits including amylose and protein contents, pasting properties of rice flour, and texture of cooked rice grains. All eating-quality traits varied widely in the germplasm accessions. Principal-components analysis (PCA) revealed that allelic differences in the Wx gene explained the largest proportion of phenotypic variation of the eating-quality traits. In 146 accessions of non-glutinous temperate japonica rice, PCA revealed that protein content and surface texture of the cooked rice grains significantly explained phenotypic variations of the eating-quality traits. An allelic difference based on simple sequence repeats, which was located near a quantitative trait locus (QTL) on the short arm of chromosome 3, was associated with differences in the eating quality of non-glutinous temperate japonica rice. These results suggest that eating quality is controlled by genetic factors, including the Wx gene and the QTL on chromosome 3, in Japanese rice accessions. These genetic factors have been consciously selected for eating quality during rice breeding programs in Japan. PMID:27162502

Variation in cooking and eating quality traits in Japanese rice germplasm accessions.

PubMed

Hori, Kiyosumi; Suzuki, Keitaro; Iijima, Ken; Ebana, Kaworu

2016-03-01

The eating quality of cooked rice is important and determines its market price and consumer acceptance. To comprehensively describe the variation of eating quality in 183 rice germplasm accessions, we evaluated 33 eating-quality traits including amylose and protein contents, pasting properties of rice flour, and texture of cooked rice grains. All eating-quality traits varied widely in the germplasm accessions. Principal-components analysis (PCA) revealed that allelic differences in the Wx gene explained the largest proportion of phenotypic variation of the eating-quality traits. In 146 accessions of non-glutinous temperate japonica rice, PCA revealed that protein content and surface texture of the cooked rice grains significantly explained phenotypic variations of the eating-quality traits. An allelic difference based on simple sequence repeats, which was located near a quantitative trait locus (QTL) on the short arm of chromosome 3, was associated with differences in the eating quality of non-glutinous temperate japonica rice. These results suggest that eating quality is controlled by genetic factors, including the Wx gene and the QTL on chromosome 3, in Japanese rice accessions. These genetic factors have been consciously selected for eating quality during rice breeding programs in Japan.

Quantitative phosphoproteomic analysis of porcine muscle within 24 h postmortem.

PubMed

Huang, Honggang; Larsen, Martin R; Palmisano, Giuseppe; Dai, Jie; Lametsch, René

2014-06-25

Protein phosphorylation can regulate most of the important processes in muscle, such as metabolism and contraction. The postmortem (PM) metabolism and rigor mortis have essential effects on meat quality. In order to identify and characterize the protein phosphorylation events involved in meat quality development, a quantitative mass spectrometry-based phosphoproteomic study was performed to analyze the porcine muscle within 24h PM using dimethyl labeling combined with the TiSH phosphopeptide enrichment strategy. In total 305 unique proteins were identified, including 160 phosphoproteins with 784 phosphorylation sites. Among these, 184 phosphorylation sites on 93 proteins had their phosphorylation levels significantly changed. The proteins involved in glucose metabolism and muscle contraction were the two largest clusters of phosphoproteins with significantly changed phosphorylation levels in muscle within 24 h PM. The high phosphorylation level of heat shock proteins (HSPs) in early PM may be an adaptive response to slaughter stress and protect muscle cell from apoptosis, as observed in the serine 84 of HSP27. This work indicated that PM muscle proteins underwent significant changes at the phosphorylation level but were relatively stable at the total protein level, suggesting that protein phosphorylation may have important roles in meat quality development through the regulation of proteins involved in glucose metabolism and muscle contraction, thereby affecting glycolysis and rigor mortis development in PM muscle. The manuscript describes the characterization of postmortem (PM) porcine muscle within 24 h postmortem from the perspective of protein phosphorylation using advanced phosphoproteomic techniques. In the study, the authors employed the dimethyl labeling combined with the TiSH phosphopeptide enrichment and LC-MS/MS strategy. This was the first high-throughput quantitative phosphoproteomic study in PM muscle of farm animals. In the work, both the proteome and phosphoproteome were analyzed, and the large number of identified peptides, phosphopeptides and phosphorylation sites can greatly enrich the current farm animal protein database. The proteins involved in glycometabolism, muscle contraction and heat shock proteins (HSPs) showed significantly changed phosphorylation levels during PM meat development. This work indicated that PM muscle proteins underwent significant changes at phosphorylation level but were relatively stable at the total protein level, suggesting that protein phosphorylation may have important roles in meat development through the regulation of proteins involved in metabolism and muscle contraction, thereby affecting glycolysis and rigor mortis development in PM muscle. The work can promote the understanding of PM muscle metabolism and meat quality development, and be helpful for future meat quality control. Copyright © 2014 Elsevier B.V. All rights reserved.

Utilization of different waste proteins to create a novel PGPR-containing bio-organic fertilizer

PubMed Central

Huang, Yan; Sun, Li; Zhao, Jianshu; Huang, Rong; Li, Rong; Shen, Qirong

2015-01-01

High-quality bio-organic fertilizers (BIOs) cannot be produced without the addition of some proteins, while many waste proteins are haphazardly disposed, causing serious environmental pollution. In this study, several waste proteins were used as additives to assist with the reproduction of the functional microbe (Bacillus amyloliquefaciens SQR9) inoculated into matured composts to produce BIOs. An optimized composition of solid-state fermentation (SSF) raw materials was predicted by response surface methodology and experimental validation. The results showed that 7.61% (w/w, DW, the same below) rapeseed meal, 8.85% expanded feather meal, 6.47% dewatered blue algal sludge and 77.07% chicken compost resulted in maximum biomass of strain SQR-9 and the maximum amount of lipopeptides 7 days after SSF. Spectroscopy experiments showed that the inner material structural changes in the novel SSF differed from the control and the novel BIO had higher dissolved organic matter. This study offers a high value-added utilization of waste proteins for producing economical but high-quality BIO. PMID:25586328

Transgenic approach to improve wheat (Triticum aestivum L.) nutritional quality.

PubMed

Tamás, Cecília; Kisgyörgy, Boglárka N; Rakszegi, Mariann; Wilkinson, Mark D; Yang, Moon-Sik; Láng, László; Tamás, László; Bedo, Zoltán

2009-07-01

An amaranth (Amaranthus hypochondriacus) albumin gene, encoding the 35-kDa AmA1 protein of the seed, with a high content of essential amino acids, was used in the biolistic transformation of bread wheat (Triticum aestivum L.) variety Cadenza. The transformation cassette carried the ama1 gene under the control of a powerful wheat endosperm-specific promoter (1Bx17 HMW-GS). Southern-blot analysis of T(1) lines confirmed the integration of the foreign gene, while RT-PCR and Western-blot analyses of the samples confirmed the transcription and translation of the transgene. The effects of the extra albumin protein on the properties of flour, produced from bulked T(2) seeds, were calculated using total protein and essential amino acid content analysis, polymeric/monomeric protein and HMW/LMW glutenin subunit ratio measurements. The results indicated that not only can essential amino acid content be increased, but some parameters associated with functional quality may also be improved because of the expression of the AmA1 protein.

Utilization of different waste proteins to create a novel PGPR-containing bio-organic fertilizer

NASA Astrophysics Data System (ADS)

Huang, Yan; Sun, Li; Zhao, Jianshu; Huang, Rong; Li, Rong; Shen, Qirong

2015-01-01

High-quality bio-organic fertilizers (BIOs) cannot be produced without the addition of some proteins, while many waste proteins are haphazardly disposed, causing serious environmental pollution. In this study, several waste proteins were used as additives to assist with the reproduction of the functional microbe (Bacillus amyloliquefaciens SQR9) inoculated into matured composts to produce BIOs. An optimized composition of solid-state fermentation (SSF) raw materials was predicted by response surface methodology and experimental validation. The results showed that 7.61% (w/w, DW, the same below) rapeseed meal, 8.85% expanded feather meal, 6.47% dewatered blue algal sludge and 77.07% chicken compost resulted in maximum biomass of strain SQR-9 and the maximum amount of lipopeptides 7 days after SSF. Spectroscopy experiments showed that the inner material structural changes in the novel SSF differed from the control and the novel BIO had higher dissolved organic matter. This study offers a high value-added utilization of waste proteins for producing economical but high-quality BIO.

N-glycan based ER molecular chaperone and protein quality control system: the calnexin binding cycle

PubMed Central

Lamriben, Lydia; Graham, Jill B.; Adams, Benjamin M.; Hebert, Daniel N.

2015-01-01

Helenius and colleagues proposed over twenty-years ago a paradigm-shifting model for how chaperone binding in the endoplasmic reticulum was mediated and controlled for a new type of molecular chaperone- the carbohydrate binding chaperones, calnexin and calreticulin. While the originally established basics for this lectin chaperone binding cycle holds true today, there has been a number of important advances that have expanded our understanding of its mechanisms of action, role in protein homeostasis, and its connection to disease states that are highlighted in this review. PMID:26676362

Application of Sigma Metrics Analysis for the Assessment and Modification of Quality Control Program in the Clinical Chemistry Laboratory of a Tertiary Care Hospital.

PubMed

Iqbal, Sahar; Mustansar, Tazeen

2017-03-01

Sigma is a metric that quantifies the performance of a process as a rate of Defects-Per-Million opportunities. In clinical laboratories, sigma metric analysis is used to assess the performance of laboratory process system. Sigma metric is also used as a quality management strategy for a laboratory process to improve the quality by addressing the errors after identification. The aim of this study is to evaluate the errors in quality control of analytical phase of laboratory system by sigma metric. For this purpose sigma metric analysis was done for analytes using the internal and external quality control as quality indicators. Results of sigma metric analysis were used to identify the gaps and need for modification in the strategy of laboratory quality control procedure. Sigma metric was calculated for quality control program of ten clinical chemistry analytes including glucose, chloride, cholesterol, triglyceride, HDL, albumin, direct bilirubin, total bilirubin, protein and creatinine, at two control levels. To calculate the sigma metric imprecision and bias was calculated with internal and external quality control data, respectively. The minimum acceptable performance was considered as 3 sigma. Westgard sigma rules were applied to customize the quality control procedure. Sigma level was found acceptable (≥3) for glucose (L2), cholesterol, triglyceride, HDL, direct bilirubin and creatinine at both levels of control. For rest of the analytes sigma metric was found <3. The lowest value for sigma was found for chloride (1.1) at L2. The highest value of sigma was found for creatinine (10.1) at L3. HDL was found with the highest sigma values at both control levels (8.8 and 8.0 at L2 and L3, respectively). We conclude that analytes with the sigma value <3 are required strict monitoring and modification in quality control procedure. In this study application of sigma rules provided us the practical solution for improved and focused design of QC procedure.

Mechanisms of protein balance in skeletal muscle.

PubMed

Anthony, T G

2016-07-01

Increased global demand for adequate protein nutrition against a backdrop of climate change and concern for animal agriculture sustainability necessitates new and more efficient approaches to livestock growth and production. Anabolic growth is achieved when rates of new synthesis exceed turnover, producing a positive net protein balance. Conversely, deterioration or atrophy of lean mass is a consequence of a net negative protein balance. During early life and periods of growth, muscle mass is driven by increases in protein synthesis at the level of mRNA translation. Throughout life, muscle mass is further influenced by degradative processes such as autophagy and the ubiquitin proteasome pathway. Multiple signal transduction networks guide and coordinate these processes alongside quality control mechanisms to maintain protein homeostasis (proteostasis). Genetics, hormones, and environmental stimuli each influence proteostasis control, altering capacity and/or efficiency of muscle growth. An overview of recent findings and current methods to assess muscle protein balance and proteostasis is presented. Current efforts to identify novel control points have the potential through selective breeding design or development of hormetic strategies to better promote growth and health span during environmental stress. Copyright © 2016 Elsevier Inc. All rights reserved.

Structural Biology of Proteins of the Multi-enzyme Assembly Human Pyruvate Dehydrogenase Complex

NASA Technical Reports Server (NTRS)

2003-01-01

Objectives and research challenges of this effort include: 1. Need to establish Human Pyruvate Dehydrogenase Complex protein crystals; 2. Need to test value of microgravity for improving crystal quality of Human Pyruvate Dehydrogenase Complex protein crystals; 3. Need to improve flight hardware in order to control and understand the effects of microgravity on crystallization of Human Pyruvate Dehydrogenase Complex proteins; 4. Need to integrate sets of national collaborations with the restricted and specific requirements of flight experiments; 5. Need to establish a highly controlled experiment in microgravity with a rigor not yet obtained; 6. Need to communicate both the rigor of microgravity experiments and the scientific value of results obtained from microgravity experiments to the national community; and 7. Need to advance the understanding of Human Pyruvate Dehydrogenase Complex structures so that scientific and commercial advance is identified for these proteins.

Study of Fluid Flow Control in Protein Crystallization using Strong Magnetic Fields

NASA Astrophysics Data System (ADS)

Ramachandran, Narayanan; Leslie, Fred; Ciszak, Ewa

2002-11-01

An important component in biotechnology, particularly in the area of protein engineering and rational drug design is the knowledge of the precise three-dimensional molecular structure of proteins. The quality of structural information obtained from X-ray diffraction methods is directly dependent on the degree of perfection of the protein crystals. As a consequence, the growth of high quality macromolecular crystals for diffraction analyses has been the central focus for biochemists, biologists, and bioengineers. Macromolecular crystals are obtained from solutions that contain the crystallizing species in equilibrium with higher aggregates, ions, precipitants, other possible phases of the protein, foreign particles, the walls of the container, and a likely host of other impurities. By changing transport modes in general, i.e., reduction of convection and sedimentation, as is achieved in "microgravity", researchers have been able to dramatically affect the movement and distribution of macromolecules in the fluid, and thus their transport, formation of crystal nuclei, and adsorption to the crystal surface. While a limited number of high quality crystals from space flights have been obtained, as the recent National Research Council (NRC) review of the NASA microgravity crystallization program pointed out, the scientific approach and research in crystallization of proteins has been mainly empirical yielding inconclusive results. We postulate that we can reduce convection in ground-based experiments and we can understand the different aspects of convection control through the use of strong magnetic fields and field gradients. Whether this limited convection in a magnetic field will provide the environment for the growth of high quality crystals is still a matter of conjecture that our research will address. The approach exploits the variation of fluid magnetic susceptibility with concentration for this purpose and the convective damping is realized by appropriately positioning the crystal growth cell so that the magnetic susceptibility force counteracts terrestrial gravity. The general objective is to test the hypothesis of convective control using a strong magnetic field and magnetic field gradient and to understand the nature of the various forces that come into play. Specifically we aim to delineate causative factors and to quantify them through experiments, analysis and numerical modeling. Once the basic understanding is obtained, the study will focus on testing the hypothesis on proteins of pyruvate dehydrogenase complex (PDC), proteins E1 and E3. Obtaining high crystal quality of these proteins is of great importance to structural biologists since their structures need to be determined. Specific goals for the investigation are: 1. To develop an understanding of convection control in diamagnetic fluids with concentration gradients through experimentation and numerical modeling. Specifically solutal buoyancy driven convection due to crystal growth will be considered. 2. To develop predictive measures for successful crystallization in a magnetic field using analyses and numerical modeling for use in future protein crystal growth experiments. This will establish criteria that can be used to estimate the efficacy of magnetic field flow damping on crystallization of candidate proteins. 3. To demonstrate the understanding of convection damping by high magnetic fields to a class of proteins that is of interest and whose structure is as yet not determined. 4. To compare quantitatively, the quality of the grown crystals with and without a magnetic field. X-ray diffraction techniques will be used for the comparative studies. In a preliminary set of experiments, we studied crystal dissolution effects in a 5 Tesla magnet available at NASA Marshall Space Flight Center (MSFC). Using a Schlieren setup, a 1mm crystal of Alum (Aluminum-Potassium Sulfate) was introduced in a 75% saturated solution and the resulting dissolution plume was observed. The experiment was conducted both in the presence and absence of a magnetic field gradient. The magnet produces a gradient field of approx. 1 Tesla2/cm. Image analysis of the recorded images indicated an enhanced plume velocity that was of the order of the measurement limit. For this experiment, both the gradient and gravity fields are in the same direction resulting in an enhanced effective gravity that tends to accelerate the observed plume velocity. While the results are not conclusive, pending further tests, it clearly points out the inadequacy of the MSFC magnet for conducting protein crystallization experiments and the need for a stronger magnet. In spacebased experiments, however, where the gravitational effects are small, only a weak magnetic field will be required to control or mitigate the effects of convective contamination.

Study of Fluid Flow Control in Protein Crystallization using Strong Magnetic Fields

NASA Technical Reports Server (NTRS)

Ramachandran, Narayanan; Leslie, Fred; Ciszak, Ewa

2002-01-01

An important component in biotechnology, particularly in the area of protein engineering and rational drug design is the knowledge of the precise three-dimensional molecular structure of proteins. The quality of structural information obtained from X-ray diffraction methods is directly dependent on the degree of perfection of the protein crystals. As a consequence, the growth of high quality macromolecular crystals for diffraction analyses has been the central focus for biochemists, biologists, and bioengineers. Macromolecular crystals are obtained from solutions that contain the crystallizing species in equilibrium with higher aggregates, ions, precipitants, other possible phases of the protein, foreign particles, the walls of the container, and a likely host of other impurities. By changing transport modes in general, i.e., reduction of convection and sedimentation, as is achieved in "microgravity", researchers have been able to dramatically affect the movement and distribution of macromolecules in the fluid, and thus their transport, formation of crystal nuclei, and adsorption to the crystal surface. While a limited number of high quality crystals from space flights have been obtained, as the recent National Research Council (NRC) review of the NASA microgravity crystallization program pointed out, the scientific approach and research in crystallization of proteins has been mainly empirical yielding inconclusive results. We postulate that we can reduce convection in ground-based experiments and we can understand the different aspects of convection control through the use of strong magnetic fields and field gradients. Whether this limited convection in a magnetic field will provide the environment for the growth of high quality crystals is still a matter of conjecture that our research will address. The approach exploits the variation of fluid magnetic susceptibility with concentration for this purpose and the convective damping is realized by appropriately positioning the crystal growth cell so that the magnetic susceptibility force counteracts terrestrial gravity. The general objective is to test the hypothesis of convective control using a strong magnetic field and magnetic field gradient and to understand the nature of the various forces that come into play. Specifically we aim to delineate causative factors and to quantify them through experiments, analysis and numerical modeling. Once the basic understanding is obtained, the study will focus on testing the hypothesis on proteins of pyruvate dehydrogenase complex (PDC), proteins E1 and E3. Obtaining high crystal quality of these proteins is of great importance to structural biologists since their structures need to be determined. Specific goals for the investigation are: 1. To develop an understanding of convection control in diamagnetic fluids with concentration gradients through experimentation and numerical modeling. Specifically solutal buoyancy driven convection due to crystal growth will be considered. 2. To develop predictive measures for successful crystallization in a magnetic field using analyses and numerical modeling for use in future protein crystal growth experiments. This will establish criteria that can be used to estimate the efficacy of magnetic field flow damping on crystallization of candidate proteins. 3. To demonstrate the understanding of convection damping by high magnetic fields to a class of proteins that is of interest and whose structure is as yet not determined. 4. To compare quantitatively, the quality of the grown crystals with and without a magnetic field. X-ray diffraction techniques will be used for the comparative studies. In a preliminary set of experiments, we studied crystal dissolution effects in a 5 Tesla magnet available at NASA Marshall Space Flight Center (MSFC). Using a Schlieren setup, a 1mm crystal of Alum (Aluminum-Potassium Sulfate) was introduced in a 75% saturated solution and the resulting dissolution plume was observed. The experiment was conducted both in the presence and absence of a magnetic field gradient. The magnet produces a gradient field of approx. 1 Tesla2/cm. Image analysis of the recorded images indicated an enhanced plume velocity that was of the order of the measurement limit. For this experiment, both the gradient and gravity fields are in the same direction resulting in an enhanced effective gravity that tends to accelerate the observed plume velocity. While the results are not conclusive, pending further tests, it clearly points out the inadequacy of the MSFC magnet for conducting protein crystallization experiments and the need for a stronger magnet. In spacebased experiments, however, where the gravitational effects are small, only a weak magnetic field will be required to control or mitigate the effects of convective contamination.

Expression of P53 protein after exposure to ionizing radiation

NASA Astrophysics Data System (ADS)

Salazar, A. M.; Salvador, C.; Ruiz-Trejo, C.; Ostrosky, P.; Brandan, M. E.

2001-10-01

One of the most important tumor suppressor genes is p53 gene, which is involved in apoptotic cell death, cell differentiation and cell cycle arrest. The expression of p53 gene can be evaluated by determining the presence of P53 protein in cells using Western Blot assay with a chemiluminescent method. This technique has shown variabilities that are due to biological factors. Film developing process can influence the quality of the p53 bands obtained. We irradiated tumor cell lines and human peripheral lymphocytes with 137Cs and 60Co gamma rays to standardize irradiation conditions, to compare ionizing radiation with actinomycin D and to reduce the observed variability of P53 protein induction levels. We found that increasing radiation doses increase P53 protein induction while it decreases viability. We also conclude that ionizing radiation could serve as a positive control for Western Blot analysis of protein P53. In addition, our results show that the developing process may play an important role in the quality of P53 protein bands and data interpretation.

Quality, functionality, and shelf life of fermented meat and meat products: A review.

PubMed

Kumar, Pavan; Chatli, M K; Verma, Akhilesh K; Mehta, Nitin; Malav, O P; Kumar, Devendra; Sharma, Neelesh

2017-09-02

Fermentation of meat is a traditional preservation method used widely for improving quality and shelf life of fermented meat products. Fermentation of meat causes a number of physical, biochemical, and microbial changes, which eventually impart functional properties, sensory characteristics, and nutritional aspects to these products and inhibit the growth of various pathogenic and spoilage microorganisms. These changes include acidification (carbohydrate catabolism), solubilization and gelation of myofibrillar and sarcoplasmic proteins of muscle, degradation of proteins and lipids, reduction of nitrate into nitrite, formation of nitrosomyoglobin, and dehydration. Dry-fermented sausages are increasingly being used as carrier of probiotics. The production of biogenic amines during fermentation can be controlled by selecting proper starter cultures and other preventive measures such as quality of raw materials, hygienic measures, temperature, etc.

Impact of 5'-amp-activated Protein Kinase on Male Gonad and Spermatozoa Functions.

PubMed

Nguyen, Thi Mong Diep

2017-01-01

As we already know, the male reproductive system requires less energetic investment than the female one. Nevertheless, energy balance is an important feature for spermatozoa production in the testis and for spermatozoa properties after ejaculation. The 5'-AMP-activated protein kinase, AMPK, is a sensor of cell energy, that regulates many metabolic pathways and that has been recently shown to control spermatozoa quality and functions. It is indeed involved in the regulation of spermatozoa quality through its action on the proliferation of testicular somatic cells (Sertoli and Leydig), on spermatozoa motility and acrosome reaction. It also favors spermatozoa quality through the management of lipid peroxidation and antioxidant enzymes. I review here the most recent data available on the roles of AMPK in vertebrate spermatozoa functions.

In Search of a Cure for Proteostasis-Addicted Cancer: A AAA Target Revealed.

PubMed

Xia, Di; Ye, Yihong

2015-11-09

Tumorigenesis is often associated with an unbalanced protein homeostasis (proteostasis) network, which sensitizes cancer cells to drugs targeting protein quality control (PQC) regulators. In this issue of Cancer Cell, Anderson and colleagues investigated the anti-cancer activity of a new class of inhibitor against a multi-functional ATPase essential for proteostasis maintenance. Copyright © 2015 Elsevier Inc. All rights reserved.

Identification of species- and tissue-specific proteins using proteomic strategy

NASA Astrophysics Data System (ADS)

Chernukha, I. M.; Vostrikova, N. L.; Kovalev, L. I.; Shishkin, S. S.; Kovaleva, M. A.; Manukhin, Y. S.

2017-09-01

Proteomic technologies have proven to be very effective for detecting biochemical changes in meat products, such as changes in tissue- and species-specific proteins. In the tissues of cattle, pig, horse and camel M. longissimus dorsi both tissue- and species specific proteins were detected using two dimensional electrophoresis. Species-specific isoforms of several muscle proteins were also identified. The identified and described proteins of cattle, pig, horse and camel skeletal muscles (including mass spectra of the tryptic peptides) were added to the national free access database “Muscle organ proteomics”. This research has enabled the development of new highly sensitive technologies for meat product quality control against food fraud.

Application of sigma metrics for the assessment of quality control in clinical chemistry laboratory in Ghana: A pilot study.

PubMed

Afrifa, Justice; Gyekye, Seth A; Owiredu, William K B A; Ephraim, Richard K D; Essien-Baidoo, Samuel; Amoah, Samuel; Simpong, David L; Arthur, Aaron R

2015-01-01

Sigma metrics provide a uniquely defined scale with which we can assess the performance of a laboratory. The objective of this study was to assess the internal quality control (QC) in the clinical chemistry laboratory of the University of Cape Cost Hospital (UCC) using the six sigma metrics application. We used commercial control serum [normal (L1) and pathological (L2)] for validation of quality control. Metabolites (glucose, urea, and creatinine), lipids [triglycerides (TG), total cholesterol, high-density lipoprotein cholesterol (HDL-C)], enzymes [alkaline phosphatase (ALP), alanine aminotransferase (AST)], electrolytes (sodium, potassium, chloride) and total protein were assessed. Between-day imprecision (CVs), inaccuracy (Bias) and sigma values were calculated for each control level. Apart from sodium (2.40%, 3.83%), chloride (2.52% and 2.51%) for both L1 and L2 respectively, and glucose (4.82%), cholesterol (4.86%) for L2, CVs for all other parameters (both L1 and L2) were >5%. Four parameters (HDL-C, urea, creatinine and potassium) achieved sigma levels >1 for both controls. Chloride and sodium achieved sigma levels >1 for L1 but <1 for L2. In contrast, cholesterol, total protein and AST achieved sigma levels <1 for L1 but >1 for L2. Glucose and ALP achieved a sigma level >1 for both control levels whereas TG achieved a sigma level >2 for both control levels. Unsatisfactory sigma levels (<3) where achieved for all parameters using both control levels, this shows instability and low consistency of results. There is the need for detailed assessment of the analytical procedures and the strengthening of the laboratory control systems in order to achieve effective six sigma levels for the laboratory.

Application of sigma metrics for the assessment of quality control in clinical chemistry laboratory in Ghana: A pilot study

PubMed Central

Afrifa, Justice; Gyekye, Seth A.; Owiredu, William K. B. A.; Ephraim, Richard K. D.; Essien-Baidoo, Samuel; Amoah, Samuel; Simpong, David L.; Arthur, Aaron R.

2015-01-01

Background: Sigma metrics provide a uniquely defined scale with which we can assess the performance of a laboratory. The objective of this study was to assess the internal quality control (QC) in the clinical chemistry laboratory of the University of Cape Cost Hospital (UCC) using the six sigma metrics application. Materials and Methods: We used commercial control serum [normal (L1) and pathological (L2)] for validation of quality control. Metabolites (glucose, urea, and creatinine), lipids [triglycerides (TG), total cholesterol, high-density lipoprotein cholesterol (HDL-C)], enzymes [alkaline phosphatase (ALP), alanine aminotransferase (AST)], electrolytes (sodium, potassium, chloride) and total protein were assessed. Between-day imprecision (CVs), inaccuracy (Bias) and sigma values were calculated for each control level. Results: Apart from sodium (2.40%, 3.83%), chloride (2.52% and 2.51%) for both L1 and L2 respectively, and glucose (4.82%), cholesterol (4.86%) for L2, CVs for all other parameters (both L1 and L2) were >5%. Four parameters (HDL-C, urea, creatinine and potassium) achieved sigma levels >1 for both controls. Chloride and sodium achieved sigma levels >1 for L1 but <1 for L2. In contrast, cholesterol, total protein and AST achieved sigma levels <1 for L1 but >1 for L2. Glucose and ALP achieved a sigma level >1 for both control levels whereas TG achieved a sigma level >2 for both control levels. Conclusion: Unsatisfactory sigma levels (<3) where achieved for all parameters using both control levels, this shows instability and low consistency of results. There is the need for detailed assessment of the analytical procedures and the strengthening of the laboratory control systems in order to achieve effective six sigma levels for the laboratory. PMID:25657495

Can organic and transgenic soy be used as a substitute for animal protein by rats?

PubMed

Soares, L L; Lucas, A M M; Boaventura, G T

2005-04-01

We evaluated the protein quality of organic and transgenic soy fed to rats throughout life. Thirty female Wistar rats were divided into three groups (N = 10): organic soy group (OSG) receiving organic soy-based diet, genetically modified soy group (GMSG) receiving transgenic soy-based diet, and a control group (CG) receiving casein-based diet. All animals received water and isocaloric diet (10% protein), ad libitum for 291 days. After this, the weight of GMSG animals (290.9 +/- 9.1 g) was significantly lower (P <= 0.04) than CG (323.2 +/- 7.9 g). The weight of OSG (302.2 +/- 8.7 g) was between that of the GMSG and the CG. Protein intake was similar for OSG (308.4 +/- 6.8 g) and GMSG (301.5 +/- 2.5 g), and significantly lower (P <= 0.0005) than the CG (358.4 +/- 8.1 g). Growth rate was similar for all groups: OSG (0.80 +/- 0.02 g), GMSG (0.81 +/- 0.03 g) and CG (0.75 +/- 0.02 g). In addition to providing a good protein intake and inducing less weight gain, both types of soy were utilized in a manner similar to that of casein, suggesting that the protein quality of soy is similar to that of the standard protein casein. The groups fed soy-based diet gained less weight, which may be considered to be beneficial for health. We conclude that organic and transgenic soy can be fed throughout life to rats in place of animal protein, because contain high quality protein and do not cause a marked increase in body weight.

Deg proteases and their role in protein quality control and processing in different subcellular compartments of the plant cell.

PubMed

Schuhmann, Holger; Adamska, Iwona

2012-05-01

Degradation of periplasmic proteins (Deg)/high temperature requirement A (HtrA) proteases are ATP-independent serine endopeptidases found in almost every organism. Database searches revealed that 16 Deg paralogues are encoded by the genome of Arabidopsis thaliana, six of which were experimentally shown to be located in chloroplasts, one in peroxisomes, one in mitochondria and one in the nucleus. Two more Deg proteases are predicted to reside in chloroplasts, five in mitochondria (one of them with a dual chloroplastidial/mitochondrial localization) and the subcellular location of one protein is uncertain. This review summarizes the current knowledge on the role of Deg proteases in maintaining protein homeostasis and protein processing in various subcompartments of the plant cell. The chloroplast Deg proteases are the best examined so far, especially with respect to their role in the degradation of photodamaged photosynthetic proteins and in biogenesis of photosystem II (PSII). A combined action of thylakoid lumen and stroma Deg proteases in the primary cleavage of photodamaged D1 protein from PSII reaction centre is discussed on the basis of a recently resolved crystal structure of plant Deg1. The peroxisomal Deg protease is a processing enzyme responsible for the cleavage of N-terminal peroxisomal targeting signals (PTSs). A. thaliana mutants lacking this enzyme show reduced peroxisomal β-oxidation, indicating for the first time the impact of protein processing on peroxisomal functions in plants. Much less data is available for mitochondrial and nuclear Deg proteases. Based on the available expression data we hypothesize a role in general protein quality control and during acquired heat resistance. Copyright © Physiologia Plantarum 2011.

Genetic control of wheat quality: interactions between chromosomal regions determining protein content and composition, dough rheology, and sponge and dough baking properties.

PubMed

Mann, Gulay; Diffey, Simon; Cullis, Brian; Azanza, Fermin; Martin, David; Kelly, Alison; McIntyre, Lynne; Schmidt, Adele; Ma, Wujun; Nath, Zena; Kutty, Ibrahim; Leyne, P Emmett; Rampling, Lynette; Quail, Ken J; Morell, Matthew K

2009-05-01

While the genetic control of wheat processing characteristics such as dough rheology is well understood, limited information is available concerning the genetic control of baking parameters, particularly sponge and dough (S&D) baking. In this study, a quantitative trait loci (QTL) analysis was performed using a population of doubled haploid lines derived from a cross between Australian cultivars Kukri x Janz grown at sites across different Australian wheat production zones (Queensland in 2001 and 2002 and Southern and Northern New South Wales in 2003) in order to examine the genetic control of protein content, protein expression, dough rheology and sponge and dough baking performance. The study highlighted the inconsistent genetic control of protein content across the test sites, with only two loci (3A and 7A) showing QTL at three of the five sites. Dough rheology QTL were highly consistent across the 5 sites, with major effects associated with the Glu-B1 and Glu-D1 loci. The Glu-D1 5 + 10 allele had consistent effects on S&D properties across sites; however, there was no evidence for a positive effect of the high dough strength Glu-B1-al allele at Glu-B1. A second locus on 5D had positive effects on S&D baking at three of five sites. This study demonstrated that dough rheology measurements were poor predictors of S&D quality. In the absence of robust predictive tests, high heritability values for S&D demonstrate that direct selection is the current best option for achieving genetic gain in this product category.

ProXL (Protein Cross-Linking Database): A Platform for Analysis, Visualization, and Sharing of Protein Cross-Linking Mass Spectrometry Data

PubMed Central

2016-01-01

ProXL is a Web application and accompanying database designed for sharing, visualizing, and analyzing bottom-up protein cross-linking mass spectrometry data with an emphasis on structural analysis and quality control. ProXL is designed to be independent of any particular software pipeline. The import process is simplified by the use of the ProXL XML data format, which shields developers of data importers from the relative complexity of the relational database schema. The database and Web interfaces function equally well for any software pipeline and allow data from disparate pipelines to be merged and contrasted. ProXL includes robust public and private data sharing capabilities, including a project-based interface designed to ensure security and facilitate collaboration among multiple researchers. ProXL provides multiple interactive and highly dynamic data visualizations that facilitate structural-based analysis of the observed cross-links as well as quality control. ProXL is open-source, well-documented, and freely available at https://github.com/yeastrc/proxl-web-app. PMID:27302480

ProXL (Protein Cross-Linking Database): A Platform for Analysis, Visualization, and Sharing of Protein Cross-Linking Mass Spectrometry Data.

PubMed

Riffle, Michael; Jaschob, Daniel; Zelter, Alex; Davis, Trisha N

2016-08-05

ProXL is a Web application and accompanying database designed for sharing, visualizing, and analyzing bottom-up protein cross-linking mass spectrometry data with an emphasis on structural analysis and quality control. ProXL is designed to be independent of any particular software pipeline. The import process is simplified by the use of the ProXL XML data format, which shields developers of data importers from the relative complexity of the relational database schema. The database and Web interfaces function equally well for any software pipeline and allow data from disparate pipelines to be merged and contrasted. ProXL includes robust public and private data sharing capabilities, including a project-based interface designed to ensure security and facilitate collaboration among multiple researchers. ProXL provides multiple interactive and highly dynamic data visualizations that facilitate structural-based analysis of the observed cross-links as well as quality control. ProXL is open-source, well-documented, and freely available at https://github.com/yeastrc/proxl-web-app .

High-Fructose Consumption Impairs the Redox System and Protein Quality Control in the Brain of Syrian Hamsters: Therapeutic Effects of Melatonin.

PubMed

Bermejo-Millo, Juan Carlos; Guimarães, Marcela Rodrigues Moreira; de Luxán-Delgado, Beatriz; Potes, Yaiza; Pérez-Martínez, Zulema; Díaz-Luis, Andrea; Caballero, Beatriz; Solano, Juan José; Vega-Naredo, Ignacio; Coto-Montes, Ana

2018-02-28

Although numerous studies have demonstrated the harmful effect of excessive fructose consumption at the systemic level, there is little information on its effects in the central nervous system. The purpose of the present work was to study the cellular alterations related to oxidative stress and protein quality control systems induced by a high-fructose diet in the brain of Syrian hamsters and their possible attenuation by exogenous melatonin. High-fructose intake induced type II diabetes together with oxidative damage, led to alterations of the unfolded protein response by activating the eIF2α branch, and impaired the macroautophagic machinery in the brain, favoring the accumulation of aggregates labeled for selective degradation and neurodegeneration markers such as β-amyloid (1-42), tau-p-S199, and tau-p-S404. Melatonin attenuated the manifestation of type II diabetes and reduced oxidative stress, deactivated eIF2α, and decreased tau-p-S404 levels in the brain of animals fed a high-fructose diet.

A Rationale for System-Dependent Advantages and Disadvantages of Solution Crystal Growth at Low Gravity

NASA Technical Reports Server (NTRS)

Rosenberger, Franz; Vekilov, Peter G.; Lin, Hong; Alexander, J. Iwan D.

1997-01-01

Protein crystallization experiments at reduced gravity have yielded crystals that, depending on the specific material, are either superior or inferior in their structural perfection compared to counterparts grown at normal gravity. A reduction of the crystals' quality due to their growth at low gravity cannot be understood from existing models. Our experimental investigations of the ground-based crystallization of the protein lysozyme have revealed pronounced unsteady growth layer dynamics and associated defect formation under steady external conditions. Through scaling analysis and numerical simulations we show that the observed fluctuations originate from the coupling of bulk transport with non-linear interface kinetics under mixed kinetics-transport control of the growth rate. The amplitude of the fluctuations is smallest when either transport or interfacial kinetics dominate the control of the crystallization process. Thus, depending on the specific system, crystal quality may be improved by either enhancing or suppressing the transport in the solution. These considerations provide, for the first time, a material-dependent rationale for the advantages, as well as the disadvantages, of reduced gravity for (protein) crystallization.

Protein quality control in the early secretory pathway

PubMed Central

Anelli, Tiziana; Sitia, Roberto

2008-01-01

Eukaryotic cells are able to discriminate between native and non-native polypeptides, selectively transporting the former to their final destinations. Secretory proteins are scrutinized at the endoplasmic reticulum (ER)–Golgi interface. Recent findings reveal novel features of the underlying molecular mechanisms, with several chaperone networks cooperating in assisting the maturation of complex proteins and being selectively induced to match changing synthetic demands. ‘Public' and ‘private' chaperones, some of which enriched in specializes subregions, operate for most or selected substrates, respectively. Moreover, sequential checkpoints are distributed along the early secretory pathway, allowing efficiency and fidelity in protein secretion. PMID:18216874

The rational development of molecularly imprinted polymer-based sensors for protein detection.

PubMed

Whitcombe, Michael J; Chianella, Iva; Larcombe, Lee; Piletsky, Sergey A; Noble, James; Porter, Robert; Horgan, Adrian

2011-03-01

The detection of specific proteins as biomarkers of disease, health status, environmental monitoring, food quality, control of fermenters and civil defence purposes means that biosensors for these targets will become increasingly more important. Among the technologies used for building specific recognition properties, molecularly imprinted polymers (MIPs) are attracting much attention. In this critical review we describe many methods used for imprinting recognition for protein targets in polymers and their incorporation with a number of transducer platforms with the aim of identifying the most promising approaches for the preparation of MIP-based protein sensors (277 references).

Chemical properties and sensory quality of ice cream fortified with fish protein.

PubMed

Shaviklo, Gholam Reza; Thorkelsson, Gudjon; Sveinsdottir, Kolbrun; Rafipour, Fereidon

2011-05-01

Fish protein powder is a functional ingredient that can be used for enhancing the nutritional value of food products. In this study the effect of fortification with different levels of fish protein powder (FP) on chemical properties and sensory quality of Persian ice cream with 0, 30 and 50 g kg(-1) FP during storage at - 18 °C for 4 months was investigated. Ice creams fortified with 50 and 30 g kg(-1) FP had significantly higher protein and solid-non-fat content than ice cream with 0% FP or 83, 69 and 51 g kg(-1) protein and 215, 204 and 181 g kg(-1) solid non-fat, respectively. All products had the same levels of fat, lactose, acidity and pH. They had similar sensory quality after production except for colour, but sensory properties of fortified samples changed significantly after 2 months of storage. Colour faded, cohesiveness decreased, sandiness/coarseness increased, sweetness decreased and fish flavour and off-odour increased. The control ice cream scored highest for additives odour and flavour. Development of ice cream fortified with fish protein powder could be an effective way to enhance nutritional and functional value of ice cream. But studies on storage stability, consumers' acceptance and attitudes are recommended if companies are planning to do so. Copyright © 2011 Society of Chemical Industry.

The Ubiquitin Ligase CHIP Prevents SirT6 Degradation through Noncanonical Ubiquitination

PubMed Central

Ronnebaum, Sarah M.; Wu, Yaxu; McDonough, Holly

2013-01-01

The ubiquitin ligase CHIP (carboxyl terminus of Hsp70-interacting protein) regulates protein quality control, and CHIP deletion accelerates aging and reduces the life span in mice. Here, we reveal a mechanism for CHIP's influence on longevity by demonstrating that CHIP stabilizes the sirtuin family member SirT6, a lysine deacetylase/ADP ribosylase involved in DNA repair, metabolism, and longevity. In CHIP-deficient cells, SirT6 protein half-life is substantially reduced due to increased proteasome-mediated degradation, but CHIP overexpression in these cells increases SirT6 protein expression without affecting SirT6 transcription. CHIP noncanonically ubiquitinates SirT6 at K170, which stabilizes SirT6 and prevents SirT6 canonical ubiquitination by other ubiquitin ligases. In CHIP-depleted cells, SirT6 K170 mutation increases SirT6 half-life and prevents proteasome-mediated degradation. The global decrease in SirT6 expression in the absence of CHIP is associated with decreased SirT6 promoter occupancy, which increases histone acetylation and promotes downstream gene transcription in CHIP-depleted cells. Cells lacking CHIP are hypersensitive to DNA-damaging agents, but DNA repair and cell viability are rescued by enforced expression of SirT6. The discovery of this CHIP-SirT6 interaction represents a novel protein-stabilizing mechanism and defines an intersection between protein quality control and epigenetic regulation to influence pathways that regulate the biology of aging. PMID:24043303

Protein Oxidation and Sensory Quality of Brine-Injected Pork Loins Added Ascorbate or Extracts of Green Tea or Maté during Chill-Storage in High-Oxygen Modified Atmosphere

PubMed Central

Tørngren, Mari Ann

2018-01-01

Background: Ascorbate is often applied to enhance stability and robustness of brine-injected pork chops sold for retail, but may affect protein oxidation, while plant extracts are potential substitutes. Methods: Brine-injected pork chops (weight-gain ~12%, NaCl ~0.9%) prepared with ascorbate (225 ppm), green tea extract (25 ppm gallic acid equivalents (GAE)), or maté extract (25 ppm GAE) stored (5 °C, seven days) in high-oxygen atmosphere packaging (MAP: 80% O2 and 20% CO2) were analyzed for color changes, sensory quality, and protein oxidation compared to a control without antioxidant. Results: No significant differences were observed for green tea and maté extracts as compared to ascorbate when evaluated based on lipid oxidation derived off-flavors, except for stale flavor, which maté significantly reduced. All treatments increased the level of the protein oxidation product, α-aminoadipic semialdehyde as compared to the control, and ascorbate was further found to increase thiol loss and protein cross-linking, with a concomitant decrease in the sensory perceived tenderness. Conclusions: Green tea and maté were found to equally protect against lipid oxidation derived off-flavors, and maté showed less prooxidative activity towards proteins as compared to ascorbate, resulting in more tender meat. Maté is a valuable substitute for ascorbate in brine-injected pork chops. PMID:29342928

Small heat shock proteins protect against {alpha}-synuclein-induced toxicity and aggregation

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

Outeiro, Tiago Fleming; Klucken, Jochen; Strathearn, Katherine E.

Protein misfolding and inclusion formation are common events in neurodegenerative diseases, such as Parkinson's disease (PD), Alzheimer's disease (AD) or Huntington's disease (HD). {alpha}-Synuclein (aSyn) is the main protein component of inclusions called Lewy bodies (LB) which are pathognomic of PD, Dementia with Lewy bodies (DLB), and other diseases collectively known as LB diseases. Heat shock proteins (HSPs) are one class of the cellular quality control system that mediate protein folding, remodeling, and even disaggregation. Here, we investigated the role of the small heat shock proteins Hsp27 and {alpha}B-crystallin, in LB diseases. We demonstrate, via quantitative PCR, that Hsp27 messengermore » RNA levels are {approx}2-3-fold higher in DLB cases compared to control. We also show a corresponding increase in Hsp27 protein levels. Furthermore, we found that Hsp27 reduces aSyn-induced toxicity by {approx}80% in a culture model while {alpha}B-crystallin reduces toxicity by {approx}20%. In addition, intracellular inclusions were immunopositive for endogenous Hsp27, and overexpression of this protein reduced aSyn aggregation in a cell culture model.« less

Parkin and PINK1 function in a vesicular trafficking pathway regulating mitochondrial quality control

PubMed Central

McLelland, Gian-Luca; Soubannier, Vincent; Chen, Carol X; McBride, Heidi M; Fon, Edward A

2014-01-01

Mitochondrial dysfunction has long been associated with Parkinson's disease (PD). Parkin and PINK1, two genes associated with familial PD, have been implicated in the degradation of depolarized mitochondria via autophagy (mitophagy). Here, we describe the involvement of parkin and PINK1 in a vesicular pathway regulating mitochondrial quality control. This pathway is distinct from canonical mitophagy and is triggered by the generation of oxidative stress from within mitochondria. Wild-type but not PD-linked mutant parkin supports the biogenesis of a population of mitochondria-derived vesicles (MDVs), which bud off mitochondria and contain a specific repertoire of cargo proteins. These MDVs require PINK1 expression and ultimately target to lysosomes for degradation. We hypothesize that loss of this parkin- and PINK1-dependent trafficking mechanism impairs the ability of mitochondria to selectively degrade oxidized and damaged proteins leading, over time, to the mitochondrial dysfunction noted in PD. PMID:24446486

Unfolded Protein Response of the Endoplasmic Reticulum in Tumor Progression and Immunogenicity

PubMed Central

Yoo, Yoon Seon; Han, Hye Gyeong

2017-01-01

The endoplasmic reticulum (ER) is a pivotal regulator of folding, quality control, trafficking, and targeting of secreted and transmembrane proteins, and accordingly, eukaryotic cells have evolved specialized machinery to ensure that the ER enables these proteins to acquire adequate folding and maturation in the presence of intrinsic and extrinsic insults. This adaptive capacity of the ER to intrinsic and extrinsic perturbations is important for maintaining protein homeostasis, which is termed proteostasis. Failure in adaptation to these perturbations leads to accumulation of misfolded or unassembled proteins in the ER, which is termed ER stress, resulting in the activation of unfolded protein response (UPR) of the ER and the execution of ER-associated degradation (ERAD) to restore homeostasis. Furthermore, both of the two axes play key roles in the control of tumor progression, inflammation, immunity, and aging. Therefore, understanding UPR of the ER and subsequent ERAD will provide new insights into the pathogenesis of many human diseases and contribute to therapeutic intervention in these diseases. PMID:29430279

Programming the composition of polymer blend particles for controlled immunity towards individual protein antigens.

PubMed

Zhan, Xi; Shen, Hong

2015-05-28

In order for a more precise control over the quality and quantity of immune responses stimulated by synthetic particle-based vaccines, it is critical to control the colloidal stability of particles and the release of protein antigens in both extracellular space and intracellular compartments. Different proteins exhibit different sizes, charges and solubilities. This study focused on modulating the release and colloidal stability of proteins with varied isoelectric points. A polymer particle delivery platform made from the blend of three polymers, poly(lactic-co-glycolic acid) (PLGA) and two random pH-sensitive copolymers, were developed. Our study demonstrated its programmability with respective to individual proteins. We showed the colloidal stability of particles at neutral environment and the release of each individual protein at different pH environments were dependent on the ratio of two charge polymers. Subsequently, two antigenic proteins, ovalbumin (OVA) and Type 2 Herpes Simplex Virus (HSV-2) glycoprotein D (gD) protein, were incorporated into particles with systematically varied compositions. We demonstrated that the level of in vitro CD8(+) T cell and in vivo immune responses were dependent on the ratio of two charged polymers, which correlated well with the release of proteins. This study provided a promising design framework of pH-responsive synthetic vaccines for protein antigens of interest. Copyright © 2015 Elsevier Ltd. All rights reserved.

Ataxia and hypogonadism caused by the loss of ubiquitin ligase activity of the U box protein CHIP.

PubMed

Shi, Chang-He; Schisler, Jonathan C; Rubel, Carrie E; Tan, Song; Song, Bo; McDonough, Holly; Xu, Lei; Portbury, Andrea L; Mao, Cheng-Yuan; True, Cadence; Wang, Rui-Hao; Wang, Qing-Zhi; Sun, Shi-Lei; Seminara, Stephanie B; Patterson, Cam; Xu, Yu-Ming

2014-02-15

Gordon Holmes syndrome (GHS) is a rare Mendelian neurodegenerative disorder characterized by ataxia and hypogonadism. Recently, it was suggested that disordered ubiquitination underlies GHS though the discovery of exome mutations in the E3 ligase RNF216 and deubiquitinase OTUD4. We performed exome sequencing in a family with two of three siblings afflicted with ataxia and hypogonadism and identified a homozygous mutation in STUB1 (NM_005861) c.737C→T, p.Thr246Met, a gene that encodes the protein CHIP (C-terminus of HSC70-interacting protein). CHIP plays a central role in regulating protein quality control, in part through its ability to function as an E3 ligase. Loss of CHIP function has long been associated with protein misfolding and aggregation in several genetic mouse models of neurodegenerative disorders; however, a role for CHIP in human neurological disease has yet to be identified. Introduction of the Thr246Met mutation into CHIP results in a loss of ubiquitin ligase activity measured directly using recombinant proteins as well as in cell culture models. Loss of CHIP function in mice resulted in behavioral and reproductive impairments that mimic human ataxia and hypogonadism. We conclude that GHS can be caused by a loss-of-function mutation in CHIP. Our findings further highlight the role of disordered ubiquitination and protein quality control in the pathogenesis of neurodegenerative disease and demonstrate the utility of combining whole-exome sequencing with molecular analyses and animal models to define causal disease polymorphisms.

Ataxia and hypogonadism caused by the loss of ubiquitin ligase activity of the U box protein CHIP

PubMed Central

Shi, Chang-He; Schisler, Jonathan C.; Rubel, Carrie E.; Tan, Song; Song, Bo; McDonough, Holly; Xu, Lei; Portbury, Andrea L.; Mao, Cheng-Yuan; True, Cadence; Wang, Rui-Hao; Wang, Qing-Zhi; Sun, Shi-Lei; Seminara, Stephanie B.; Patterson, Cam; Xu, Yu-Ming

2014-01-01

Gordon Holmes syndrome (GHS) is a rare Mendelian neurodegenerative disorder characterized by ataxia and hypogonadism. Recently, it was suggested that disordered ubiquitination underlies GHS though the discovery of exome mutations in the E3 ligase RNF216 and deubiquitinase OTUD4. We performed exome sequencing in a family with two of three siblings afflicted with ataxia and hypogonadism and identified a homozygous mutation in STUB1 (NM_005861) c.737C→T, p.Thr246Met, a gene that encodes the protein CHIP (C-terminus of HSC70-interacting protein). CHIP plays a central role in regulating protein quality control, in part through its ability to function as an E3 ligase. Loss of CHIP function has long been associated with protein misfolding and aggregation in several genetic mouse models of neurodegenerative disorders; however, a role for CHIP in human neurological disease has yet to be identified. Introduction of the Thr246Met mutation into CHIP results in a loss of ubiquitin ligase activity measured directly using recombinant proteins as well as in cell culture models. Loss of CHIP function in mice resulted in behavioral and reproductive impairments that mimic human ataxia and hypogonadism. We conclude that GHS can be caused by a loss-of-function mutation in CHIP. Our findings further highlight the role of disordered ubiquitination and protein quality control in the pathogenesis of neurodegenerative disease and demonstrate the utility of combining whole-exome sequencing with molecular analyses and animal models to define causal disease polymorphisms. PMID:24113144

Physiological and environmental control of yeast prions

PubMed Central

Chernova, Tatiana A.; Wilkinson, Keith D.; Chernoff, Yury O.

2014-01-01

Prions are self-perpetuating protein isoforms that cause fatal and incurable neurodegenerative disease in mammals. Recent evidence indicates that a majority of human proteins involved in amyloid and neural inclusion disorders possess at least some prion properties. In lower eukaryotes, such as yeast, prions act as epigenetic elements, which increase phenotypic diversity by altering a range of cellular processes. While some yeast prions are clearly pathogenic, it is also postulated that prion formation could be beneficial in variable environmental conditions. Yeast and mammalian prions have similar molecular properties. Crucial cellular factors and conditions influencing prion formation and propagation were uncovered in the yeast models. Stress-related chaperones, protein quality control deposits, degradation pathways and cytoskeletal networks control prion formation and propagation in yeast. Environmental stresses trigger prion formation and loss, supposedly acting via influencing intracellular concentrations of the prion-inducing proteins, and/or by localizing prionogenic proteins to the prion induction sites via heterologous ancillary helpers. Physiological and environmental modulation of yeast prions points to new opportunities for pharmacological intervention and/or prophylactic measures targeting general cellular systems rather than the properties of individual amyloids and prions. PMID:24236638

TRIM Family Proteins: Roles in Autophagy, Immunity, and Carcinogenesis.

PubMed

Hatakeyama, Shigetsugu

2017-04-01

Tripartite motif (TRIM) family proteins, most of which have E3 ubiquitin ligase activities, have various functions in cellular processes including intracellular signaling, development, apoptosis, protein quality control, innate immunity, autophagy, and carcinogenesis. The ubiquitin system is one of the systems for post-translational modifications, which play crucial roles not only as markers for degradation of target proteins by the proteasome but also as regulators of protein-protein interactions and of the activation of enzymes. Accumulating evidence has shown that TRIM family proteins have unique, important roles and that their dysregulation causes several diseases classified as cancer, immunological disease, or developmental disorders. In this review we focus on recent emerging topics on TRIM proteins in the regulation of autophagy, innate immunity, and carcinogenesis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mitochondria drive autophagy pathology via microtubule disassembly: a new hypothesis for Parkinson disease.

PubMed

Arduíno, Daniela M; Esteves, A Raquel; Cardoso, Sandra Morais

2013-01-01

Neurons are exquisitely dependent on quality control systems to maintain a healthy intracellular environment. A permanent assessment of protein and organelle "quality" allows a coordinated action between repair and clearance of damage proteins and dysfunctional organelles. Impairments in the intracellular clearance mechanisms in long-lived postmitotic cells, like neurons, result in the progressive accumulation of damaged organelles and aggregates of aberrant proteins. Using cells bearing Parkinson disease (PD) patients' mitochondria, we demonstrated that aberrant accumulation of autophagosomes in PD, commonly interpreted as an abnormal induction of autophagy, is instead due to defective autophagic clearance. This defect is a consequence of alterations in the microtubule network driven by mitochondrial dysfunction that hinder mitochondria and autophagosome trafficking. We uncover mitochondria and microtubule-directed traffic as main players in the regulation of autophagy in PD.

A BAG3 chaperone complex maintains cardiomyocyte function during proteotoxic stress

PubMed Central

Judge, Luke M.; Perez-Bermejo, Juan A.; Truong, Annie; Ribeiro, Alexandre J.S.; Yoo, Jennie C.; Jensen, Christina L.; Mandegar, Mohammad A.; Huebsch, Nathaniel; Kaake, Robyn M.; So, Po-Lin; Srivastava, Deepak; Krogan, Nevan J.

2017-01-01

Molecular chaperones regulate quality control in the human proteome, pathways that have been implicated in many diseases, including heart failure. Mutations in the BAG3 gene, which encodes a co-chaperone protein, have been associated with heart failure due to both inherited and sporadic dilated cardiomyopathy. Familial BAG3 mutations are autosomal dominant and frequently cause truncation of the coding sequence, suggesting a heterozygous loss-of-function mechanism. However, heterozygous knockout of the murine BAG3 gene did not cause a detectable phenotype. To model BAG3 cardiomyopathy in a human system, we generated an isogenic series of human induced pluripotent stem cells (iPSCs) with loss-of-function mutations in BAG3. Heterozygous BAG3 mutations reduced protein expression, disrupted myofibril structure, and compromised contractile function in iPSC-derived cardiomyocytes (iPS-CMs). BAG3-deficient iPS-CMs were particularly sensitive to further myofibril disruption and contractile dysfunction upon exposure to proteasome inhibitors known to cause cardiotoxicity. We performed affinity tagging of the endogenous BAG3 protein and mass spectrometry proteomics to further define the cardioprotective chaperone complex that BAG3 coordinates in the human heart. Our results establish a model for evaluating protein quality control pathways in human cardiomyocytes and their potential as therapeutic targets and susceptibility factors for cardiac drug toxicity. PMID:28724793

A BAG3 chaperone complex maintains cardiomyocyte function during proteotoxic stress.

PubMed

Judge, Luke M; Perez-Bermejo, Juan A; Truong, Annie; Ribeiro, Alexandre Js; Yoo, Jennie C; Jensen, Christina L; Mandegar, Mohammad A; Huebsch, Nathaniel; Kaake, Robyn M; So, Po-Lin; Srivastava, Deepak; Pruitt, Beth L; Krogan, Nevan J; Conklin, Bruce R

2017-07-20

Molecular chaperones regulate quality control in the human proteome, pathways that have been implicated in many diseases, including heart failure. Mutations in the BAG3 gene, which encodes a co-chaperone protein, have been associated with heart failure due to both inherited and sporadic dilated cardiomyopathy. Familial BAG3 mutations are autosomal dominant and frequently cause truncation of the coding sequence, suggesting a heterozygous loss-of-function mechanism. However, heterozygous knockout of the murine BAG3 gene did not cause a detectable phenotype. To model BAG3 cardiomyopathy in a human system, we generated an isogenic series of human induced pluripotent stem cells (iPSCs) with loss-of-function mutations in BAG3. Heterozygous BAG3 mutations reduced protein expression, disrupted myofibril structure, and compromised contractile function in iPSC-derived cardiomyocytes (iPS-CMs). BAG3-deficient iPS-CMs were particularly sensitive to further myofibril disruption and contractile dysfunction upon exposure to proteasome inhibitors known to cause cardiotoxicity. We performed affinity tagging of the endogenous BAG3 protein and mass spectrometry proteomics to further define the cardioprotective chaperone complex that BAG3 coordinates in the human heart. Our results establish a model for evaluating protein quality control pathways in human cardiomyocytes and their potential as therapeutic targets and susceptibility factors for cardiac drug toxicity.

Structure and functions of the chaperone-like p97/CDC48 in plants.

PubMed

Bègue, Hervé; Jeandroz, Sylvain; Blanchard, Cécile; Wendehenne, David; Rosnoblet, Claire

2017-01-01

The chaperone-like p97 is a member of the AAA+ ATPase enzyme family that contributes to numerous cellular activities. P97 has been broadly studied in mammals (VCP/p97) and yeasts (CDC48: Cell Division Cycle 48/p97) and numerous investigations highlighted that this protein is post-translationally regulated, is structured in homohexamer and interacts with partners and cofactors that direct it to distinct cellular signalization pathway including protein quality control and degradation, cell cycle regulation, genome stability, vesicular trafficking, autophagy and immunity. p97 is also conserved in plants (CDC48) but its functions are less understood. In the present review we intended to present the state of the art of the structure, regulation and functions of CDC48 in plants. Evidence accumulated underline that CDC48 plays a crucial role in development, cell cycle regulation and protein turnover in plants. Furthermore, its involvement in plant immunity has recently emerged and first interacting partners have been identified, shedding light on its putative cellular activities. Identification of emerging functions of CDC48 in plants opens new roads of research in immunity and provides new insights into the mechanisms of protein quality control. Copyright © 2016 Elsevier B.V. All rights reserved.

Chronic exposure to emissions from photocopiers in copy shops causes oxidative stress and systematic inflammation among photocopier operators in India.

PubMed

Elango, Nithya; Kasi, Vallikkannu; Vembhu, Bhuvaneswari; Poornima, Jeyanthi Govindasamy

2013-09-11

We assessed indoor air quality in photocopier centers and investigated whether occupational exposure to emissions from photocopiers is associated with decline in lung function or changes in haematological parameters, oxidative stress and inflammatory status. Indoor air quality was monitored in five photocopier centers. Pulmonary function was assessed by spirometry in 81 photocopier operators (64 male and 17 female) and 43 healthy control (31 male and 12 female) subjects. Hematological status, serum thio-barbituric acid reactive substances (TBARS), total ferric reducing antioxidant capacity (FRAC), leukotriene B4 (LTB4), 8-isoprostane, C reactive protein (CRP), interleukin 8 (IL-8), clara cell protein (CC-16), intercellular adhesion molecule 1 (ICAM-1) and eosinophilic cationic protein (ECP) were analyzed. Relationships between cumulative exposure, lung function and inflammatory markers were assessed. PM10 and PM2.5 were above the permissible levels in all the photocopier centers, whereas the levels of carbon monoxide, nitrogen oxides, ozone, sulphur dioxide, lead, arsenic, nickel, ammonia, benzene and benzo(a)pyrene were within Indian ambient air quality standards. Lung function was similar in the photocopier operators and control subjects. Serum TBARS was significantly higher and FRAC was lower among photocopier operators when compared to healthy controls. Plasma IL-8, LTB4, ICAM-1 and ECP were significantly higher in the photocopier exposed group. Photocopiers emit high levels of particulate matter. Long term exposure to emissions from photocopiers was not associated with decreased lung function, but resulted in high oxidative stress and systemic inflammation leading to high risk of cardiovascular diseases.

Chronic exposure to emissions from photocopiers in copy shops causes oxidative stress and systematic inflammation among photocopier operators in India

PubMed Central

2013-01-01

Background We assessed indoor air quality in photocopier centers and investigated whether occupational exposure to emissions from photocopiers is associated with decline in lung function or changes in haematological parameters, oxidative stress and inflammatory status. Methods Indoor air quality was monitored in five photocopier centers. Pulmonary function was assessed by spirometry in 81 photocopier operators (64 male and 17 female) and 43 healthy control (31 male and 12 female) subjects. Hematological status, serum thio-barbituric acid reactive substances (TBARS), total ferric reducing antioxidant capacity (FRAC), leukotriene B4 (LTB4), 8-isoprostane, C reactive protein (CRP), interleukin 8 (IL-8), clara cell protein (CC-16), intercellular adhesion molecule 1 (ICAM-1) and eosinophilic cationic protein (ECP) were analyzed. Relationships between cumulative exposure, lung function and inflammatory markers were assessed. Results PM10 and PM2.5 were above the permissible levels in all the photocopier centers, whereas the levels of carbon monoxide, nitrogen oxides, ozone, sulphur dioxide, lead, arsenic, nickel, ammonia, benzene and benzo(a)pyrene were within Indian ambient air quality standards. Lung function was similar in the photocopier operators and control subjects. Serum TBARS was significantly higher and FRAC was lower among photocopier operators when compared to healthy controls. Plasma IL-8, LTB4, ICAM-1 and ECP were significantly higher in the photocopier exposed group. Conclusions Photocopiers emit high levels of particulate matter. Long term exposure to emissions from photocopiers was not associated with decreased lung function, but resulted in high oxidative stress and systemic inflammation leading to high risk of cardiovascular diseases. PMID:24025094

Mapping monomeric threading to protein-protein structure prediction.

PubMed

Guerler, Aysam; Govindarajoo, Brandon; Zhang, Yang

2013-03-25

The key step of template-based protein-protein structure prediction is the recognition of complexes from experimental structure libraries that have similar quaternary fold. Maintaining two monomer and dimer structure libraries is however laborious, and inappropriate library construction can degrade template recognition coverage. We propose a novel strategy SPRING to identify complexes by mapping monomeric threading alignments to protein-protein interactions based on the original oligomer entries in the PDB, which does not rely on library construction and increases the efficiency and quality of complex template recognitions. SPRING is tested on 1838 nonhomologous protein complexes which can recognize correct quaternary template structures with a TM score >0.5 in 1115 cases after excluding homologous proteins. The average TM score of the first model is 60% and 17% higher than that by HHsearch and COTH, respectively, while the number of targets with an interface RMSD <2.5 Å by SPRING is 134% and 167% higher than these competing methods. SPRING is controlled with ZDOCK on 77 docking benchmark proteins. Although the relative performance of SPRING and ZDOCK depends on the level of homology filters, a combination of the two methods can result in a significantly higher model quality than ZDOCK at all homology thresholds. These data demonstrate a new efficient approach to quaternary structure recognition that is ready to use for genome-scale modeling of protein-protein interactions due to the high speed and accuracy.

Health effects of protein intake in healthy adults: a systematic literature review

PubMed Central

Pedersen, Agnes N.; Kondrup, Jens; Børsheim, Elisabet

2013-01-01

The purpose of this systematic review is to assess the evidence behind the dietary requirement of protein and to assess the health effects of varying protein intake in healthy adults. The literature search covered the years 2000–2011. Prospective cohort, case-control, and intervention studies were included. Out of a total of 5,718 abstracts, 412 full papers were identified as potentially relevant, and after careful scrutiny, 64 papers were quality graded as A (highest), B, or C. The grade of evidence was classified as convincing, probable, suggestive or inconclusive. The evidence is assessed as: probable for an estimated average requirement of 0.66 g good-quality protein/kg body weight (BW)/day based on nitrogen balance studies, suggestive for a relationship between increased all-cause mortality risk and long-term low-carbohydrate–high-protein (LCHP) diets; but inconclusive for a relationship between all-cause mortality risk and protein intake per se; suggestive for an inverse relationship between cardiovascular mortality and vegetable protein intake; inconclusive for relationships between cancer mortality and cancer diseases, respectively, and protein intake; inconclusive for a relationship between cardiovascular diseases and total protein intake; suggestive for an inverse relationship between blood pressure (BP) and vegetable protein; probable to convincing for an inverse relationship between soya protein intake and LDL cholesterol; inconclusive for a relationship between protein intake and bone health, energy intake, BW control, body composition, renal function, and risk of kidney stones, respectively; suggestive for a relationship between increased risk of type 2 diabetes (T2D) and long-term LCHP-high-fat diets; inconclusive for impact of physical training on protein requirement; and suggestive for effect of physical training on whole-body protein retention. In conclusion, the evidence is assessed as probable regarding the estimated requirement based on nitrogen balance studies, and suggestive to inconclusive for protein intake and mortality and morbidity. Vegetable protein intake was associated with decreased risk in many studies. Potentially adverse effects of a protein intake exceeding 20–23 E% remain to be investigated. PMID:23908602

Bap31 enhances the ER export and quality control of human class I MHC molecules

PubMed Central

Ladasky, John J.; Boyle, Sarah; Seth, Malini; Li, Hewang; Pentcheva, Tsvetelina; Abe, Fumiyoshi; Steinberg, Steven J.; Edidin, Michael

2006-01-01

The assembly of class I MHC molecules and their export from the endoplasmic reticulum is governed by chaperones and accessory proteins. We present evidence that the putative cargo receptor protein Bap31 participates in the transport and the quality control of human class I molecules. Transfection of the human adenocarcinoma cell line HeLa with YFP-Bap31 chimeras increased surface levels of class I in a dose-dependent manner, by as much as 3.7-fold. The increase in surface class I resulted from an increase in the rate of export of newly-synthesized class I molecules to the cell surface and from an increase in the stability of the exported molecules. We propose that Bap31 performs quality control on class I molecules in two distinct phases: first, by exporting peptide-loaded class I molecules to the ERGIC and second, by retrieving class I molecules which have lost peptides in the acidic post-ER environment. This function of Bap31 is conditional or redundant, since we find that Bap31 deficiency does not reduce surface class I levels. Overexpression of the Bap31 homolog, Bap29, decreases surface class levels in HeLa, indicating that it does not substitute for Bap31. PMID:17056546

Improving transcriptome construction in non-model organisms: integrating manual and automated gene definition in Emiliania huxleyi.

PubMed

Feldmesser, Ester; Rosenwasser, Shilo; Vardi, Assaf; Ben-Dor, Shifra

2014-02-22

The advent of Next Generation Sequencing technologies and corresponding bioinformatics tools allows the definition of transcriptomes in non-model organisms. Non-model organisms are of great ecological and biotechnological significance, and consequently the understanding of their unique metabolic pathways is essential. Several methods that integrate de novo assembly with genome-based assembly have been proposed. Yet, there are many open challenges in defining genes, particularly where genomes are not available or incomplete. Despite the large numbers of transcriptome assemblies that have been performed, quality control of the transcript building process, particularly on the protein level, is rarely performed if ever. To test and improve the quality of the automated transcriptome reconstruction, we used manually defined and curated genes, several of them experimentally validated. Several approaches to transcript construction were utilized, based on the available data: a draft genome, high quality RNAseq reads, and ESTs. In order to maximize the contribution of the various data, we integrated methods including de novo and genome based assembly, as well as EST clustering. After each step a set of manually curated genes was used for quality assessment of the transcripts. The interplay between the automated pipeline and the quality control indicated which additional processes were required to improve the transcriptome reconstruction. We discovered that E. huxleyi has a very high percentage of non-canonical splice junctions, and relatively high rates of intron retention, which caused unique issues with the currently available tools. While individual tools missed genes and artificially joined overlapping transcripts, combining the results of several tools improved the completeness and quality considerably. The final collection, created from the integration of several quality control and improvement rounds, was compared to the manually defined set both on the DNA and protein levels, and resulted in an improvement of 20% versus any of the read-based approaches alone. To the best of our knowledge, this is the first time that an automated transcript definition is subjected to quality control using manually defined and curated genes and thereafter the process is improved. We recommend using a set of manually curated genes to troubleshoot transcriptome reconstruction.

Proteomic analysis of differentially expressed proteins in kidneys of brain dead rabbits.

PubMed

Li, Ling; Li, Ning; He, Chongxiang; Huang, Wei; Fan, Xiaoli; Zhong, Zibiao; Wang, Yanfeng; Ye, Qifa

2017-07-01

A large number of previous clinical studies have reported a delayed graft function for brain dead donors, when compared with living relatives or cadaveric organ transplantations. However, there is no accurate method for the quality evaluation of kidneys from brain‑dead donors. In the present study, two‑dimensional gel electrophoresis and MALDI‑TOF MS‑based comparative proteomic analysis were conducted to profile the differentially‑expressed proteins between brain death and the control group renal tissues. A total of 40 age‑ and sex‑matched rabbits were randomly divided into donation following brain death (DBD) and control groups. Following the induction of brain death via intracranial progressive pressure, the renal function and the morphological alterations were measured 2, 6 and 8 h afterwards. The differentially expressed proteins were detected from renal histological evidence at 6 h following brain death. Although 904±19 protein spots in control groups and 916±25 in DBD groups were identified in the two‑dimensional gel electrophoresis, >2‑fold alterations were identified by MALDI‑TOF MS and searched by NCBI database. The authors successfully acquired five downregulated proteins, these were: Prohibitin (isoform CRA_b), beta-1,3‑N-acetylgalactosaminyltransferase 1, Annexin A5, superoxide dismutase (mitochondrial) and cytochrome b‑c1 complex subunit 1 (mitochondrial precursor). Conversely, the other five upregulated proteins were: PRP38 pre‑mRNA processing factor 38 (yeast) domain containing A, calcineurin subunit B type 1, V‑type proton ATPase subunit G 1, NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 10 and peroxiredoxin‑3 (mitochondrial). Immunohistochemical results revealed that the expressions of prohibitin (PHB) were gradually increased in a time‑dependent manner. The results indicated that there were alterations in levels of several proteins in the kidneys of those with brain death, even if the primary function and the morphological changes were not obvious. PHB may therefore be a novel biomarker for primary quality evaluation of kidneys from brain‑dead donors.

Proteomic analysis of differentially expressed proteins in kidneys of brain dead rabbits

PubMed Central

Li, Ling; Li, Ning; He, Chongxiang; Huang, Wei; Fan, Xiaoli; Zhong, Zibiao; Wang, Yanfeng; Ye, Qifa

2017-01-01

A large number of previous clinical studies have reported a delayed graft function for brain dead donors, when compared with living relatives or cadaveric organ transplantations. However, there is no accurate method for the quality evaluation of kidneys from brain-dead donors. In the present study, two-dimensional gel electrophoresis and MALDI-TOF MS-based comparative proteomic analysis were conducted to profile the differentially-expressed proteins between brain death and the control group renal tissues. A total of 40 age- and sex-matched rabbits were randomly divided into donation following brain death (DBD) and control groups. Following the induction of brain death via intracranial progressive pressure, the renal function and the morphological alterations were measured 2, 6 and 8 h afterwards. The differentially expressed proteins were detected from renal histological evidence at 6 h following brain death. Although 904±19 protein spots in control groups and 916±25 in DBD groups were identified in the two-dimensional gel electrophoresis, >2-fold alterations were identified by MALDI-TOF MS and searched by NCBI database. The authors successfully acquired five downregulated proteins, these were: Prohibitin (isoform CRA_b), beta-1,3-N-acetylgalactosaminyltransferase 1, Annexin A5, superoxide dismutase (mitochondrial) and cytochrome b-c1 complex subunit 1 (mitochondrial precursor). Conversely, the other five upregulated proteins were: PRP38 pre-mRNA processing factor 38 (yeast) domain containing A, calcineurin subunit B type 1, V-type proton ATPase subunit G 1, NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 10 and peroxiredoxin-3 (mitochondrial). Immunohistochemical results revealed that the expressions of prohibitin (PHB) were gradually increased in a time-dependent manner. The results indicated that there were alterations in levels of several proteins in the kidneys of those with brain death, even if the primary function and the morphological changes were not obvious. PHB may therefore be a novel biomarker for primary quality evaluation of kidneys from brain-dead donors. PMID:28534953

Protein and amino acid bioavailability of extruded dog food with protein meals of different quality using growing mink () as a model.

PubMed

Tjernsbekk, M T; Tauson, A-H; Matthiesen, C F; Ahlstrøm, Ø

2016-09-01

The present study evaluated growing mink () as a model for dietary protein quality assessment of protein meals used in extruded dog foods. Three foods with similar CP content but of different protein quality were produced using different protein meals. The protein meals varied with respect to CP digestibility and AA composition and included lamb meal (LBM), poultry meal (PM), and fish meal (FM) with low, intermediate, and high protein quality, respectively. Nitrogen balance, BW gain, protein efficiency ratio (PER), and apparent total tract digestibility (ATTD) were used as measures of protein and AA bioavailability in growing mink. Standardized ileal digestibility (SID) was used to measure protein and AA bioavailability in adult dogs (). The mink study (3 × 3 Latin square design) included 12 kits aged 8 to 11 wk. The dog study included 12 dogs divided in 3 groups allocated to 1 of the experimental diets. The growing mink responded in accordance with the different AA supply between diets, as determined by the first limiting AA. The LBM diet deviated from the other diets with lower ( < 0.001) values for N retention, BW gain, and PER, and the diets differed ( < 0.001) in ATTD of CP and all AA, except for hydroxyproline. Retention of N was 0.66, 1.04, and 1.18 g·kg·d; BW gain was 8.2, 26.8, and 35.3 g/d; PER was 0.38, 1.39, and 1.71; and ATTD of CP was 66.8, 73.8, and 82.1% for the LBM, PM, and FM diets, respectively. In dogs, SID of CP and AA differed ( ≤ 0.017) between diets and was generally lowest for the LBM diet, intermediate for the PM diet, and greatest for the FM diet. For CP, SID was 71.5, 80.2, and 87.0% for the LBM, PM, and FM diets, respectively. The contents of digestible CP and AA (based on SID) covered the minimal requirement for adult dogs set by the NRC for all diets, except for the content of digestible Met + Cys in the LBM diet. Despite this, dietary content of Met + Cys in the LBM diet agreed with the recommended level set by the NRC and the Association of American Feed Control Officials for adult dogs but was below the level recommended by the European Pet Food Industry Federation. It was concluded that growth studies with mink kits can provide valuable information in protein quality assessment of extruded dog foods. Furthermore, the study showed that to ensure nutritional adequacy of dog food and to be able to compare protein quality of dog foods, information on AA composition and digestibility is crucial.

Biopharmaceutical production: Applications of surface plasmon resonance biosensors.

PubMed

Thillaivinayagalingam, Pranavan; Gommeaux, Julien; McLoughlin, Michael; Collins, David; Newcombe, Anthony R

2010-01-15

Surface plasmon resonance (SPR) permits the quantitative analysis of therapeutic antibody concentrations and impurities including bacteria, Protein A, Protein G and small molecule ligands leached from chromatography media. The use of surface plasmon resonance has gained popularity within the biopharmaceutical industry due to the automated, label free, real time interaction that may be exploited when using this method. The application areas to assess protein interactions and develop analytical methods for biopharmaceutical downstream process development, quality control, and in-process monitoring are reviewed. 2009 Elsevier B.V. All rights reserved.

Microgravity

NASA Image and Video Library

2004-04-15

The Commercial Vapor Diffusion Apparatus will be used to perform 128 individual crystal growth investigations for commercial and science research. These experiments will grow crystals of several different proteins, including HIV-1 Protease Inhibitor, Glycogen Phosphorylase A, and NAD Synthetase. The Commercial Vapor Diffusion Apparatus supports multiple commercial investigations within a controlled environment. The goal of the Commercial Protein Crystal Growth payload on STS-95 is to grow large, high-quality crystals of several different proteins of interest to industry, and to continue to refine the technology and procedures used in microgravity for this important commercial research.

Impact of Aging and Exercise on Mitochondrial Quality Control in Skeletal Muscle

PubMed Central

Kim, Yuho; Triolo, Matthew

2017-01-01

Mitochondria are characterized by its pivotal roles in managing energy production, reactive oxygen species, and calcium, whose aging-related structural and functional deteriorations are observed in aging muscle. Although it is still unclear how aging alters mitochondrial quality and quantity in skeletal muscle, dysregulation of mitochondrial biogenesis and dynamic controls has been suggested as key players for that. In this paper, we summarize current understandings on how aging regulates muscle mitochondrial biogenesis, while focusing on transcriptional regulations including PGC-1α, AMPK, p53, mtDNA, and Tfam. Further, we review current findings on the muscle mitochondrial dynamic systems in aging muscle: fusion/fission, autophagy/mitophagy, and protein import. Next, we also discuss how endurance and resistance exercises impact on the mitochondrial quality controls in aging muscle, suggesting possible effective exercise strategies to improve/maintain mitochondrial health. PMID:28656072

Significance of biological resource collection and tumor tissue bank creation.

PubMed

Yu, Ying-Yan; Zhu, Zheng-Gang

2010-01-15

Progress in the molecular oncology of gastrointestinal carcinomas depends on high quality cancer tissues for research. Recent acceleration on new technological platforms as well as the "omics" revolution increases the demands on tissues and peripheral blood for research at the DNA, mRNA and protein levels. Tissue bank creation emerges as a priority. Tumor tissue banks are facilities that are organized to collect, store and distribute samples of tumor and normal tissue for further use in basic and translational cancer research. The samples are generally obtained immediately after excision, prior to fixation, to ensure optimal preservation of proteins and nucleic acids. It is possible for surgeons or pathologists to collect fresh tissue prospectively during their routine dissection procedures. Most tissue banks are "project-driven" tumor banks, which are specialized collections of tumor samples on which their research is based. Systematic collection of all available tumor tissue is much rarer. High quality tissue banks need the collaboration of clinicians and basic scientists, but also the informed consent of patients and ethical approval. Through the standard operation procedure, snap frozen fresh tissue collection, storage and quality control for cryopreserved tissues are the pivotal factors on tissue bank construction and maintaining. The purpose of the tissue bank creation is enhancing the quality and speed on both the basic and translational research on gastrointestinal cancer. The quality assurance and quality control are handled based on reviewing HE staining slides or touch imprint cytology by pathologists.

Nutritional value of milk and meat products derived from cloning.

PubMed

Tomé, Daniel; Dubarry, Michel; Fromentin, Gilles

2004-01-01

The development and use of milk and meat products derived from cloning depends on their safety and on the nutritional advantages they can confer to the products as perceived by consumers. The development of such products thus implies (i) to demonstrate their safety and security, (ii) to show that their nutritional value is equivalent to the traditional products, and (iii) to identify the conditions under which cloning could allow additional nutritional and health benefit in comparison to traditional products for the consumers. Both milk and meat products are a source of high quality protein as determined from their protein content and essential amino acid profile. Milk is a source of calcium, phosphorus, zinc, magnesium and vitamin B2 and B12. Meat is a source of iron, zinc and vitamin B12. An important issue regarding the nutritional quality of meat and milk is the level and quality of fat which usually present a high content in saturated fat and some modification of the fat fraction could improve the nutritional quality of the products. The role of the dietary proteins as potential allergens has to be taken into account and an important aspect regarding this question is to evaluate whether the cloning does not produce the appearance of novel allergenic structures. The presence of bio-activities associated to specific components of milk (lactoferrin, immunoglobulins, growth factors, anti-microbial components) also represents a promising development. Preliminary results obtained in rats fed cow's milk or meat-based diets prepared from control animals or from animals derived from cloning did not show any difference between control and cloning-derived products.

Protein Homeostasis in Amyotrophic Lateral Sclerosis: Therapeutic Opportunities?

PubMed Central

Webster, Christopher P.; Smith, Emma F.; Shaw, Pamela J.; De Vos, Kurt J.

2017-01-01

Protein homeostasis (proteostasis), the correct balance between production and degradation of proteins, is essential for the health and survival of cells. Proteostasis requires an intricate network of protein quality control pathways (the proteostasis network) that work to prevent protein aggregation and maintain proteome health throughout the lifespan of the cell. Collapse of proteostasis has been implicated in the etiology of a number of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), the most common adult onset motor neuron disorder. Here, we review the evidence linking dysfunctional proteostasis to the etiology of ALS and discuss how ALS-associated insults affect the proteostasis network. Finally, we discuss the potential therapeutic benefit of proteostasis network modulation in ALS. PMID:28512398

APID interactomes: providing proteome-based interactomes with controlled quality for multiple species and derived networks

PubMed Central

Alonso-López, Diego; Gutiérrez, Miguel A.; Lopes, Katia P.; Prieto, Carlos; Santamaría, Rodrigo; De Las Rivas, Javier

2016-01-01

APID (Agile Protein Interactomes DataServer) is an interactive web server that provides unified generation and delivery of protein interactomes mapped to their respective proteomes. This resource is a new, fully redesigned server that includes a comprehensive collection of protein interactomes for more than 400 organisms (25 of which include more than 500 interactions) produced by the integration of only experimentally validated protein–protein physical interactions. For each protein–protein interaction (PPI) the server includes currently reported information about its experimental validation to allow selection and filtering at different quality levels. As a whole, it provides easy access to the interactomes from specific species and includes a global uniform compendium of 90,379 distinct proteins and 678,441 singular interactions. APID integrates and unifies PPIs from major primary databases of molecular interactions, from other specific repositories and also from experimentally resolved 3D structures of protein complexes where more than two proteins were identified. For this purpose, a collection of 8,388 structures were analyzed to identify specific PPIs. APID also includes a new graph tool (based on Cytoscape.js) for visualization and interactive analyses of PPI networks. The server does not require registration and it is freely available for use at http://apid.dep.usal.es. PMID:27131791

Efficacy and safety of protein supplements for U.S. Armed Forces personnel: consensus statement.

PubMed

Pasiakos, Stefan M; Austin, Krista G; Lieberman, Harris R; Askew, E Wayne

2013-11-01

To provide evidence-based guidance regarding the efficacy and safety of dietary protein supplement (PS) use by members of the U.S. Armed Forces, a panel of internationally recognized experts in the fields of protein metabolism and dietary supplement research was convened by the Department of Defense Center Alliance for Dietary Supplement Research and the U.S. Army Medical Research and Material Command. To develop a consensus statement, potential benefits, risks, and strategies to optimize military performance through PS use were considered in the context of specific warfighter populations and occupational demands. To maintain muscle mass, strength, and performance during periods of substantial metabolic demand and concomitant negative energy balance the panel recommended that warfighters consume 1.5-2.0 g · kg(-1) · d(-1) of protein. However, if metabolic demand is low, such as in garrison, protein intake should equal the current Military Dietary Reference Intake (0.8-1.5 g · kg(-1) · d(-1)). Although PS use generally appears to be safe for healthy adults, warfighters should be educated on PS quality, given quality-control and contamination concerns with commercial dietary supplements. To achieve recommended protein intakes, the panel strongly urges consumption of high-quality protein-containing whole foods. However, when impractical, the use of PSs (20-25 g per serving or 0.25-0.3 g · kg(-1) per meal), particularly after periods of strenuous physical activity (e.g., military training, combat patrols, and exercise), is acceptable. The committee acknowledges the need for further study of protein requirements for extreme, military-specific environmental conditions and whether unique metabolic stressors associated with military service alter protein requirements for aging warfighters.

An application of mass spectrometry for quality control of biologicals: Highly sensitive profiling of plasma residuals in human plasma-derived immunoglobulin.

PubMed

Limonier, Franck; Van Steendam, Katleen; Waeterloos, Geneviève; Brusselmans, Koen; Sneyers, Myriam; Deforce, Dieter

2017-01-30

Thromboembolic events (TEE) associated to trace amounts of plasmatic activated coagulation factor XI (FXIa) in administrated immunoglobulin (Ig) have recently raised concerns and hence there is a need for highly sensitive profiling of residual plasma source proteins. This study aims to consider LC-ESI-QTOF data-dependent acquisition in combination with sample fractionation for this purpose. Sample fractionation proved mandatory to enable identification of plasma residuals. Two approaches were compared: Ig depletion with protein G - protein A affinity chromatography and low-abundant protein enrichment with a combinatorial peptide ligand library (ProteoMiner™, Bio-Rad). The latter allowed a higher number of identifications. Highly sensitive detection of prothrombotic FXIa was assessed with confident identification of a 1ng/mg spike. Moreover, different residuals compositions were profiled for various commercial Ig products. Using a quantitative label free analysis, a TEE-positive Ig batch was distinguished from other regular Ig products, with increased levels of FXIa but also other unique proteins. This could have prevented the recently observed TEE problems with Ig. The method is a convenient tool to better characterize Ig products after any plasma pool or manufacture process change, gaining insights in the product quality profile without any prior information required. This study characterized residual plasma proteins in Ig products, using bottom-up LC-MS/MS with conventional data-dependent acquisition, preceded by sample fractionation. Without any prior information or target-specific development, >30 proteins were identified in a commercial Ig product. Quality control relevance was demonstrated with the identification of FXIa spiked at 1ng/mg in Ig, which is below the minimal thrombotic dose of 3ng/mg observed in an in vivo model. Relative label-free quantitation highlighted significant differences in normalized abundances of residual proteins between Ig products. A TEE-positive batch was distinguished by unique profile of residual proteins, including FXIa but also various blood stream-regulator proteins (fibrinogen, angiotensinogen, antithrombin-III, complement component C8, …). Those results emphasize that MS screening is a relevant first-line test to prevent any undesired concentration of plasma impurities after a plasma pool or manufacturing process change. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Antibody mechanisms implicated in digestive disturbances following ingestion of soya protein in calves and piglets.

PubMed Central

Barratt, M E; Strachan, P J; Porter, P

1978-01-01

Serum antibody responses to ingested aqueous alcohol-extracted soya proteins were studied in thirty-six pre-ruminant calves. Characterization of this antibody showed it to be predominantly a complement-fixing IgG1 preciptin. No evidence of tolerance was seen; previously sensitized calves responded to reintroduction of a soya diet with marked increases in antibody levels. The soya antigen was shown to be resistant to proteolysis and, to a lesser degree, to the microbial action of rumen fluid. Biopsy studies showed that the feeding of soya protein resulted in morphological disturbances to the villi and lamina propria of the intestine. Physiological studies by Thirty-Vella loop perfusion in the pig showed that soya protein solutions resulted in significant inhibition of flow rates. The effect was only observed after previous sensitization with the soya antigen. This study shows the necessity of applying immunological criteria to the quality control of soya bean processing in order to ensure that the sensitizing agent is eliminated and the nutritional qualities of soya protein concentrates are optimized. Images FIG. 2a FIG. 2b PMID:565686

How and why do toxic conformers of aberrant proteins accumulate during ageing?

PubMed

Josefson, Rebecca; Andersson, Rebecca; Nyström, Thomas

2017-07-15

Ageing can be defined as a gradual decline in cellular and physical functions accompanied by an increased sensitivity to the environment and risk of death. The increased risk of mortality is causally connected to a gradual, intracellular accumulation of so-called ageing factors, of which damaged and aggregated proteins are believed to be one. Such aggregated proteins also contribute to several age-related neurodegenerative disorders e.g. Alzheimer's, Parkinson's, and Huntington's diseases, highlighting the importance of protein quality control (PQC) in ageing and its associated diseases. PQC consists of two interrelated systems: the temporal control system aimed at refolding, repairing, and/or removing aberrant proteins and their aggregates and the spatial control system aimed at harnessing the potential toxicity of aberrant proteins by sequestering them at specific cellular locations. The accumulation of toxic conformers of aberrant proteins during ageing is often declared to be a consequence of an incapacitated temporal PQC system-i.e. a gradual decline in the activity of chaperones and proteases. Here, we review the current knowledge on PQC in relation to ageing and highlight that the breakdown of both temporal and spatial PQC may contribute to ageing and thus comprise potential targets for therapeutic interventions of the ageing process. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Quantitative evaluation of plasma after methylene blue and white light treatment in four Chinese blood centers.

PubMed

Chunhui, Yang; Guohui, Bian; Hong, Yang; Xiaopu, Xiao; Zherong, Bai; Mingyuan, Wang; Xinsheng, Zhang; Juanjuan, Wang; Changqing, Li; Wuping, Li

2013-12-01

Pathogen reduction technology is an important process in the blood safety system, including solvent/detergent treatment, filtration and methylene blue-photochemical technology (MB-PCT). To investigate the quality of MB-PCT-treated plasma, plasma samples from four Chinese blood centers were analyzed over 12 months of storage to determine the recovery of activities and levels of various plasma proteins. Ten plasma units each from the Suzhou, Yancheng, Chongqing and Shandong blood centers were divided into two aliquots. One was subjected to treatment with one of two methylene blue-photochemical technology instruments and the other was used as control. The treated and untreated sample pairs were stored at -30°C. The recovery rates of coagulation factors, inhibitor proteins, total protein, immunoglobulins, and complement proteins were measured at different time points after storage. The mean recovery of most proteins exceeded 80% after MB treatment. The exceptions were factor XI and fibrinogen, of which only 71.3-74% and 69.0-92.3% were retained during storage. The two equipment types differed in terms of residual MB concentration in the plasma samples (0.18 μM and 0.29 μM, respectively). They had similar protein recovery rates at 0.5 month but differed at later time points. The four blood centers differed significantly with regard to factor II, V, VIII and fibrinogen activities. Only the samples from the Shandong blood center met the methylene blue treated fresh frozen plasma requirement. The major factor that influenced the quality of the MB-FFP samples was the time taken between blood collection and storage. Methylene blue treated plasma showed reduced coagulation factor (CF) activity and protein levels. The MB treatment-induced damage to the proteins was acceptable at the four Chinese blood centers, but the quality of the MB-treated plasma in general was not satisfactory. The main factor affecting plasma quality may be the duration of the collection and processing. Copyright © 2013 Elsevier Ltd. All rights reserved.

Proteome analysis of Physcomitrella patens exposed to progressive dehydration and rehydration.

PubMed

Cui, Suxia; Hu, Jia; Guo, Shilei; Wang, Jie; Cheng, Yali; Dang, Xinxing; Wu, Lili; He, Yikun

2012-01-01

Physcomitrella patens is an extremely dehydration-tolerant moss. However, the molecular basis of its responses to loss of cellular water remains unclear. A comprehensive proteomic analysis of dehydration- and rehydration-responsive proteins has been conducted using quantitative two-dimensional difference in-gel electrophoresis (2D-DIGE), and traditional 2-D gel electrophoresis (2-DE) combined with MALDI TOF/TOF MS. Of the 216 differentially-expressed protein spots, 112 and 104 were dehydration- and rehydration-responsive proteins, respectively. The functional categories of the most differentially-expressed proteins were seed maturation, defence, protein synthesis and quality control, and energy production. Strikingly, most of the late embryogenesis abundant (LEA) proteins were expressed at a basal level under control conditions and their synthesis was strongly enhanced by dehydration, a pattern that was confirmed by RT-PCR. Actinoporins, phosphatidylethanolamine-binding protein, arabinogalactan protein, and phospholipase are the likely dominant players in the defence system. In addition, 24 proteins of unknown function were identified as novel dehydration- or rehydration-responsive proteins. Our data indicate that Physcomitrella adopts a rapid protein response mechanism to cope with dehydration in its leafy-shoot and basal expression levels of desiccation-tolerant proteins are rapidly upgraded at high levels under stress. This mechanism appears similar to that seen in angiosperm seeds.

Proteome analysis of Physcomitrella patens exposed to progressive dehydration and rehydration

PubMed Central

Cui, Suxia; Hu, Jia; Guo, Shilei; Wang, Jie; Cheng, Yali; Dang, Xinxing; Wu, Lili; He, Yikun

2012-01-01

Physcomitrella patens is an extremely dehydration-tolerant moss. However, the molecular basis of its responses to loss of cellular water remains unclear. A comprehensive proteomic analysis of dehydration- and rehydration-responsive proteins has been conducted using quantitative two-dimensional difference in-gel electrophoresis (2D-DIGE), and traditional 2-D gel electrophoresis (2-DE) combined with MALDI TOF/TOF MS. Of the 216 differentially-expressed protein spots, 112 and 104 were dehydration- and rehydration-responsive proteins, respectively. The functional categories of the most differentially-expressed proteins were seed maturation, defence, protein synthesis and quality control, and energy production. Strikingly, most of the late embryogenesis abundant (LEA) proteins were expressed at a basal level under control conditions and their synthesis was strongly enhanced by dehydration, a pattern that was confirmed by RT-PCR. Actinoporins, phosphatidylethanolamine-binding protein, arabinogalactan protein, and phospholipase are the likely dominant players in the defence system. In addition, 24 proteins of unknown function were identified as novel dehydration- or rehydration-responsive proteins. Our data indicate that Physcomitrella adopts a rapid protein response mechanism to cope with dehydration in its leafy-shoot and basal expression levels of desiccation-tolerant proteins are rapidly upgraded at high levels under stress. This mechanism appears similar to that seen in angiosperm seeds. PMID:21994173

Nutritional Quality and Physicochemical Characteristics of Defatted Bovine Liver Treated by Supercritical Carbon Dioxide and Organic Solvent

PubMed Central

Kang, Sung-Won; Kim, Hye-Min; Rahman, M. Shafiur; Kim, Ah-Na; Yang, Han-Sul

2017-01-01

Defatted bovine liver (DBL) is a potential source of protein and minerals. Supercritical carbon dioxide (SC-CO2) and a traditional organic solvent method were used to remove lipid from bovine liver, and the quality characteristics of a control bovine liver (CBL), bovine liver defatted by SC-CO2 (DBLSC-CO2) at different pressures, and bovine liver defatted by organic solvent (DBL-OS) were compared. The DBLSC-CO2 samples had significantly higher (p<0.05) protein, amino acid, carbohydrate, and fiber contents than CBL and DBL-OS. There was a higher yield of lipid from CBL when using SC-CO2 than the organic solvent method. SDS-PAGE analysis demonstrated that the CBL and DBLSC-CO2 had protein bands of a similar intensity and area, whereas DBL-OS appeared extremely poor bands or no bands due to the degradation of proteins, particularly in the 50 to 75 kDa and 20 to 25 kDa molecular weight ranges. In addition, DBLSC-CO2 was shown to have superior functional properties in terms of total soluble content, water and oil absorption, and foaming and emulsification properties. Therefore, SC-CO2 treatment offers a nutritionally and environmentally friendly approach for the removal of lipid from high protein food sources. In addition, SC-CO2 may be a better substitute of traditional organic solvent extraction for producing more stable and high quality foods with high-protein, fat-free, and low calorie contents. PMID:28316468

Nutritional Quality and Physicochemical Characteristics of Defatted Bovine Liver Treated by Supercritical Carbon Dioxide and Organic Solvent.

PubMed

Kang, Sung-Won; Kim, Hye-Min; Rahman, M Shafiur; Kim, Ah-Na; Yang, Han-Sul; Choi, Sung-Gil

2017-01-01

Defatted bovine liver (DBL) is a potential source of protein and minerals. Supercritical carbon dioxide (SC-CO 2 ) and a traditional organic solvent method were used to remove lipid from bovine liver, and the quality characteristics of a control bovine liver (CBL), bovine liver defatted by SC-CO 2 (DBLSC-CO 2 ) at different pressures, and bovine liver defatted by organic solvent (DBL-OS) were compared. The DBLSC-CO 2 samples had significantly higher ( p <0.05) protein, amino acid, carbohydrate, and fiber contents than CBL and DBL-OS. There was a higher yield of lipid from CBL when using SC-CO 2 than the organic solvent method. SDS-PAGE analysis demonstrated that the CBL and DBLSC-CO 2 had protein bands of a similar intensity and area, whereas DBL-OS appeared extremely poor bands or no bands due to the degradation of proteins, particularly in the 50 to 75 kDa and 20 to 25 kDa molecular weight ranges. In addition, DBLSC-CO 2 was shown to have superior functional properties in terms of total soluble content, water and oil absorption, and foaming and emulsification properties. Therefore, SC-CO 2 treatment offers a nutritionally and environmentally friendly approach for the removal of lipid from high protein food sources. In addition, SC-CO 2 may be a better substitute of traditional organic solvent extraction for producing more stable and high quality foods with high-protein, fat-free, and low calorie contents.

Improvements in the order, isotropy and electron density of glypican-1 crystals by controlled dehydration.

PubMed

Awad, Wael; Svensson Birkedal, Gabriel; Thunnissen, Marjolein M G M; Mani, Katrin; Logan, Derek T

2013-12-01

The use of controlled dehydration for improvement of protein crystal diffraction quality is increasing in popularity, although there are still relatively few documented examples of success. A study has been carried out to establish whether controlled dehydration could be used to improve the anisotropy of crystals of the core protein of the human proteoglycan glypican-1. Crystals were subjected to controlled dehydration using the HC1 device. The optimal protocol for dehydration was developed by careful investigation of the following parameters: dehydration rate, final relative humidity and total incubation time Tinc. Of these, the most important was shown to be Tinc. After dehydration using the optimal protocol the crystals showed significantly reduced anisotropy and improved electron density, allowing the building of previously disordered parts of the structure.

[Effects of shading at different phases of grain-filling on wheat grain protein components contents and processing quality].

PubMed

Shi, Yu; Chen, Mao-xue; Yu, Zhen-wen; Xu, Zhen-zhu

2011-10-01

Taking three wheat cultivars Jimai 20 (strong gluten), Taishan 23 (medium gluten), and Ningmai 9 (weak gluten) as test materials, a field experiment was conducted to examine the effects of shading at different phases of grain-filling on the grain protein components contents and processing quality. Four treatments were installed, i. e., no shading (S0), shading at early grain-filling phase (from 0 day after anthesis (DAA) to 11 DAA; S1), shading at medium grain-filling phase (from 12 DAA to 23 DAA; S2), and shading at late grain-filling phase (from 24 DAA to 35 DAA; S3). No significant differences were observed in the grain albumin+globulin contents of the three cultivars among the four treatments. Shading increased the grain HMW-GS, LMW-GS, gluten, glutenin, and total protein contents of Jimai 20 and Taishan 23 significantly, and the increments were higher in treatment S2 than in other shading treatments. Treatments S2 and S3 increased the grain protein components contents of Ningmai 9 significantly. Comparing with the control, shading decreased the grain yield significantly, but increased the dough development time, dough stability time, and sedimentation volume, especially for treatment S2, which suggested that the wheat grain quality had a close relationship with the light intensity at medium phase of grain-filling. Overall, the regulation effect of shading at grain-filling stage on the wheat grain yield, grain protein components contents, and indices values of grain processing quality for the test cultivars was in the order of Jimai 20 > Taishan 23 > Ningmai 9.

Roller milled black gram (Phaseolus mungo) semolina and its influence on the quality characteristics of high protein pasta.

PubMed

Rajiv, Jyotsna; Milind; Inamdar, Aashitosh A; Sakhare, Suresh; Venkateswara Rao, G

2015-04-01

Black gram (Phaseolus mungo) was roller milled into semolina (BGS) and was substituted at 25, 50 and 75 % levels in vermicelli making in this investigation. There was an increase in ash and protein content as the inclusion of BGS in blends increased. The quality characteristics of pasta showed marginal increase in cooking loss up to 50 % level of BGS. The firmness value did not change much up to 50 % BGS in pasta. At 75 % level of BGS, the cooking loss and stickiness value were highest (6.10 % and 0. 90 N) whereas firmness value and overall quality score were lowest (4 N and 27.5/40) indicating that the pasta had mushy, indiscrete, sticky strands and had a prominent beany odour making it unacceptable. Hence 50 % BGS was considered optimum in vermicelli. The pasta made with 50 % BGS inclusion had a protein and dietary fiber content of 15.30 % and 8 % as against the control value of 11.30 and 4.20 % respectively.

Meta-analysis: protein and energy supplementation in older people.

PubMed

Milne, Anne C; Avenell, Alison; Potter, Jan

2006-01-03

Protein and energy undernutrition is common in older people, and further deterioration may occur during illness. To assess whether oral protein and energy supplementation improves clinical and nutritional outcomes for older people in the hospital, in an institution, or in the community. Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE, HealthStar, CINAHL, BIOSIS, and CAB abstracts. The authors included English- and non-English-language studies and hand-searched journals, contacted manufacturers, and sought information from trialists. The date of the most recent search of CENTRAL and MEDLINE is June 2005. Randomized and quasi-randomized controlled trials of oral protein and energy supplementation compared with placebo or control treatment in older people. Two reviewers independently assessed trials for inclusion, extracted data, and assessed trial quality. Differences were resolved by consensus. Fifty-five trials were included (n = 9187 randomly assigned participants). For patients in short-term care hospitals who were given oral supplements, evidence suggested fewer complications (Peto odds ratio, 0.72 [95% CI, 0.53 to 0.97]) and reduced mortality (Peto odds ratio, 0.66 [CI, 0.49 to 0.90]) for those undernourished at baseline. Few studies reported evidence that suggested any change in mortality, morbidity, or function for those given supplements at home. Ten trials reported gastrointestinal disturbances, such as nausea, vomiting, and diarrhea, with oral supplements. The quality of most studies, as reported, was poor, particularly for concealment of allocation and blinding of outcome assessors. Many studies were too small or the follow-up time was too short to detect a statistically significant change in clinical outcome. The clinical results are dominated by 1 very large recent trial in patients with stroke. Although this was a high-quality trial, few participants were undernourished at baseline. Oral nutritional supplements can improve nutritional status and seem to reduce mortality and complications for undernourished elderly patients in the hospital. Current evidence does not support routine supplementation for older people at home or for well-nourished older patients in any setting.

Whey protein, amino acids, and vitamin D supplementation with physical activity increases fat-free mass and strength, functionality, and quality of life and decreases inflammation in sarcopenic elderly.

PubMed

Rondanelli, Mariangela; Klersy, Catherine; Terracol, Gilles; Talluri, Jacopo; Maugeri, Roberto; Guido, Davide; Faliva, Milena A; Solerte, Bruno S; Fioravanti, Marisa; Lukaski, Henry; Perna, Simone

2016-03-01

Interventions to attenuate the adverse effects of age-related loss of skeletal muscle and function include increased physical activity and nutritional supplementation. This study tested the hypothesis that nutritional supplementation with whey protein (22 g), essential amino acids (10.9 g, including 4 g leucine), and vitamin D [2.5 μg (100 IU)] concurrent with regular, controlled physical activity would increase fat-free mass, strength, physical function, and quality of life, and reduce the risk of malnutrition in sarcopenic elderly persons. A total of 130 sarcopenic elderly people (53 men and 77 women; mean age: 80.3 y) participated in a 12-wk randomized, double-blind, placebo-controlled supplementation trial. All participants concurrently took part in a controlled physical activity program. We examined body composition with dual-energy X-ray absorptiometry, muscle strength with a handgrip dynamometer, and blood biochemical indexes of nutritional and health status, and evaluated global nutritional status, physical function, and quality of life before and after the 12 wk of intervention. Compared with physical activity and placebo, supplementation plus physical activity increased fat-free mass (1.7-kg gain, P < 0.001), relative skeletal muscle mass (P = 0.009), android distribution of fat (P = 0.021), handgrip strength (P = 0.001), standardized summary scores for physical components (P = 0.030), activities of daily living (P = 0.001), mini nutritional assessment (P = 0.003), and insulin-like growth factor I (P = 0.002), and lowered C-reactive protein (P = 0.038). Supplementation with whey protein, essential amino acids, and vitamin D, in conjunction with age-appropriate exercise, not only boosts fat-free mass and strength but also enhances other aspects that contribute to well-being in sarcopenic elderly. This trial was registered at clinicaltrials.gov as NCT02402608. © 2016 American Society for Nutrition.

Genotypic-dependent effects of N fertilizer, glutathione, silicon, zinc, and selenium on proteomic profiles, amino acid contents, and quality of rice genotypes with contrasting grain Cd accumulation.

PubMed

Cao, Fangbin; Fu, Manman; Wang, Runfeng; Cheng, Wangda; Zhang, Guoping; Wu, Feibo

2017-07-01

Soil heavy metal (HM) contamination has posed a serious problem for safe food production. For restricting the translocation of HM into grain, many proteins were regulated to involve in the process. To identify these proteins, 2D-based proteomic analysis was carried out using different rice genotypes with distinct Cd accumulation in grains and as affected by an alleviating regulator (AR) in field experiments. AR application improved grain quality, with increased contents in Glu, Cys, His, Pro, and protein. Twenty-six low-grain HM accumulation-associated protein species were identified and categorized as physiological functions via two-dimensional gel electrophoresis (2DE) and mass spectrometry. Among these proteins, 8, 9, and 9 proteins exhibited higher accumulation, lower accumulation, and unchanged accumulation, respectively, in Xiushui817 (low accumulator) vs R8097 (high accumulator) under control conditions but showed differential accumulation patterns after AR application. These proteins included sucrose synthase 3, alanine aminotransferase, glutelin, cupin family protein, and zinc finger CCCH domain-containing protein 32. The differential expression of these protein species might contribute to decreased HM accumulation in grain via decreasing the protein accumulation which had high affinity to HM or regulating energy metabolism and signal transduction. Our findings provide valuable insights into the mechanisms of low-grain HM accumulation in rice and possible utilization of candidate protein species in developing low-grain HM accumulation genotypes.

Large-Scale Interlaboratory Study to Develop, Analytically Validate and Apply Highly Multiplexed, Quantitative Peptide Assays to Measure Cancer-Relevant Proteins in Plasma*

PubMed Central

Abbatiello, Susan E.; Schilling, Birgit; Mani, D. R.; Zimmerman, Lisa J.; Hall, Steven C.; MacLean, Brendan; Albertolle, Matthew; Allen, Simon; Burgess, Michael; Cusack, Michael P.; Gosh, Mousumi; Hedrick, Victoria; Held, Jason M.; Inerowicz, H. Dorota; Jackson, Angela; Keshishian, Hasmik; Kinsinger, Christopher R.; Lyssand, John; Makowski, Lee; Mesri, Mehdi; Rodriguez, Henry; Rudnick, Paul; Sadowski, Pawel; Sedransk, Nell; Shaddox, Kent; Skates, Stephen J.; Kuhn, Eric; Smith, Derek; Whiteaker, Jeffery R.; Whitwell, Corbin; Zhang, Shucha; Borchers, Christoph H.; Fisher, Susan J.; Gibson, Bradford W.; Liebler, Daniel C.; MacCoss, Michael J.; Neubert, Thomas A.; Paulovich, Amanda G.; Regnier, Fred E.; Tempst, Paul; Carr, Steven A.

2015-01-01

There is an increasing need in biology and clinical medicine to robustly and reliably measure tens to hundreds of peptides and proteins in clinical and biological samples with high sensitivity, specificity, reproducibility, and repeatability. Previously, we demonstrated that LC-MRM-MS with isotope dilution has suitable performance for quantitative measurements of small numbers of relatively abundant proteins in human plasma and that the resulting assays can be transferred across laboratories while maintaining high reproducibility and quantitative precision. Here, we significantly extend that earlier work, demonstrating that 11 laboratories using 14 LC-MS systems can develop, determine analytical figures of merit, and apply highly multiplexed MRM-MS assays targeting 125 peptides derived from 27 cancer-relevant proteins and seven control proteins to precisely and reproducibly measure the analytes in human plasma. To ensure consistent generation of high quality data, we incorporated a system suitability protocol (SSP) into our experimental design. The SSP enabled real-time monitoring of LC-MRM-MS performance during assay development and implementation, facilitating early detection and correction of chromatographic and instrumental problems. Low to subnanogram/ml sensitivity for proteins in plasma was achieved by one-step immunoaffinity depletion of 14 abundant plasma proteins prior to analysis. Median intra- and interlaboratory reproducibility was <20%, sufficient for most biological studies and candidate protein biomarker verification. Digestion recovery of peptides was assessed and quantitative accuracy improved using heavy-isotope-labeled versions of the proteins as internal standards. Using the highly multiplexed assay, participating laboratories were able to precisely and reproducibly determine the levels of a series of analytes in blinded samples used to simulate an interlaboratory clinical study of patient samples. Our study further establishes that LC-MRM-MS using stable isotope dilution, with appropriate attention to analytical validation and appropriate quality control measures, enables sensitive, specific, reproducible, and quantitative measurements of proteins and peptides in complex biological matrices such as plasma. PMID:25693799

Protein disulfide isomerases in the endoplasmic reticulum promote anchorage-independent growth of breast cancer cells.

PubMed

Wise, Randi; Duhachek-Muggy, Sara; Qi, Yue; Zolkiewski, Michal; Zolkiewska, Anna

2016-06-01

Metastatic breast cancer cells are exposed to stress of detachment from the extracellular matrix (ECM). Cultured breast cancer cells that survive this stress and are capable of anchorage-independent proliferation form mammospheres. The purpose of this study was to explore a link between mammosphere growth, ECM gene expression, and the protein quality control system in the endoplasmic reticulum (ER). We compared the mRNA and protein levels of ER folding factors in SUM159PT and MCF10DCIS.com breast cancer cells grown as mammospheres versus adherent conditions. Publicly available gene expression data for mammospheres formed by primary breast cancer cells and for circulating tumor cells (CTCs) were analyzed to assess the status of ECM/ER folding factor genes in clinically relevant samples. Knock-down of selected protein disulfide isomerase (PDI) family members was performed to examine their roles in SUM159PT mammosphere growth. We found that cells grown as mammospheres had elevated expression of ECM genes and ER folding quality control genes. CTC gene expression data for an index patient indicated that upregulation of ECM and ER folding factor genes occurred at the time of acquired therapy resistance and disease progression. Knock-down of PDI, ERp44, or ERp57, three members of the PDI family with elevated protein levels in mammospheres, in SUM159PT cells partially inhibited the mammosphere growth. Thus, breast cancer cell survival and growth under detachment conditions require enhanced assistance of the ER protein folding machinery. Targeting ER folding factors, in particular members of the PDI family, may improve the therapeutic outcomes in metastatic breast cancer.

Starch and protein analysis of wheat bread enriched with phenolics-rich sprouted wheat flour.

PubMed

Świeca, Michał; Dziki, Dariusz; Gawlik-Dziki, Urszula

2017-08-01

Wheat flour in the bread formula was replaced with sprouted wheat flour (SF) characterized by enhanced nutraceutical properties, at 5%, 10%, 15% and 20% levels. The addition of SF slightly increased the total protein content; however, it decreased their digestibility. Some qualitative and quantitative changes in the electrophoretic pattern of proteins were also observed; especially, in the bands corresponding with 27kDa and 15-17kDa proteins. These results were also confirmed by SE-HPLC technique, where a significant increase in the content of proteins and peptides (molecular masses <20kDa) was determined for breads with 20% of SF. Bread enriched with sprouted wheat flour had more resistant starch, but less total starch, compared to control bread. The highest in vitro starch digestibility was determined for the control bread. The studied bread with lowered nutritional value but increased nutritional quality can be used for special groups of consumers (obese, diabetic). Copyright © 2017 Elsevier Ltd. All rights reserved.

Effect of Fortification with Fish (Pseudophycis bachus) Powder on Nutritional Quality of Durum Wheat Pasta.

PubMed

Desai, Ajay S; Brennan, Margaret A; Brennan, Charles S

2018-04-17

This paper investigates the nutraceutical (phenolic content and antioxidant activity) and nutritional potential (protein and starch digestibility) of supplementation of durum wheat semolina with 5–20% fish powder ( Pseudophycis bachus ). In general, all enriched pasta with fish powder showed a significant decrease ( p < 0.05) in reducing sugar released during an in vitro digestion, and reductions in standardized area under the curve (AUC) values, compared to control pasta. The potentially bioaccessible fraction of pasta enriched with 20% fish powder (FP) was characterized as having a 177–191% increase in phenolic content and a 145–556% higher antiradical activity. Elevation of these parameters in fortified pasta was accompanied by interaction of wheat starch, protein, and fish powder protein. Supplementation of fish powder also influenced protein digestibility (a reduction from 84.60% for control pasta to 80.80% for pasta with 20% fish powder). Fortification improved the nutraceutical and nutritional potential of the studied pasta with the effects depending on factors including protein-starch-phenolic interactions.

Proteomic responses of fruits to environmental stresses

PubMed Central

Chan, Zhulong

2012-01-01

Fruits and vegetables are extremely susceptible to decay and easily lose commercial value after harvest. Different strategies have been developed to control postharvest decay and prevent quality deterioration during postharvest storage, including cold storage, controlled atmosphere (CA), and application of biotic and abiotic stimulus. In this review, mechanisms related to protein level responses of host side and pathogen side were characterized. Protein extraction protocols have been successfully developed for recalcitrant, low protein content fruit tissues. Comparative proteome profiling and functional analysis revealed that defense related proteins, energy metabolism, and antioxidant pathway played important roles in fruits in response to storage conditions and exogenous elicitor treatments. Secretome of pathogenic fungi has been well-investigated and the results indicated that hydrolytic enzymes were the key virulent factors for the pathogen infection. These protein level changes shed new light on interaction among fruits, pathogens, and environmental conditions. Potential postharvest strategies to reduce risk of fruit decay were further proposed based on currently available proteomic data. PMID:23335934

The Strength of Family Ties: Perceptions of Network Relationship Quality and Levels of C-Reactive Proteins in the North Texas Heart Study.

PubMed

Uchino, Bert N; Ruiz, John M; Smith, Timothy W; Smyth, Joshua M; Taylor, Daniel J; Allison, Matthew; Ahn, Chul

2015-10-01

Although the quality of one's social relationships has been linked to important physical health outcomes, less work has been conducted examining family and friends that differ in their underlying positivity and negativity. The main aim of this study was to examine the association between supportive, aversive, and ambivalent family/friends with levels of C-reactive proteins. Three hundred participants from the North Texas Heart Study completed the social relationships index and a blood draw to assess high-sensitivity C-reactive proteins (hs-CRPs). After standard controls, the number of supportive family members predicted lower hs-CRP levels, whereas the number of ambivalent family members predicted higher hs-CRP levels. These links were independent of depressive symptoms and perceived stress. These data highlight the importance of considering specific types of relationships and their underlying positive and negative aspects in research on social ties and physical health.

CDSbank: taxonomy-aware extraction, selection, renaming and formatting of protein-coding DNA or amino acid sequences.

PubMed

Hazes, Bart

2014-02-28

Protein-coding DNA sequences and their corresponding amino acid sequences are routinely used to study relationships between sequence, structure, function, and evolution. The rapidly growing size of sequence databases increases the power of such comparative analyses but it makes it more challenging to prepare high quality sequence data sets with control over redundancy, quality, completeness, formatting, and labeling. Software tools for some individual steps in this process exist but manual intervention remains a common and time consuming necessity. CDSbank is a database that stores both the protein-coding DNA sequence (CDS) and amino acid sequence for each protein annotated in Genbank. CDSbank also stores Genbank feature annotation, a flag to indicate incomplete 5' and 3' ends, full taxonomic data, and a heuristic to rank the scientific interest of each species. This rich information allows fully automated data set preparation with a level of sophistication that aims to meet or exceed manual processing. Defaults ensure ease of use for typical scenarios while allowing great flexibility when needed. Access is via a free web server at http://hazeslab.med.ualberta.ca/CDSbank/. CDSbank presents a user-friendly web server to download, filter, format, and name large sequence data sets. Common usage scenarios can be accessed via pre-programmed default choices, while optional sections give full control over the processing pipeline. Particular strengths are: extract protein-coding DNA sequences just as easily as amino acid sequences, full access to taxonomy for labeling and filtering, awareness of incomplete sequences, and the ability to take one protein sequence and extract all synonymous CDS or identical protein sequences in other species. Finally, CDSbank can also create labeled property files to, for instance, annotate or re-label phylogenetic trees.

Effects of host nutrition on virulence and fitness of entomopathogenic nematodes: Lipid- and protein-based supplements in Tenebrio molitor diets

PubMed Central

Shapiro-Ilan, David; Rojas, M. Guadalupe; Morales-Ramos, Juan A.; Lewis, Edwin E.; Tedders, W. Louis

2008-01-01

Entomopathogenic nematodes, Heterorhabditis indica and Steinernema riobrave, were tested for virulence and reproductive yield in Tenebrio molitor that were fed wheat bran diets with varying lipid- and protein-based supplements. Lipid supplements were based on 20% canola oil, peanut, pork or salmon, or a low lipid control (5% canola). Protein treatments consisted of basic supplement ingredients plus 0, 10, or 20% egg white; a bran-only control was also included. Some diet supplements had positive effects on nematode quality, whereas others had negative or neutral effects. All supplements with 20% lipids except canola oil caused increased T. molitor susceptibility to H. indica, whereas susceptibility to S. riobrave was not affected. Protein supplements did not affect host susceptibility, and neither lipid nor protein diet supplements affected reproductive capacity of either nematode species. Subsequently, we determined the pest control efficacy of progeny of nematodes that had been reared through T. molitor from different diets against Diaprepes abbreviatus and Otiorhynchus sulcatus. All nematode treatments reduced insect survival relative to the control (water only). Nematodes originating from T. molitor diets with the 0% or 20% protein exhibited lower efficacy versus D. abbreviatus than the intermediate level of protein (10%) or bran-only treatments. Nematodes originating from T. molitor lipid or control diets did not differ in virulence. Our research indicates that nutritional content of an insect host diet can affect host susceptibility to entomopathogenic nematodes and nematode fitness; therefore, host media could conceivably be optimized to increase in vivo nematode production efficiency. PMID:19259513

Exposure to bisphenol A (BPA) in Wistar rats reduces sperm quality with disruption of ERK signal pathway.

PubMed

Li, Juan; Mao, Rui; Zhou, Qin; Ding, Ling; Tao, Jin; Ran, Mao-Mei; Gao, Er-Sheng; Yuan, Wei; Wang, Jin-Tao; Hou, Li-Fang

2016-01-01

Bisphenol A (BPA) is an estrogenic environmental toxin widely used in the production of plastics and ubiquitous human exposure to this chemical has been proposed to be a potential risk to human health. Exposure to BPA can negatively impact sperm quality. However, the mechanism remains largely unknown. The objectives of this study were to assess the role of BPA on sperm quality and explore the possible mechanisms. The Wistar male rats (aged 28 days) were administered BPA by oral gavage for 28 days at dose of 50, 100 and 200 mg/kg/day; meanwhile, the negative control with corn oil (0 mg/kg/day BPA) and positive control with E2 at the dose of 100 μg/kg/day. The sperm density, sperm activity and sperm survival rate were analyzed byCASA system, and the sperm abnormality rate was analyzed by improved Papanicolaou stained. The protein expression levels of Src/p-Src, ERK1/2, p-ERK1/2 and CREB/p-CREB were detected by Western bolt. The results showed that the body weight gain, testes weight, testis coefficient, sperm density, sperm activity, sperm survival rate and protein expression levels of p-ERK1, p-ERK2 and p-CREB decreased, but the sperm abnormality rate increased with increasing BPA concentrations. There were positive correlations between sperm density, sperm activity and sperm survival rate with protein expression levels of p-ERK1, p-ERK2 and p-CREB, and negative correlations between sperm abnormality rate with the protein expression levels of p-ERK1, p-ERK2 and p-CREB. Results from the structural equation model demonstrated that BPA retained a significant negative effect to p-ERK, whereas p-ERK retained a significant positive effect to sperm quality and acted as the mediate variable. This study provides a novel insight regarding the potential role of p-ERK1 and p-ERK2 protein kinase on reproductive toxicity of BPA. The adverse effects of BPA on adult male sperm quality may be through the induction of the disruption of ERK signal pathway. However, additional research is needed to confirm our findings and to further test the suggested potential mechanisms.

A large-scale and robust dynamic MRM study of colorectal cancer biomarkers.

PubMed

You, Jia; Kao, Athit; Dillon, Roslyn; Croner, Lisa J; Benz, Ryan; Blume, John E; Wilcox, Bruce

2018-06-25

Over the past 20 years, mass spectrometry (MS) has emerged as a dynamic tool for proteomics biomarker discovery. However, published MS biomarker candidates often do not translate to the clinic, failing during attempts at independent replication. The cause can be shortcomings in study design, sample quality, assay quantitation, and/or quality/process control. To address these shortcomings, we developed an MS workflow in accordance with Tier 2 measurement requirements for targeted peptides, defined by the Clinical Proteomic Tumor Analysis Consortium (CPTAC) "fit-for-purpose" approach, using dynamic multiple reaction monitoring (dMRM) which measures specific peptide transitions during predefined retention time (RT) windows. We describe the development of a robust multipex dMRM assay measuring 641 proteotypic peptides from 392 colorectal cancer (CRC) related proteins, and the procedures to track and handle sample processing and instrument variation over a four-month study, during which the assay measured blood samples from 1045 patients with CRC symptoms. After data collection, transitions were filtered by signal quality metrics before entering receiver operating characteristic (ROC) analysis. The results demonstrated CRC signal carried by 127 proteins in the symptomatic population. The workflow might be further developed to build Tier 1 assays for clinical tests identifying symptomatic individuals at elevated risk of CRC. We developed a dMRM MS method with the rigor of a Tier 2 assay as defined by the CPTAC 'fit for purpose approach' [1]. Using quality and process control procedures, the assay was used to quantify 641 proteotypic peptides representing 392 CRC-related proteins in plasma from 1045 CRC-symptomatic patients. To our knowledge, this is the largest MRM method applied to the largest study to date. The results showed that 127 of the proteins carried univariate CRC signal in the symptomatic population. This large number of single biomarkers bodes well for future development of multivariate classifiers to distinguish CRC in the symptomatic population. Copyright © 2018. Published by Elsevier B.V.

Countering Solutal Buoyant Convection with High Magnetic Fields

NASA Technical Reports Server (NTRS)

Ramachandran, N.; Leslie, F. W.

2002-01-01

An important component in biotechnology, particularly in the area of protein engineering and rational drug design is the knowledge of the precise three-dimensional molecular structure of proteins. The quality of structural information obtained from X-ray diffraction methods is directly dependent on the degree of perfection of the protein crystals. As a consequence, the growth of high quality macromolecular crystals for diffraction analyses has been the central focus for biochemist, biologists, and bioengineers. Macromolecular crystals are obtained from solutions that contain the crystallizing species in equilibrium with higher aggregates, ions, precipitant, other possible phases of the protein, foreign particles, the walls of the container, and a likely host of other impurities. By changing transport modes in general, i.e., reduction of convection and sedimentation, as is achieved in microgravity, we have been able to dramatically effect the movement and distribution of macromolecules in the fluid, and thus their transport, formation of crystal nuclei, and adsorption to the crystal surface. While a limited number of high quality crystals from space flights have been obtained, as the recent National Research Council (NRC) review of the NASA microgravity crystallization program pointed out, the scientific approach and research in crystallization of proteins has been mainly empirical yielding inconclusive results. We postulate that we can reduce convection in ground-based experiments and we can understand the different aspects of convection control through the use of strong magnetic fields and field gradients. We postulate that limited convection in a magnetic field will provide the environment for the growth of high quality crystals. The approach exploits the variation of fluid magnetic susceptibility with concentration for this purpose and the convective damping is realized by appropriately positioning the crystal growth cell so that the magnetic susceptibility force counteracts terrestrial gravity. The general objective is to test the hypothesis of convective control using a strong magnetic field and magnetic field gradient and to understand the nature of the various forces that come into play. Specifically we aim to delineate causative factors and to quantify them through experiments, analysis and numerical modeling. The paper will report on the current status of the investigation and discuss results from the experimental and modeling efforts.

Non-conventional approaches to food processing in CELSS. I - Algal proteins: Characterization and process optimization

NASA Technical Reports Server (NTRS)

Nakhost, Z.; Karel, M.; Krukonis, V. J.

1987-01-01

Protein isolate obtained from green algae (Scenedesmus obliquus) cultivated under controlled conditions was characterized. Molecular weight determination of fractionated algal proteins using SDS-polyacrylamide gel electrophoresis revealed a wide spectrum of molecular weights ranging from 15,000 to 220,000. Isoelectric points of dissociated proteins were in the range of 3.95 to 6.20. Amino acid composition of protein isolate compared favorably with FAO standards. High content of essential amino acids leucine, valine, phenylalanine and lysine makes algal protein isolate a high quality component of CELSS diets. To optimize the removal of algal lipids and pigments supercritical carbon dioxide extraction (with and without ethanol as a co-solvent) was used. Addition of ethanol to supercritical CO2 resulted in more efficient removal of algal lipids and produced protein isolate with a good yield and protein recovery. The protein isolate extracted by the above mixture had an improved water solubility.

Non-conventional approaches to food processing in CELSS, 1. Algal proteins: Characterization and process optimization

NASA Technical Reports Server (NTRS)

Nakhost, Z.; Karel, M.; Krukonis, V. J.

1987-01-01

Protein isolate obtained from green algae cultivated under controlled conditions was characterized. Molecular weight determination of fractionated algal proteins using SDS-polyacrylamide gel electrophoresis revealed a wide spectrum of molecular weights ranging from 15,000 to 220,000. Isoelectric points of dissociated proteins were in the range of 3.95 to 6.20. Amino acid composition of protein isolate compared favorably with FAO standards. High content of essential amino acids leucine, valine, phenylalanine and lysine make algal protein isolate a high quality component of closed ecological life support system diets. To optimize the removal of algal lipids and pigments supercritical carbon dioxide extraction (with and without ethanol as a co-solvent) was used. Addition of ethanol to supercritical carbon dioxide resulted in more efficient removal of algal lipids and produced protein isolate with a good yield and protein recovery. The protein isolate extracted by the above mixture had an improved water solubility.

Changes of Protein Turnover in Aging Caenorhabditis elegans

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

Dhondt, Ineke; Petyuk, Vladislav A.; Bauer, Sophie

Protein turnover rates severely decline in aging organisms, including C. elegans. However, limited information is available on turnover dynamics at the individual protein level during aging. We followed changes in protein turnover at one-day resolution using a multiple-pulse 15Nlabeling and accurate mass spectrometry approach. Forty percent of the proteome shows gradual slowdown in turnover with age, whereas only few proteins show increased turnover. Decrease in protein turnover was consistent for only a minority of functionally related protein subsets, including tubulins and vitellogenins, whereas randomly diverging turnover patterns with age were the norm. Our data suggests increased heterogeneity of protein turnovermore » of the translation machinery, whereas protein turnover of ubiquitin-proteasome and antioxidant systems are well-preserved over time. Hence, we presume that maintenance of quality control mechanisms is a protective strategy in aging worms, although the ultimate proteome collapse is inescapable.« less

Analysis of pollen load based on color, physicochemical composition and botanical source.

PubMed

Modro, Anna F H; Silva, Izabel C; Luz, Cynthia F P; Message, Dejair

2009-06-01

Pollen load samples from 10 hives of Apis mellifera (L.) were analyzed based on their physicochemical composition and botanical source, considering color as a parameter for quality control. In seven samples it was possible to establish the occurrence of more than 80% of a single pollen type, characterizing them as unifloral but with protein content variation. One of the samples was exclusively composed of saprophytic fungi (Cladosporium sp.). Comparing the mean results of the fungi loads with those of the nutritional components of pollen load, the former presented higher protein, mineral matter and dry matter and lower organic matter, ethereal extract and total carbohydrate values. The monochromatic samples met the physicochemical specifications regulating pollen load quality. The results showed that homogeneous coloration of the pollen load was not found to be a good indication of unifloral pollen, confirming the importance of physicochemical analysis and melissopalynological analysis for characterization of the quality of commercial pollen load.

Viral Evasion and Manipulation of Host RNA Quality Control Pathways

PubMed Central

2016-01-01

Viruses have evolved diverse strategies to maximize the functional and coding capacities of their genetic material. Individual viral RNAs are often used as substrates for both replication and translation and can contain multiple, sometimes overlapping open reading frames. Further, viral RNAs engage in a wide variety of interactions with both host and viral proteins to modify the activities of important cellular factors and direct their own trafficking, packaging, localization, stability, and translation. However, adaptations increasing the information density of small viral genomes can have unintended consequences. In particular, viral RNAs have developed features that mark them as potential targets of host RNA quality control pathways. This minireview focuses on ways in which viral RNAs run afoul of the cellular mRNA quality control and decay machinery, as well as on strategies developed by viruses to circumvent or exploit cellular mRNA surveillance. PMID:27226372

Viral Evasion and Manipulation of Host RNA Quality Control Pathways.

PubMed

Hogg, J Robert

2016-08-15

Viruses have evolved diverse strategies to maximize the functional and coding capacities of their genetic material. Individual viral RNAs are often used as substrates for both replication and translation and can contain multiple, sometimes overlapping open reading frames. Further, viral RNAs engage in a wide variety of interactions with both host and viral proteins to modify the activities of important cellular factors and direct their own trafficking, packaging, localization, stability, and translation. However, adaptations increasing the information density of small viral genomes can have unintended consequences. In particular, viral RNAs have developed features that mark them as potential targets of host RNA quality control pathways. This minireview focuses on ways in which viral RNAs run afoul of the cellular mRNA quality control and decay machinery, as well as on strategies developed by viruses to circumvent or exploit cellular mRNA surveillance. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Effect of probiotics and thyme essential oil on the essential amino acid content of the broiler chicken meat.

PubMed

Alfaig, Ebrahim; Angelovičova, Maria; Kral, Martin; Bučko, Ondrej

2014-01-01

Differences in the types and percentages of essential amino acids (EAAs) in food could influence the value of protein consumed and proteins with a high content of EAAs are the most important components of poultry meat. The use of probiotics for meat and carcass quality improvement has been questioned, while feed supplementation with thyme essential oil (TEO) could be considered as useful natural supplement to be applied in the poultry industry to improve meat quality. Day-old broilers Ross 308 (n = 400) were randomly divided into four groups based on the feed supplement as follows: control, probiotics 0.05%, TEO 0.05% and combination of probiotics and TEO, while the fattening period was 42 days. Six birds of both sexes from each group were selected as a sample, slaughtered and then stored (-18°C) for 6 months till the analysis. The muscular homogeneous sample (50 g) from the breast and thigh of each sample bird was analysed by the Fourier Transform Infrared Spectroscopy method using the device Nicolet 6700. The essential amino acids content was determined and the quality indicators include chemical score, amino acid score, EAA index and biological value were calculated. The obtained results show that for all the tested EAAs of the breast and thigh muscles, the content numerically increased gradually and progressively within the groups as the control scored the minimum followed by the probiotics group, then the combination group and finally the TEO group which scored the highest results. It can be concluded that the TEO promoted the increase of all the EAAs and consequently the quality indicators with significant different compared with the control group and significantly different for some EAAs and quality indicators compared with the probiotics group and the combination group.

The effects of actomyosin disruptors on the mechanical integrity of the avian crystalline lens

PubMed Central

Won, Gah-Jone; Fudge, Douglas S.

2015-01-01

Purpose: Actin and myosin within the crystalline lens maintain the structural integrity of lens fiber cells and form a hexagonal lattice cradling the posterior surface of the lens. The actomyosin network was pharmacologically disrupted to examine the effects on lenticular biomechanics and optical quality. Methods: One lens of 7-day-old White Leghorn chickens was treated with 10 µM of a disruptor and the other with 0.01% dimethyl sulfoxide (vehicle). Actin, myosin, and myosin light chain kinase (MLCK) disruptors were used. The stiffness and the optical quality of the control and treated lenses were measured. Western blotting and confocal imaging were used to confirm that treatment led to a disruption of the actomyosin network. The times for the lenses to recover stiffness to match the control values were also measured. Results: Disruptor-treated lenses were significantly less stiff than their controls (p≤0.0274 for all disruptors). The disruptors led to changes in the relative protein amounts as well as the distributions of proteins within the lattice. However, the disruptors did not affect the clarity of the lenses (p≥0.4696 for all disruptors), nor did they affect spherical aberration (p = 0.02245). The effects of all three disruptors were reversible, with lenses recovering from treatment with actin, myosin, and MLCK disruptors after 4 h, 1 h, and 8 min, respectively. Conclusions: Cytoskeletal protein disruptors led to a decreased stiffness of the lens, and the effects were reversible. Optical quality was mostly unaffected, but the long-term consequences remain unclear. Our results raise the possibility that the mechanical properties of the avian lens may be actively regulated in vivo via adjustments to the actomyosin lattice. PMID:25684975

The effects of actomyosin disruptors on the mechanical integrity of the avian crystalline lens.

PubMed

Won, Gah-Jone; Fudge, Douglas S; Choh, Vivian

2015-01-01

Actin and myosin within the crystalline lens maintain the structural integrity of lens fiber cells and form a hexagonal lattice cradling the posterior surface of the lens. The actomyosin network was pharmacologically disrupted to examine the effects on lenticular biomechanics and optical quality. One lens of 7-day-old White Leghorn chickens was treated with 10 µM of a disruptor and the other with 0.01% dimethyl sulfoxide (vehicle). Actin, myosin, and myosin light chain kinase (MLCK) disruptors were used. The stiffness and the optical quality of the control and treated lenses were measured. Western blotting and confocal imaging were used to confirm that treatment led to a disruption of the actomyosin network. The times for the lenses to recover stiffness to match the control values were also measured. Disruptor-treated lenses were significantly less stiff than their controls (p≤0.0274 for all disruptors). The disruptors led to changes in the relative protein amounts as well as the distributions of proteins within the lattice. However, the disruptors did not affect the clarity of the lenses (p≥0.4696 for all disruptors), nor did they affect spherical aberration (p = 0.02245). The effects of all three disruptors were reversible, with lenses recovering from treatment with actin, myosin, and MLCK disruptors after 4 h, 1 h, and 8 min, respectively. Cytoskeletal protein disruptors led to a decreased stiffness of the lens, and the effects were reversible. Optical quality was mostly unaffected, but the long-term consequences remain unclear. Our results raise the possibility that the mechanical properties of the avian lens may be actively regulated in vivo via adjustments to the actomyosin lattice.

Regulation of endoplasmic reticulum turnover by selective autophagy.

PubMed

Khaminets, Aliaksandr; Heinrich, Theresa; Mari, Muriel; Grumati, Paolo; Huebner, Antje K; Akutsu, Masato; Liebmann, Lutz; Stolz, Alexandra; Nietzsche, Sandor; Koch, Nicole; Mauthe, Mario; Katona, Istvan; Qualmann, Britta; Weis, Joachim; Reggiori, Fulvio; Kurth, Ingo; Hübner, Christian A; Dikic, Ivan

2015-06-18

The endoplasmic reticulum (ER) is the largest intracellular endomembrane system, enabling protein and lipid synthesis, ion homeostasis, quality control of newly synthesized proteins and organelle communication. Constant ER turnover and modulation is needed to meet different cellular requirements and autophagy has an important role in this process. However, its underlying regulatory mechanisms remain unexplained. Here we show that members of the FAM134 reticulon protein family are ER-resident receptors that bind to autophagy modifiers LC3 and GABARAP, and facilitate ER degradation by autophagy ('ER-phagy'). Downregulation of FAM134B protein in human cells causes an expansion of the ER, while FAM134B overexpression results in ER fragmentation and lysosomal degradation. Mutant FAM134B proteins that cause sensory neuropathy in humans are unable to act as ER-phagy receptors. Consistently, disruption of Fam134b in mice causes expansion of the ER, inhibits ER turnover, sensitizes cells to stress-induced apoptotic cell death and leads to degeneration of sensory neurons. Therefore, selective ER-phagy via FAM134 proteins is indispensable for mammalian cell homeostasis and controls ER morphology and turnover in mice and humans.

Different Dietary Protein and PUFA Interventions Alter the Fatty Acid Concentrations, but Not the Meat Quality, of Porcine Muscle

PubMed Central

Dannenberger, Dirk; Nuernberg, Karin; Nuernberg, Gerd; Priepke, Antje

2012-01-01

The present study investigated the effect of a reduced protein diet in combination with different vegetable oils (sunflower seed oil or linseed oil) on carcass traits, meat quality and fatty acid profile in porcine muscle. Forty male Landrace pigs were allocated into four experimental groups (each n = 8) and one control group (n = 8) at a live weight of approximately 60 kg. The pigs were fed ad libitum from 60 kg to 100 kg live weight and restricted to 2.8 kg/day until they reached 120 kg. In contrast to other studies, the intramuscular fat content (IMF) did not increase in animals of groups fed a reduced protein diet and vegetable oils. The IMF ranged between 1.2% and 1.4%. The growth performance and meat quality of the longissimus muscle was not affected by the diet, but the average daily gain (ADG) and drip loss were affected. The muscle fatty acid concentrations were significantly affected by the diet, resulting in higher n-3 FA concentrations up to 113 mg/100 g muscle and lower n-6/n-3 PUFA ratio for pigs fed linseed oil-containing high- and reduced protein diets, compared to sunflower seed oil-containing diets. PMID:23112912

Protein quality control in protection against systolic overload cardiomyopathy: the long term role of small heat shock proteins

PubMed Central

Kumarapeli, Asangi R K; Horak, Kathleen; Wang, Xuejun

2010-01-01

Molecular chaperones represent the first line of defense of intracellular protein quality control. As a major constituent of molecular chaperones, heat shock proteins (HSP) are known to confer cardiomyocyte short-term protection against various insults and injuries. Previously, we reported that the small HSP αB-crystallin (CryAB) attenuates cardiac hypertrophic response in mice subjected to 2 weeks of severe pressure overload. However, the long-term role of small HSPs in cardiac hypertrophy and failure has rarely been studied. The present study investigates the cardiac responses to chronic severe pressure overload in CryAB/HSPB2 germ line ablated (KO) and cardiac-specific CryAB overexpressingtransgenic (TG) mice. Pressure overload was induced by transverse aortic constriction in KO, TG, and non-transgenic wild type (NTG) control mice and 10 weeks later molecular, cellular, and whole organ level hypertrophic responses were analyzed. As we previously described, CryAB/HSPB2 KO mice showed abnormal baseline cardiac physiology that worsened into a restrictive cardiomyopathic phenotype with aging. Severe pressure overload in these mice led to rapid deterioration of heart function and development of congestive cardiac failure. Contrary to their short term protective phenotype, CryAB TG mice showed no significant effects on cardiac hypertrophic responses and very modest improvement of hemodynamics during chronic systolic overload. These findings indicate that small HSPs CryAB and/or HSPB2 are essential to maintain cardiac structure and function but overex-pression of CryAB is not sufficient to confer a sustained protection against chronic systolic overload. PMID:20733949

Xanthan gum as a fat replacer in goshtaba-a traditional meat product of India: effects on quality and oxidative stability.

PubMed

Rather, Sajad A; Masoodi, F A; Akhter, Rehana; Gani, Adil; Wani, S M; Malik, A H

2015-12-01

Goshtaba is a restructured meat product of Kashmiri wazwan prepared from meat emulsion with added fat (20 %), salt, spices and condiments and cooked in the curd. The present study was undertaken for the development of low fat goshtaba with the addition of xanthan gum as a fat replacer and was evaluated for proximate composition, pH, colour, lipid and protein oxidation, texture, microstructure and sensory properties. Low fat goshtaba formulations containing xanthan gum were higher in protein and moisture contents but, lower in fat content and pH value than the high fat control (p < 0.05). Colour evaluation revealed that high fat goshtaba had significantly higher L* value, but lower a* value than its low fat counterparts (p < 0.05). The significant decrease of TBARS values, protein carbonyls and loss of protein sulphydryl groups in low fat goshtaba formulations reflects the potential antioxidant activity of xanthan gum (p < 0.05). Hardness was significantly higher in high fat control but, cohesiveness, gumminess, and chewiness did not show any significant difference. Springiness increased with the increasing concentration of xanthan gum (0.5-1.5 %) and was higher in low fat product containing 1.5 % xanthan gum. SEM results indicate that xanthan gum lead to formation of an additional gel network which holds more water. Sensory evaluation revealed that goshtaba product with 0.5 % xanthan gum had quality characteristics that were similar to the control product containing 20 % fat.

Protein quality control in protection against systolic overload cardiomyopathy: the long term role of small heat shock proteins.

PubMed

Kumarapeli, Asangi R K; Horak, Kathleen; Wang, Xuejun

2010-07-21

Molecular chaperones represent the first line of defense of intracellular protein quality control. As a major constituent of molecular chaperones, heat shock proteins (HSP) are known to confer cardiomyocyte short-term protection against various insults and injuries. Previously, we reported that the small HSP alphaB-crystallin (CryAB) attenuates cardiac hypertrophic response in mice subjected to 2 weeks of severe pressure overload. However, the long-term role of small HSPs in cardiac hypertrophy and failure has rarely been studied. The present study investigates the cardiac responses to chronic severe pressure overload in CryAB/HSPB2 germ line ablated (KO) and cardiac-specific CryAB overexpressingtransgenic (TG) mice. Pressure overload was induced by transverse aortic constriction in KO, TG, and non-transgenic wild type (NTG) control mice and 10 weeks later molecular, cellular, and whole organ level hypertrophic responses were analyzed. As we previously described, CryAB/HSPB2 KO mice showed abnormal baseline cardiac physiology that worsened into a restrictive cardiomyopathic phenotype with aging. Severe pressure overload in these mice led to rapid deterioration of heart function and development of congestive cardiac failure. Contrary to their short term protective phenotype, CryAB TG mice showed no significant effects on cardiac hypertrophic responses and very modest improvement of hemodynamics during chronic systolic overload. These findings indicate that small HSPs CryAB and/or HSPB2 are essential to maintain cardiac structure and function but overex-pression of CryAB is not sufficient to confer a sustained protection against chronic systolic overload.

Whey Protein Components - Lactalbumin and Lactoferrin - Improve Energy Balance and Metabolism.

PubMed

Zapata, Rizaldy C; Singh, Arashdeep; Pezeshki, Adel; Nibber, Traj; Chelikani, Prasanth K

2017-08-30

Whey protein promotes weight loss and improves diabetic control, however, less is known of its bioactive components that produce such benefits. We compared the effects of normal protein (control) diet with high protein diets containing whey, or its fractions lactalbumin and lactoferrin, on energy balance and metabolism. Diet-induced obese rats were randomized to isocaloric diets: Control, Whey, Lactalbumin, Lactoferrin, or pair-fed to lactoferrin. Whey and lactalbumin produced transient hypophagia, whereas lactoferrin caused prolonged hypophagia; the hypophagia was likely due to decreased preference. Lactalbumin decreased weight and fat gain. Notably, lactoferrin produced sustained weight and fat loss, and attenuated the reduction in energy expenditure associated with calorie restriction. Lactalbumin and lactoferrin decreased plasma leptin and insulin, and lactalbumin increased peptide YY. Whey, lactalbumin and lactoferrin improved glucose clearance partly through differential upregulation of glucoregulatory transcripts in the liver and skeletal muscle. Interestingly, lactalbumin and lactoferrin decreased hepatic lipidosis partly through downregulation of lipogenic and/or upregulation of β-oxidation transcripts, and differentially modulated cecal bacterial populations. Our findings demonstrate that protein quantity and quality are important for improving energy balance. Dietary lactalbumin and lactoferrin improved energy balance and metabolism, and decreased adiposity, with the effects of lactoferrin being partly independent of caloric intake.

Improvement in the traditional processing method and nutritional quality of traditional extruded cassava-based snack (modified Ajogun).

PubMed

Obadina, Adewale O; Oyewole, Olusola B; Williams, Oluwasolabomi E

2013-07-01

This study was carried out to investigate and improve the traditional processing method and nutritional quality of the traditional cassava snack (Ajogun). Cassava root (Manihot esculenta Crantz L.) of TME 419 variety was processed into mash (40% moisture content). The cassava mash was mixed into different blends to produce fried traditional "Ajogun", fried and baked extrudates (modified Ajogun) as snacks. These products were analyzed to determine the proximate composition including carbohydrate, fat, protein, fiber, ash, and moisture contents and functional properties such as bulk density. The results obtained for the moisture, fat, protein, and ash contents showed significant difference (P < 0.05) between the control sample and the extrudates. However, there was no significant difference (P > 0.05) in the carbohydrate and fiber contents between the three samples. There was no significant difference (P > 0.05) in the bulk density of the snacks. Also, sensory evaluation was carried out on the cassava-based snacks using the 9-point hedonic scale to determine the degree of acceptability. Results obtained showed significant difference (P < 0.05) between the extrudates and control sample in terms of appearance, taste, flavor, color, aroma, texture, and overall acceptability. The highest acceptability level of the product was at 8.04 for the control sample (traditional Ajogun). This study has shown that "Ajogun", which is a lesser known cassava product, is rich in protein and fat.

Protein Quality Control by Molecular Chaperones in Neurodegeneration

PubMed Central

Ciechanover, Aaron; Kwon, Yong Tae

2017-01-01

Protein homeostasis (proteostasis) requires the timely degradation of misfolded proteins and their aggregates by protein quality control (PQC), of which molecular chaperones are an essential component. Compared with other cell types, PQC in neurons is particularly challenging because they have a unique cellular structure with long extensions. Making it worse, neurons are postmitotic, i.e., cannot dilute toxic substances by division, and, thus, are highly sensitive to misfolded proteins, especially as they age. Failure in PQC is often associated with neurodegenerative diseases, such as Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD), and prion disease. In fact, many neurodegenerative diseases are considered to be protein misfolding disorders. To prevent the accumulation of disease-causing aggregates, neurons utilize a repertoire of chaperones that recognize misfolded proteins through exposed hydrophobic surfaces and assist their refolding. If such an effort fails, chaperones can facilitate the degradation of terminally misfolded proteins through either the ubiquitin (Ub)-proteasome system (UPS) or the autophagy-lysosome system (hereafter autophagy). If soluble, the substrates associated with chaperones, such as Hsp70, are ubiquitinated by Ub ligases and degraded through the proteasome complex. Some misfolded proteins carrying the KFERQ motif are recognized by the chaperone Hsc70 and delivered to the lysosomal lumen through a process called, chaperone-mediated autophagy (CMA). Aggregation-prone misfolded proteins that remain unprocessed are directed to macroautophagy in which cargoes are collected by adaptors, such as p62/SQSTM-1/Sequestosome-1, and delivered to the autophagosome for lysosomal degradation. The aggregates that have survived all these refolding/degradative processes can still be directly dissolved, i.e., disaggregated by chaperones. Studies have shown that molecular chaperones alleviate the pathogenic symptoms by neurodegeneration-causing protein aggregates. Chaperone-inducing drugs and anti-aggregation drugs are actively exploited for beneficial effects on symptoms of disease. Here, we discuss how chaperones protect misfolded proteins from aggregation and mediate the degradation of terminally misfolded proteins in collaboration with cellular degradative machinery. The topics also include therapeutic approaches to improve the expression and turnover of molecular chaperones and to develop anti-aggregation drugs. PMID:28428740

Automated classification of immunostaining patterns in breast tissue from the human protein atlas.

PubMed

Swamidoss, Issac Niwas; Kårsnäs, Andreas; Uhlmann, Virginie; Ponnusamy, Palanisamy; Kampf, Caroline; Simonsson, Martin; Wählby, Carolina; Strand, Robin

2013-01-01

The Human Protein Atlas (HPA) is an effort to map the location of all human proteins (http://www.proteinatlas.org/). It contains a large number of histological images of sections from human tissue. Tissue micro arrays (TMA) are imaged by a slide scanning microscope, and each image represents a thin slice of a tissue core with a dark brown antibody specific stain and a blue counter stain. When generating antibodies for protein profiling of the human proteome, an important step in the quality control is to compare staining patterns of different antibodies directed towards the same protein. This comparison is an ultimate control that the antibody recognizes the right protein. In this paper, we propose and evaluate different approaches for classifying sub-cellular antibody staining patterns in breast tissue samples. The proposed methods include the computation of various features including gray level co-occurrence matrix (GLCM) features, complex wavelet co-occurrence matrix (CWCM) features, and weighted neighbor distance using compound hierarchy of algorithms representing morphology (WND-CHARM)-inspired features. The extracted features are used into two different multivariate classifiers (support vector machine (SVM) and linear discriminant analysis (LDA) classifier). Before extracting features, we use color deconvolution to separate different tissue components, such as the brownly stained positive regions and the blue cellular regions, in the immuno-stained TMA images of breast tissue. We present classification results based on combinations of feature measurements. The proposed complex wavelet features and the WND-CHARM features have accuracy similar to that of a human expert. Both human experts and the proposed automated methods have difficulties discriminating between nuclear and cytoplasmic staining patterns. This is to a large extent due to mixed staining of nucleus and cytoplasm. Methods for quantification of staining patterns in histopathology have many applications, ranging from antibody quality control to tumor grading.

Physiological and genotype-specific factors associated with grain quality changes in rice exposed to high ozone.

PubMed

Jing, Liquan; Dombinov, Vitalij; Shen, Shibo; Wu, Yanzhen; Yang, Lianxin; Wang, Yunxia; Frei, Michael

2016-03-01

Rising tropospheric ozone concentrations in Asia affect the yield and quality of rice. This study investigated ozone-induced changes in rice grain quality in contrasting rice genotypes, and explored the associated physiological processes during the reproductive growth phase. The ozone sensitive variety Nipponbare and a breeding line (L81) containing two tolerance QTLs in Nipponbare background were exposed to 100 ppb ozone (8 h per day) or control conditions throughout their growth. Ozone affected grain chalkiness and protein concentration and composition. The percentage of chalky grains was significantly increased in Nipponbare but not in L81. Physiological measurements suggested that grain chalkiness was associated with a drop in foliar carbohydrate and nitrogen levels during grain filling, which was less pronounced in the tolerant L81. Grain total protein concentration was significantly increased in the ozone treatment, although the albumin fraction (water soluble protein) decreased. The increase in protein was more pronounced in L81, due to increases in the glutelin fraction in this genotype. Amino acids responded differently to the ozone treatment. Three essential amino acids (leucine, methionine and threonine) showed significant increases, while seven showed significant treatment by genotype interactions, mostly due to more positive responses in L81. The trend of increased grain protein was in contrast to foliar nitrogen levels, which were negatively affected by ozone. A negative correlation between grain protein and foliar nitrogen in ozone stress indicated that higher grain protein cannot be explained by a concentration effect in all tissues due to lower biomass production. Rather, ozone exposure affected the nitrogen distribution, as indicated by altered foliar activity of the enzymes involved in nitrogen metabolism, such as glutamine synthetase and glutamine-2-oxoglutarate aminotransferase. Our results demonstrate differential responses of grain quality to ozone due to the presence of tolerance QTL, and partly explain the underlying physiological processes. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Unfolded Protein Response in Chronic Obstructive Pulmonary Disease

PubMed Central

2016-01-01

Accumulation of nonfunctional and potentially cytotoxic, misfolded proteins in chronic obstructive pulmonary disease (COPD) is believed to contribute to lung cell apoptosis, inflammation, and autophagy. Because of its fundamental role as a quality control system in protein metabolism, the “unfolded protein response” (UPR) is of potential importance in the pathogenesis of COPD. The UPR comprises a series of transcriptional, translational, and post-translational processes that decrease protein synthesis while enhancing protein folding capacity and protein degradation. Several studies have suggested that the UPR contributes to lung cell apoptosis and lung inflammation in at least some subjects with human COPD. However, information on the prevalence of the UPR in subjects with COPD, the lung cells that manifest a UPR, and the role of the UPR in the pathogenesis of COPD is extremely limited and requires additional study. PMID:27115948

The Unfolded Protein Response in Chronic Obstructive Pulmonary Disease.

PubMed

Kelsen, Steven G

2016-04-01

Accumulation of nonfunctional and potentially cytotoxic, misfolded proteins in chronic obstructive pulmonary disease (COPD) is believed to contribute to lung cell apoptosis, inflammation, and autophagy. Because of its fundamental role as a quality control system in protein metabolism, the "unfolded protein response" (UPR) is of potential importance in the pathogenesis of COPD. The UPR comprises a series of transcriptional, translational, and post-translational processes that decrease protein synthesis while enhancing protein folding capacity and protein degradation. Several studies have suggested that the UPR contributes to lung cell apoptosis and lung inflammation in at least some subjects with human COPD. However, information on the prevalence of the UPR in subjects with COPD, the lung cells that manifest a UPR, and the role of the UPR in the pathogenesis of COPD is extremely limited and requires additional study.

Protein Separation by Capillary Gel Electrophoresis: A Review

PubMed Central

Zhu, Zaifang; Lu, Joann J.; Liu, Shaorong

2011-01-01

Capillary gel electrophoresis (CGE) has been used for protein separation for more than two decades. Due to the technology advancement, current CGE methods are becoming more and more robust and reliable for protein analysis, and some of the methods have been routinely used for the analysis of protein-based pharmaceuticals and quality controls. In light of this progress, we survey 147 papers related to CGE separations of proteins and present an overview of this technology. We first introduce briefly the early development of CGE. We then review the methodology, in which we specifically describe the matrices, coatings, and detection strategies used in CGE. CGE using microfabricated channels and incorporation of CGE with two-dimensional protein separations are also discussed in this section. We finally present a few representative applications of CGE for separating proteins in real-world samples. PMID:22122927

The Commercial Vapor Diffusion Apparatus (CVDA) STS-95

NASA Technical Reports Server (NTRS)

2004-01-01

The Commercial Vapor Diffusion Apparatus will be used to perform 128 individual crystal growth investigations for commercial and science research. These experiments will grow crystals of several different proteins, including HIV-1 Protease Inhibitor, Glycogen Phosphorylase A, and NAD Synthetase. The Commercial Vapor Diffusion Apparatus supports multiple commercial investigations within a controlled environment. The goal of the Commercial Protein Crystal Growth payload on STS-95 is to grow large, high-quality crystals of several different proteins of interest to industry, and to continue to refine the technology and procedures used in microgravity for this important commercial research.

Hydrogen-Exchange Mass Spectrometry for the Study of Intrinsic Disorder in Proteins

PubMed Central

Balasubramaniam, Deepa; Komives, Elizabeth A.

2012-01-01

Amide hydrogen/deuterium exchange detected by mass spectrometry (HXMS) is seeing wider use for the identification of intrinsically disordered parts of proteins. In this review, we discuss examples of how discovery of intrinsically disordered regions and their removal can aid in structure determination, biopharmaceutical quality control, the characterization of how posttranslational modifications affect weak structuring of disordered regions, the study of coupled folding and binding, and the characterization of amyloid formation. PMID:23099262

The Arabidopsis endoplasmic reticulum associated degradation pathways are involved in the regulation of heat stress response

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

Li, Lin-Mao; University of Chinese Academy of Sciences, Beijing; Lü, Shi-You

Abstracts: The Cytosolic Protein Response (CPR) in the cytosol and the Unfolded Protein Response (UPR) and ER-associated degradation (ERAD) in the endoplasmic reticulum are major pathways of the cellular proteostasis network. However, despite years of effort, how these protein quality control systems coordinated in vivo remains largely unknown, particularly in plants. In this study, the roles of two evolutionarily conserved ERAD pathways (DOA10 and HRD1) in heat stress response were investigated through reverse genetic approaches in Arabidopsis. Phenotypic analysis of the mutants showed that the two ERAD pathways additively play negative roles in heat tolerance, which was demonstrated by higher survivalmore » rate and lower electrolyte leakage in the loss of function mutants compared to the wild type plants. Importantly, gene expression analysis revealed that the mutant plants showed elevated transcriptional regulation of several downstream genes, including those encoding CPR and UPR marker genes, under both basal and heat stress conditions. Finally, multiple components of ERAD genes exhibited rapid response to increasing temperature. Taken together, our data not only unravels key insights into the crosstalk between different protein quality control processes, but also provides candidate genes to genetically improve plant heat tolerance in the future. - Highlights: • ERAD pathways cooperatively regulate plant thermotolerance. • ERAD pathways cooperatively regulate UPR and CPR. • ERAD components gene expression are upregulated by heat stress.« less

Molecular mechanism of ER stress-induced pre-emptive quality control involving association of the translocon, Derlin-1, and HRD1.

PubMed

Kadowaki, Hisae; Satrimafitrah, Pasjan; Takami, Yasunari; Nishitoh, Hideki

2018-05-09

The maintenance of endoplasmic reticulum (ER) homeostasis is essential for cell function. ER stress-induced pre-emptive quality control (ERpQC) helps alleviate the burden to a stressed ER by limiting further protein loading. We have previously reported the mechanisms of ERpQC, which includes a rerouting step and a degradation step. Under ER stress conditions, Derlin family proteins (Derlins), which are components of ER-associated degradation, reroute specific ER-targeting proteins to the cytosol. Newly synthesized rerouted polypeptides are degraded via the cytosolic chaperone Bag6 and the AAA-ATPase p97 in the ubiquitin-proteasome system. However, the mechanisms by which ER-targeting proteins are rerouted from the ER translocation pathway to the cytosolic degradation pathway and how the E3 ligase ubiquitinates ERpQC substrates remain unclear. Here, we show that ERpQC substrates are captured by the carboxyl-terminus region of Derlin-1 and ubiquitinated by the HRD1 E3 ubiquitin ligase prior to degradation. Moreover, HRD1 forms a large ERpQC-related complex composed of Sec61α and Derlin-1 during ER stress. These findings indicate that the association of the degradation factor HRD1 with the translocon and the rerouting factor Derlin-1 may be necessary for the smooth and effective clearance of ERpQC substrates.

Meat Quality Derived from High Inclusion of a Micro-Alga or Insect Meal as an Alternative Protein Source in Poultry Diets: A Pilot Study

PubMed Central

Neumann, Carmen; Velten, Susanne; Liebert, Frank

2018-01-01

The effects on meat quality resulting from alternative dietary protein sources (Spirulina and Hermetia meal) in poultry diets are studied to determine the overall suitability of these ingredients considering state-of-the-art packaging practices—highly oxygenated modified atmosphere packaging (HiOx MAP). We monitored standard slaughterhouse parameters, such as live weight, carcass weight, dressed yield, and pH at 20 min and 24 h post mortem. In addition, we studied the effects that 3 and 7-day storage in HiOx MAP has on the overall product physico-chemical and sensory properties. In addition to previously supported effects of HiOx MAP, we found that meat quality could be improved when Spirulina replaces 50% of the soy protein in broiler diets; however, this substitution results in a dark reddish-yellowish meat colour. On the other hand, the substitution with Hermetia larval meal results in a product that does not differ from the standard fed control group, with the exception that the breast filet has a more intense flavour that decreases over storage time. All-in-all Spirulina and Hermetia meal have the potential to replace soybean meal in broiler diets without deteriorating meat quality. PMID:29518048

Dietary calcium deficiency in laying ducks impairs eggshell quality by suppressing shell biomineralization.

PubMed

Chen, Wei; Zhao, Fei; Tian, Zhi Mei; Zhang, Han Xing; Ruan, Dong; Li, Yan; Wang, Shuang; Zheng, Chun Tian; Lin, Ying Cai

2015-10-01

The objective of this study was to determine the effects of dietary calcium deficiency on the process of shell formation. Four hundred and fifty female ducks (Anas platyrhynchos) at 22 weeks were randomly assigned to three groups. Ducks were fed one of two calcium-deficient diets (containing 1.8% or 0.38% calcium, respectively) or a calcium-adequate control diet (containing 3.6% calcium) for 67 days (depletion period) and then all ducks were fed a calcium-adequate diet for an additional 67 days (repletion period). Compared with the calcium-adequate control, the average shell thickness, egg shell weight, breaking strength, mammillae density and mammillary knob thickness of shell from ducks that consumed the diet with 0.38% calcium were significantly decreased (P<0.05) during the depletion period, accompanied by reduced tibia quality. The mRNA expression of both secreted phosphoprotein 1 (SPP1) and carbonic anhydrase 2 (CA2) in the uterus was decreased after feeding calcium-deficient diets (1.8% or 0.38% calcium). mRNA transcripts of calbindin 1 (CALB1), an important protein responsible for calcium transport, and the matrix protein genes ovocalyxin-32 (OCX-32) and ovocleidin-116 (OC-116) were reduced in ducks fed 0.38% calcium but not 1.8% calcium. Plasma estradiol concentration was decreased by both of the calcium-deficient diets (P<0.05). The impaired shell quality and suppressed functional proteins involved in shell formation could be reversed by repletion of dietary calcium. The results of the present study suggest that dietary calcium deficiency negatively affects eggshell quality and microarchitecture, probably by suppressing shell biomineralization. © 2015. Published by The Company of Biologists Ltd.

Ube2w and ataxin-3 coordinately regulate the ubiquitin ligase CHIP

PubMed Central

Scaglione, K. Matthew; Zavodszky, Eszter; Todi, Sokol V.; Patury, Srikanth; Xu, Ping; Rodríguez-Lebrón, Edgardo; Fischer, Svetlana; Konen, John; Djarmati, Ana; Peng, Junmin; Gestwicki, Jason E.; Paulson, Henry L.

2011-01-01

Summary The mechanisms by which ubiquitin ligases are regulated remain poorly understood. Here we describe a series of molecular events that coordinately regulate CHIP, a neuroprotective E3 implicated in protein quality control. Through their opposing activities, the initiator E2, Ube2w, and the specialized deubiquitinating enzyme (DUB), ataxin-3, participate in initiating, regulating and terminating the CHIP ubiquitination cycle. Monoubiquitination of CHIP by Ube2w stabilizes the interaction between CHIP and ataxin-3, which through its DUB activity limits the length of chains attached to CHIP substrates. Upon completion of substrate ubiquitination ataxin-3 deubiquitinates CHIP, effectively terminating the reaction. Our results suggest that functional pairing of E3s with ataxin-3 or similar DUBs represents an important point of regulation in ubiquitin-dependent protein quality control. In addition, the results shed light on disease pathogenesis in SCA3, a neurodegenerative disorder caused by polyglutamine expansion in ataxin-3. PMID:21855799

EOL-1, the Homolog of the Mammalian Dom3Z, Regulates Olfactory Learning in C. elegans

PubMed Central

Shen, Yu; Zhang, Jiangwen; Calarco, John A.

2014-01-01

Learning is an essential function of the nervous system. However, our understanding of molecular underpinnings of learning remains incomplete. Here, we characterize a conserved protein EOL-1 that regulates olfactory learning in Caenorhabditis elegans. A recessive allele of eol-1 (enhanced olfactory learning) learns better to adjust its olfactory preference for bacteria foods and eol-1 acts in the URX sensory neurons to regulate learning. The mammalian homolog of EOL-1, Dom3Z, which regulates quality control of pre-mRNAs, can substitute the function of EOL-1 in learning regulation, demonstrating functional conservation between these homologs. Mutating the residues of Dom3Z that are critical for its enzymatic activity, and the equivalent residues in EOL-1, abolishes the function of these proteins in learning. Together, our results provide insights into the function of EOL-1/Dom3Z and suggest that its activity in pre-mRNA quality control is involved in neural plasticity. PMID:25274815

Fish gelatin combined with chitosan coating inhibits myofibril degradation of golden pomfret (Trachinotus blochii) fillet during cold storage.

PubMed

Feng, Xiao; Bansal, Nidhi; Yang, Hongshun

2016-06-01

Coating of gelatin and chitosan can improve fish fillet's quality, but the mechanism is not clear. Chitosan/gelatin coatings significantly prevented deterioration of golden pomfret fillet at 4 °C. Chitosan with 7.2% gelatin group showed the best effect on preserving the length of myofibril, which remained greater than 15 μm at day 17 of storage, while for control, chitosan and chitosan combined with 3.6% gelatin group, it was 5.03, 10.04 and 9.02 μm, respectively. The MALDI-TOF MS result revealed that the coatings slowed down the protein deterioration of fillet. On days 13 and 17, the myosin light chain and myoglobin in control group degraded, while the two proteins still existed in chitosan/gelatin coated groups. Overall, the chitosan with 7.2% gelatin coating had the best effect on preserving fillet's quality during storage. The coating may exert its protective effect via inhibiting myofibril degradation within fillet. Copyright © 2016 Elsevier Ltd. All rights reserved.

A novel cell line generated using the CRISPR/Cas9 technology as universal quality control material for KRAS G12V mutation testing.

PubMed

Jia, Shiyu; Zhang, Rui; Lin, Guigao; Peng, Rongxue; Gao, Peng; Han, Yanxi; Fu, Yu; Ding, Jiansheng; Wu, Qisheng; Zhang, Kuo; Xie, Jiehong; Li, Jinming

2018-06-01

KRAS mutations are the key indicator for EGFR monoclonal antibody-targeted therapy and acquired drug resistance, and their accurate detection is critical to the clinical decision-making of colorectal cancer. However, no proper quality control material is available for the current detection methods, particularly next-generation sequencing (NGS). The ideal quality control material for NGS needs to provide both the tumor mutation gene and the matched background genomic DNA, which is uncataloged in public databases, to accurately distinguish germline polymorphisms and somatic mutations. We developed a novel KRAS G12V mutant cell line using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technique to make up for the deficiencies in existing quality control material and further validated the feasibility of the cell line as quality control material by amplification refractory mutation system (ARMS), Sanger sequencing, digital PCR (dPCR), and NGS. We verified that the edited cell line specifically had the G12V mutation, and the validation results presented a high consistency among the four methods of detection. The three cell lines screened contained the G12V mutation and the mutation allele fractions of G12V-1, G12V-2, and G12V-3 were 52.01%, 82.06%, and 17.29%, respectively. The novel KRAS G12V cell line generated using the CRISPR/Cas9 gene editing system is suitable as a quality control material for all current detection methods and provides a new direction in the development of quality control material. © 2018 Wiley Periodicals, Inc.

Biochemical and proteomic analysis of grape berries (Vitis labruscana) during cold storage upon postharvest salicylic acid treatment.

PubMed

Cai, Han; Yuan, Xiaozhuan; Pan, Jiaojiao; Li, Huan; Wu, Ziming; Wang, Yun

2014-10-15

Salicylic acid (SA) treatment has been widely used to maintain fruit quality during postharvest storage. To elucidate the molecular mechanism related to this treatment, the effect of SA treatment on fruit quality as well as protein expression profiles of grape berries (Vitis labruscana cv. Kyoho) during the subsequent cold storage was evaluated. As expected, SA treatment inhibited postharvest loss and chilling damage by reducing fruit softening and membrane damage and slowing weight loss. A gel-based proteomic approach was designed to screen for differentially expressed proteins in SA-treated and control grape berries. A total of 69 differentially accumulated proteins were successfully identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, which can be functionally classified into eight categories. Among these proteins, antioxidant enzymes including ascorbate peroxidase, oxidoreductase, and glutathione S-transferase were induced, and the abundances of several defense-related proteins, such as heat shock protein (HSP) and temperature-induced lipocalin, were up-regulated by SA treatment. In addition, proteins involved in carbohydrate catabolism and energy production were also induced by SA treatment. Interpretation of the data for differential accumulation of proteins revealed that the effect of SA on reducing postharvest losses and chilling damage of grape berries during cold storage may be due to activated defense responses and carbohydrate metabolism and higher levels of energy status.

Comparison of Protein Immunoprecipitation-Multiple Reaction Monitoring with ELISA for Assay of Biomarker Candidates in Plasma

PubMed Central

2013-01-01

Quantitative analysis of protein biomarkers in plasma is typically done by ELISA, but this method is limited by the availability of high-quality antibodies. An alternative approach is protein immunoprecipitation combined with multiple reaction monitoring mass spectrometry (IP-MRM). We compared IP-MRM to ELISA for the analysis of six colon cancer biomarker candidates (metalloproteinase inhibitor 1 (TIMP1), cartilage oligomeric matrix protein (COMP), thrombospondin-2 (THBS2), endoglin (ENG), mesothelin (MSLN) and matrix metalloproteinase-9 (MMP9)) in plasma from colon cancer patients and noncancer controls. Proteins were analyzed by multiplex immunoprecipitation from plasma with the ELISA capture antibodies, further purified by SDS-PAGE, digested and analyzed by stable isotope dilution MRM. IP-MRM provided linear responses (r = 0.978–0.995) between 10 and 640 ng/mL for the target proteins spiked into a “mock plasma” matrix consisting of 60 mg/mL bovine serum albumin. Measurement variation (coefficient of variation at the limit of detection) for IP-MRM assays ranged from 2.3 to 19%, which was similar to variation for ELISAs of the same samples. IP-MRM and ELISA measurements for all target proteins except ENG were highly correlated (r = 0.67–0.97). IP-MRM with high-quality capture antibodies thus provides an effective alternative method to ELISA for protein quantitation in biological fluids. PMID:24224610

Quality control of murine monoclonal antibodies using isoelectric focusing affinity immunoblot analysis

NASA Technical Reports Server (NTRS)

Hamilton, Robert G.; Rodkey, L. Scott; Reimer, Charles B.

1987-01-01

The quality control of murine hybridoma secretory products has been performed using two approaches for isoelectric focusing affinity immunoblot analysis: (1) a method in which antigen-coated nitrocellulose is placed on top of an acrylamide gel containing isoelectrically focused ascites to bind the antigen specific monoclonal antibody; and (2) a method in which focused ascite proteins were passively blotted onto nitrocellulose and specific monoclonal antibodies were detected with enzyme-conjugated antigen. Analysis by both methods of batches of ascites containing antihuman IgG antibodies that were produced by six hybridomas permitted effective monitoring of immunoreactive antibodies for pI microheterogeneity.

Protecting the proteome: Eukaryotic cotranslational quality control pathways

PubMed Central

2014-01-01

The correct decoding of messenger RNAs (mRNAs) into proteins is an essential cellular task. The translational process is monitored by several quality control (QC) mechanisms that recognize defective translation complexes in which ribosomes are stalled on substrate mRNAs. Stalled translation complexes occur when defects in the mRNA template, the translation machinery, or the nascent polypeptide arrest the ribosome during translation elongation or termination. These QC events promote the disassembly of the stalled translation complex and the recycling and/or degradation of the individual mRNA, ribosomal, and/or nascent polypeptide components, thereby clearing the cell of improper translation products and defective components of the translation machinery. PMID:24535822

Feeding value of whole raw soya beans as a protein supplement for beef cattle consuming low-quality forages.

PubMed

Arelovich, H M; Lagrange, S; Torre, R; Martinez, M F; Laborde, H E

2018-02-01

Experiments (Exp) I and II were conducted to compare raw whole soya beans (WSB), roasted (rWSB) or other protein sources as supplements of low-quality forages fed ad libitum to beef cattle, upon DM intake (DMI), ruminal and blood parameters, and animal performance. Exp I: treatments for wheat straw fed to four ruminally cannulated steers were (i) Control-WS: no supplement; (ii) WSB-WS: whole soya beans; (iii) rWSB-WS: roasted WSB; and (iv) SBM-WS: soybean meal-wheat midds mixture; all fed at 1.4 kg DM/day. Exp II: 12 steers grazed deferred grain sorghum (DS) receiving these treatments: (i) Control-DS: no supplement; (ii) WSB-DS: 1.26 kg DM/day whole soya beans; and (iii) SFM-DS: 1.35 kg DM/day of sunflower meal. In Exp I, WS DMI resulted 47, 52 and 41% greater for WSB-WS, rWSB-WS and SBM-WS, respectively, than Control-WS (p < .05). In Exp II, the DMI of DS was unaffected by supplementation; a substitution of DS by supplement was found for WSB-DS (p < .05); however, total diet and digestible DMI increased with supplementation (p < .05). Rumen pH in Exp I remained unaffected by supplementation, but N-NH 3 as well as blood urea-N in Exp II increased (p < .05). In Exp II, average daily weight gains improved similarly with both supplements compared with Control-DS. Additionally, feed-to-gain ratio decreased (p < .05), being lower for WSB-DS (8.3) vs. SFM-DS (9.9). Roasting effects of WSB as a supplement for low-quality forages were not detected, and all protein sources increased total diet DMI and forage utilization. Only moderate cattle weight gains could be expected for unsupplemented DS. © 2017 Blackwell Verlag GmbH.

Control of mitochondrial biogenesis and function by the ubiquitin-proteasome system.

PubMed

Bragoszewski, Piotr; Turek, Michal; Chacinska, Agnieszka

2017-04-01

Mitochondria are pivotal organelles in eukaryotic cells. The complex proteome of mitochondria comprises proteins that are encoded by nuclear and mitochondrial genomes. The biogenesis of mitochondrial proteins requires their transport in an unfolded state with a high risk of misfolding. The mislocalization of mitochondrial proteins is deleterious to the cell. The electron transport chain in mitochondria is a source of reactive oxygen species that damage proteins. Mitochondrial dysfunction is linked to many pathological conditions and, together with the loss of cellular protein homeostasis (proteostasis), are hallmarks of ageing and ageing-related degeneration diseases. The pathogenesis of neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease, has been associated with mitochondrial and proteostasis failure. Thus, mitochondrial proteins require sophisticated surveillance mechanisms. Although mitochondria form a proteasome-exclusive compartment, multiple lines of evidence indicate a crucial role for the cytosolic ubiquitin-proteasome system (UPS) in the quality control of mitochondrial proteins. The proteasome affects mitochondrial proteins at stages of their biogenesis and maturity. The effects of the UPS go beyond the removal of damaged proteins and include the adjustment of mitochondrial proteome composition, the regulation of organelle dynamics and the protection of cellular homeostasis against mitochondrial failure. In turn, mitochondrial activity and mitochondrial dysfunction adjust the activity of the UPS, with implications at the cellular level. © 2017 The Authors.

Control of mitochondrial biogenesis and function by the ubiquitin–proteasome system

PubMed Central

Bragoszewski, Piotr; Turek, Michal

2017-01-01

Mitochondria are pivotal organelles in eukaryotic cells. The complex proteome of mitochondria comprises proteins that are encoded by nuclear and mitochondrial genomes. The biogenesis of mitochondrial proteins requires their transport in an unfolded state with a high risk of misfolding. The mislocalization of mitochondrial proteins is deleterious to the cell. The electron transport chain in mitochondria is a source of reactive oxygen species that damage proteins. Mitochondrial dysfunction is linked to many pathological conditions and, together with the loss of cellular protein homeostasis (proteostasis), are hallmarks of ageing and ageing-related degeneration diseases. The pathogenesis of neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease, has been associated with mitochondrial and proteostasis failure. Thus, mitochondrial proteins require sophisticated surveillance mechanisms. Although mitochondria form a proteasome-exclusive compartment, multiple lines of evidence indicate a crucial role for the cytosolic ubiquitin–proteasome system (UPS) in the quality control of mitochondrial proteins. The proteasome affects mitochondrial proteins at stages of their biogenesis and maturity. The effects of the UPS go beyond the removal of damaged proteins and include the adjustment of mitochondrial proteome composition, the regulation of organelle dynamics and the protection of cellular homeostasis against mitochondrial failure. In turn, mitochondrial activity and mitochondrial dysfunction adjust the activity of the UPS, with implications at the cellular level. PMID:28446709

Effects of exercise on the urinary proteome.

PubMed

Kohler, Maxie; Schänzer, Wilhelm; Thevis, Mario

2015-01-01

Exercise-induced proteinuria has been observed and studied for more than a century. It was found that different sport disciplines alter the urinary proteome in different ways. Moderate-intensity exercise results in increased glomerular filtration, meaning that medium-sized proteins are excreted in higher amounts, while high-intensity exercise of short duration also increases the excretion of low molecular weight proteins as a result of tubular dysfunction. Exhaustive exercise may lead to the excretion of hemoglobin or myoglobin, which changes the urinary proteome considerably. Studies comparing protein maps of different sport types compared to a control group showed that quality and quantity of urinary proteins are interindividually different. In addition, urine samples collected before and after exercise exhibit substantially different protein patterns even from the same person. Therefore, further studies investigating the urinary proteome are desirable. As the variation of protein content and composition in urine are generally much higher than in other matrices, respective studies need to be well controlled and homogenous groups of volunteers should be chosen. In addition to the sport-related physiological and biochemical interest, exercise-induced protein changes also need to be considered for biomarker measurements from urine samples for kidney or other diseases.

Emerging Evidence for the Importance of Dietary Protein Source on Glucoregulatory Markers and Type 2 Diabetes: Different Effects of Dairy, Meat, Fish, Egg, and Plant Protein Foods.

PubMed

Comerford, Kevin B; Pasin, Gonca

2016-07-23

Observational studies provide evidence that a higher intake of protein from plant-based foods and certain animal-based foods is associated with a lower risk for type 2 diabetes. However, there are few distinguishable differences between the glucoregulatory qualities of the proteins in plant-based foods, and it is likely their numerous non-protein components (e.g., fibers and phytochemicals) that drive the relationship with type 2 diabetes risk reduction. Conversely, the glucoregulatory qualities of the proteins in animal-based foods are extremely divergent, with a higher intake of certain animal-based protein foods showing negative effects, and others showing neutral or positive effects on type 2 diabetes risk. Among the various types of animal-based protein foods, a higher intake of dairy products (such as milk, yogurt, cheese and whey protein) consistently shows a beneficial relationship with glucose regulation and/or type 2 diabetes risk reduction. Intervention studies provide evidence that dairy proteins have more potent effects on insulin and incretin secretion compared to other commonly consumed animal proteins. In addition to their protein components, such as insulinogenic amino acids and bioactive peptides, dairy products also contain a food matrix rich in calcium, magnesium, potassium, trans-palmitoleic fatty acids, and low-glycemic index sugars-all of which have been shown to have beneficial effects on aspects of glucose control, insulin secretion, insulin sensitivity and/or type 2 diabetes risk. Furthermore, fermentation and fortification of dairy products with probiotics and vitamin D may improve a dairy product's glucoregulatory effects.

Direct bone morphogenetic protein 2 and Indian hedgehog gene transfer for articular cartilage repair using bone marrow coagulates.

PubMed

Sieker, J T; Kunz, M; Weißenberger, M; Gilbert, F; Frey, S; Rudert, M; Steinert, A F

2015-03-01

Bone morphogenetic protein 2 (BMP-2, encoded by BMP2) and Indian hedgehog protein (IHH, encoded by IHH) are well known regulators of chondrogenesis and chondrogenic hypertrophy. Despite being a potent chondrogenic factor BMP-2 was observed to induce chondrocyte hypertrophy in osteoarthritis (OA), growth plate cartilage and adult mesenchymal stem cells (MSCs). IHH might induce chondrogenic differentiation through different intracellular signalling pathways without inducing subsequent chondrocyte hypertrophy. The primary objective of this study is to test the efficacy of direct BMP2 and IHH gene delivery via bone marrow coagulates to influence histological repair cartilage quality in vivo. Vector-laden autologous bone marrow coagulates with 10(11) adenoviral vector particles encoding BMP2, IHH or the Green fluorescent protein (GFP) were delivered to 3.2 mm osteochondral defects in the trochlea of rabbit knees. After 13 weeks the histological repair cartilage quality was assessed using the ICRS II scoring system and the type II collagen positive area. IHH treatment resulted in superior histological repair cartilage quality than GFP controls in all of the assessed parameters (with P < 0.05 in five of 14 assessed parameters). Results of BMP2 treatment varied substantially, including severe intralesional bone formation in two of six joints after 13 weeks. IHH gene transfer is effective to improve repair cartilage quality in vivo, whereas BMP2 treatment, carried the risk intralesional bone formation. Therefore IHH protein can be considered as an attractive alternative candidate growth factor for further preclinical research and development towards improved treatments for articular cartilage defects. Copyright © 2014 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

MQAPRank: improved global protein model quality assessment by learning-to-rank.

PubMed

Jing, Xiaoyang; Dong, Qiwen

2017-05-25

Protein structure prediction has achieved a lot of progress during the last few decades and a greater number of models for a certain sequence can be predicted. Consequently, assessing the qualities of predicted protein models in perspective is one of the key components of successful protein structure prediction. Over the past years, a number of methods have been developed to address this issue, which could be roughly divided into three categories: single methods, quasi-single methods and clustering (or consensus) methods. Although these methods achieve much success at different levels, accurate protein model quality assessment is still an open problem. Here, we present the MQAPRank, a global protein model quality assessment program based on learning-to-rank. The MQAPRank first sorts the decoy models by using single method based on learning-to-rank algorithm to indicate their relative qualities for the target protein. And then it takes the first five models as references to predict the qualities of other models by using average GDT_TS scores between reference models and other models. Benchmarked on CASP11 and 3DRobot datasets, the MQAPRank achieved better performances than other leading protein model quality assessment methods. Recently, the MQAPRank participated in the CASP12 under the group name FDUBio and achieved the state-of-the-art performances. The MQAPRank provides a convenient and powerful tool for protein model quality assessment with the state-of-the-art performances, it is useful for protein structure prediction and model quality assessment usages.

S-nitrosylation drives cell senescence and aging in mammals by controlling mitochondrial dynamics and mitophagy.

PubMed

Rizza, Salvatore; Cardaci, Simone; Montagna, Costanza; Di Giacomo, Giuseppina; De Zio, Daniela; Bordi, Matteo; Maiani, Emiliano; Campello, Silvia; Borreca, Antonella; Puca, Annibale A; Stamler, Jonathan S; Cecconi, Francesco; Filomeni, Giuseppe

2018-04-10

S -nitrosylation, a prototypic redox-based posttranslational modification, is frequently dysregulated in disease. S -nitrosoglutathione reductase (GSNOR) regulates protein S -nitrosylation by functioning as a protein denitrosylase. Deficiency of GSNOR results in tumorigenesis and disrupts cellular homeostasis broadly, including metabolic, cardiovascular, and immune function. Here, we demonstrate that GSNOR expression decreases in primary cells undergoing senescence, as well as in mice and humans during their life span. In stark contrast, exceptionally long-lived individuals maintain GSNOR levels. We also show that GSNOR deficiency promotes mitochondrial nitrosative stress, including excessive S -nitrosylation of Drp1 and Parkin, thereby impairing mitochondrial dynamics and mitophagy. Our findings implicate GSNOR in mammalian longevity, suggest a molecular link between protein S -nitrosylation and mitochondria quality control in aging, and provide a redox-based perspective on aging with direct therapeutic implications.

Study on the effect of different concentration of Spirulina platensis paste added into dried noodle to its quality characteristics

NASA Astrophysics Data System (ADS)

Agustini, T. W.; Ma'ruf, W. F.; Widayat; Wibowo, B. A.; Hadiyanto

2017-02-01

High nutritional content of microalgae S. platensis is very good for human health especially for its protein and β-carotene. These compounds can be used for improving quality of food from nutritional point of view. This study was designed to observe the effect of different concentration of S. platensis paste to dried noodle quality based on physical, chemical and sensory perspectives. Material used in this study was S. platensis powder obtained from CV Neoalgae, Sukoharjo. The best treatment observed from preliminary study was then used for further study and compared to control treatment (without added S. platensis paste). The dried noodle resulted was then analyzed for elasticity, chemical (β-carotene, water, protein, ash, fat and carbohydrate) and sensory (hedonic). Parametric data obtained were analyzed using t-Test while non-parametric data was analyzed using Kruskal-Wallis followed by Multiple Comparison test. The study showed that dried noodle added with 9% S. platensis paste give significant effect (P<0.05) on elasticity; β-carotene, water, protein, ash, fat and carbohydrate and also sensory (hedonic). Dried noodle without added S. platensis paste has lower in β-carotene and protein compared to that of added S. platensis paste with the value of 0.06 mg/100 gr and 17.51 mg/100gr (β-carotene) and 8.88% and 38.6% (protein), respectively. Those, chemical composition of dried noodle added with S. platensis comply with Indonesian National Standard in term of protein, water, and ash content with sensory of 7.26 ≤ μ ≤ 7.63. Addition of S. platensis can significantly increase the nutritional of dried noodle especially protein (by 4 times) and β-carotene.

Chemotherapy-Related Toxicity, Nutritional Status and Quality of Life in Precachectic Oncologic Patients with, or without, High Protein Nutritional Support. A Prospective, Randomized Study.

PubMed

Ziętarska, Monika; Krawczyk-Lipiec, Joanna; Kraj, Leszek; Zaucha, Renata; Małgorzewicz, Sylwia

2017-10-11

Cancer disease is usually associated with impaired nutritional status, which is one of the factors contributing to deterioration of the results of surgery, chemotherapy or radiotherapy. The aim of the study was to determine whether nutritional support with high protein (ONS) in adult oncologic patients in the first step of cancer cachexia-asymptomatic precachexia, has an influence on the toxicity of systemic therapy. However, secondary endpoints were established: to determine whether high protein ONS influences the nutritional status, the quality of life, and the performance status. A total of 114 persons aged 40-84 years old with colorectal cancer were examined. Based on the randomization, 47 patients were qualified to the interventional group (ONS group) and 48 to Control group. To evaluate the nutritional status NRS-2002 (Nutritional Risk Screening), SGA (Subjective Global Assessment), SCRINIO (SCReenIng the Nutritional status In Oncology) Working Group classification, VAS (Visual Analog Scale) for appetite was used. FAACT (Functional Assessment of Anorexia/Cachexia Therapy) questionnaire was used for assessment of the quality of life. The health status of patients was evaluated based on the Karnofsky Performance Scale. Anthropometric measurements were done. Severe complications of chemotherapy, which caused the end of treatment, a slight complication of the gastrointestinal tract such as diarrhea grade 2 according to ECOG (Eastern Cooperative Oncology Group) score regardless of the studied group, were observed. There were no statistical differences in the number and severity of the observed complications, i.e., neutropenia, leucopenia, thrombocytopenia, anemia, abdominal pain, nausea and vomiting, and diarrhea. During the follow-up the significant changes of SGA, VAS, albumin and prealbumin were observed between groups. In the ONS group an improvement in nutritional status was noticed (increased appetite VAS, p = 0.05; increased points in SGA, p = 0.015, and increased levels of albumin and prealbumin, p = 0.05). In Control group nutritional status was stable during observation. The performance status and quality of life were stable in both groups. No statistical differences between groups (ONS vs. Control) in the numbers for disqualification, resignation, delay in treatment, or dose reduction were observed. Results of the study did not indicate that nutritional support in precachectic oncologic patients influenced the toxicity of systemic therapy. High protein nutritional support improved nutritional status assessed by SGA, VAS for appetite, albumin, and prealbumin. The performance status and quality of life were stable throughout the observation and were not changed under the supplementation.

Chemotherapy-Related Toxicity, Nutritional Status and Quality of Life in Precachectic Oncologic Patients with, or without, High Protein Nutritional Support. A Prospective, Randomized Study

PubMed Central

Ziętarska, Monika; Krawczyk-Lipiec, Joanna; Kraj, Leszek; Zaucha, Renata; Małgorzewicz, Sylwia

2017-01-01

Background: Cancer disease is usually associated with impaired nutritional status, which is one of the factors contributing to deterioration of the results of surgery, chemotherapy or radiotherapy. Objectives: The aim of the study was to determine whether nutritional support with high protein (ONS) in adult oncologic patients in the first step of cancer cachexia—asymptomatic precachexia, has an influence on the toxicity of systemic therapy. However, secondary endpoints were established: to determine whether high protein ONS influences the nutritional status, the quality of life, and the performance status. Materials and Methods: A total of 114 persons aged 40–84 years old with colorectal cancer were examined. Based on the randomization, 47 patients were qualified to the interventional group (ONS group) and 48 to Control group. To evaluate the nutritional status NRS-2002 (Nutritional Risk Screening), SGA (Subjective Global Assessment), SCRINIO (SCReenIng the Nutritional status In Oncology) Working Group classification, VAS (Visual Analog Scale) for appetite was used. FAACT (Functional Assessment of Anorexia/Cachexia Therapy) questionnaire was used for assessment of the quality of life. The health status of patients was evaluated based on the Karnofsky Performance Scale. Anthropometric measurements were done. Results: Severe complications of chemotherapy, which caused the end of treatment, a slight complication of the gastrointestinal tract such as diarrhea grade 2 according to ECOG (Eastern Cooperative Oncology Group) score regardless of the studied group, were observed. There were no statistical differences in the number and severity of the observed complications, i.e., neutropenia, leucopenia, thrombocytopenia, anemia, abdominal pain, nausea and vomiting, and diarrhea. During the follow-up the significant changes of SGA, VAS, albumin and prealbumin were observed between groups. In the ONS group an improvement in nutritional status was noticed (increased appetite VAS, p = 0.05; increased points in SGA, p = 0.015, and increased levels of albumin and prealbumin, p = 0.05). In Control group nutritional status was stable during observation. The performance status and quality of life were stable in both groups. No statistical differences between groups (ONS vs. Control) in the numbers for disqualification, resignation, delay in treatment, or dose reduction were observed. Conclusions: Results of the study did not indicate that nutritional support in precachectic oncologic patients influenced the toxicity of systemic therapy. High protein nutritional support improved nutritional status assessed by SGA, VAS for appetite, albumin, and prealbumin. The performance status and quality of life were stable throughout the observation and were not changed under the supplementation. PMID:29019951

Genome-Wide Association Mapping and Genomic Selection for Alfalfa (Medicago sativa) Forage Quality Traits

PubMed Central

Pecetti, Luciano; Brummer, E. Charles; Palmonari, Alberto; Tava, Aldo

2017-01-01

Genetic progress for forage quality has been poor in alfalfa (Medicago sativa L.), the most-grown forage legume worldwide. This study aimed at exploring opportunities for marker-assisted selection (MAS) and genomic selection of forage quality traits based on breeding values of parent plants. Some 154 genotypes from a broadly-based reference population were genotyped by genotyping-by-sequencing (GBS), and phenotyped for leaf-to-stem ratio, leaf and stem contents of protein, neutral detergent fiber (NDF) and acid detergent lignin (ADL), and leaf and stem NDF digestibility after 24 hours (NDFD), of their dense-planted half-sib progenies in three growing conditions (summer harvest, full irrigation; summer harvest, suspended irrigation; autumn harvest). Trait-marker analyses were performed on progeny values averaged over conditions, owing to modest germplasm × condition interaction. Genomic selection exploited 11,450 polymorphic SNP markers, whereas a subset of 8,494 M. truncatula-aligned markers were used for a genome-wide association study (GWAS). GWAS confirmed the polygenic control of quality traits and, in agreement with phenotypic correlations, indicated substantially different genetic control of a given trait in stems and leaves. It detected several SNPs in different annotated genes that were highly linked to stem protein content. Also, it identified a small genomic region on chromosome 8 with high concentration of annotated genes associated with leaf ADL, including one gene probably involved in the lignin pathway. Three genomic selection models, i.e., Ridge-regression BLUP, Bayes B and Bayesian Lasso, displayed similar prediction accuracy, whereas SVR-lin was less accurate. Accuracy values were moderate (0.3–0.4) for stem NDFD and leaf protein content, modest for leaf ADL and NDFD, and low to very low for the other traits. Along with previous results for the same germplasm set, this study indicates that GBS data can be exploited to improve both quality traits (by genomic selection or MAS) and forage yield. PMID:28068350

Genome-Wide Association Mapping and Genomic Selection for Alfalfa (Medicago sativa) Forage Quality Traits.

PubMed

Biazzi, Elisa; Nazzicari, Nelson; Pecetti, Luciano; Brummer, E Charles; Palmonari, Alberto; Tava, Aldo; Annicchiarico, Paolo

2017-01-01

Genetic progress for forage quality has been poor in alfalfa (Medicago sativa L.), the most-grown forage legume worldwide. This study aimed at exploring opportunities for marker-assisted selection (MAS) and genomic selection of forage quality traits based on breeding values of parent plants. Some 154 genotypes from a broadly-based reference population were genotyped by genotyping-by-sequencing (GBS), and phenotyped for leaf-to-stem ratio, leaf and stem contents of protein, neutral detergent fiber (NDF) and acid detergent lignin (ADL), and leaf and stem NDF digestibility after 24 hours (NDFD), of their dense-planted half-sib progenies in three growing conditions (summer harvest, full irrigation; summer harvest, suspended irrigation; autumn harvest). Trait-marker analyses were performed on progeny values averaged over conditions, owing to modest germplasm × condition interaction. Genomic selection exploited 11,450 polymorphic SNP markers, whereas a subset of 8,494 M. truncatula-aligned markers were used for a genome-wide association study (GWAS). GWAS confirmed the polygenic control of quality traits and, in agreement with phenotypic correlations, indicated substantially different genetic control of a given trait in stems and leaves. It detected several SNPs in different annotated genes that were highly linked to stem protein content. Also, it identified a small genomic region on chromosome 8 with high concentration of annotated genes associated with leaf ADL, including one gene probably involved in the lignin pathway. Three genomic selection models, i.e., Ridge-regression BLUP, Bayes B and Bayesian Lasso, displayed similar prediction accuracy, whereas SVR-lin was less accurate. Accuracy values were moderate (0.3-0.4) for stem NDFD and leaf protein content, modest for leaf ADL and NDFD, and low to very low for the other traits. Along with previous results for the same germplasm set, this study indicates that GBS data can be exploited to improve both quality traits (by genomic selection or MAS) and forage yield.

Expression of heat shock protein 70 in transport-stressed broiler pectoralis major muscle and its relationship with meat quality.

PubMed

Xing, T; Wang, M F; Han, M Y; Zhu, X S; Xu, X L; Zhou, G H

2017-09-01

Omics research has indicated that heat shock protein 70 (HSP70) is a potential biomarker of meat quality. However, the specific changes and the potential role of HSP70 in postmortem meat quality development need to be further defined. In this study, Arbor Acres broiler chickens (n=126) were randomly categorized into three treatment groups of unstressed control (C), 0.5-h transport (T) and subsequent water shower spray following transport (T/W). Each treatment consisted of six replicates with seven birds each. The birds were transported according to a designed protocol. The pectoralis major (PM) muscles of the transport-stressed broilers were categorized as normal and pale, soft and exudative (PSE)-like muscle samples according to L* and pH24 h values to test the expression and location of HSP70. Results revealed that the activities of plasma creatine kinase and lactate dehydrogenase increased significantly (P<0.05) in normal and PSE-like muscle samples after transportation. The mRNA expression of HSP70 in normal muscle samples increased significantly (P<0.05) compared with that in the controls after stress. The protein expression of HSP70 increased significantly in normal muscle samples and decreased significantly (P<0.05) in PSE-like muscles. Immuno-fluorescence showed that HSP70 was present in the cytoplasm and on surface membranes of PM muscle cells in the normal samples following stress. Meanwhile, HSP70 was present on the surface membranes and extracellular matrix but was barely visible in the cytoplasm of the PSE-like samples. Principal component analysis showed high correlations between HSP70 and meat quality and stress indicators. In conclusion, this research suggests that the variation in HSP70 expression may provide a novel insight into the pathways underlying meat quality development.

Shotgun label-free quantitative proteomics of developing peanut (Arachis hypogaea L.) seed

USDA-ARS?s Scientific Manuscript database

Legume seeds and peanuts, in particular, are an inexpensive source of plant proteins and edible oil. Owing to their importance in global food security, it is necessary to understand the genetic, biochemical, and physiological mechanisms controlling seed quality and nutritive attributes. A comprehens...

Trypanosoma cruzi Calreticulin Is a Lectin That Binds Monoglucosylated Oligosaccharides but Not Protein Moieties of Glycoproteins

PubMed Central

Labriola, Carlos; Cazzulo, Juan J.; Parodi, Armando J.

1999-01-01

Trypanosoma cruzi is a protozoan parasite that belongs to an early branch in evolution. Although it lacks several features of the pathway of protein N-glycosylation and oligosaccharide processing present in the endoplasmic reticulum of higher eukaryotes, it displays UDP-Glc:glycoprotein glucosyltransferase and glucosidase II activities. It is herewith reported that this protozoan also expresses a calreticulin-like molecule, the third component of the quality control of glycoprotein folding. No calnexin-encoding gene was detected. Recombinant T. cruzi calreticulin specifically recognized free monoglucosylated high-mannose-type oligosaccharides. Addition of anti-calreticulin serum to extracts obtained from cells pulse–chased with [35S]Met plus [35S]Cys immunoprecipitated two proteins that were identified as calreticulin and the lysosomal proteinase cruzipain (a major soluble glycoprotein). The latter but not the former protein disappeared from immunoprecipitates upon chasing cells. Contrary to what happens in mammalian cells, addition of the glucosidase II inhibitor 1-deoxynojirimycin promoted calreticulin–cruzipain interaction. This result is consistent with the known pathway of protein N-glycosylation and oligosaccharide processing occurring in T. cruzi. A treatment of the calreticulin-cruzipain complexes with endo-β-N-acetylglucosaminidase H either before or after addition of anti-calreticulin serum completely disrupted calreticulin–cruzipain interaction. In addition, mature monoglucosylated but not unglucosylated cruzipain isolated from lysosomes was found to interact with recombinant calreticulin. It was concluded that the quality control of glycoprotein folding appeared early in evolution, and that T. cruzi calreticulin binds monoglucosylated oligosaccharides but not the protein moiety of cruzipain. Furthermore, evidence is presented indicating that glucosyltransferase glucosylated cruzipain at its last folding stages. PMID:10233151

Trypanosoma cruzi calreticulin is a lectin that binds monoglucosylated oligosaccharides but not protein moieties of glycoproteins.

PubMed

Labriola, C; Cazzulo, J J; Parodi, A J

1999-05-01

Trypanosoma cruzi is a protozoan parasite that belongs to an early branch in evolution. Although it lacks several features of the pathway of protein N-glycosylation and oligosaccharide processing present in the endoplasmic reticulum of higher eukaryotes, it displays UDP-Glc:glycoprotein glucosyltransferase and glucosidase II activities. It is herewith reported that this protozoan also expresses a calreticulin-like molecule, the third component of the quality control of glycoprotein folding. No calnexin-encoding gene was detected. Recombinant T. cruzi calreticulin specifically recognized free monoglucosylated high-mannose-type oligosaccharides. Addition of anti-calreticulin serum to extracts obtained from cells pulse-chased with [35S]Met plus [35S]Cys immunoprecipitated two proteins that were identified as calreticulin and the lysosomal proteinase cruzipain (a major soluble glycoprotein). The latter but not the former protein disappeared from immunoprecipitates upon chasing cells. Contrary to what happens in mammalian cells, addition of the glucosidase II inhibitor 1-deoxynojirimycin promoted calreticulin-cruzipain interaction. This result is consistent with the known pathway of protein N-glycosylation and oligosaccharide processing occurring in T. cruzi. A treatment of the calreticulin-cruzipain complexes with endo-beta-N-acetylglucosaminidase H either before or after addition of anti-calreticulin serum completely disrupted calreticulin-cruzipain interaction. In addition, mature monoglucosylated but not unglucosylated cruzipain isolated from lysosomes was found to interact with recombinant calreticulin. It was concluded that the quality control of glycoprotein folding appeared early in evolution, and that T. cruzi calreticulin binds monoglucosylated oligosaccharides but not the protein moiety of cruzipain. Furthermore, evidence is presented indicating that glucosyltransferase glucosylated cruzipain at its last folding stages.

The de-ubiquitinating enzyme ataxin-3 does not modulate disease progression in a knock-in mouse model of Huntington disease.

PubMed

Zeng, Li; Tallaksen-Greene, Sara J; Wang, Bo; Albin, Roger L; Paulson, Henry L

2013-01-01

Ataxin-3 is a deubiquitinating enzyme (DUB) that participates in ubiquitin-dependent protein quality control pathways and, based on studies in model systems, may be neuroprotective against toxic polyglutamine proteins such as the Huntington's disease (HD) protein, huntingtin (htt). HD is one of at least nine polyglutamine neurodegenerative diseases in which disease-causing proteins accumulate in ubiquitin-positive inclusions within neurons. In studies crossing mice null for ataxin-3 to an established HD knock-in mouse model (HdhQ200), we tested whether loss of ataxin-3 alters disease progression, perhaps by impairing the clearance of mutant htt or the ubiquitination of inclusions. While loss of ataxin-3 mildly exacerbated age-dependent motor deficits, it did not alter inclusion formation, ubiquitination of inclusions or levels of mutant or normal htt. Ataxin-3, itself a polyglutamine-containing protein with multiple ubiquitin binding domains, was not observed to localize to htt inclusions. Changes in neurotransmitter receptor binding known to occur in HD knock-in mice also were not altered by the loss of ataxin-3, although we unexpectedly observed increased GABAA receptor binding in the striatum of HdhQ200 mice, which has not previously been noted. Finally, we confirmed that CNS levels of hsp70 are decreased in HD mice as has been reported in other HD mouse models, regardless of the presence or absence of ataxin-3. We conclude that while ataxin-3 may participate in protein quality control pathways, it does not critically regulate the handling of mutant htt or contribute to major features of disease pathogenesis in HD.

Improved Function With Enhanced Protein Intake per Meal: A Pilot Study of Weight Reduction in Frail, Obese Older Adults.

PubMed

Porter Starr, Kathryn N; Pieper, Carl F; Orenduff, Melissa C; McDonald, Shelley R; McClure, Luisa B; Zhou, Run; Payne, Martha E; Bales, Connie W

2016-10-01

Obesity is a significant cause of functional limitations in older adults; yet, concerns that weight reduction could diminish muscle along with fat mass have impeded progress toward an intervention. Meal-based enhancement of protein intake could protect function and/or lean mass but has not been studied during geriatric obesity reduction. In this 6-month randomized controlled trial, 67 obese (body mass index ≥30kg/m(2)) older (≥60 years) adults with a Short Physical Performance Battery score of 4-10 were randomly assigned to a traditional (Control) weight loss regimen or one with higher protein intake (>30g) at each meal (Protein). All participants were prescribed a hypo-caloric diet, and weighed and provided dietary guidance weekly. Physical function (Short Physical Performance Battery) and lean mass (BOD POD), along with secondary measures, were assessed at 0, 3, and 6 months. At the 6-month endpoint, there was significant (p < .001) weight loss in both the Control (-7.5±6.2kg) and Protein (-8.7±7.4kg) groups. Both groups also improved function but the increase in the Protein (+2.4±1.7 units; p < .001) was greater than in the Control (+0.9±1.7 units; p < .01) group (p = .02). Obese, functionally limited older adults undergoing a 6-month weight loss intervention with a meal-based enhancement of protein quantity and quality lost similar amounts of weight but had greater functional improvements relative to the Control group. If confirmed, this dietary approach could have important implications for improving the functional status of this vulnerable population (ClinicalTrials.gov identifier: NCT01715753). © The Author 2016. Published by Oxford University Press on behalf of The Gerontological Society of America.

Improved Function With Enhanced Protein Intake per Meal: A Pilot Study of Weight Reduction in Frail, Obese Older Adults

PubMed Central

Pieper, Carl F.; Orenduff, Melissa C.; McDonald, Shelley R.; McClure, Luisa B.; Zhou, Run; Payne, Martha E.; Bales, Connie W.

2016-01-01

Abstract Background: Obesity is a significant cause of functional limitations in older adults; yet, concerns that weight reduction could diminish muscle along with fat mass have impeded progress toward an intervention. Meal-based enhancement of protein intake could protect function and/or lean mass but has not been studied during geriatric obesity reduction. Methods: In this 6-month randomized controlled trial, 67 obese (body mass index ≥30kg/m2) older (≥60 years) adults with a Short Physical Performance Battery score of 4–10 were randomly assigned to a traditional (Control) weight loss regimen or one with higher protein intake (>30g) at each meal (Protein). All participants were prescribed a hypo-caloric diet, and weighed and provided dietary guidance weekly. Physical function (Short Physical Performance Battery) and lean mass (BOD POD), along with secondary measures, were assessed at 0, 3, and 6 months. Results: At the 6-month endpoint, there was significant (p < .001) weight loss in both the Control (−7.5±6.2kg) and Protein (−8.7±7.4kg) groups. Both groups also improved function but the increase in the Protein (+2.4±1.7 units; p < .001) was greater than in the Control (+0.9±1.7 units; p < .01) group (p = .02). Conclusion: Obese, functionally limited older adults undergoing a 6-month weight loss intervention with a meal-based enhancement of protein quantity and quality lost similar amounts of weight but had greater functional improvements relative to the Control group. If confirmed, this dietary approach could have important implications for improving the functional status of this vulnerable population (ClinicalTrials.gov identifier: NCT01715753). PMID:26786203

Protein single-model quality assessment by feature-based probability density functions.

PubMed

Cao, Renzhi; Cheng, Jianlin

2016-04-04

Protein quality assessment (QA) has played an important role in protein structure prediction. We developed a novel single-model quality assessment method-Qprob. Qprob calculates the absolute error for each protein feature value against the true quality scores (i.e. GDT-TS scores) of protein structural models, and uses them to estimate its probability density distribution for quality assessment. Qprob has been blindly tested on the 11th Critical Assessment of Techniques for Protein Structure Prediction (CASP11) as MULTICOM-NOVEL server. The official CASP result shows that Qprob ranks as one of the top single-model QA methods. In addition, Qprob makes contributions to our protein tertiary structure predictor MULTICOM, which is officially ranked 3rd out of 143 predictors. The good performance shows that Qprob is good at assessing the quality of models of hard targets. These results demonstrate that this new probability density distribution based method is effective for protein single-model quality assessment and is useful for protein structure prediction. The webserver of Qprob is available at: http://calla.rnet.missouri.edu/qprob/. The software is now freely available in the web server of Qprob.

Identification of genetic loci associated with crude protein and mineral concentrations in alfalfa (Medicago sativa) using association mapping.

PubMed

Jia, Congjun; Wu, Xinming; Chen, Min; Wang, Yunqi; Liu, Xiqiang; Gong, Pan; Xu, Qingfang; Wang, Xuemin; Gao, Hongwen; Wang, Zan

2017-06-06

Alfalfa (Medicago sativa) is one of the most important legume forage species in China and many other countries of the world. It provides a quality source of proteins and minerals to animals. Genetic underpinnings for these important traits, however, are elusive. An alfalfa (M. sativa) association mapping study for six traits, namely crude protein (CP), rumen undegraded protein (RUP), and four mineral elements (Ca, K, Mg and P), was conducted in three consecutive years using a large collection encompassing 336 genotypes genotyped with 85 simple sequence repeat (SSR) markers. All the traits were significantly influenced by genotype, environment, and genotype × environment interaction. Eight-five significant associations (P < 0.005) were identified. Among these, five associations with Ca were repeatedly observed and six co-localized associations were identified. The identified marker alleles significantly associated with the traits provided important information for understanding genetic controls of alfalfa quality. The markers could be used in assisting selection for the individual traits in breeding populations for developing new alfalfa cultivars.

Development of proteome-wide binding reagents for research and diagnostics.

PubMed

Taussig, Michael J; Schmidt, Ronny; Cook, Elizabeth A; Stoevesandt, Oda

2013-12-01

Alongside MS, antibodies and other specific protein-binding molecules have a special place in proteomics as affinity reagents in a toolbox of applications for determining protein location, quantitative distribution and function (affinity proteomics). The realisation that the range of research antibodies available, while apparently vast is nevertheless still very incomplete and frequently of uncertain quality, has stimulated projects with an objective of raising comprehensive, proteome-wide sets of protein binders. With progress in automation and throughput, a remarkable number of recent publications refer to the practical possibility of selecting binders to every protein encoded in the genome. Here we review the requirements of a pipeline of production of protein binders for the human proteome, including target prioritisation, antigen design, 'next generation' methods, databases and the approaches taken by ongoing projects in Europe and the USA. While the task of generating affinity reagents for all human proteins is complex and demanding, the benefits of well-characterised and quality-controlled pan-proteome binder resources for biomedical research, industry and life sciences in general would be enormous and justify the effort. Given the technical, personnel and financial resources needed to fulfil this aim, expansion of current efforts may best be addressed through large-scale international collaboration. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Protein quality and growth in malnourished children

USDA-ARS?s Scientific Manuscript database

Protein quality refers to the amounts and ratios of essential amino acids in a food. Two methods most commonly used for determining protein quality are the protein digestibility-corrected amino acid score (PDCAAS) and the digestible indispensible amino acid score (DIAAS). To use existing literature ...

Automated quantitative assessment of proteins' biological function in protein knowledge bases.

PubMed

Mayr, Gabriele; Lepperdinger, Günter; Lackner, Peter

2008-01-01

Primary protein sequence data are archived in databases together with information regarding corresponding biological functions. In this respect, UniProt/Swiss-Prot is currently the most comprehensive collection and it is routinely cross-examined when trying to unravel the biological role of hypothetical proteins. Bioscientists frequently extract single entries and further evaluate those on a subjective basis. In lieu of a standardized procedure for scoring the existing knowledge regarding individual proteins, we here report about a computer-assisted method, which we applied to score the present knowledge about any given Swiss-Prot entry. Applying this quantitative score allows the comparison of proteins with respect to their sequence yet highlights the comprehension of functional data. pfs analysis may be also applied for quality control of individual entries or for database management in order to rank entry listings.

Effects of tea polyphenols on the post-mortem integrity of large yellow croaker (Pseudosciaena crocea) fillet proteins.

PubMed

Zhao, Jin; Lv, Weijin; Wang, Jinlin; Li, Jianrong; Liu, Xiaoxiang; Zhu, Junli

2013-12-01

Tea polyphenols (TP) are known to be important for the post-mortem deterioration of fish muscle and can enhance food quality. To shed light on the influence of TP on the status of large yellow croaker muscle proteins, control and treated fillets (0.1% TP, 0.2% TP and 0.3% TP, w/v) were analysed periodically for myofibrillar protein functional properties (Ca(2+)-ATPase activity, surface hydrophobicity, total sulfhydryl content, emulsion stability index and rheological behaviour). Degradation of the myofibrillar protein myosin could be clearly observed; several proteins were also observed to vary in abundance following post-mortem storage for 25 days. The present study offers new evidence that TP have an effective impact on muscle protein integrity post-mortem. Copyright © 2013 Elsevier Ltd. All rights reserved.

Heat Shock Proteins and Autophagy Pathways in Neuroprotection: From Molecular Bases to Pharmacological Interventions

PubMed Central

Penke, Botond; Bogár, Ferenc; Sántha, Miklós; Tóth, Melinda E.; Vígh, László

2018-01-01

Neurodegenerative diseases (NDDs) such as Alzheimer’s disease, Parkinson’s disease and Huntington’s disease (HD), amyotrophic lateral sclerosis, and prion diseases are all characterized by the accumulation of protein aggregates (amyloids) into inclusions and/or plaques. The ubiquitous presence of amyloids in NDDs suggests the involvement of disturbed protein homeostasis (proteostasis) in the underlying pathomechanisms. This review summarizes specific mechanisms that maintain proteostasis, including molecular chaperons, the ubiquitin-proteasome system (UPS), endoplasmic reticulum associated degradation (ERAD), and different autophagic pathways (chaperon mediated-, micro-, and macro-autophagy). The role of heat shock proteins (Hsps) in cellular quality control and degradation of pathogenic proteins is reviewed. Finally, putative therapeutic strategies for efficient removal of cytotoxic proteins from neurons and design of new therapeutic targets against the progression of NDDs are discussed. PMID:29361800

Quantitative evaluation of protein conformation in pharmaceuticals using cross-linking reactions coupled with LC-MS/MS analysis.

PubMed

Yamaguchi, Hideto; Hirakura, Yutaka; Shirai, Hiroki; Mimura, Hisashi; Toyo'oka, Toshimasa

2011-06-01

The need for a simple and high-throughput method for identifying the tertiary structure of protein pharmaceuticals has increased. In this study, a simple method for mapping the protein fold is proposed for use as a complementary quality test. This method is based on cross-linking a protein using a [bis(sulfosuccinimidyl)suberate (BS(3))], followed by peptide mapping by LC-MS. Consensus interferon (CIFN) was used as the model protein. The tryptic map obtained via liquid chromatography tandem mass spectroscopy (LC-MS/MS) and the mass mapping obtained via matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy were used to identify cross-linked peptides. While LC-MS/MS analyses found that BS(3) formed cross-links in the loop region of the protein, which was regarded as the biologically active site, sodium dodecyl-sulfate polyacrylamide gel electrophoresis demonstrated that cross-linking occurred within a protein molecule, but not between protein molecules. The occurrence of cross-links at the active site depends greatly on the conformation of the protein, which is determined by the denaturing conditions. Quantitative evaluation of the tertiary structure of CIFN was thus possible by monitoring the amounts of cross-linked peptides generated. Assuming that background information is available at the development stage, this method may be applicable to process development as a complementary test for quality control. Copyright © 2011 Elsevier B.V. All rights reserved.

Interactome of two diverse RNA granules links mRNA localization to translational repression in neurons.

PubMed

Fritzsche, Renate; Karra, Daniela; Bennett, Keiryn L; Ang, Foong Yee; Heraud-Farlow, Jacki E; Tolino, Marco; Doyle, Michael; Bauer, Karl E; Thomas, Sabine; Planyavsky, Melanie; Arn, Eric; Bakosova, Anetta; Jungwirth, Kerstin; Hörmann, Alexandra; Palfi, Zsofia; Sandholzer, Julia; Schwarz, Martina; Macchi, Paolo; Colinge, Jacques; Superti-Furga, Giulio; Kiebler, Michael A

2013-12-26

Transport of RNAs to dendrites occurs in neuronal RNA granules, which allows local synthesis of specific proteins at active synapses on demand, thereby contributing to learning and memory. To gain insight into the machinery controlling dendritic mRNA localization and translation, we established a stringent protocol to biochemically purify RNA granules from rat brain. Here, we identified a specific set of interactors for two RNA-binding proteins that are known components of neuronal RNA granules, Barentsz and Staufen2. First, neuronal RNA granules are much more heterogeneous than previously anticipated, sharing only a third of the identified proteins. Second, dendritically localized mRNAs, e.g., Arc and CaMKIIα, associate selectively with distinct RNA granules. Third, our work identifies a series of factors with known roles in RNA localization, translational control, and RNA quality control that are likely to keep localized transcripts in a translationally repressed state, often in distinct types of RNPs. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

The burden of trisomy 21 disrupts the proteostasis network in Down syndrome

PubMed Central

Rauniyar, Abhishek K.; Jiang, Hua; Liggett, L. Alexander; Maclean, Kenneth N.

2017-01-01

Down syndrome (DS) is a genetic disorder caused by trisomy of chromosome 21. Abnormalities in chromosome number have the potential to lead to disruption of the proteostasis network (PN) and accumulation of misfolded proteins. DS individuals suffer from several comorbidities, and we hypothesized that disruption of proteostasis could contribute to the observed pathology and decreased cell viability in DS. Our results confirm the presence of a disrupted PN in DS, as several of its elements, including the unfolded protein response, chaperone system, and proteasomal degradation exhibited significant alterations compared to euploid controls in both cell and mouse models. Additionally, when cell models were treated with compounds that promote disrupted proteostasis, we observed diminished levels of cell viability in DS compared to controls. Collectively our findings provide a cellular-level characterization of PN dysfunction in DS and an improved understanding of the potential pathogenic mechanisms contributing to disrupted cellular physiology in DS. Lastly, this study highlights the future potential of designing therapeutic strategies that mitigate protein quality control dysfunction. PMID:28430800

Differential proteomics reveals the hallmarks of seed development in common bean (Phaseolus vulgaris L.).

PubMed

Parreira, J R; Bouraada, J; Fitzpatrick, M A; Silvestre, S; Bernardes da Silva, A; Marques da Silva, J; Almeida, A M; Fevereiro, P; Altelaar, A F M; Araújo, S S

2016-06-30

Common bean (Phaseolus vulgaris L.) is one of the most consumed staple foods worldwide. Little is known about the molecular mechanisms controlling seed development. This study aims to comprehensively describe proteome dynamics during seed development of common bean. A high-throughput gel-free proteomics approach (LC-MS/MS) was conducted on seeds at 10, 20, 30 and 40days after anthesis, spanning from late embryogenesis until desiccation. Of the 418 differentially accumulated proteins identified, 255 were characterized, most belonging to protein metabolism. An accumulation of proteins belonging to the MapMan functional categories of "protein", "glycolysis", "TCA", "DNA", "RNA", "cell" and "stress" were found at early seed development stages, reflecting an extensive metabolic activity. In the mid stages, accumulation of storage, signaling, starch synthesis and cell wall-related proteins stood out. In the later stages, an increase in proteins related to redox, protein degradation/modification/folding and nucleic acid metabolisms reflect that seed desiccation-resistance mechanisms were activated. Our study unveils new clues to understand the regulation of seed development mediated by post-translational modifications and maintenance of genome integrity. This knowledge enhances the understanding on seed development molecular mechanisms that may be used in the design and selection of common bean seeds with desired quality traits. Common bean (P. vulgaris) is an important source of proteins and carbohydrates worldwide. Despite the agronomic and economic importance of this pulse, knowledge on common bean seed development is limited. Herein, a gel-free high throughput methodology was used to describe the proteome changes during P. vulgaris seed development. Data obtained will enhance the knowledge on the molecular mechanisms controlling this grain legume seed development and may be used in the design and selection of common bean seeds with desired quality traits. Results may be extrapolated to other pulses. Copyright © 2016 Elsevier B.V. All rights reserved.

P19-S Managing Proteomics Data from Data Generation and Data Warehousing to Central Data Repository and Journal Reviewing Processes

PubMed Central

Thiele, H.; Glandorf, J.; Koerting, G.; Reidegeld, K.; Blüggel, M.; Meyer, H.; Stephan, C.

2007-01-01

In today’s proteomics research, various techniques and instrumentation bioinformatics tools are necessary to manage the large amount of heterogeneous data with an automatic quality control to produce reliable and comparable results. Therefore a data-processing pipeline is mandatory for data validation and comparison in a data-warehousing system. The proteome bioinformatics platform ProteinScape has been proven to cover these needs. The reprocessing of HUPO BPP participants’ MS data was done within ProteinScape. The reprocessed information was transferred into the global data repository PRIDE. ProteinScape as a data-warehousing system covers two main aspects: archiving relevant data of the proteomics workflow and information extraction functionality (protein identification, quantification and generation of biological knowledge). As a strategy for automatic data validation, different protein search engines are integrated. Result analysis is performed using a decoy database search strategy, which allows the measurement of the false-positive identification rate. Peptide identifications across different workflows, different MS techniques, and different search engines are merged to obtain a quality-controlled protein list. The proteomics identifications database (PRIDE), as a public data repository, is an archiving system where data are finally stored and no longer changed by further processing steps. Data submission to PRIDE is open to proteomics laboratories generating protein and peptide identifications. An export tool has been developed for transferring all relevant HUPO BPP data from ProteinScape into PRIDE using the PRIDE.xml format. The EU-funded ProDac project will coordinate the development of software tools covering international standards for the representation of proteomics data. The implementation of data submission pipelines and systematic data collection in public standards–compliant repositories will cover all aspects, from the generation of MS data in each laboratory to the conversion of all the annotating information and identifications to a standardized format. Such datasets can be used in the course of publishing in scientific journals.

Simultaneous quantification of the viral antigens hemagglutinin and neuraminidase in influenza vaccines by LC-MSE.

PubMed

Creskey, Marybeth C; Li, Changgui; Wang, Junzhi; Girard, Michel; Lorbetskie, Barry; Gravel, Caroline; Farnsworth, Aaron; Li, Xuguang; Smith, Daryl G S; Cyr, Terry D

2012-07-06

Current methods for quality control of inactivated influenza vaccines prior to regulatory approval include determining the hemagglutinin (HA) content by single radial immunodiffusion (SRID), verifying neuraminidase (NA) enzymatic activity, and demonstrating that the levels of the contaminant protein ovalbumin are below a set threshold of 1 μg/dose. The SRID assays require the availability of strain-specific reference HA antigens and antibodies, the production of which is a potential rate-limiting step in vaccine development and release, particularly during a pandemic. Immune responses induced by neuraminidase also contribute to protection from infection; however, the amounts of NA antigen in influenza vaccines are currently not quantified or standardized. Here, we report a method for vaccine analysis that yields simultaneous quantification of HA and NA levels much more rapidly than conventional HA quantification techniques, while providing additional valuable information on the total protein content. Enzymatically digested vaccine proteins were analyzed by LC-MS(E), a mass spectrometric technology that allows absolute quantification of analytes, including the HA and NA antigens, other structural influenza proteins and chicken egg proteins associated with the manufacturing process. This method has potential application for increasing the accuracy of reference antigen standards and for validating label claims for HA content in formulated vaccines. It can also be used to monitor NA and chicken egg protein content in order to monitor manufacturing consistency. While this is a useful methodology with potential for broad application, we also discuss herein some of the inherent limitations of this approach and the care and caution that must be taken in its use as a tool for absolute protein quantification. The variations in HA, NA and chicken egg protein concentrations in the vaccines analyzed in this study are indicative of the challenges associated with the current manufacturing and quality control testing procedures. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

An approach to including protein quality when assessing the net contribution of livestock to human food supply.

PubMed

Ertl, P; Knaus, W; Zollitsch, W

2016-11-01

The production of protein from animal sources is often criticized because of the low efficiency of converting plant protein from feeds into protein in the animal products. However, this critique does not consider the fact that large portions of the plant-based proteins fed to animals may be human-inedible and that the quality of animal proteins is usually superior as compared with plant proteins. The aim of the present study was therefore to assess changes in protein quality in the course of the transformation of potentially human-edible plant proteins into animal products via livestock production; data from 30 Austrian dairy farms were used as a case study. A second aim was to develop an approach for combining these changes with quantitative aspects (e.g. with the human-edible feed conversion efficiency (heFCE), defined as kilogram protein in the animal product divided by kilogram potentially human-edible protein in the feeds). Protein quality of potentially human-edible inputs and outputs was assessed using the protein digestibility-corrected amino acid score and the digestible indispensable amino acid score, two methods proposed by the Food and Agriculture Organization of the United Nations to describe the nutritional value of proteins for humans. Depending on the method used, protein scores were between 1.40 and 1.87 times higher for the animal products than for the potentially human-edible plant protein input on a barn-gate level (=protein quality ratio (PQR)). Combining the PQR of 1.87 with the heFCE for the same farms resulted in heFCE×PQR of 2.15. Thus, considering both quantity and quality, the value of the proteins in the animal products for human consumption (in this case in milk and beef) is 2.15 times higher than that of proteins in the potentially human-edible plant protein inputs. The results of this study emphasize the necessity of including protein quality changes resulting from the transformation of plant proteins to animal proteins when evaluating the net contribution of livestock to the human food supply. Furthermore, these differences in protein quality might also need to be considered when choosing a functional unit for the assessment of environmental impacts of the production of different proteins.

Quality characteristics, structural changes, and storage stability of semi-dried noodles induced by moderate dehydration: understanding the quality changes in semi-dried noodles.

PubMed

Li, Man; Zhu, Ke-Xue; Sun, Qing-Jie; Amza, Tidjani; Guo, Xiao-Na; Zhou, Hui-Ming

2016-03-01

Based on the critical water content (for noodle deterioration) concluded previously, high-temperature-short-time (HTST; 105-135°C) and medium-temperature-long-time (MTLT; 45-75°C) dehydrations were introduced in this study to produce semi-dried noodles. The effects of HTST and MTLT on the quality parameters of semi-dried noodles, as well as noodle structure, storage stability, and changes in starch and protein components were thoroughly investigated. Differential scanning calorimeter (DSC) and birefringent analysis presented few starch gelatinization (approximately 30%) in HTST dehydrated noodles. Scanning electron microscopy (SEM) images showed more compact noodle surface, with uniform pores in the cross section, probably due to enhanced protein-starch combination after HTST dehydration. Meanwhile, HTST induced protein polymerizations in semi-dried noodles, mainly by -SH-S-S interchange, and resulted in significantly (P<0.05) reduced cooking loss. Furthermore, HTST noodles showed higher microbial and color stability. Shelf-life of dehydrated samples at 120°C was extended to 5days from 1day of the control. Copyright © 2015 Elsevier Ltd. All rights reserved.

[Effect of increased protein content on nutritional and sensory quality of cookies].

PubMed

Pérez, Santiago Rafael; Osella, Carlos Alberto; Torre, Maria Adela de la; Sánchez, Hugo Diego

2008-12-01

The objective of this work was to study the effect of soy flour and whey protein concentrate (WPC) on cookies quality. An optimal recipe showing improved protein quality and content as well as acceptable sensory quality was defined taking into account the results obtained. Rotary moulded cookie formulation adaptable to lamination and cutting in pilot plant was used. Wheat flour from this formulation was partially replaced by whey protein concentrate and full fat soy flour. Second order models were employed to generate response surfaces for: total protein, lysine by 16 grams of total nitrogen, lysine by 100 grams of sample, loss of lysine during processing and sensory evaluation of cookies. We could obtain an effect on available lysine value when water content was increased in the formulation because a delay in the Maillard reaction. The optimal formulation contains 13% of full fat soy flour, 3% of whey protein concentrate and 23% of water. The results demonstrated that the protein content and the protein quality of the supplemented flours were increased when soy flour was added in the formulation of cookies. On other hand, protein content was increased but protein quality was decreased when WPC was used, because of available lysine loss.

Changes in tissue protein synthesis are involved in regulating urea synthesis in rats given proteins of different quality.

PubMed

Tujioka, Kazuyo; Lyou, Sunok; Sano, Atushi; Hayase, Kazutoshi; Yokogoshi, Hidehiko

2004-10-01

The purpose of present study was to determine whether the regulation of urea synthesis is mediated through changes in supply of amino acids by protein synthesis and whether the concentration of ammonia, or activities of amino acid catabolizing enzymes, regulate urea synthesis when the dietary protein quality is manipulated. Experiments were done on three groups of rats given diets containing 10 g gluten, 10 g casein or 10 g whole egg protein/100 g for 10 d. The urinary excretion of urea, and the liver concentrations of glutamate, serine and alanine increased with a decrease in quality of dietary protein. The fractional and absolute rates of protein synthesis in tissues declined with the decrease in quality of dietary protein quality. The ammonia concentration in plasma and liver, and activities of hepatic amino acid catabolizing enzymes was not related to urea excretion under these conditions. These results suggest that the lower protein synthesis seen in tissues of rats given the lower quality of protein is likely to be one of the factors to increasing the supply of amino acids and stimulating urea synthesis.

Selective aggregation of the splicing factor Hsh155 suppresses splicing upon genotoxic stress.

PubMed

Mathew, Veena; Tam, Annie S; Milbury, Karissa L; Hofmann, Analise K; Hughes, Christopher S; Morin, Gregg B; Loewen, Christopher J R; Stirling, Peter C

2017-12-04

Upon genotoxic stress, dynamic relocalization events control DNA repair as well as alterations of the transcriptome and proteome, enabling stress recovery. How these events may influence one another is only partly known. Beginning with a cytological screen of genome stability proteins, we find that the splicing factor Hsh155 disassembles from its partners and localizes to both intranuclear and cytoplasmic protein quality control (PQC) aggregates under alkylation stress. Aggregate sequestration of Hsh155 occurs at nuclear and then cytoplasmic sites in a manner that is regulated by molecular chaperones and requires TORC1 activity signaling through the Sfp1 transcription factor. This dynamic behavior is associated with intron retention in ribosomal protein gene transcripts, a decrease in splicing efficiency, and more rapid recovery from stress. Collectively, our analyses suggest a model in which some proteins evicted from chromatin and undergoing transcriptional remodeling during stress are targeted to PQC sites to influence gene expression changes and facilitate stress recovery. © 2017 Mathew et al.

Controlled In Meso Phase Crystallization – A Method for the Structural Investigation of Membrane Proteins

PubMed Central

Kubicek, Jan; Schlesinger, Ramona; Baeken, Christian; Büldt, Georg; Schäfer, Frank; Labahn, Jörg

2012-01-01

We investigated in meso crystallization of membrane proteins to develop a fast screening technology which combines features of the well established classical vapor diffusion experiment with the batch meso phase crystallization, but without premixing of protein and monoolein. It inherits the advantages of both methods, namely (i) the stabilization of membrane proteins in the meso phase, (ii) the control of hydration level and additive concentration by vapor diffusion. The new technology (iii) significantly simplifies in meso crystallization experiments and allows the use of standard liquid handling robots suitable for 96 well formats. CIMP crystallization furthermore allows (iv) direct monitoring of phase transformation and crystallization events. Bacteriorhodopsin (BR) crystals of high quality and diffraction up to 1.3 Å resolution have been obtained in this approach. CIMP and the developed consumables and protocols have been successfully applied to obtain crystals of sensory rhodopsin II (SRII) from Halobacterium salinarum for the first time. PMID:22536388

Multifunctional Mitochondrial AAA Proteases

PubMed Central

Glynn, Steven E.

2017-01-01

Mitochondria perform numerous functions necessary for the survival of eukaryotic cells. These activities are coordinated by a diverse complement of proteins encoded in both the nuclear and mitochondrial genomes that must be properly organized and maintained. Misregulation of mitochondrial proteostasis impairs organellar function and can result in the development of severe human diseases. ATP-driven AAA+ proteins play crucial roles in preserving mitochondrial activity by removing and remodeling protein molecules in accordance with the needs of the cell. Two mitochondrial AAA proteases, i-AAA and m-AAA, are anchored to either face of the mitochondrial inner membrane, where they engage and process an array of substrates to impact protein biogenesis, quality control, and the regulation of key metabolic pathways. The functionality of these proteases is extended through multiple substrate-dependent modes of action, including complete degradation, partial processing, or dislocation from the membrane without proteolysis. This review discusses recent advances made toward elucidating the mechanisms of substrate recognition, handling, and degradation that allow these versatile proteases to control diverse activities in this multifunctional organelle. PMID:28589125

Multifunctional Mitochondrial AAA Proteases.

PubMed

Glynn, Steven E

2017-01-01

Mitochondria perform numerous functions necessary for the survival of eukaryotic cells. These activities are coordinated by a diverse complement of proteins encoded in both the nuclear and mitochondrial genomes that must be properly organized and maintained. Misregulation of mitochondrial proteostasis impairs organellar function and can result in the development of severe human diseases. ATP-driven AAA+ proteins play crucial roles in preserving mitochondrial activity by removing and remodeling protein molecules in accordance with the needs of the cell. Two mitochondrial AAA proteases, i-AAA and m-AAA, are anchored to either face of the mitochondrial inner membrane, where they engage and process an array of substrates to impact protein biogenesis, quality control, and the regulation of key metabolic pathways. The functionality of these proteases is extended through multiple substrate-dependent modes of action, including complete degradation, partial processing, or dislocation from the membrane without proteolysis. This review discusses recent advances made toward elucidating the mechanisms of substrate recognition, handling, and degradation that allow these versatile proteases to control diverse activities in this multifunctional organelle.

Effect of Fortification with Fish (Pseudophycis bachus) Powder on Nutritional Quality of Durum Wheat Pasta

PubMed Central

Desai, Ajay S.; Brennan, Margaret A.

2018-01-01

This paper investigates the nutraceutical (phenolic content and antioxidant activity) and nutritional potential (protein and starch digestibility) of supplementation of durum wheat semolina with 5–20% fish powder (Pseudophycis bachus). In general, all enriched pasta with fish powder showed a significant decrease (p < 0.05) in reducing sugar released during an in vitro digestion, and reductions in standardized area under the curve (AUC) values, compared to control pasta. The potentially bioaccessible fraction of pasta enriched with 20% fish powder (FP) was characterized as having a 177–191% increase in phenolic content and a 145–556% higher antiradical activity. Elevation of these parameters in fortified pasta was accompanied by interaction of wheat starch, protein, and fish powder protein. Supplementation of fish powder also influenced protein digestibility (a reduction from 84.60% for control pasta to 80.80% for pasta with 20% fish powder). Fortification improved the nutraceutical and nutritional potential of the studied pasta with the effects depending on factors including protein-starch-phenolic interactions. PMID:29673139

Designing and evaluating the MULTICOM protein local and global model quality prediction methods in the CASP10 experiment

PubMed Central

2014-01-01

Background Protein model quality assessment is an essential component of generating and using protein structural models. During the Tenth Critical Assessment of Techniques for Protein Structure Prediction (CASP10), we developed and tested four automated methods (MULTICOM-REFINE, MULTICOM-CLUSTER, MULTICOM-NOVEL, and MULTICOM-CONSTRUCT) that predicted both local and global quality of protein structural models. Results MULTICOM-REFINE was a clustering approach that used the average pairwise structural similarity between models to measure the global quality and the average Euclidean distance between a model and several top ranked models to measure the local quality. MULTICOM-CLUSTER and MULTICOM-NOVEL were two new support vector machine-based methods of predicting both the local and global quality of a single protein model. MULTICOM-CONSTRUCT was a new weighted pairwise model comparison (clustering) method that used the weighted average similarity between models in a pool to measure the global model quality. Our experiments showed that the pairwise model assessment methods worked better when a large portion of models in the pool were of good quality, whereas single-model quality assessment methods performed better on some hard targets when only a small portion of models in the pool were of reasonable quality. Conclusions Since digging out a few good models from a large pool of low-quality models is a major challenge in protein structure prediction, single model quality assessment methods appear to be poised to make important contributions to protein structure modeling. The other interesting finding was that single-model quality assessment scores could be used to weight the models by the consensus pairwise model comparison method to improve its accuracy. PMID:24731387

Designing and evaluating the MULTICOM protein local and global model quality prediction methods in the CASP10 experiment.

PubMed

Cao, Renzhi; Wang, Zheng; Cheng, Jianlin

2014-04-15

Protein model quality assessment is an essential component of generating and using protein structural models. During the Tenth Critical Assessment of Techniques for Protein Structure Prediction (CASP10), we developed and tested four automated methods (MULTICOM-REFINE, MULTICOM-CLUSTER, MULTICOM-NOVEL, and MULTICOM-CONSTRUCT) that predicted both local and global quality of protein structural models. MULTICOM-REFINE was a clustering approach that used the average pairwise structural similarity between models to measure the global quality and the average Euclidean distance between a model and several top ranked models to measure the local quality. MULTICOM-CLUSTER and MULTICOM-NOVEL were two new support vector machine-based methods of predicting both the local and global quality of a single protein model. MULTICOM-CONSTRUCT was a new weighted pairwise model comparison (clustering) method that used the weighted average similarity between models in a pool to measure the global model quality. Our experiments showed that the pairwise model assessment methods worked better when a large portion of models in the pool were of good quality, whereas single-model quality assessment methods performed better on some hard targets when only a small portion of models in the pool were of reasonable quality. Since digging out a few good models from a large pool of low-quality models is a major challenge in protein structure prediction, single model quality assessment methods appear to be poised to make important contributions to protein structure modeling. The other interesting finding was that single-model quality assessment scores could be used to weight the models by the consensus pairwise model comparison method to improve its accuracy.

Proteomic analysis of early-stage embryos: implications for egg quality in hapuku (Polyprion oxygeneios).

PubMed

Kohn, Yair Y; Symonds, Jane E; Kleffmann, Torsten; Nakagawa, Shinichi; Lagisz, Malgorzata; Lokman, P Mark

2015-12-01

In order to develop biomarkers that may help predict the egg quality of captive hapuku (Polyprion oxygeneios) and provide potential avenues for its manipulation, the present study (1) sequenced the proteome of early-stage embryos using isobaric tag for relative and absolute quantification analysis, and (2) aimed to establish the predictive value of the abundance of identified proteins with regard to egg quality through regression analysis. Egg quality was determined for eight different egg batches by blastomere symmetry scores. In total, 121 proteins were identified and assigned to one of nine major groups according to their function/pathway. A mixed-effects model analysis revealed a decrease in relative protein abundance that correlated with (decreasing) egg quality in one major group (heat-shock proteins). No differences were found in the other protein groups. Linear regression analysis, performed for each identified protein separately, revealed seven proteins that showed a significant decrease in relative abundance with reduced blastomere symmetry: two correlates that have been named in other studies (vitellogenin, heat-shock protein-70) and a further five new candidate proteins (78 kDa glucose-regulated protein, elongation factor-2, GTP-binding nuclear protein Ran, iduronate 2-sulfatase and 6-phosphogluconate dehydrogenase). Notwithstanding issues associated with multiple statistical testing, we conclude that these proteins, and especially iduronate 2-sulfatase and the generic heat-shock protein group, could serve as biomarkers of egg quality in hapuku.

Protein quality and quantity and insulin control of mammary gland glucose utilization during lactation

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

Masor, M.L.

1987-01-01

Virgin Sprague-Dawley rats were bred, and fed laboratory stock (STOCK), 13% casein plus methionine, 13% wheat gluten, or 5% casein plus methionine through gestation and 4 days of lactation. Diets were switched at parturition to determine the effects of dietary protein quality and quantity fed during gestation and/or lactation on insulin stimulation of mammary glucose utilization. On day 20 of gestation (20G) and day 4 of lactation (4L) the right inguinal-abdominal mammary glands were removed, and acini and tissue slices were incubated in Krebs buffer with or without insulin containing (U-/sup 14/C)-glucose and 5mM glucose for 1 hour at 37/degrees/C.more » Glucose incorporation into CO/sub 2/, lipid and lactose was determined. Glucose incorporation into CO/sub 2/ and lipid, but not lactose was stimulated by insulin in mammary slices. Diet effects on glucose utilization in acini were confirmed in slices for basal and insulin stimulated levels. Treatment affected the absolute increase of insulin stimulation. Regression analysis significantly correlated pup weight gain with total glucose utilization. Poor dietary protein quality and quantity fed during gestation impaired both overall response of mammary glucose utilization to insulin stimulation, and mammary development during pregnancy. Improving protein value at parturition did not overcome those deficits by 4L.« less

Optimizing the Combination of Smoking and Boiling on Quality of Korean Traditional Boiled Loin (M. longissimus dorsi)

PubMed Central

Choi, Yun-Sang; Kim, Hyun-Wook; Kim, Young-Boong; Kim, Cheon-Jei

2015-01-01

The combined effects of smoking and boiling on the proximate composition, technological quality traits, shear force, and sensory characteristics of the Korean traditional boiled loin were studied. Cooking loss, processing loss, and shear force were lower in the smoked/boiled samples than those in the control (without smoking treatment) (p<0.05). The results showed that the boiled loin samples between the control and treatment did not differ significantly in protein, fat, or ash contents, or pH values (p>0.05). The treated samples had higher score for overall acceptability than the control (p<0.05). Thus, these results show that the Korean traditional boiled loin treated with smoking for 60 min before boiling had improved physicochemical properties and sensory characteristics. PMID:26761822

Improvement in the traditional processing method and nutritional quality of traditional extruded cassava-based snack (modified Ajogun)

PubMed Central

Obadina, Adewale O; Oyewole, Olusola B; Williams, Oluwasolabomi E

2013-01-01

This study was carried out to investigate and improve the traditional processing method and nutritional quality of the traditional cassava snack (Ajogun). Cassava root (Manihot esculenta Crantz L.) of TME 419 variety was processed into mash (40% moisture content). The cassava mash was mixed into different blends to produce fried traditional “Ajogun”, fried and baked extrudates (modified Ajogun) as snacks. These products were analyzed to determine the proximate composition including carbohydrate, fat, protein, fiber, ash, and moisture contents and functional properties such as bulk density. The results obtained for the moisture, fat, protein, and ash contents showed significant difference (P < 0.05) between the control sample and the extrudates. However, there was no significant difference (P > 0.05) in the carbohydrate and fiber contents between the three samples. There was no significant difference (P > 0.05) in the bulk density of the snacks. Also, sensory evaluation was carried out on the cassava-based snacks using the 9-point hedonic scale to determine the degree of acceptability. Results obtained showed significant difference (P < 0.05) between the extrudates and control sample in terms of appearance, taste, flavor, color, aroma, texture, and overall acceptability. The highest acceptability level of the product was at 8.04 for the control sample (traditional Ajogun). This study has shown that “Ajogun”, which is a lesser known cassava product, is rich in protein and fat. PMID:24804039

Bt crops benefit natural enemies to control non-target pests

PubMed Central

Tian, Jun-Ce; Yao, Ju; Long, Li-Ping; Romeis, Jörg; Shelton, Anthony M.

2015-01-01

Crops producing insecticidal crystal (Cry) proteins from Bacillus thuringiensis (Bt) control important lepidopteran pests. However, pests such as aphids not susceptible to Cry proteins may require other integrated pest management (IPM) tactics, including biological control. We fed aphids on Bt and non-Bt plants and analyzed the Bt protein residue in aphids and compared the effects of Bt plants and a pyrethroid, lambda-cyhalothrin, on the performance of three natural enemies (predators: Coleomegilla maculata and Eupeodes americanus; parasitoid Aphidius colemani) of the green peach aphid, Myzus persicae. No Bt protein residues in aphids were detected and no significant differences were recorded in the performance of pyrethroid-resistant aphids that fed on Bt broccoli expressing Cry1Ab or Cry1C, or on non-Bt broccoli plants treated or not treated with the pyrethroid. This indicated the aphids were not affected by the Cry proteins or the pyrethroid, thus removing any effect of prey quality. Tri-trophic experiments demonstrated that no C. maculata and E. americanus survived consumption of pyrethroid-treated aphids and that ovipositional behavior of A. colemani was impaired when provided with pyrethroid-treated aphids. In contrast, natural enemies were not affected when fed aphids reared on Bt broccoli, thus demonstrating the safety of these Bt plants for IPM. PMID:26559133

Bt crops benefit natural enemies to control non-target pests.

PubMed

Tian, Jun-Ce; Yao, Ju; Long, Li-Ping; Romeis, Jörg; Shelton, Anthony M

2015-11-12

Crops producing insecticidal crystal (Cry) proteins from Bacillus thuringiensis (Bt) control important lepidopteran pests. However, pests such as aphids not susceptible to Cry proteins may require other integrated pest management (IPM) tactics, including biological control. We fed aphids on Bt and non-Bt plants and analyzed the Bt protein residue in aphids and compared the effects of Bt plants and a pyrethroid, lambda-cyhalothrin, on the performance of three natural enemies (predators: Coleomegilla maculata and Eupeodes americanus; parasitoid Aphidius colemani) of the green peach aphid, Myzus persicae. No Bt protein residues in aphids were detected and no significant differences were recorded in the performance of pyrethroid-resistant aphids that fed on Bt broccoli expressing Cry1Ab or Cry1C, or on non-Bt broccoli plants treated or not treated with the pyrethroid. This indicated the aphids were not affected by the Cry proteins or the pyrethroid, thus removing any effect of prey quality. Tri-trophic experiments demonstrated that no C. maculata and E. americanus survived consumption of pyrethroid-treated aphids and that ovipositional behavior of A. colemani was impaired when provided with pyrethroid-treated aphids. In contrast, natural enemies were not affected when fed aphids reared on Bt broccoli, thus demonstrating the safety of these Bt plants for IPM.

Utilization of non-conventional systems for conversion of biomass to food components: Recovery optimization and characterizations of algal proteins and lipids

NASA Technical Reports Server (NTRS)

Karel, M.; Nakhost, Z.

1986-01-01

Protein isolate obtained from green algae (Scenedesmus obliquus) cultivated under controlled conditions was characterized. Molecular weight determination of fractionated algal proteins using SDS-polyacrylamide gel electrophoresis revealed a wide spectrum of molecular weights ranging from 15,000 to 220,000. Isoelectric points of dissociated proteins were in the range of 3.95 to 6.20. Amino acid composition of protein isolate compared favorably with FAO standards. High content of essential amino acids leucine, valine, phenylalanine and lysine makes algal protein isolate a high quality component of closed environment life support system (CELSS) diets. To optimize the removal of algal lipids and pigments supercritical carbon dioxide extraction (with and without ethanol as a co-solvent) was used. Addition of ethanol to supercritical CO2 resulted in more efficient removal of algal lipids and produced protein isolate with a good yield and protein recovery. The protein isolate extracted by the above mixture had an improved water solubility.

Role of AAA(+)-proteins in peroxisome biogenesis and function.

PubMed

Grimm, Immanuel; Erdmann, Ralf; Girzalsky, Wolfgang

2016-05-01

Mutations in the PEX1 gene, which encodes a protein required for peroxisome biogenesis, are the most common cause of the Zellweger spectrum diseases. The recognition that Pex1p shares a conserved ATP-binding domain with p97 and NSF led to the discovery of the extended family of AAA+-type ATPases. So far, four AAA+-type ATPases are related to peroxisome function. Pex6p functions together with Pex1p in peroxisome biogenesis, ATAD1/Msp1p plays a role in membrane protein targeting and a member of the Lon-family of proteases is associated with peroxisomal quality control. This review summarizes the current knowledge on the AAA+-proteins involved in peroxisome biogenesis and function.

Nutritional improvement of corn pasta-like product with broad bean (Vicia faba) and quinoa (Chenopodium quinoa).

PubMed

Giménez, M A; Drago, S R; Bassett, M N; Lobo, M O; Sammán, N C

2016-05-15

In this study, the nutritional quality of pasta-like product (spaghetti-type), made with corn (Zea mays) flour enriched with 30% broad bean (Vicia faba) flour and 20% of quinoa (Chenopodium quinoa) flour, was determined. Proximate chemical composition and iron, zinc and dietary fiber were determined. A biological assay was performed to assess the protein value using net protein utilization (NPU), true digestibility (TD) and protein digestibility-corrected amino acid score (PDCAAS). Iron and zinc availability were estimated by measuring dialyzable mineral fraction (%Da) resulting from in vitro gastrointestinal digestion. Nutritionally improved, gluten-free spaghetti (NIS) showed significantly increased NPU and decreased TD compared with a non-enriched control sample. One NIS-portion supplied 10-20% of recommended fiber daily intake. Addition of quinoa flour had a positive effect on the FeDa% as did broad bean flour on ZnDa%. EDTA increased Fe- and ZnDa% in all NIS-products, but it also impaired sensorial quality. Copyright © 2016. Published by Elsevier Ltd.

Quality assessment of protein model-structures based on structural and functional similarities.

PubMed

Konopka, Bogumil M; Nebel, Jean-Christophe; Kotulska, Malgorzata

2012-09-21

Experimental determination of protein 3D structures is expensive, time consuming and sometimes impossible. A gap between number of protein structures deposited in the World Wide Protein Data Bank and the number of sequenced proteins constantly broadens. Computational modeling is deemed to be one of the ways to deal with the problem. Although protein 3D structure prediction is a difficult task, many tools are available. These tools can model it from a sequence or partial structural information, e.g. contact maps. Consequently, biologists have the ability to generate automatically a putative 3D structure model of any protein. However, the main issue becomes evaluation of the model quality, which is one of the most important challenges of structural biology. GOBA--Gene Ontology-Based Assessment is a novel Protein Model Quality Assessment Program. It estimates the compatibility between a model-structure and its expected function. GOBA is based on the assumption that a high quality model is expected to be structurally similar to proteins functionally similar to the prediction target. Whereas DALI is used to measure structure similarity, protein functional similarity is quantified using standardized and hierarchical description of proteins provided by Gene Ontology combined with Wang's algorithm for calculating semantic similarity. Two approaches are proposed to express the quality of protein model-structures. One is a single model quality assessment method, the other is its modification, which provides a relative measure of model quality. Exhaustive evaluation is performed on data sets of model-structures submitted to the CASP8 and CASP9 contests. The validation shows that the method is able to discriminate between good and bad model-structures. The best of tested GOBA scores achieved 0.74 and 0.8 as a mean Pearson correlation to the observed quality of models in our CASP8 and CASP9-based validation sets. GOBA also obtained the best result for two targets of CASP8, and one of CASP9, compared to the contest participants. Consequently, GOBA offers a novel single model quality assessment program that addresses the practical needs of biologists. In conjunction with other Model Quality Assessment Programs (MQAPs), it would prove useful for the evaluation of single protein models.

Protein hydrolysate of salted duck egg white as a substitute of phosphate and its effect on quality of Pacific white shrimp (Litopenaeus vannamei).

PubMed

Kaewmanee, Thammarat; Benjakul, Soottawat; Visessanguan, Wonnop

2009-10-01

Protein hydrolysate from salted egg white (PHSEW) with different degrees of hydrolysis (DH) (3%, 6%, and 9%) was produced using pepsin. Disappearance of proteins with molecular weight (MW) of 108 and 85 kDa with the concomitant formation of proteins with MW of 23, 20, 13, and 5 kDa was observed in PHSEW. The use of PHSEW for quality improvement of Pacific white shrimp (Litopenaeus vannamei) was investigated. Shrimp soaked in 4% NaCl containing 7% PHSEW and 2.5% mixed phosphates (0.625% sodium acid pyrophosphate [SAPP] and 1.875% tetrasodium pyrophosphate [TSPP]) had the highest cooking yield with the lowest cooking loss (P < 0.05), compared with shrimps with other treatments. Nevertheless, no difference in weight gain was obtained in comparison with those treated with 4% NaCl containing 3.5% mixed phosphate (P > 0.05). Cooked shrimp treated with 4% NaCl containing 7% PHSEW and 2.5% mixed phosphate or those treated with 4% NaCl containing 3.5% mixed phosphate had the higher score of appearance, texture, and overall likeness but less shear force, in comparison with the control (no treatment) (P < 0.05). Microstructure study revealed that muscle fibers of cooked shrimp from both treatments had the swollen fibrils and gaps, while the control had the swollen compact structure. Therefore, use of PHSEW could reduce phosphate residue in shrimps without an adverse effect on sensory properties.

Glycosylation-related genes in NS0 cells are insensitive to moderately elevated ammonium concentrations

PubMed Central

Brodsky, Arthur Nathan; Caldwell, Mary; Bae, Sooneon; Harcum, Sarah W.

2014-01-01

NS0 and Chinese hamster ovary (CHO) cell lines are used to produce recombinant proteins for human therapeutics; however, ammonium accumulation can negatively impact cell growth, recombinant protein production, and protein glycosylation. To improve product quality and decrease costs, the relationship between ammonium and protein glycosylation needs to be elucidated. While ammonium has been shown to adversely affect glycosylation-related gene expression in CHO cells, NS0 studies have not been performed. Therefore, this study sought to determine if glycosylation in NS0 cells were ammonium-sensitive at the gene expression level. Using a DNA microarray that contained mouse glycosylation-related and housekeeping genes, the of these genes was analysed in response to various culture conditions – elevated ammonium, elevated salt, and elevated ammonium with proline. Surprisingly, no significant differences in gene expression levels were observed between the control and these conditions. Further, the elevated ammonium cultures were analysed using real-time quantitative reverse transcriptase PCR (qRT-PCR) for key glycosylation genes, and the qRT-PCR results corroborated the DNA microarray results, demonstrating that NS0 cells are ammonium-insensitive at the gene expression level. Since NS0 are known to have elevated nucleotide sugar pools under ammonium stress, and none of the genes directly responsible for these metabolic pools were changed, consequently cellular control at the translational or substrate-level must be responsible for the universally observed decreased glycosylation quality under elevated ammonium. PMID:25062658

Regulators of Mitochondrial Quality Control Differs in Subcutaneous Fat of Metabolically Healthy and Unhealthy Obese Monkeys

PubMed Central

Kavanagh, Kylie; Davis, Ashley T; Peters, Diane E; Le Grand, Andre; Bharadwaj, Manish S; Molina, Anthony JA

2016-01-01

Objectives Obesity exists with and without accompanying cardiometabolic disease, termed metabolically unhealthy obesity (MUO) and healthy obesity respectively (MHO). Underlying differences in the ability of subcutaneous (SQ) fat to respond to nutrient excess is emerging as a key pathway. We aimed to document the first spontaneous animal model of MHO and MUO and differences in SQ adipose tissue. Methods Vervet monkeys (Chlorocebus aethiops; n=171) were screened for Metabolic Syndrome. A subset of MHO and MUO monkeys (n=6/group) had SQ fat biopsies collected for histologic evaluations and examination of key mitochondrial proteins. Results Obesity was seen in 20% of monkeys, and within this population, 31% were healthy which mirrors human prevalence estimates. MUO monkeys had more than 60% lower adiponectin concentrations despite similar fat cell size, uncoupling protein 3, and activated macrophage abundance. However, alternatively activated/anti-inflammatory macrophages were 70% lower. Deficiencies of 50% or more in mitochondrial quality control regulators, and selected mitochondrial fission and fusion markers were observed in the SQ fat of MUO monkeys despite comparable mitochondrial content. Conclusions We characterized a novel and translatable spontaneously obese animal model of healthy and unhealthy obesity, occurring independently of dietary factors. Differences in mitochondrial quality and inflammatory cell populations of subcutaneous fat may underpin divergent metabolic health. PMID:28236433

Regulators of mitochondrial quality control differ in subcutaneous fat of metabolically healthy and unhealthy obese monkeys.

PubMed

Kavanagh, Kylie; Davis, Ashley T; Peters, Diane E; LeGrand, Andre C; Bharadwaj, Manish S; Molina, Anthony J A

2017-04-01

Obesity exists with and without accompanying cardiometabolic disease, termed metabolically unhealthy obesity (MUO) and healthy obesity (MHO), respectively. Underlying differences in the ability of subcutaneous (SQ) fat to respond to nutrient excess are emerging as a key pathway. This study aimed to document the first spontaneous animal model of MHO and MUO and differences in SQ adipose tissue. Vervet monkeys (Chlorocebus aethiops; N = 171) were screened for metabolic syndrome. A subset of MHO and MUO monkeys (n = 6/group) had SQ fat biopsies collected for histological evaluations and examination of key mitochondrial proteins. Obesity was seen in 20% of monkeys, and within this population, 31% were healthy, which mirrors human prevalence estimates. MUO monkeys had more than 60% lower adiponectin concentrations despite similar fat cell size, uncoupling protein 3, and activated macrophage abundance. However, alternatively activated/anti-inflammatory macrophages were 70% lower. Deficiencies of 50% or more in mitochondrial quality control regulators and selected mitochondrial fission and fusion markers were observed in the SQ fat of MUO monkeys despite comparable mitochondrial content. A novel and translatable spontaneously obese animal model of MHO and MUO, occurring independently of dietary factors, was characterized. Differences in mitochondrial quality and inflammatory cell populations of subcutaneous fat may underpin divergent metabolic health. © 2017 The Obesity Society.

Endoplasmic Reticulum Protein Quality Control Is Determined by Cooperative Interactions between Hsp/c70 Protein and the CHIP E3 Ligase*

PubMed Central

Matsumura, Yoshihiro; Sakai, Juro; Skach, William R.

2013-01-01

The C terminus of Hsp70 interacting protein (CHIP) E3 ligase functions as a key regulator of protein quality control by binding the C-terminal (M/I)EEVD peptide motif of Hsp/c70(90) with its N-terminal tetratricopeptide repeat (TPR) domain and facilitating polyubiquitination of misfolded client proteins via its C-terminal catalytic U-box. Using CFTR as a model client, we recently showed that the duration of the Hsc70-client binding cycle is a primary determinant of stability. However, molecular features that control CHIP recruitment to Hsp/c70, and hence the fate of the Hsp/c70 client, remain unknown. To understand how CHIP recognizes Hsp/c70, we utilized a dominant negative mutant in which loss of a conserved proline in the U-box domain (P269A) eliminates E3 ligase activity. In a cell-free reconstituted ER-associated degradation system, P269A CHIP inhibited Hsc70-dependent CFTR ubiquitination and degradation in a dose-dependent manner. Optimal inhibition required both the TPR and the U-box, indicating cooperativity between the two domains. Neither the wild type nor the P269A mutant changed the extent of Hsc70 association with CFTR nor the dissociation rate of the Hsc70-CFTR complex. However, the U-box mutation stimulated CHIP binding to Hsc70 while promoting CHIP oligomerization. CHIP binding to Hsc70 binding was also stimulated by the presence of an Hsc70 client with a preference for the ADP-bound state. Thus, the Hsp/c70 (M/I)EEVD motif is not a simple anchor for the TPR domain. Rather CHIP recruitment involves reciprocal allosteric interactions between its TPR and U-box domains and the substrate-binding and C-terminal domains of Hsp/c70. PMID:23990462

Blood derived products in pediatrics: New laboratory tools for optimizing potency assignment and reducing side effects.

PubMed

Amiral, Jean; Seghatchian, Jerard

2017-04-01

Neonates and children can develop rare bleeding disorders due to congenital/acquired coagulation Factor deficiencies, or allo-immune/autoimmune complications, or can undergo surgeries at high haemorrhagic risk. They then need specialized transfusion of blood components/products, or purified blood extracted products or recombinant proteins. Blood-derived therapies conventionally used for management of affected infants with genetic/acquired deficiencies, bleeding problems (coagulation Factor reduced or missing) or thrombotic disorders (reduced or missing anticoagulant proteins) pose some additional risks. These remedial therapies can cause tolerance when used very early in life and, sometimes needed, repeatedly. The introduction of recombinant proteins has allowed manufacturers to produce large amounts of the proteins usually present at very low concentration in blood. This has also changed the risk pattern of plasma-extracted products, especially in terms of continual reduction of viral transmission. Many efforts have been made over these past decades to reduce the risks associated with the use of all these products in terms of viral and bacterial safety, as well as immune disorders but they are not the objective of this article. Other associated side effects are the presence of undesired activities in blood products, which can produce thrombotic events or adverse reactions. The progressive introduction of blood derived products has greatly improved the prognosis and quality of life of affected patients. This concerns whole blood, but also blood cell concentrates, mainly platelets and red blood cells, plasma, while the blood extracted products are increasingly replaced by recombinant proteins. All these therapeutic products, i.e. blood extracted drugs, improve health and quality of life for hemophiliac's A or B, or patients with auto/allo-immune thrombocytopenias or with rare bleeding disorders, and those with thrombotic events occurring in childhood, which are mainly due to Protein C or Protein S deficiencies (congenital or acquired). Progress in analytical methods and biotechnology allow better control of the manufacturing processes for all blood derived or plasma extracted products and recombinant proteins, and contribute to improved manufacturing processes to minimize the occurrence of side effects. These adverse events can be due to the aging of the blood cell concentrate with release of their granule content, and generation of EVs, which can produce anaphylactic reactions and risk of thrombosis, but also to the presence of activated coagulation Factors in purified products, such as Factor Xia as recently identified in immunoglobulin concentrates. Characterization and measurement of contaminant products is of special usefulness during product preparation and for optimization of manufacturing processes for purified extracted products, but also for recombinant proteins. The pharmaceutical industry introduces these new methods for validating manufacturing processes, or for quality control assessments. The objective is first to warrant the full quality and safety of the lots produced, and assure the highest efficacy with the lowest risks when used in patients. For cell concentrates and fresh blood, storage conditions are critical and measurement of analytes such as EVs or Annexin V allows evaluation of quality of each individual transfused pouch. In addition to all the rules around viral and bacterial transmission risk, and immune tolerance, our available laboratory methods contribute to reducing the side effects of blood cell concentrates and derived plasma products, as well as those of the therapeutic recombinant proteins. Copyright © 2017 Elsevier Ltd. All rights reserved.

Regulated Proteolysis in Bacteria.

PubMed

Mahmoud, Samar A; Chien, Peter

2018-06-20

Regulated proteolysis is a vital process that affects all living things. Bacteria use energy-dependent AAA+ proteases to power degradation of misfolded and native regulatory proteins. Given that proteolysis is an irreversible event, specificity and selectivity in degrading substrates are key. Specificity is often augmented through the use of adaptors that modify the inherent specificity of the proteolytic machinery. Regulated protein degradation is intricately linked to quality control, cell-cycle progression, and physiological transitions. In this review, we highlight recent work that has shed light on our understanding of regulated proteolysis in bacteria. We discuss the role AAA+ proteases play during balanced growth as well as how these proteases are deployed during changes in growth. We present examples of how protease selectivity can be controlled in increasingly complex ways. Finally, we describe how coupling a core recognition determinant to one or more modifying agents is a general theme for regulated protein degradation.

Emerging Evidence for the Importance of Dietary Protein Source on Glucoregulatory Markers and Type 2 Diabetes: Different Effects of Dairy, Meat, Fish, Egg, and Plant Protein Foods

PubMed Central

Comerford, Kevin B.; Pasin, Gonca

2016-01-01

Observational studies provide evidence that a higher intake of protein from plant-based foods and certain animal-based foods is associated with a lower risk for type 2 diabetes (T2DM). However, there are few distinguishable differences between the glucoregulatory qualities of the proteins in plant-based foods, and it is likely their numerous non-protein components (e.g., fibers and phytochemicals) that drive the relationship with T2DM risk reduction. Conversely, the glucoregulatory qualities of the proteins in animal-based foods are extremely divergent, with a higher intake of certain animal-based protein foods showing negative effects, and others showing neutral or positive effects on T2DM risk. Among the various types of animal-based protein foods, a higher intake of dairy products (such as milk, yogurt, cheese and whey protein) consistently shows a beneficial relationship with glucose regulation and/or T2DM risk reduction. Intervention studies provide evidence that dairy proteins have more potent effects on insulin and incretin secretion compared to other commonly consumed animal proteins. In addition to their protein components, such as insulinogenic amino acids and bioactive peptides, dairy products also contain a food matrix rich in calcium, magnesium, potassium, trans-palmitoleic fatty acids, and low-glycemic index sugars—all of which have been shown to have beneficial effects on aspects of glucose control, insulin secretion, insulin sensitivity and/or T2DM risk. Furthermore, fermentation and fortification of dairy products with probiotics and vitamin D may improve a dairy product’s glucoregulatory effects. PMID:27455320

The Application of Clove Extract Protects Chinese-style Sausages against Oxidation and Quality Deterioration

PubMed Central

Peng, Xinyan

2017-01-01

This study was conducted to evaluate the effects of clove extract (CE) (0.25%, 0.5%, 1%, and 2%) on the oxidative stability and quality deterioration of Chinese-style sausage stored for 21 d at 4°C. The addition of clove extract to sausages significantly retarded increases in Thiobarbituric Reactive Substances (TBARS) values (p<0.05), while also controlling the production of protein carbonyls (p<0.05). However, the addition of clove extract promoted reduced thiol group content in sausages (p<0.05). Sausages amended with clove extract also had decreased L* values (p<0.05) and increased a* values (p<0.05) when compared with the control. Similarly, texture deterioration was retarded in sausage containing added clove extract when compared with the control during refrigerated storage. Moreover, the addition of clove extract had no negative effects on the sensory properties of sausages. These results suggested that clove extract was effective at protecting sausages from oxidation and quality deterioration during refrigerated storage for 21 d. PMID:28316478

The Application of Clove Extract Protects Chinese-style Sausages against Oxidation and Quality Deterioration.

PubMed

Zhang, Huiyun; Peng, Xinyan; Li, Xinling; Wu, Jingjuan; Guo, Xinyu

2017-01-01

This study was conducted to evaluate the effects of clove extract (CE) (0.25%, 0.5%, 1%, and 2%) on the oxidative stability and quality deterioration of Chinese-style sausage stored for 21 d at 4°C. The addition of clove extract to sausages significantly retarded increases in Thiobarbituric Reactive Substances (TBARS) values ( p <0.05), while also controlling the production of protein carbonyls ( p <0.05). However, the addition of clove extract promoted reduced thiol group content in sausages ( p <0.05). Sausages amended with clove extract also had decreased L* values ( p <0.05) and increased a* values ( p <0.05) when compared with the control. Similarly, texture deterioration was retarded in sausage containing added clove extract when compared with the control during refrigerated storage. Moreover, the addition of clove extract had no negative effects on the sensory properties of sausages. These results suggested that clove extract was effective at protecting sausages from oxidation and quality deterioration during refrigerated storage for 21 d.

DeepQA: improving the estimation of single protein model quality with deep belief networks.

PubMed

Cao, Renzhi; Bhattacharya, Debswapna; Hou, Jie; Cheng, Jianlin

2016-12-05

Protein quality assessment (QA) useful for ranking and selecting protein models has long been viewed as one of the major challenges for protein tertiary structure prediction. Especially, estimating the quality of a single protein model, which is important for selecting a few good models out of a large model pool consisting of mostly low-quality models, is still a largely unsolved problem. We introduce a novel single-model quality assessment method DeepQA based on deep belief network that utilizes a number of selected features describing the quality of a model from different perspectives, such as energy, physio-chemical characteristics, and structural information. The deep belief network is trained on several large datasets consisting of models from the Critical Assessment of Protein Structure Prediction (CASP) experiments, several publicly available datasets, and models generated by our in-house ab initio method. Our experiments demonstrate that deep belief network has better performance compared to Support Vector Machines and Neural Networks on the protein model quality assessment problem, and our method DeepQA achieves the state-of-the-art performance on CASP11 dataset. It also outperformed two well-established methods in selecting good outlier models from a large set of models of mostly low quality generated by ab initio modeling methods. DeepQA is a useful deep learning tool for protein single model quality assessment and protein structure prediction. The source code, executable, document and training/test datasets of DeepQA for Linux is freely available to non-commercial users at http://cactus.rnet.missouri.edu/DeepQA/ .

Effects of varying ruminally undegradable protein supplementation on forage digestion, nitrogen metabolism, and urea kinetics in Nellore cattle fed low-quality tropical forage.

PubMed

Batista, E D; Detmann, E; Titgemeyer, E C; Valadares Filho, S C; Valadares, R F D; Prates, L L; Rennó, L N; Paulino, M F

2016-01-01

Effects of supplemental RDP and RUP on nutrient digestion, N metabolism, urea kinetics, and muscle protein degradation were evaluated in Nellore heifers () consuming low-quality signal grass hay (5% CP and 80% NDF, DM basis). Five ruminally and abomasally cannulated Nellore heifers (248 ± 9 kg) were used in a 5 × 5 Latin square. Treatments were the control (no supplement) and RDP supplementation to meet 100% of the RDP requirement plus RUP provision to supply 0, 50, 100, or 150% of the RUP requirement. Supplemental RDP (casein plus NPN) was ruminally dosed twice daily, and RUP supply (casein) was continuously infused abomasally. Jugular infusion of [NN]-urea with measurement of enrichment in urine was used to evaluate urea kinetics. The ratio of urinary 3-methylhistidine to creatinine was used to estimate skeletal muscle protein degradation. Forage NDF intake (2.48 kg/d) was not affected ( ≥ 0.37) by supplementation, but supplementation did increase ruminal NDF digestion ( < 0.01). Total N intake (by design) and N retention increased ( < 0.001) with supplementation and also linearly increased with RUP provision. Urea entry rate and gastrointestinal entry rate of urea were increased by supplementation ( < 0.001). Supplementation with RUP linearly increased ( = 0.02) urea entry rate and tended ( = 0.07) to linearly increase gastrointestinal entry rate of urea. Urea use for anabolic purposes tended ( = 0.07) to be increased by supplementation, and RUP provision also tended ( = 0.08) to linearly increase the amount of urea used for anabolism. The fraction of recycled urea N incorporated into microbial N was greater ( < 0.001) for control (22%) than for supplemented (9%) heifers. Urinary 3-methylhistidine:creatinine of control heifers was more than double that of supplemented heifers ( < 0.001). Control heifers reabsorbed a greater ( < 0.001) fraction of urea from the renal tubule than did supplemented heifers. Overall, unsupplemented heifers had greater mobilization of AA from myofibrillar protein, which provided N for urea synthesis and subsequent recycling. Supplemental RUP, when RDP was supplied, not only increased N retention but also supported increased urea N recycling and increased ruminal microbial protein synthesis.

The binary protein-protein interaction landscape of Escherichia coli

PubMed Central

Rajagopala, Seesandra V.; Vlasblom, James; Arnold, Roland; Franca-Koh, Jonathan; Pakala, Suman B.; Phanse, Sadhna; Ceol, Arnaud; Häuser, Roman; Siszler, Gabriella; Wuchty, Stefan; Emili, Andrew; Babu, Mohan; Aloy, Patrick; Pieper, Rembert; Uetz, Peter

2014-01-01

Efforts to map the Escherichia coli interactome have identified several hundred macromolecular complexes, but direct binary protein-protein interactions (PPIs) have not been surveyed on a large scale. Here we performed yeast two-hybrid screens of 3,305 baits against 3,606 preys (~70% of the E. coli proteome) in duplicate to generate a map of 2,234 interactions, approximately doubling the number of known binary PPIs in E. coli. Integration of binary PPIs and genetic interactions revealed functional dependencies among components involved in cellular processes, including envelope integrity, flagellum assembly and protein quality control. Many of the binary interactions that could be mapped within multi-protein complexes were informative regarding internal topology and indicated that interactions within complexes are significantly more conserved than those interactions connecting different complexes. This resource will be useful for inferring bacterial gene function and provides a draft reference of the basic physical wiring network of this evolutionarily significant model microbe. PMID:24561554

Nutritional and functional evaluation of wheat flour cookies supplemented with gram flour.

PubMed

Yousaf, Ali A; Ahmed, Anwaar; Ahmad, Asif; Hameed, Tabassum; Randhawa, Muhammad Atif; Hayat, Imran; Khalid, Nauman

2013-02-01

Protein-enriched cookies were prepared by supplementing gram flour into wheat flour at levels of 0%, 10%, 20%, 30%, 40% and 50% and analysed for physicochemical properties. The protein quality of the cookies was assessed by feeding gram flour-supplemented cookies to albino rats for 10 days. The supplementation resulted in a significant increase in protein, fat, crude fibre and ash contents of the cookies. The thickness and spread factor of cookies differ significantly while non-significant effect was observed in the width of the cookies. The protein efficiency ratio, net protein utilization, biological value and true digestibility differed significantly among diets containing cookies with gram flour fed to rats. Cookies with 30% substitution of straight grade flour and gram flour produced acceptable cookies as compared to control. The cookies containing 40-50% gram flour were best regarded as protein bioavailability for rats.

Mass spectrometry based biomarker discovery, verification, and validation--quality assurance and control of protein biomarker assays.

PubMed

Parker, Carol E; Borchers, Christoph H

2014-06-01

In its early years, mass spectrometry (MS)-based proteomics focused on the cataloging of proteins found in different species or different tissues. By 2005, proteomics was being used for protein quantitation, typically based on "proteotypic" peptides which act as surrogates for the parent proteins. Biomarker discovery is usually done by non-targeted "shotgun" proteomics, using relative quantitation methods to determine protein expression changes that correlate with disease (output given as "up-or-down regulation" or "fold-increases"). MS-based techniques can also perform "absolute" quantitation which is required for clinical applications (output given as protein concentrations). Here we describe the differences between these methods, factors that affect the precision and accuracy of the results, and some examples of recent studies using MS-based proteomics to verify cancer-related biomarkers. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Exotic QTL improve grain quality in the tri-parental wheat population SW84

PubMed Central

Nedelkou, Ioanna-Pavlina; Maurer, Andreas; Schubert, Anne; Léon, Jens

2017-01-01

Developing the tri-parental exotic wheat population SW84 Genetic diversity of cultivated wheat was markedly reduced, first, during domestication and, second, since the onset of modern elite breeding. There is an increasing demand for utilizing genetic resources to increase genetic diversity and, simultaneously, to improve agronomic performance of cultivated wheat. To locate favorable effects of exotic wheat alleles, we developed the tri-parental wheat population SW84. The population was derived from crossing the hexaploid spring wheat cultivars Triso and Devon with one synthetic exotic donor accession, Syn084L, followed by two rounds of backcrossing and three rounds of selfing. SW84 consists of 359 BC2F4 lines, split into two families, D84 (Devon*Syn084L) and T84 (Triso*Syn084L). Studying the genetic control of grain quality in SW84 As a case study, grain quality of SW84 was studied in replicated field trials. Transgressive segregation was observed for all studied grain quality traits by evaluating SW84 for two years at two locations under low and high nitrogen supply. Subsequently, a genome-wide association study (GWAS) was carried out based on genomic data derived from a 90k Infinium iSELECT single nucleotide polymorphism (SNP) array. In total, GWAS yielded 37 marker-trait associations, summarized to 16 quantitative trait loci (QTL). These SNPs indicate genetic regulators of grain protein content, grain hardness, sedimentation value and sedimentation ratio. The majority of exotic QTL alleles (75%) exerted favorable effects, increasing grain protein content and sedimentation value in ten and two cases, respectively. For instance, two exotic QTL alleles were associated with a substantial increase of grain protein content and sedimentation value by 1.09% and 7.31 ml, respectively. This finding confirms the potential of exotic germplasm to improve grain quality in cultivated wheat. So far, the molecular nature of most of the detected QTL is unknown. However, two QTL correspond to known genes controlling grain quality: The major QTL on chromosome 6B, increasing grain protein content by 0.70%, on average, co-localizes with the NAM-B1 gene, known to control grain protein content as well as iron and zinc content. Likewise, the major QTL on chromosome 5D, reducing grain hardness by 8.98%, on average, co-localizes with the gene for puroindoline b (Pinb-D1) at the Ha locus. In total, 13 QTL were detected across families, whereas one and three QTL were exclusively detected in families D84 and T84, respectively. Likewise, ten QTL were detected across nitrogen treatments, whereas one and five QTL were exclusively detected under low and high N treatments, respectively. Our data indicate that most effects in SW84 act across families and N levels. Merging of data from two families or two N treatments may, thus, be considered in association studies to increase sample size and, as a result, QTL detection power. Utilizing favorable exotic QTL alleles in wheat breeding Our study serves as a model how favorable exotic QTL alleles can be located in exotic germplasm of wheat. In future, the localized favorable exotic QTL alleles will be utilized in wheat breeding programs to simultaneously improve grain quality and selectively expand genetic diversity of the elite wheat gene pool. PMID:28686676

Challenges in Routine Implementation and Quality Control of Rapid Diagnostic Tests for Malaria–Rufiji District, Tanzania

PubMed Central

McMorrow, Meredith L.; Masanja, M. Irene; Abdulla, Salim M. K.; Kahigwa, Elizeus; Kachur, S. Patrick

2018-01-01

Rapid diagnostic tests (RDTs) represent an alternative to microscopy for malaria diagnosis and have shown high sensitivity and specificity in a variety of study settings. Current World Health Organization (WHO) guidelines for quality control of RDTs provide detailed instructions on pre-field testing, but offer little guidance for quality assurance once RDTs are deployed in health facilities. From September 2006 to April 2007, we introduced a histidine-rich protein II (HRP2)-based RDT (Paracheck) for suspected malaria cases five years of age and older in nine health facilities in Rufiji District, Tanzania, to assess sensitivity and specificity of RDTs in routine use at rural health facilities. Thick blood smears were collected for all patients tested with RDTs and stained and read by laboratory personnel in each facility. Thick smears were subsequently reviewed by a reference microscopist to determine RDT sensitivity and specificity. In all nine health facilities, there were significant problems with the quality of staining and microscopy. Sensitivity and specificity of RDTs were difficult to assess given the poor quality of routine blood smear staining. Mean operational sensitivity of RDTs based on reference microscopy was 64.8%, but varied greatly by health facility, range 18.8–85.9%. Sensitivity of RDTs increased with increasing parasite density. Specificity remained high at 87.8% despite relatively poor slide quality. Institution of quality control of RDTs based on poor quality blood smear staining may impede reliable measurement of sensitivity and specificity and undermine confidence in the new diagnostic. There is an urgent need for the development of alternative quality control procedures for rapid diagnostic tests that can be performed at the facility level. PMID:18784230

Analysis of high accuracy, quantitative proteomics data in the MaxQB database.

PubMed

Schaab, Christoph; Geiger, Tamar; Stoehr, Gabriele; Cox, Juergen; Mann, Matthias

2012-03-01

MS-based proteomics generates rapidly increasing amounts of precise and quantitative information. Analysis of individual proteomic experiments has made great strides, but the crucial ability to compare and store information across different proteome measurements still presents many challenges. For example, it has been difficult to avoid contamination of databases with low quality peptide identifications, to control for the inflation in false positive identifications when combining data sets, and to integrate quantitative data. Although, for example, the contamination with low quality identifications has been addressed by joint analysis of deposited raw data in some public repositories, we reasoned that there should be a role for a database specifically designed for high resolution and quantitative data. Here we describe a novel database termed MaxQB that stores and displays collections of large proteomics projects and allows joint analysis and comparison. We demonstrate the analysis tools of MaxQB using proteome data of 11 different human cell lines and 28 mouse tissues. The database-wide false discovery rate is controlled by adjusting the project specific cutoff scores for the combined data sets. The 11 cell line proteomes together identify proteins expressed from more than half of all human genes. For each protein of interest, expression levels estimated by label-free quantification can be visualized across the cell lines. Similarly, the expression rank order and estimated amount of each protein within each proteome are plotted. We used MaxQB to calculate the signal reproducibility of the detected peptides for the same proteins across different proteomes. Spearman rank correlation between peptide intensity and detection probability of identified proteins was greater than 0.8 for 64% of the proteome, whereas a minority of proteins have negative correlation. This information can be used to pinpoint false protein identifications, independently of peptide database scores. The information contained in MaxQB, including high resolution fragment spectra, is accessible to the community via a user-friendly web interface at http://www.biochem.mpg.de/maxqb.

Analysis of Flow Cytometry DNA Damage Response Protein Activation Kinetics Following X-rays and High Energy Iron Nuclei Exposure

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

Universities Space Research Association; Chappell, Lori J.; Whalen, Mary K.

2010-12-15

We developed a mathematical method to analyze flow cytometry data to describe the kinetics of {gamma}H2AX and pATF2 phosphorylations ensuing various qualities of low dose radiation in normal human fibroblast cells. Previously reported flow cytometry kinetic results for these DSB repair phospho-proteins revealed that distributions of intensity were highly skewed, severely limiting the detection of differences in the very low dose range. Distributional analysis reveals significant differences between control and low dose samples when distributions are compared using the Kolmogorov-Smirnov test. Radiation quality differences are found in the distribution shapes and when a nonlinear model is used to relate dosemore » and time to the decay of the mean ratio of phosphoprotein intensities of irradiated samples to controls. We analyzed cell cycle phase and radiation quality dependent characteristic repair times and residual phospho-protein levels with these methods. Characteristic repair times for {gamma}H2AX were higher following Fe nuclei as compared to X-rays in G1 cells (4.5 {+-} 0.46 h vs 3.26 {+-} 0.76 h, respectively), and in S/G2 cells (5.51 {+-} 2.94 h vs 2.87 {+-} 0.45 h, respectively). The RBE in G1 cells for Fe nuclei relative to X-rays for {gamma}H2AX was 2.05 {+-} 0.61 and 5.02 {+-} 3.47, at 2 h and 24-h postirradiation, respectively. For pATF2, a saturation effect is observed with reduced expression at high doses, especially for Fe nuclei, with much slower characteristic repair times (>7 h) compared to X-rays. RBEs for pATF2 were 0.66 {+-} 0.13 and 1.66 {+-} 0.46 at 2 h and 24 h, respectively. Significant differences in {gamma}H2AX and pATF2 levels comparing irradiated samples to control were noted even at the lowest dose analyzed (0.05 Gy) using these methods of analysis. These results reveal that mathematical models can be applied to flow cytometry data to uncover important and subtle differences following exposure to various qualities of low dose radiation.« less

Impact of Processing on the Protein Quality of Pinto Bean (Phaseolus vulgaris) and Buckwheat (Fagopyrum esculentum Moench) Flours and Blends, As Determined by in Vitro and in Vivo Methodologies.

PubMed

Nosworthy, Matthew G; Franczyk, Adam; Zimoch-Korzycka, Anna; Appah, Paulyn; Utioh, Alphonsus; Neufeld, Jason; House, James D

2017-05-17

Blending of protein sources can increase protein quality by compensating for limiting amino acids present in individual sources, whereas processing grain flours by extrusion or baking can also alter protein quality. To determine the effect of baking and extrusion on the protein quality of blended flours from buckwheat and pinto beans, a rodent bioassay was performed and compared to an in vitro method of protein quality determination. Overall, extruded products had higher protein efficiency ratio values, increased digestibility, and greater protein digestibility corrected amino acid score (PDCAAS) values than baked products, with the extruded buckwheat/pinto blend having the greatest PDCAAS value of the experimental diets investigated. A correlation was found between both digestibility and PDCAAS values generated from in vitro and in vivo methods. The use of in vitro digestibility analysis should be investigated as a potential replacement for the current rodent assay for nutrient content claim purposes.

ChiLin: a comprehensive ChIP-seq and DNase-seq quality control and analysis pipeline.

PubMed

Qin, Qian; Mei, Shenglin; Wu, Qiu; Sun, Hanfei; Li, Lewyn; Taing, Len; Chen, Sujun; Li, Fugen; Liu, Tao; Zang, Chongzhi; Xu, Han; Chen, Yiwen; Meyer, Clifford A; Zhang, Yong; Brown, Myles; Long, Henry W; Liu, X Shirley

2016-10-03

Transcription factor binding, histone modification, and chromatin accessibility studies are important approaches to understanding the biology of gene regulation. ChIP-seq and DNase-seq have become the standard techniques for studying protein-DNA interactions and chromatin accessibility respectively, and comprehensive quality control (QC) and analysis tools are critical to extracting the most value from these assay types. Although many analysis and QC tools have been reported, few combine ChIP-seq and DNase-seq data analysis and quality control in a unified framework with a comprehensive and unbiased reference of data quality metrics. ChiLin is a computational pipeline that automates the quality control and data analyses of ChIP-seq and DNase-seq data. It is developed using a flexible and modular software framework that can be easily extended and modified. ChiLin is ideal for batch processing of many datasets and is well suited for large collaborative projects involving ChIP-seq and DNase-seq from different designs. ChiLin generates comprehensive quality control reports that include comparisons with historical data derived from over 23,677 public ChIP-seq and DNase-seq samples (11,265 datasets) from eight literature-based classified categories. To the best of our knowledge, this atlas represents the most comprehensive ChIP-seq and DNase-seq related quality metric resource currently available. These historical metrics provide useful heuristic quality references for experiment across all commonly used assay types. Using representative datasets, we demonstrate the versatility of the pipeline by applying it to different assay types of ChIP-seq data. The pipeline software is available open source at https://github.com/cfce/chilin . ChiLin is a scalable and powerful tool to process large batches of ChIP-seq and DNase-seq datasets. The analysis output and quality metrics have been structured into user-friendly directories and reports. We have successfully compiled 23,677 profiles into a comprehensive quality atlas with fine classification for users.

Characterisation of zinc-binding domains of peroxisomal RING finger proteins using size exclusion chromatography/inductively coupled plasma-mass spectrometry.

PubMed

Koellensperger, Gunda; Daubert, Simon; Erdmann, Ralf; Hann, Stephan; Rottensteiner, Hanspeter

2007-11-01

We determined the zinc binding stoichiometry of peroxisomal RING finger proteins by measuring sulfur/metal ratios using inductively coupled plasma-mass spectrometry coupled to size exclusion chromatography, a strategy that provides a fast and quantitative overview on the binding of metals in proteins. As a quality control, liquid chromatography-electrospray ionisation-time of flight-mass spectrometry was used to measure the molar masses of the intact proteins. The RING fingers of Pex2p, Pex10p, and Pex12p showed a stoichiometry of 2.0, 2.1, and 1.2 mol zinc/mol protein, respectively. Thus, Pex2p and Pex10p possess a typical RING domain with two coordinated zinc atoms, whereas that of Pex12p coordinates only a single zinc atom.

Msp1 Is a Membrane Protein Dislocase for Tail-Anchored Proteins.

PubMed

Wohlever, Matthew L; Mateja, Agnieszka; McGilvray, Philip T; Day, Kasey J; Keenan, Robert J

2017-07-20

Mislocalized tail-anchored (TA) proteins of the outer mitochondrial membrane are cleared by a newly identified quality control pathway involving the conserved eukaryotic protein Msp1 (ATAD1 in humans). Msp1 is a transmembrane AAA-ATPase, but its role in TA protein clearance is not known. Here, using purified components reconstituted into proteoliposomes, we show that Msp1 is both necessary and sufficient to drive the ATP-dependent extraction of TA proteins from the membrane. A crystal structure of the Msp1 cytosolic region modeled into a ring hexamer suggests that active Msp1 contains a conserved membrane-facing surface adjacent to a central pore. Structure-guided mutagenesis of the pore residues shows that they are critical for TA protein extraction in vitro and for functional complementation of an msp1 deletion in yeast. Together, these data provide a molecular framework for Msp1-dependent extraction of mislocalized TA proteins from the outer mitochondrial membrane. Copyright © 2017 Elsevier Inc. All rights reserved.

Bioengineered protein-based nanocage for drug delivery.

PubMed

Lee, Eun Jung; Lee, Na Kyeong; Kim, In-San

2016-11-15

Nature, in its wonders, presents and assembles the most intricate and delicate protein structures and this remarkable phenomenon occurs in all kingdom and phyla of life. Of these proteins, cage-like multimeric proteins provide spatial control to biological processes and also compartmentalizes compounds that may be toxic or unstable and avoids their contact with the environment. Protein-based nanocages are of particular interest because of their potential applicability as drug delivery carriers and their perfect and complex symmetry and ideal physical properties, which have stimulated researchers to engineer, modify or mimic these qualities. This article reviews various existing types of protein-based nanocages that are used for therapeutic purposes, and outlines their drug-loading mechanisms and bioengineering strategies via genetic and chemical functionalization. Through a critical evaluation of recent advances in protein nanocage-based drug delivery in vitro and in vivo, an outlook for de novo and in silico nanocage design, and also protein-based nanocage preclinical and future clinical applications will be presented. Copyright © 2016 Elsevier B.V. All rights reserved.

Comparison of Anastrepha ludens (Diptera: Tephritidae) Bisexual and Genetic Sexing (Tapachula-7) Strains: Effect of Hypoxia, Fly Density, Chilling Period, and Food Type on Fly Quality.

PubMed

Arredondo, José; Ruiz, Lía; Hernández, Emilio; Montoya, Pablo; Díaz-Fleischer, Francisco

2016-04-01

The use of genetic sexing strain (GSS) insects in the sterile insect technique (SIT) makes necessary the revision of quality parameters of some stressful steps used during the packing process for aerial release because of possible differences in tolerance between fly strains. Here, we determined the effect of three periods of hypoxia (12, 24, and 36 h at pupal stage), three cage densities (1.0, 1.3, and 1.5 flies/cm2), two different foods (protein/sugar (1/24) and Mubarqui), and three chilling times (20 min [control], 90, and 180 min) on the quality parameters of flies of two Anastrepha ludens (Loew) strains (bisexual and GSS Tapachula-7). In general, the response to stressful conditions of both fly strains was qualitatively equivalent but quantitatively different, as flies of both strains responded equally to the stressful factors; however, flies of Tapachula-7 exhibited lower quality parameters than the control flies. Thus, hypoxia affected the flying ability but not the emergence or longevity of flies. The food type affected the adult weight; protein/sugar produced heavier flies that also survived longer and had a greater mating propensity. Flies under the lowest density were better fliers that those at the other two densities. Increasing chilling time reduced flight ability but not longevity or mating propensity. The implications of these findings for the use of A. ludens GSS in SIT programs are discussed herein.

Realizing the promise of reverse phase protein arrays for clinical, translational, and basic research: a workshop report: the RPPA (Reverse Phase Protein Array) society.

PubMed

Akbani, Rehan; Becker, Karl-Friedrich; Carragher, Neil; Goldstein, Ted; de Koning, Leanne; Korf, Ulrike; Liotta, Lance; Mills, Gordon B; Nishizuka, Satoshi S; Pawlak, Michael; Petricoin, Emanuel F; Pollard, Harvey B; Serrels, Bryan; Zhu, Jingchun

2014-07-01

Reverse phase protein array (RPPA) technology introduced a miniaturized "antigen-down" or "dot-blot" immunoassay suitable for quantifying the relative, semi-quantitative or quantitative (if a well-accepted reference standard exists) abundance of total protein levels and post-translational modifications across a variety of biological samples including cultured cells, tissues, and body fluids. The recent evolution of RPPA combined with more sophisticated sample handling, optical detection, quality control, and better quality affinity reagents provides exquisite sensitivity and high sample throughput at a reasonable cost per sample. This facilitates large-scale multiplex analysis of multiple post-translational markers across samples from in vitro, preclinical, or clinical samples. The technical power of RPPA is stimulating the application and widespread adoption of RPPA methods within academic, clinical, and industrial research laboratories. Advances in RPPA technology now offer scientists the opportunity to quantify protein analytes with high precision, sensitivity, throughput, and robustness. As a result, adopters of RPPA technology have recognized critical success factors for useful and maximum exploitation of RPPA technologies, including the following: preservation and optimization of pre-analytical sample quality, application of validated high-affinity and specific antibody (or other protein affinity) detection reagents, dedicated informatics solutions to ensure accurate and robust quantification of protein analytes, and quality-assured procedures and data analysis workflows compatible with application within regulated clinical environments. In 2011, 2012, and 2013, the first three Global RPPA workshops were held in the United States, Europe, and Japan, respectively. These workshops provided an opportunity for RPPA laboratories, vendors, and users to share and discuss results, the latest technology platforms, best practices, and future challenges and opportunities. The outcomes of the workshops included a number of key opportunities to advance the RPPA field and provide added benefit to existing and future participants in the RPPA research community. The purpose of this report is to share and disseminate, as a community, current knowledge and future directions of the RPPA technology. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Protein quality and the protein to carbohydrate ratio within a high fat diet influences energy balance and the gut microbiota in C57BL/6J mice.

PubMed

McAllan, Liam; Skuse, Peter; Cotter, Paul D; O'Connor, Paula; Cryan, John F; Ross, R Paul; Fitzgerald, Gerald; Roche, Helen M; Nilaweera, Kanishka N

2014-01-01

Macronutrient quality and composition are important determinants of energy balance and the gut microbiota. Here, we investigated how changes to protein quality (casein versus whey protein isolate; WPI) and the protein to carbohydrate (P/C) ratio within a high fat diet (HFD) impacts on these parameters. Mice were fed a low fat diet (10% kJ) or a high fat diet (HFD; 45% kJ) for 21 weeks with either casein (20% kJ, HFD) or WPI at 20%, 30% or 40% kJ. In comparison to casein, WPI at a similar energy content normalised energy intake, increased lean mass and caused a trend towards a reduction in fat mass (P = 0.08), but the protein challenge did not alter oxygen consumption or locomotor activity. WPI reduced HFD-induced plasma leptin and liver triacylglycerol, and partially attenuated the reduction in adipose FASN mRNA in HFD-fed mice. High throughput sequence-based analysis of faecal microbial populations revealed microbiota in the HFD-20% WPI group clustering closely with HFD controls, although WPI specifically increased Lactobacillaceae/Lactobacillus and decreased Clostridiaceae/Clostridium in HFD-fed mice. There was no effect of increasing the P/C ratio on energy intake, but the highest ratio reduced HFD-induced weight gain, fat mass and plasma triacylglycerol, non-esterified fatty acids, glucose and leptin levels, while it increased lean mass and oxygen consumption. Similar effects were observed on adipose mRNA expression, where the highest ratio reduced HFD-associated expression of UCP-2, TNFα and CD68 and increased the diet-associated expression of β3-AR, LPL, IR, IRS-1 and GLUT4. The P/C ratio also impacted on gut microbiota, with populations in the 30/40% WPI groups clustering together and away from the 20% WPI group. Taken together, our data show that increasing the P/C ratio has a dramatic effect on energy balance and the composition of gut microbiota, which is distinct from that caused by changes to protein quality.

Protein Quality and the Protein to Carbohydrate Ratio within a High Fat Diet Influences Energy Balance and the Gut Microbiota In C57BL/6J Mice

PubMed Central

McAllan, Liam; Skuse, Peter; Cotter, Paul D.; Connor, Paula O'; Cryan, John F.; Ross, R. Paul; Fitzgerald, Gerald; Roche, Helen M.; Nilaweera, Kanishka N.

2014-01-01

Macronutrient quality and composition are important determinants of energy balance and the gut microbiota. Here, we investigated how changes to protein quality (casein versus whey protein isolate; WPI) and the protein to carbohydrate (P/C) ratio within a high fat diet (HFD) impacts on these parameters. Mice were fed a low fat diet (10% kJ) or a high fat diet (HFD; 45% kJ) for 21 weeks with either casein (20% kJ, HFD) or WPI at 20%, 30% or 40% kJ. In comparison to casein, WPI at a similar energy content normalised energy intake, increased lean mass and caused a trend towards a reduction in fat mass (P = 0.08), but the protein challenge did not alter oxygen consumption or locomotor activity. WPI reduced HFD-induced plasma leptin and liver triacylglycerol, and partially attenuated the reduction in adipose FASN mRNA in HFD-fed mice. High throughput sequence-based analysis of faecal microbial populations revealed microbiota in the HFD-20% WPI group clustering closely with HFD controls, although WPI specifically increased Lactobacillaceae/Lactobacillus and decreased Clostridiaceae/Clostridium in HFD-fed mice. There was no effect of increasing the P/C ratio on energy intake, but the highest ratio reduced HFD-induced weight gain, fat mass and plasma triacylglycerol, non-esterified fatty acids, glucose and leptin levels, while it increased lean mass and oxygen consumption. Similar effects were observed on adipose mRNA expression, where the highest ratio reduced HFD-associated expression of UCP-2, TNFα and CD68 and increased the diet-associated expression of β3-AR, LPL, IR, IRS-1 and GLUT4. The P/C ratio also impacted on gut microbiota, with populations in the 30/40% WPI groups clustering together and away from the 20% WPI group. Taken together, our data show that increasing the P/C ratio has a dramatic effect on energy balance and the composition of gut microbiota, which is distinct from that caused by changes to protein quality. PMID:24520424

A fungal biofilm reactor based on metal structured packing improves the quality of a Gla::GFP fusion protein produced by Aspergillus oryzae.

PubMed

Zune, Q; Delepierre, A; Gofflot, S; Bauwens, J; Twizere, J C; Punt, P J; Francis, F; Toye, D; Bawin, T; Delvigne, F

2015-08-01

Fungal biofilm is known to promote the excretion of secondary metabolites in accordance with solid-state-related physiological mechanisms. This work is based on the comparative analysis of classical submerged fermentation with a fungal biofilm reactor for the production of a Gla::green fluorescent protein (GFP) fusion protein by Aspergillus oryzae. The biofilm reactor comprises a metal structured packing allowing the attachment of the fungal biomass. Since the production of the target protein is under the control of the promoter glaB, specifically induced in solid-state fermentation, the biofilm mode of culture is expected to enhance the global productivity. Although production of the target protein was enhanced by using the biofilm mode of culture, we also found that fusion protein production is also significant when the submerged mode of culture is used. This result is related to high shear stress leading to biomass autolysis and leakage of intracellular fusion protein into the extracellular medium. Moreover, 2-D gel electrophoresis highlights the preservation of fusion protein integrity produced in biofilm conditions. Two fungal biofilm reactor designs were then investigated further, i.e. with full immersion of the packing or with medium recirculation on the packing, and the scale-up potentialities were evaluated. In this context, it has been shown that full immersion of the metal packing in the liquid medium during cultivation allows for a uniform colonization of the packing by the fungal biomass and leads to a better quality of the fusion protein.

High resolution top-down experimental strategies on the Orbitrap platform.

PubMed

Scheffler, Kai; Viner, Rosa; Damoc, Eugen

2018-03-20

Top-down mass spectrometry (MS) strategies allow in-depth characterization of proteins by fragmentation of the entire molecule(s) inside a mass spectrometer without requiring prior proteolytic digestion. Importantly, the fragmentation techniques on commercially available mass spectrometers have become more versatile over the past decade, with different characteristics in regards to the type and wealth of fragment ions that can be obtained while preserving labile protein post-translational modifications. Due to these and other improvements, top-down MS has become of broader interest and has started to be applied in more disciplines, such as the quality control of recombinant proteins, analysis and characterization of biopharmaceuticals, and clinical biochemistry to probe protein forms as potential disease biomarkers. This article provides a technical overview and guidance for data acquisition strategies on the Orbitrap platform for single proteins and low complexity protein mixtures. A protein standard mixture composed of six recombinant proteins is also introduced and analysis strategies are discussed in detail. The article provides a detailed overview and guidance on how to choose from the variety of available methods for protein characterization by top-down analysis on the Orbitrap platform. Technical details are provided explaining important observations and phenomena when working with intact proteins and data from a number of different samples should serve to provide a solid understanding on how experiments were and should be setup and to set the right expectations on the outcome of these types of experiments. Additionally, a new intact protein standard sample is introduced that will help as a QC sample to check the instrument's hardware and method setup conditions as a requirement for obtaining high quality data from biologically relevant samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Bioengineering a Human Plasma-Based Epidermal Substitute With Efficient Grafting Capacity and High Content in Clonogenic Cells

PubMed Central

Alexaline, Maia M.; Trouillas, Marina; Nivet, Muriel; Bourreau, Emilie; Leclerc, Thomas; Duhamel, Patrick; Martin, Michele T.; Doucet, Christelle; Fortunel, Nicolas O.

2015-01-01

Cultured epithelial autografts (CEAs) produced from a small, healthy skin biopsy represent a lifesaving surgical technique in cases of full-thickness skin burn covering >50% of total body surface area. CEAs also present numerous drawbacks, among them the use of animal proteins and cells, the high fragility of keratinocyte sheets, and the immaturity of the dermal-epidermal junction, leading to heavy cosmetic and functional sequelae. To overcome these weaknesses, we developed a human plasma-based epidermal substitute (hPBES) for epidermal coverage in cases of massive burn, as an alternative to traditional CEA, and set up critical quality controls for preclinical and clinical studies. In this study, phenotypical analyses in conjunction with functional assays (clonal analysis, long-term culture, or in vivo graft) showed that our new substitute fulfills the biological requirements for epidermal regeneration. hPBES keratinocytes showed high potential for cell proliferation and subsequent differentiation similar to healthy skin compared with a well-known reference material, as ascertained by a combination of quality controls. This work highlights the importance of integrating relevant multiparameter quality controls into the bioengineering of new skin substitutes before they reach clinical development. Significance This work involves the development of a new bioengineered epidermal substitute with pertinent functional quality controls. The novelty of this work is based on this quality approach. PMID:25848122

Bioengineering a human plasma-based epidermal substitute with efficient grafting capacity and high content in clonogenic cells.

PubMed

Alexaline, Maia M; Trouillas, Marina; Nivet, Muriel; Bourreau, Emilie; Leclerc, Thomas; Duhamel, Patrick; Martin, Michele T; Doucet, Christelle; Fortunel, Nicolas O; Lataillade, Jean-Jacques

2015-06-01

Cultured epithelial autografts (CEAs) produced from a small, healthy skin biopsy represent a lifesaving surgical technique in cases of full-thickness skin burn covering >50% of total body surface area. CEAs also present numerous drawbacks, among them the use of animal proteins and cells, the high fragility of keratinocyte sheets, and the immaturity of the dermal-epidermal junction, leading to heavy cosmetic and functional sequelae. To overcome these weaknesses, we developed a human plasma-based epidermal substitute (hPBES) for epidermal coverage in cases of massive burn, as an alternative to traditional CEA, and set up critical quality controls for preclinical and clinical studies. In this study, phenotypical analyses in conjunction with functional assays (clonal analysis, long-term culture, or in vivo graft) showed that our new substitute fulfills the biological requirements for epidermal regeneration. hPBES keratinocytes showed high potential for cell proliferation and subsequent differentiation similar to healthy skin compared with a well-known reference material, as ascertained by a combination of quality controls. This work highlights the importance of integrating relevant multiparameter quality controls into the bioengineering of new skin substitutes before they reach clinical development. This work involves the development of a new bioengineered epidermal substitute with pertinent functional quality controls. The novelty of this work is based on this quality approach. ©AlphaMed Press.

RNA Surveillance: Molecular Approaches in Transcript Quality Control and their Implications in Clinical Diseases

PubMed Central

Moraes, Karen CM

2010-01-01

Production of mature mRNAs that encode functional proteins involves highly complex pathways of synthesis, processing and surveillance. At numerous steps during the maturation process, the mRNA transcript undergoes scrutiny by cellular quality control machinery. This extensive RNA surveillance ensures that only correctly processed mature mRNAs are translated and precludes production of aberrant transcripts that could encode mutant or possibly deleterious proteins. Recent advances in elucidating the molecular mechanisms of mRNA processing have demonstrated the existence of an integrated network of events, and have revealed that a variety of human diseases are caused by disturbances in the well-coordinated molecular equilibrium of these events. From a medical perspective, both loss and gain of function are relevant, and a considerable number of different diseases exemplify the importance of the mechanistic function of RNA surveillance in a cell. Here, mechanistic hallmarks of mRNA processing steps are reviewed, highlighting the medical relevance of their deregulation and how the understanding of such mechanisms can contribute to the development of therapeutic strategies. PMID:19829759

EOL-1, the homolog of the mammalian Dom3Z, regulates olfactory learning in C. elegans.

PubMed

Shen, Yu; Zhang, Jiangwen; Calarco, John A; Zhang, Yun

2014-10-01

Learning is an essential function of the nervous system. However, our understanding of molecular underpinnings of learning remains incomplete. Here, we characterize a conserved protein EOL-1 that regulates olfactory learning in Caenorhabditis elegans. A recessive allele of eol-1 (enhanced olfactory learning) learns better to adjust its olfactory preference for bacteria foods and eol-1 acts in the URX sensory neurons to regulate learning. The mammalian homolog of EOL-1, Dom3Z, which regulates quality control of pre-mRNAs, can substitute the function of EOL-1 in learning regulation, demonstrating functional conservation between these homologs. Mutating the residues of Dom3Z that are critical for its enzymatic activity, and the equivalent residues in EOL-1, abolishes the function of these proteins in learning. Together, our results provide insights into the function of EOL-1/Dom3Z and suggest that its activity in pre-mRNA quality control is involved in neural plasticity. Copyright © 2014 the authors 0270-6474/14/3413364-07$15.00/0.

The Ubiquitination of PINK1 Is Restricted to Its Mature 52-kDa Form.

PubMed

Liu, Yuhui; Guardia-Laguarta, Cristina; Yin, Jiang; Erdjument-Bromage, Hediye; Martin, Brittany; James, Michael; Jiang, Xuejun; Przedborski, Serge

2017-07-05

Along with Parkin, PINK1 plays a critical role in maintaining mitochondrial quality control. Although PINK1 is expressed constitutively, its level is kept low in healthy mitochondria by polyubiquitination and ensuing proteasomal degradation of its mature, 52 kDa, form. We show here that the target of PINK1 polyubiquitination is the mature form and is mediated by ubiquitination of a conserved lysine at position 137. Notably, the full-length protein also contains Lys-137 but is not ubiquitinated. On the basis of our data, we propose that cleavage of full-length PINK1 at Phe-104 disrupts the major hydrophobic membrane-spanning domain in the protein, inducing a conformation change in the resultant mature form that exposes Lys-137 to the cytosol for subsequent modification by the ubiquitination machinery. Thus, the balance between the full-length and mature PINK1 allows its levels to be regulated via ubiquitination of the mature form and ensures that PINK1 functions as a mitochondrial quality control factor. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Effect of weight reduction on the quality of life in obese patients with fibromyalgia syndrome: a randomized controlled trial.

PubMed

Senna, Mohammed Kamal; Sallam, Rehab Abd-El Raouf; Ashour, Hala Salah; Elarman, Mohammed

2012-11-01

The aim of the study was to examine whether weight reduction can result in improvement of fibromyalgia impact questionnaire (FIQ) in the patients with fibromyalgia syndrome (FMS). This study was a randomized controlled trial. Obese patients with fibromyalgia were randomly assigned to 6-month dietary weight loss (n = 41) and no weight loss (n = 42) groups. Patients were assessed at baseline and at 6 months. The primary outcome measure was FIQ. Secondary measures included the tender point (TP) examination, Beck Depression Inventory-II, and Pittsburg Sleep Quality Index. Compared to the control group, patients who underwent weight reduction obtained significantly better FIQ (p = 0.007), lower mean TP count (p = 0.015), and lower mean TP pain rating in the lower body (p < 0.001). Patients who lost weight had less depression and better sleep quality than the controls. Patients who lost weight had significantly lower interleukin 6 and C-reactive protein levels than those in the control group (p = 0.034 and p = 0.007, respectively). Weight loss in obese patients with FMS leads to significant improvement in the quality of life as shown by the decrease in the FIQ score. Depression, sleep quality, and tender point count are also significantly improved by weight loss in obese patients with fibromyalgia. Our results suggest that weight reduction should be a part of fibromyalgia treatment.

Do cancer proteins really interact strongly in the human protein-protein interaction network?

PubMed

Xia, Junfeng; Sun, Jingchun; Jia, Peilin; Zhao, Zhongming

2011-06-01

Protein-protein interaction (PPI) network analysis has been widely applied in the investigation of the mechanisms of diseases, especially cancer. Recent studies revealed that cancer proteins tend to interact more strongly than other categories of proteins, even essential proteins, in the human interactome. However, it remains unclear whether this observation was introduced by the bias towards more cancer studies in humans. Here, we examined this important issue by uniquely comparing network characteristics of cancer proteins with three other sets of proteins in four organisms, three of which (fly, worm, and yeast) whose interactomes are essentially not biased towards cancer or other diseases. We confirmed that cancer proteins had stronger connectivity, shorter distance, and larger betweenness centrality than non-cancer disease proteins, essential proteins, and control proteins. Our statistical evaluation indicated that such observations were overall unlikely attributed to random events. Considering the large size and high quality of the PPI data in the four organisms, the conclusion that cancer proteins interact strongly in the PPI networks is reliable and robust. This conclusion suggests that perturbation of cancer proteins might cause major changes of cellular systems and result in abnormal cell function leading to cancer. © 2011 Elsevier Ltd. All rights reserved.

Do cancer proteins really interact strongly in the human protein-protein interaction network?

PubMed Central

Xia, Junfeng; Sun, Jingchun; Jia, Peilin; Zhao, Zhongming

2011-01-01

Protein-protein interaction (PPI) network analysis has been widely applied in the investigation of the mechanisms of diseases, especially cancer. Recent studies revealed that cancer proteins tend to interact more strongly than other categories of proteins, even essential proteins, in the human interactome. However, it remains unclear whether this observation was introduced by the bias towards more cancer studies in humans. Here, we examined this important issue by uniquely comparing network characteristics of cancer proteins with three other sets of proteins in four organisms, three of which (fly, worm, and yeast) whose interactomes are essentially not biased towards cancer or other diseases. We confirmed that cancer proteins had stronger connectivity, shorter distance, and larger betweenness centrality than non-cancer disease proteins, essential proteins, and control proteins. Our statistical evaluation indicated that such observations were overall unlikely attributed to random events. Considering the large size and high quality of the PPI data in the four organisms, the conclusion that cancer proteins interact strongly in the PPI networks is reliable and robust. This conclusion suggests that perturbation of cancer proteins might cause major changes of cellular systems and result in abnormal cell function leading to cancer. PMID:21666777

Cellular Localization of Wheat High Molecular Weight Glutenin Subunits in Transgenic Rice Grain

PubMed Central

Jo, Yeong-Min; Cho, Kyoungwon; Lee, Hye-Jung; Lim, Sun-Hyung; Kim, Jin Sun; Kim, Young-Mi; Lee, Jong-Yeol

2017-01-01

Rice (Oryza sativa L.) is a primary global food cereal. However, when compared to wheat, rice has poor food processing qualities. Dough that is made from rice flour has low viscoelasticity because rice seed lacks storage proteins that are comparable to gluten protein from wheat. Thus, current research efforts aim to improve rice flour processing qualities through the transgenic expression of viscoelastic proteins in rice seeds. In this study, we characterized the transgenic expression of wheat glutenin subunits in rice seeds. The two genes 1Dx5_KK and 1Dy10_JK, which both encode wheat high-molecular-weight glutenin subunits that confer high dough elasticity, were cloned from Korean wheat cultivars KeumKang and JoKyung, respectively. These genes were inserted into binary vectors under the control of the rice endosperm-specific Glu-B1 promoter and were expressed in the high-amylose Korean rice cultivar Koami (Oryza sativa L.). Individual expression of both glutenin subunits was confirmed by SDS-PAGE and immunoblot analyses performed using T3 generation of transgenic rice seeds. The subcellular localization of 1Dx5_KK and 1Dy10_JK in the rice seed endosperm was confirmed by immunofluorescence analysis, indicating that the wheat glutenin subunits accumulate in protein body-II and novel protein body types in the rice seed. These results contribute to our understanding of engineered seed storage proteins in rice. PMID:29156580

Text Mining Improves Prediction of Protein Functional Sites

PubMed Central

Cohn, Judith D.; Ravikumar, Komandur E.

2012-01-01

We present an approach that integrates protein structure analysis and text mining for protein functional site prediction, called LEAP-FS (Literature Enhanced Automated Prediction of Functional Sites). The structure analysis was carried out using Dynamics Perturbation Analysis (DPA), which predicts functional sites at control points where interactions greatly perturb protein vibrations. The text mining extracts mentions of residues in the literature, and predicts that residues mentioned are functionally important. We assessed the significance of each of these methods by analyzing their performance in finding known functional sites (specifically, small-molecule binding sites and catalytic sites) in about 100,000 publicly available protein structures. The DPA predictions recapitulated many of the functional site annotations and preferentially recovered binding sites annotated as biologically relevant vs. those annotated as potentially spurious. The text-based predictions were also substantially supported by the functional site annotations: compared to other residues, residues mentioned in text were roughly six times more likely to be found in a functional site. The overlap of predictions with annotations improved when the text-based and structure-based methods agreed. Our analysis also yielded new high-quality predictions of many functional site residues that were not catalogued in the curated data sources we inspected. We conclude that both DPA and text mining independently provide valuable high-throughput protein functional site predictions, and that integrating the two methods using LEAP-FS further improves the quality of these predictions. PMID:22393388

Improvements in the order, isotropy and electron density of glypican-1 crystals by controlled dehydration

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

Awad, Wael; Cairo University, Cairo; Svensson Birkedal, Gabriel

The anisotropy of crystals of glypican-1 was significantly reduced by controlled dehydration using the HC1 device, allowing the building of previously disordered parts of the structure. The use of controlled dehydration for improvement of protein crystal diffraction quality is increasing in popularity, although there are still relatively few documented examples of success. A study has been carried out to establish whether controlled dehydration could be used to improve the anisotropy of crystals of the core protein of the human proteoglycan glypican-1. Crystals were subjected to controlled dehydration using the HC1 device. The optimal protocol for dehydration was developed by carefulmore » investigation of the following parameters: dehydration rate, final relative humidity and total incubation time T{sub inc}. Of these, the most important was shown to be T{sub inc}. After dehydration using the optimal protocol the crystals showed significantly reduced anisotropy and improved electron density, allowing the building of previously disordered parts of the structure.« less

Functional Analysis of the Hsp93/ClpC Chaperone at the Chloroplast Envelope1[OPEN

PubMed Central

Tanabe, Noriaki; Clarke, Adrian K.

2016-01-01

The Hsp100-type chaperone Hsp93/ClpC has crucial roles in chloroplast biogenesis. In addition to its role in proteolysis in the stroma, biochemical and genetic evidence led to the hypothesis that this chaperone collaborates with the inner envelope TIC complex to power preprotein import. Recently, it was suggested that Hsp93, working together with the Clp proteolytic core, can confer a protein quality control mechanism at the envelope. Thus, the role of envelope-localized Hsp93, and the mechanism by which it participates in protein import, remain unclear. To analyze the function of Hsp93 in protein import independently of its ClpP association, we created a mutant of Hsp93 affecting its ClpP-binding motif (PBM) (Hsp93[P-]), which is essential for the chaperone’s interaction with the Clp proteolytic core. The Hsp93[P-] construct was ineffective at complementing the pale-yellow phenotype of hsp93 Arabidopsis (Arabidopsis thaliana) mutants, indicating that the PBM is essential for Hsp93 function. As expected, the PBM mutation negatively affected the degradation activity of the stromal Clp protease. The mutation also disrupted association of Hsp93 with the Clp proteolytic core at the envelope, without affecting the envelope localization of Hsp93 itself or its association with the TIC machinery, which we demonstrate to be mediated by a direct interaction with Tic110. Nonetheless, Hsp93[P-] expression did not detectably improve the protein import efficiency of hsp93 mutant chloroplasts. Thus, our results do not support the proposed function of Hsp93 in protein import propulsion, but are more consistent with the notion of Hsp93 performing a quality control role at the point of import. PMID:26586836

Hsp70 and Hsp90 Multichaperone Complexes Sequentially Regulate Thiazide-sensitive Cotransporter Endoplasmic Reticulum-associated Degradation and Biogenesis*

PubMed Central

Donnelly, Bridget F.; Needham, Patrick G.; Snyder, Avin C.; Roy, Ankita; Khadem, Shaheen; Brodsky, Jeffrey L.; Subramanya, Arohan R.

2013-01-01

The thiazide-sensitive NaCl cotransporter (NCC) is the primary mediator of salt reabsorption in the distal convoluted tubule and is a key determinant of the blood pressure set point. Given its complex topology, NCC is inefficiently processed and prone to endoplasmic reticulum (ER)-associated degradation (ERAD), although the mechanisms governing this process remain obscure. Here, we identify factors that impact the ER quality control of NCC. Analyses of NCC immunoprecipitates revealed that the cotransporter formed complexes with the core chaperones Hsp90, Hsp70, and Hsp40. Disruption of Hsp90 function accelerated NCC degradation, suggesting that Hsp90 promotes NCC folding. In addition, two cochaperones, the C terminus of Hsp70-interacting protein (CHIP) and the Hsp70/Hsp90 organizer protein, were associated with NCC. Although CHIP, an E3 ubiquitin ligase, promoted NCC ubiquitination and ERAD, the Hsp70/Hsp90 organizer protein stabilized NCC turnover, indicating that these two proteins differentially remodel the core chaperone systems to favor cotransporter degradation and biogenesis, respectively. Adjusting the folding environment in mammalian cells via reduced temperature enhanced NCC biosynthetic trafficking, increased Hsp90-NCC interaction, and diminished binding to Hsp70. In contrast, cotransporters harboring disease-causing mutations that impair NCC biogenesis failed to escape ERAD as efficiently as the wild type protein when cells were incubated at a lower temperature. Instead, these mutants interacted more strongly with Hsp70, Hsp40, and CHIP, consistent with a role for the Hsp70/Hsp40 system in selecting misfolded NCC for ERAD. Collectively, these observations indicate that Hsp70 and Hsp90 comprise two functionally distinct ER quality control checkpoints that sequentially monitor NCC biogenesis. PMID:23482560

Proximate Composition Analysis.

PubMed

2016-01-01

The proximate composition of foods includes moisture, ash, lipid, protein and carbohydrate contents. These food components may be of interest in the food industry for product development, quality control (QC) or regulatory purposes. Analyses used may be rapid methods for QC or more accurate but time-consuming official methods. Sample collection and preparation must be considered carefully to ensure analysis of a homogeneous and representative sample, and to obtain accurate results. Estimation methods of moisture content, ash value, crude lipid, total carbohydrates, starch, total free amino acids and total proteins are put together in a lucid manner.

Initial investigation of dietitian perception of plant-based protein quality.

PubMed

Hughes, Glenna J; Kress, Kathleen S; Armbrecht, Eric S; Mukherjea, Ratna; Mattfeldt-Beman, Mildred

2014-07-01

Interest in plant-based diets is increasing, evidenced by scientific and regulatory recommendations, including Dietary Guidelines for Americans. Dietitians provide guidance in dietary protein selection but little is known about how familiar dietitians are with the quality of plant versus animal proteins or methods for measuring protein quality. Likewise, there is a need to explore their beliefs related to dietary recommendations. The aim of this study was to assess dietitians' perceptions of plant-based protein quality and to determine if these are affected by demographic factors such as age and dietary practice group (DPG) membership. This was a cross-sectional design using an online survey. The survey was sent to all members of the Missouri Dietetic Association. All completed surveys (136) were analyzed. The main outcome measures were responses to belief and knowledge questions about the protein quality of plant-based diets, along with demographic information including age and DPG membership. Descriptive statistics and frequencies were determined, and chi-square analysis was used to determine the associations between belief and knowledge responses and demographic characteristics. Responses to belief statements suggested a high level of support for plant-based diets. No associations were found between any of the belief questions and demographic factors. A majority of respondents were not familiar with protein quality determination methods that are currently recognized by global regulatory and advisory agencies. Potential barriers identified in shifting to a more plant-based diet were lack of interest and perceived difficulty. Knowledge among dietitians of plant-based protein quality in general, and methods of protein quality measurement more specifically, needs to be addressed to enhance their knowledge base for making dietary protein recommendations. Two potential avenues for training are university curricula and continuing education opportunities provided to practitioners who provide dietary advice.

Comparison of Integrated Responses to Nonlethal and Lethal Hypothermal Stress in Milkfish (Chanos chanos): A Proteomics Study.

PubMed

Chang, Chia-Hao; Tang, Cheng-Hao; Kang, Chao-Kai; Lo, Wan-Yu; Lee, Tsung-Han

Milkfish is an important aquaculture species in Taiwan, and its high mortality during cold snaps in winter usually causes huge economic losses. To understand the effect of hypothermal stress and the corresponding compensatory stress response in milkfish, this study aimed to compare liver and gill protein levels between milkfish exposed to nonlethal (18°C), lethal (16°C), and control (28°C) temperatures. Using a proteomics approach based on two-dimensional electrophoresis and nano-LC-MS/MS analysis, this study identified thirty unique protein spots from milkfish livers and gills for which protein abundance was significantly different between nonlethal, lethal, and control temperature groups. Proteins identified in the liver were classified into three different categories according to their cellular function: (1) anti-oxidative stress, (2) apoptotic pathway, and (3) cytoskeleton. Similarly, proteins identified in the gill were sorted in five different functional categories: (1) cytoskeleton, (2) immune response, (3) protein quality control, (4) energy production, and (5) intracellular homeostasis. Based on functional information derived from the identified proteins, we assumed that different levels of hypothermal stress had a different effect and induced a different cellular response. Upon nonlethal hypothermal stress, the identified proteins were involved in anti-oxidative stress and anti-inflammation pathways, suggesting that milkfish had high levels of oxidative stress in the liver and exhibited inflammation response in the gill. Upon lethal hypothermal stress, however, identified proteins were associated with apoptosis in the liver and regulation of intracellular homeostasis in the gill. The present study provided evidence to illustrate different multi-physiological responses to nonlethal and lethal hypothermal stress in milkfish livers and gills.

Comparison of Integrated Responses to Nonlethal and Lethal Hypothermal Stress in Milkfish (Chanos chanos): A Proteomics Study

PubMed Central

Chang, Chia-Hao; Tang, Cheng-Hao; Kang, Chao-Kai; Lo, Wan-Yu; Lee, Tsung-Han

2016-01-01

Milkfish is an important aquaculture species in Taiwan, and its high mortality during cold snaps in winter usually causes huge economic losses. To understand the effect of hypothermal stress and the corresponding compensatory stress response in milkfish, this study aimed to compare liver and gill protein levels between milkfish exposed to nonlethal (18°C), lethal (16°C), and control (28°C) temperatures. Using a proteomics approach based on two-dimensional electrophoresis and nano-LC-MS/MS analysis, this study identified thirty unique protein spots from milkfish livers and gills for which protein abundance was significantly different between nonlethal, lethal, and control temperature groups. Proteins identified in the liver were classified into three different categories according to their cellular function: (1) anti-oxidative stress, (2) apoptotic pathway, and (3) cytoskeleton. Similarly, proteins identified in the gill were sorted in five different functional categories: (1) cytoskeleton, (2) immune response, (3) protein quality control, (4) energy production, and (5) intracellular homeostasis. Based on functional information derived from the identified proteins, we assumed that different levels of hypothermal stress had a different effect and induced a different cellular response. Upon nonlethal hypothermal stress, the identified proteins were involved in anti-oxidative stress and anti-inflammation pathways, suggesting that milkfish had high levels of oxidative stress in the liver and exhibited inflammation response in the gill. Upon lethal hypothermal stress, however, identified proteins were associated with apoptosis in the liver and regulation of intracellular homeostasis in the gill. The present study provided evidence to illustrate different multi-physiological responses to nonlethal and lethal hypothermal stress in milkfish livers and gills. PMID:27657931

Quality assessment of protein model-structures based on structural and functional similarities

PubMed Central

2012-01-01

Background Experimental determination of protein 3D structures is expensive, time consuming and sometimes impossible. A gap between number of protein structures deposited in the World Wide Protein Data Bank and the number of sequenced proteins constantly broadens. Computational modeling is deemed to be one of the ways to deal with the problem. Although protein 3D structure prediction is a difficult task, many tools are available. These tools can model it from a sequence or partial structural information, e.g. contact maps. Consequently, biologists have the ability to generate automatically a putative 3D structure model of any protein. However, the main issue becomes evaluation of the model quality, which is one of the most important challenges of structural biology. Results GOBA - Gene Ontology-Based Assessment is a novel Protein Model Quality Assessment Program. It estimates the compatibility between a model-structure and its expected function. GOBA is based on the assumption that a high quality model is expected to be structurally similar to proteins functionally similar to the prediction target. Whereas DALI is used to measure structure similarity, protein functional similarity is quantified using standardized and hierarchical description of proteins provided by Gene Ontology combined with Wang's algorithm for calculating semantic similarity. Two approaches are proposed to express the quality of protein model-structures. One is a single model quality assessment method, the other is its modification, which provides a relative measure of model quality. Exhaustive evaluation is performed on data sets of model-structures submitted to the CASP8 and CASP9 contests. Conclusions The validation shows that the method is able to discriminate between good and bad model-structures. The best of tested GOBA scores achieved 0.74 and 0.8 as a mean Pearson correlation to the observed quality of models in our CASP8 and CASP9-based validation sets. GOBA also obtained the best result for two targets of CASP8, and one of CASP9, compared to the contest participants. Consequently, GOBA offers a novel single model quality assessment program that addresses the practical needs of biologists. In conjunction with other Model Quality Assessment Programs (MQAPs), it would prove useful for the evaluation of single protein models. PMID:22998498

Initial ground experiments of silkworm cultures living on different feedstock for provision of high quality animal protein for human in space

NASA Astrophysics Data System (ADS)

Yang, Yunan; Tang, Liman; Tong, Ling; Liu, Yang; Liu, Hong; Li, Xiaomin

2010-09-01

Silkworm could be an alternative to provide edible animal protein in Controlled Ecological Life Support System (CELSS) for long-term manned space missions. Silkworms can consume non-edible plant residue and convert plant nutrients to high quality edible animal protein for astronauts. The preliminary investigation of silkworm culture was carried out in earth environment. The silkworms were fed with artificial silkworm diet and the leaves of stem lettuce ( Lactuca sativa L. var. angustana Irish) separately and the nutritional structure of silkworm was investigated and compared, The culture experiments showed that: (1) Stem lettuce leaves could be used as food of silkworm. The protein content of silkworm fed with lettuce leaves can reach 70% of dry mass. (2) The protein content of silkworm powder produced by the fifth instar silkworm larvae was 70%, which was similar to the protein content of silkworm pupae. The powder of the fifth instar silkworm larvae can be utilized by astronaut. (3) The biotransformation rate of silkworm larvae between the third instar and the fifth instar could reach above 70%. The biotransformation cycle of silkworm was determined as 24 days. (4) Using the stem lettuce leaves to raise silkworm, the coarse fiber content of silkworm excrements reached about 33%. The requirements of space silkworm culture equipment, feeding approaches and feeding conditions were also preliminarily designed and calculated. It is estimated that 2.2 m 3 of culture space could satisfy daily animal protein demand for seven astronauts.

Inpatient rehabilitation improves functional capacity, peripheral muscle strength and quality of life in patients with community-acquired pneumonia: a randomised trial.

PubMed

José, Anderson; Dal Corso, Simone

2016-04-01

Among people who are hospitalised for community-acquired pneumonia, does an inpatient exercise-based rehabilitation program improve functional outcomes, symptoms, quality of life and length of hospital stay more than a respiratory physiotherapy regimen? Randomised trial with concealed allocation, intention-to-treat analysis and blinding of some outcomes. Forty-nine adults hospitalised for community-acquired pneumonia. The experimental group (n=32) underwent a physical training program that included warm-up, stretching, peripheral muscle strength training and walking at a controlled speed for 15 minutes. The control group (n=17) underwent a respiratory physiotherapy regimen that included percussion, vibrocompression, respiratory exercises and free walking. The intervention regimens lasted 8 days. The primary outcome was the Glittre Activities of Daily Living test, which assesses the time taken to complete a series of functional tasks (eg, rising from a chair, walking, stairs, lifting and bending). Secondary outcomes were distance walked in the incremental shuttle walk test, peripheral muscle strength, quality of life, dyspnoea, lung function, C-reactive protein and length of hospital stay. Measures were taken 1 day before and 1 day after the intervention period. There was greater improvement in the experimental group than in the control group on the Glittre Activities of Daily Living test (mean between-group difference 39 seconds, 95% CI 20 to 59) and the incremental shuttle walk test (mean between-group difference 130 m, 95% CI 77 to 182). There were also significantly greater improvements in quality of life, dyspnoea and peripheral muscle strength in the experimental group than in the control group. There were no between-group differences in lung function, C-reactive protein or length of hospital stay. The improvement in functional outcomes after an inpatient rehabilitation program was greater than the improvement after standard respiratory physiotherapy. The exercise training program led to greater benefits in functional capacity, peripheral muscle strength, dyspnoea and quality of life. ClinicalTrials.gov, NCT02103400. Copyright © 2016 Australian Physiotherapy Association. Published by Elsevier B.V. All rights reserved.

Up-Regulation of Autophagy in Small Intestine Paneth Cells in Response to Total-Body gamma-Irradiation

DTIC Science & Technology

2009-01-01

standard error of the mean (SEM). Analysis of variance procedures with Tukey post hoc correction examined the existence and nature of temporal trends ...apoptosis. Cell 2006;126:121–134. 20. Yorimitsu T, Klionsky DJ. Eating the enoplasmic reticulum: quality control by autophagy. Trends Cell Biol 2007;17...oxide signaling to iron- regulatory protein: direct control of ferritin mRNA translation and transferrin receptor mRNA stability in transfected

Cross-system excision of chaperone-mediated proteolysis in chaperone-assisted recombinant protein production

PubMed Central

Martínez-Alonso, Mónica; Villaverde, Antonio

2010-01-01

Main Escherichia coli cytosolic chaperones such as DnaK are key components of the control quality network designed to minimize the prevalence of polypeptides with aberrant conformations. This is achieved by both favoring refolding activities but also stimulating proteolytic degradation of folding reluctant species. This last activity is responsible for the decrease of the proteolytic stability of recombinant proteins when co-produced along with DnaK, where an increase in solubility might be associated to a decrease in protein yield. However, when DnaK and its co-chaperone DnaJ are co-produced in cultured insect cells or whole insect larvae (and expectedly, in other heterologous hosts), only positive, folding-related effects of these chaperones are observed, in absence of proteolysis-mediated reduction of recombinant protein yield. PMID:21326941

Ubiquilin-1 and protein quality control in Alzheimer disease.

PubMed

El Ayadi, Amina; Stieren, Emily S; Barral, José M; Boehning, Darren

2013-01-01

Single nucleotide polymorphisms in the ubiquilin-1 gene may confer risk for late-onset Alzheimer disease (AD). We have shown previously that ubiquilin-1 functions as a molecular chaperone for the amyloid precursor protein (APP) and that protein levels of ubiquilin-1 are decreased in the brains of AD patients. We have recently found that ubiquilin-1 regulates APP trafficking and subsequent secretase processing by stimulating non-degradative ubiquitination of a single lysine residue in the cytosolic domain of APP. Thus, ubiquilin-1 plays a central role in regulating APP biosynthesis, trafficking and ultimately toxicity. As ubiquilin-1 and other ubiquilin family members have now been implicated in the pathogenesis of numerous neurodegenerative diseases, these findings provide mechanistic insights into the central role of ubiquilin proteins in maintaining neuronal proteostasis.

Effects of dietary oregano essential oil and vitamin E supplementation on meat quality, stress response and intestinal morphology in pigs following transport stress.

PubMed

Zou, Yi; Hu, Xiao Ming; Zhang, Ting; Wei, Hong Kui; Zhou, Yuan Fei; Zhou, Zhong Xin; Peng, Jian

2017-02-14

This study investigates the effects of dietary oregano essential oil (OEO) and vitamin E (Vit E) supplementation on meat quality, stress response and intestinal morphology in pigs following transport stress. A total of 288 finishing pigs were randomly assigned to three groups: a basal diet or a basal diet supplemented either with 200 mg/kg Vit E or 25 mg/kg OEO. After a 28-day feeding trial, total of 132 finishing pigs according diet and transport stress were assigned to one of four treatment groups: 1) control treatment without transport stress (Control group), 2) control treatment with 5-hr transport stress (Negative group), 3) Vit E treatment with 5-hr transport stress and 4) OEO treatment with 5-hr transport stress. Transport stress pigs had lower muscle 45 min pH (pHi) and higher drip loss than control pigs. Dietary OEO and Vit E supplementation significantly increased 45min pH under transport stress, and the OEO groups produced lower 24-hr drip loss values (P<0.05) than that of pigs from the negative group. The OEO-supplemented pigs showed decreased serum levels of creatine kinase (CK) and cortisol (P<0.05), and decreased Hsp 27 (heat shock protein 27) and Hsp 70 (heat shock protein 70) mRNA expression in the muscle (P<0.05). Additionally, histological analysis revealed intestinal epithelial damage in transport stress pigs that was reversed by dietary supplementation with OEO. In conclusion, supplementation with dietary OEO may be superior to supplementation with dietary Vit E in alleviating the meat quality, stress response and intestinal morphology of pigs after challenge due to transportation stress.

Effects of dietary oregano essential oil and vitamin E supplementation on meat quality, stress response and intestinal morphology in pigs following transport stress

PubMed Central

ZOU, Yi; HU, Xiao Ming; ZHANG, Ting; WEI, Hong Kui; ZHOU, Yuan Fei; ZHOU, Zhong Xin; PENG, Jian

2016-01-01

This study investigates the effects of dietary oregano essential oil (OEO) and vitamin E (Vit E) supplementation on meat quality, stress response and intestinal morphology in pigs following transport stress. A total of 288 finishing pigs were randomly assigned to three groups: a basal diet or a basal diet supplemented either with 200 mg/kg Vit E or 25 mg/kg OEO. After a 28-day feeding trial, total of 132 finishing pigs according diet and transport stress were assigned to one of four treatment groups: 1) control treatment without transport stress (Control group), 2) control treatment with 5-hr transport stress (Negative group), 3) Vit E treatment with 5-hr transport stress and 4) OEO treatment with 5-hr transport stress. Transport stress pigs had lower muscle 45 min pH (pHi) and higher drip loss than control pigs. Dietary OEO and Vit E supplementation significantly increased 45min pH under transport stress, and the OEO groups produced lower 24-hr drip loss values (P<0.05) than that of pigs from the negative group. The OEO-supplemented pigs showed decreased serum levels of creatine kinase (CK) and cortisol (P<0.05), and decreased Hsp 27 (heat shock protein 27) and Hsp 70 (heat shock protein 70) mRNA expression in the muscle (P<0.05). Additionally, histological analysis revealed intestinal epithelial damage in transport stress pigs that was reversed by dietary supplementation with OEO. In conclusion, supplementation with dietary OEO may be superior to supplementation with dietary Vit E in alleviating the meat quality, stress response and intestinal morphology of pigs after challenge due to transportation stress. PMID:27916788

Effectiveness of treatments for infantile colic: systematic review.

PubMed

Lucassen, P L; Assendelft, W J; Gubbels, J W; van Eijk, J T; van Geldrop, W J; Neven, A K

1998-05-23

To evaluate the effectiveness of diets, drug treatment, and behavioural interventions on infantile colic in trials with crying or the presence of colic as the primary outcome measure. Controlled clinical trials identified by a highly sensitive search strategy in Medline (1966-96), Embase (1986-95), and the Cochrane Controlled Trials Register, in combination with reference checking for further relevant publications. Keywords were crying and colic. Two independent assessors selected controlled trials with interventions lasting at least 3 days that included infants younger than 6 months who cried excessively. Methodological quality was assessed by two assessors independently with a quality assessment scale (range 0-5). Effect sizes were calculated as percentage success. Effect sizes of trials using identical interventions were pooled using a random effects model. 27 controlled trials were identified. Elimination of cows' milk protein was effective when substituted by hypoallergenic formula milks (effect size 0.22 (95% confidence interval 0.09 to 0.34)). The effectiveness of substitution by soy formula milks was unclear when only trials of good methodological quality were considered. The benefit of eliminating cows' milk protein was not restricted to highly selected populations. Dicyclomine was effective (effect size 0.46 (0.33 to 0.60)), but serious side effects have been reported. The advice to reduce stimulation was beneficial (effect size 0.48 (0.23 to 0.74)), whereas the advice to increase carrying and holding seemed not to reduce crying. No benefit was shown for simethicone. Uncertainty remained about the effectiveness of low lactose formula milks. Infantile colic should preferably be treated by advising carers to reduce stimulation and with a one week trial of a hypoallergenic formula milk.

Whey protein isolate/cellulose nanofibre/TiO2 nanoparticle/rosemary essential oil nanocomposite film: Its effect on microbial and sensory quality of lamb meat and growth of common foodborne pathogenic bacteria during refrigeration.

PubMed

Alizadeh Sani, Mahmood; Ehsani, Ali; Hashemi, Mohammad

2017-06-19

The use of biodegradable nanocomposite films in active packaging is of great importance since they can have a controlled release of antimicrobial compounds. This study was conducted to evaluate the efficacy of whey protein isolate (WPI)/cellulose nanofibre (CNF) nanocomposite films containing 1.0% (w/w) titanium dioxide (TiO 2 ) and 2.0% (w/v) rosemary essential oil (REO) in preserving the microbial and sensory quality of lamb meat during the storage at 4±1°C. Initially, the best concentration of each compound to be added to the film was determined by micro-dilution and disc diffusion methods. The microbial and sensory properties of lamb meat were controlled in two groups (control and treatment) over 15days of storage. Then, the samples were analysed for total viable count (TVC), Pseudomonas spp. count, Enterobacteriaceae count, Lactic acid bacteria (LAB) count, inoculated Staphylococcus aureus count, Listeria monocytogenes count, and Escherichia coli O 157 :H 7 count. Microbial analysis and nine-point hedonic scale was applied for the sensory analysis. Results indicated that the use of nanocomposite films significantly reduced the bacterial counts of treatment group. Higher inhibition effect was observed on Gram-positive bacteria than on Gram-negative bacteria (P<0.05). The microbial and sensory evaluations also showed that the use of nanocomposite films significantly increased the shelf life of treated meat (15days) compared to the control meat (6days). Based on the results of this study, the edible nanocomposite films were effective in preserving the microbial and sensory qualities of lamb meat; therefore, this application is recommended in meat especially red meat. Copyright © 2017 Elsevier B.V. All rights reserved.

Preservation of protein clefts in comparative models.

PubMed

Piedra, David; Lois, Sergi; de la Cruz, Xavier

2008-01-16

Comparative, or homology, modelling of protein structures is the most widely used prediction method when the target protein has homologues of known structure. Given that the quality of a model may vary greatly, several studies have been devoted to identifying the factors that influence modelling results. These studies usually consider the protein as a whole, and only a few provide a separate discussion of the behaviour of biologically relevant features of the protein. Given the value of the latter for many applications, here we extended previous work by analysing the preservation of native protein clefts in homology models. We chose to examine clefts because of their role in protein function/structure, as they are usually the locus of protein-protein interactions, host the enzymes' active site, or, in the case of protein domains, can also be the locus of domain-domain interactions that lead to the structure of the whole protein. We studied how the largest cleft of a protein varies in comparative models. To this end, we analysed a set of 53507 homology models that cover the whole sequence identity range, with a special emphasis on medium and low similarities. More precisely we examined how cleft quality - measured using six complementary parameters related to both global shape and local atomic environment, depends on the sequence identity between target and template proteins. In addition to this general analysis, we also explored the impact of a number of factors on cleft quality, and found that the relationship between quality and sequence identity varies depending on cleft rank amongst the set of protein clefts (when ordered according to size), and number of aligned residues. We have examined cleft quality in homology models at a range of seq.id. levels. Our results provide a detailed view of how quality is affected by distinct parameters and thus may help the user of comparative modelling to determine the final quality and applicability of his/her cleft models. In addition, the large variability in model quality that we observed within each sequence bin, with good models present even at low sequence identities (between 20% and 30%), indicates that properly developed identification methods could be used to recover good cleft models in this sequence range.

Assessing reliability of protein-protein interactions by integrative analysis of data in model organisms.

PubMed

Lin, Xiaotong; Liu, Mei; Chen, Xue-wen

2009-04-29

Protein-protein interactions play vital roles in nearly all cellular processes and are involved in the construction of biological pathways such as metabolic and signal transduction pathways. Although large-scale experiments have enabled the discovery of thousands of previously unknown linkages among proteins in many organisms, the high-throughput interaction data is often associated with high error rates. Since protein interaction networks have been utilized in numerous biological inferences, the inclusive experimental errors inevitably affect the quality of such prediction. Thus, it is essential to assess the quality of the protein interaction data. In this paper, a novel Bayesian network-based integrative framework is proposed to assess the reliability of protein-protein interactions. We develop a cross-species in silico model that assigns likelihood scores to individual protein pairs based on the information entirely extracted from model organisms. Our proposed approach integrates multiple microarray datasets and novel features derived from gene ontology. Furthermore, the confidence scores for cross-species protein mappings are explicitly incorporated into our model. Applying our model to predict protein interactions in the human genome, we are able to achieve 80% in sensitivity and 70% in specificity. Finally, we assess the overall quality of the experimentally determined yeast protein-protein interaction dataset. We observe that the more high-throughput experiments confirming an interaction, the higher the likelihood score, which confirms the effectiveness of our approach. This study demonstrates that model organisms certainly provide important information for protein-protein interaction inference and assessment. The proposed method is able to assess not only the overall quality of an interaction dataset, but also the quality of individual protein-protein interactions. We expect the method to continually improve as more high quality interaction data from more model organisms becomes available and is readily scalable to a genome-wide application.

Short term effects on bone quality associated with consumption of soy protein isolate and other dietary protein sources in rapidly growing female rats

USDA-ARS?s Scientific Manuscript database

Beneficial effects of soy protein consumption on bone quality have been reported. The effects of other dietary protein sources such as whey protein hydrolysate (WPH) and rice protein isolate (RPI) on bone growth has been less well examined. The current study compared effects of feeding soy protein i...

Quinoa seed quality response to sodium chloride and sodium sulfate salinity

USDA-ARS?s Scientific Manuscript database

Quinoa (Chenopodium Quinoa Willd.) is a pseudocereal which has high protein content, but also provides high quality protein. The essential amino acids are balanced; protein efficiency ratio and true protein digestibility are comparable to those of casein, which considered as a good protein resource....

Gene networks in the synthesis and deposition of protein polymers during grain development of wheat.

PubMed

She, Maoyun; Ye, Xingguo; Yan, Yueming; Howit, C; Belgard, M; Ma, Wujun

2011-03-01

As the amino acid storing organelle, the protein bodies provide nutrients for embryo development, seed germination and early seedling growth through storage proteolysis in cereal plants, such as wheat and rice. In protein bodies, the monomeric and polymeric prolamins, i.e. gliadins and glutenins, form gluten and play a key role in determining dough functionality and end-product quality of wheat. The formation of intra- and intermolecular bonds, including disulphide and tyrosine bonds, in and between prolamins confers cohesivity, viscosity, elasticity and extensibility to wheat dough during mixing and processing. In this review, we summarize recent progress in wheat gluten research with a focus on the fundamental molecular biological aspects, including transcriptional regulation on genes coding for prolamin components, biosynthesis, deposition and secretion of protein polymers, formation of protein bodies, genetic control of seed storage proteins, the transportation of the protein bodies and key enzymes for determining the formation of disulphide bonds of prolamin polymers.

Development of an automated large-scale protein-crystallization and monitoring system for high-throughput protein-structure analyses.

PubMed

Hiraki, Masahiko; Kato, Ryuichi; Nagai, Minoru; Satoh, Tadashi; Hirano, Satoshi; Ihara, Kentaro; Kudo, Norio; Nagae, Masamichi; Kobayashi, Masanori; Inoue, Michio; Uejima, Tamami; Oda, Shunichiro; Chavas, Leonard M G; Akutsu, Masato; Yamada, Yusuke; Kawasaki, Masato; Matsugaki, Naohiro; Igarashi, Noriyuki; Suzuki, Mamoru; Wakatsuki, Soichi

2006-09-01

Protein crystallization remains one of the bottlenecks in crystallographic analysis of macromolecules. An automated large-scale protein-crystallization system named PXS has been developed consisting of the following subsystems, which proceed in parallel under unified control software: dispensing precipitants and protein solutions, sealing crystallization plates, carrying robot, incubators, observation system and image-storage server. A sitting-drop crystallization plate specialized for PXS has also been designed and developed. PXS can set up 7680 drops for vapour diffusion per hour, which includes time for replenishing supplies such as disposable tips and crystallization plates. Images of the crystallization drops are automatically recorded according to a preprogrammed schedule and can be viewed by users remotely using web-based browser software. A number of protein crystals were successfully produced and several protein structures could be determined directly from crystals grown by PXS. In other cases, X-ray quality crystals were obtained by further optimization by manual screening based on the conditions found by PXS.

S-nitrosylation of the thioredoxin-like domains of protein disulfide isomerase and its role in neurodegenerative conditions.

NASA Astrophysics Data System (ADS)

Conway, Myra; Harris, Matthew

2015-04-01

Correct protein folding and inhibition of protein aggregation is facilitated by a cellular ‘quality control system’ that engages a network of protein interactions including molecular chaperones and the ubiquitin proteasome system. Key chaperones involved in these regulatory mechanisms are the protein disulphide isomerases (PDI) and their homologues, predominantly expressed in the endoplasmic reticulum of most tissues. Redox changes that disrupt ER homeostasis can lead to modification of these enzymes or chaperones with the loss of their proposed neuroprotective role resulting in an increase in protein misfolding. Misfolded protein aggregates have been observed in several disease states and are considered to play a pivotal role in the pathogenesis of neurodegenerative conditions such as Alzheimer’s disease, Parkinson’s disease, and Amyotrophic Lateral sclerosis. This review will focus on the importance of the thioredoxin-like -CGHC- active site of PDI and how our understanding of this structural motif will play a key role in unravelling the pathogenic mechanisms that underpin these neurodegenerative conditions.

Comparative study of the quality characteristics of defatted soy flour treated by supercritical carbon dioxide and organic solvent.

PubMed

Kang, Sung-Won; Rahman, M Shafiur; Kim, Ah-Na; Lee, Kyo-Yeon; Park, Chan-Yang; Kerr, William L; Choi, Sung-Gil

2017-07-01

Defatted soy flour is a potential source of food protein, amino acids, ash and isoflavones. The supercritical carbon dioxide (SC-CO 2 ) and a traditional organic solvent extraction methods were used to remove fat from soy flour, and the quality characteristics of a control soy flour (CSF), defatted soy flour by SC-CO 2 (DSFSC-CO 2 ) and defatted soy flour by an organic solvent (DSF-OS) were compared. The SC-CO 2 process was carried out at a constant temperature of 45 °C, and a pressure of 40 MPa for 3 h with a CO 2 flow rate of 30 g/min. The DSFSC-CO 2 had significantly higher protein, ash, and amino acids content than CSF and DSF-OS. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis demonstrated that CSF and DSFSC-CO 2 had protein bands of similar intensity and area that indicated no denaturation of protein, whereas DSF-OS showed diffuse bands or no bands due to protein denaturation. In addition to higher nutritional value and protein contents, DSFSC-CO 2 showed superior functional properties in terms of total soluble solids content, water and oil absorption, emulsifying and foaming capacity. The SC-CO 2 method offers a nutritionally and environmentally friendly alternative extraction processing approach for the removal of oil from high-protein food sources. It has a great potential for producing high-protein fat-free, and low-calorie content diet than the traditional organic solvent extraction method.

Development of a Premium Quality Plasma-derived IVIg (IQYMUNE®) Utilizing the Principles of Quality by Design-A Worked-through Case Study.

PubMed

Paolantonacci, Philippe; Appourchaux, Philippe; Claudel, Béatrice; Ollivier, Monique; Dennett, Richard; Siret, Laurent

2018-01-01

Polyvalent human normal immunoglobulins for intravenous use (IVIg), indicated for rare and often severe diseases, are complex plasma-derived protein preparations. A quality by design approach has been used to develop the Laboratoire Français du Fractionnement et des Biotechnologies new-generation IVIg, targeting a high level of purity to generate an enhanced safety profile while maintaining a high level of efficacy. A modular approach of quality by design was implemented consisting of five consecutive steps to cover all the stages from the product design to the final product control strategy.A well-defined target product profile was translated into 27 product quality attributes that formed the basis of the process design. In parallel, a product risk analysis was conducted and identified 19 critical quality attributes among the product quality attributes. Process risk analysis was carried out to establish the links between process parameters and critical quality attributes. Twelve critical steps were identified, and for each of these steps a risk mitigation plan was established.Among the different process risk mitigation exercises, five process robustness studies were conducted at qualified small scale with a design of experiment approach. For each process step, critical process parameters were identified and, for each critical process parameter, proven acceptable ranges were established. The quality risk management and risk mitigation outputs, including verification of proven acceptable ranges, were used to design the process verification exercise at industrial scale.Finally, the control strategy was established using a mix, or hybrid, of the traditional approach plus elements of the quality by design enhanced approach, as illustrated, to more robustly assign material and process controls and in order to securely meet product specifications.The advantages of this quality by design approach were improved process knowledge for industrial design and process validation and a clear justification of the process and product specifications as a basis for control strategy and future comparability exercises. © PDA, Inc. 2018.

Nutrition and physical activity for the prevention and treatment of age-related sarcopenia.

PubMed

Bosaeus, Ingvar; Rothenberg, Elisabet

2016-05-01

Sarcopenia, defined as loss of skeletal muscle mass and function, is associated with adverse outcomes such as physical disability, impaired quality of life and increased mortality. Several mechanisms are involved in the development of sarcopenia. Potentially modifiable factors include nutrition and physical activity. Protein metabolism is central to the nutritional issues, along with other potentially modifying nutritional factors as energy balance and vitamin D status. An increasing but still incomplete knowledge base has generated recent recommendations on an increased protein intake in the elderly. Several factors beyond the total amount of protein consumed emerge as potentially important in this context. A recent summit examined three hypotheses: (1) A meal threshold; habitually consuming 25-30 g protein at breakfast, lunch and dinner provides sufficient protein to effectively stimulate muscle protein anabolism; (2) Protein quality; including high-quality protein at each meal improves postprandial muscle protein synthesis; and (3) performing physical activity in close temporal proximity to a high-quality protein meal enhances muscle anabolism. Optimising the potential for muscle protein anabolism by consuming an adequate amount of high-quality protein at each meal, in combination with physical activity, appears as a promising strategy to prevent or delay the onset of sarcopenia. However, results of interventions are inconsistent, and well-designed, standardised studies evaluating exercise or nutrition interventions are needed before guidelines can be developed for the prevention and treatment of age-related sarcopenia.

Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex

PubMed Central

Lyumkis, Dmitry; Oliveira dos Passos, Dario; Tahara, Erich B.; Webb, Kristofor; Bennett, Eric J.; Vinterbo, Staal; Potter, Clinton S.; Carragher, Bridget; Joazeiro, Claudio A. P.

2014-01-01

All organisms have evolved mechanisms to manage the stalling of ribosomes upon translation of aberrant mRNA. In eukaryotes, the large ribosomal subunit-associated quality control complex (RQC), composed of the listerin/Ltn1 E3 ubiquitin ligase and cofactors, mediates the ubiquitylation and extraction of ribosome-stalled nascent polypeptide chains for proteasomal degradation. How RQC recognizes stalled ribosomes and performs its functions has not been understood. Using single-particle cryoelectron microscopy, we have determined the structure of the RQC complex bound to stalled 60S ribosomal subunits. The structure establishes how Ltn1 associates with the large ribosomal subunit and properly positions its E3-catalytic RING domain to mediate nascent chain ubiquitylation. The structure also reveals that a distinguishing feature of stalled 60S particles is an exposed, nascent chain-conjugated tRNA, and that the Tae2 subunit of RQC, which facilitates Ltn1 binding, is responsible for selective recognition of stalled 60S subunits. RQC components are engaged in interactions across a large span of the 60S subunit surface, connecting the tRNA in the peptidyl transferase center to the distally located nascent chain tunnel exit. This work provides insights into a mechanism linking translation and protein degradation that targets defective proteins immediately after synthesis, while ignoring nascent chains in normally translating ribosomes. PMID:25349383

Mieap, a p53-Inducible Protein, Controls Mitochondrial Quality by Repairing or Eliminating Unhealthy Mitochondria

PubMed Central

Kitamura, Noriaki; Nakamura, Yasuyuki; Miyamoto, Yuji; Miyamoto, Takafumi; Kabu, Koki; Yoshida, Masaki; Futamura, Manabu; Ichinose, Shizuko; Arakawa, Hirofumi

2011-01-01

Maintenance of healthy mitochondria prevents aging, cancer, and a variety of degenerative diseases that are due to the result of defective mitochondrial quality control (MQC). Recently, we discovered a novel mechanism for MQC, in which Mieap induces intramitochondrial lysosome-like organella that plays a critical role in the elimination of oxidized mitochondrial proteins (designated MALM for Mieap-induced accumulation of lysosome-like organelles within mitochondria). However, a large part of the mechanisms for MQC remains unknown. Here, we report additional mechanisms for Mieap-regulated MQC. Reactive oxygen species (ROS) scavengers completely inhibited MALM. A mitochondrial outer membrane protein NIX interacted with Mieap in a ROS-dependent manner via the BH3 domain of NIX and the coiled-coil domain of Mieap. Deficiency of NIX also completely impaired MALM. When MALM was inhibited, Mieap induced vacuole-like structures (designated as MIV for Mieap-induced vacuole), which engulfed and degraded the unhealthy mitochondria by accumulating lysosomes. The inactivation of p53 severely impaired both MALM and MIV generation, leading to accumulation of unhealthy mitochondria. These results suggest that (1) mitochondrial ROS and NIX are essential factors for MALM, (2) MIV is a novel mechanism for lysosomal degradation of mitochondria, and (3) the p53-Mieap pathway plays a pivotal role in MQC by repairing or eliminating unhealthy mitochondria via MALM or MIV generation, respectively. PMID:21264228

Role of Autophagy in Metabolic Syndrome-Associated Heart Disease

PubMed Central

Ren, Sidney Y.; Xu, Xihui

2014-01-01

Metabolic syndrome (MetS) is a constellation of multiple metabolic risk factors including abdominal obesity, glucose intolerance, insulin resistance, dyslipidemia and hypertension. Over the past decades, the prevalence of metabolic syndrome has increased dramatically, imposing a devastating, pandemic health threat. More importantly, individuals with metabolic syndrome are at an increased risk of diabetes mellitus and overall cardiovascular diseases. One of the common comorbidities of metabolic syndrome is heart anomalies leading to the loss of cardiomyocytes, cardiac dysfunction and ultimately heart failure. Up-to-date, a plethora cell signaling pathways have been postulated for the pathogenesis of cardiac complications in obesity including lipotoxicity, inflammation, oxidative stress, apoptosis and sympathetic overactivation although the precise mechanism of action underscoring obesity-associated heart dysfunction remains elusive. Recent evidence has indicated a potential role of protein quality control in components of metabolic syndrome. Within the protein quality control system, the autophagy-lysosome pathway is an evolutionarily conserved pathway responsible for bulk degradation of large intracellular organelles and protein aggregates. Autophagy has been demonstrated to play an indispensible role in the maintenance of cardiac geometry and function under both physiological and pathological conditions. Accumulating studies have demonstrated that autophagy plays a pivotal role in the etiology of cardiac anomalies under obesity and metabolic syndrome. In this mini review, we will discuss on how autophagy is involved in the regulation of cardiac function in obesity and metabolic syndrome. PMID:24810277

Crystallization of proteins by dynamic control of supersaturation. Ph.D. Thesis Semiannual Status Report, 21 Mar. - 20 Sep. 1990

NASA Technical Reports Server (NTRS)

Wilson, Lori June

1990-01-01

The growth of protein crystals is known to be the limiting factor in the determination of the three-dimensional structures of most proteins. It is expected that the kinetics of supersaturation, which is directly related to solvent evaporation, will affect protein crystal growth and nucleation and accordingly determine the quality, number, size, and morphology of the crystals. With a technique that controls the evaporation of solvent from a protein solution with N2(g) it is possible to determine the effect of different evaporation profiles on hen egg white lysozyme crystals. Hen egg white lysozyme was chosen as the model protein because it crystallizes easily and has solubility data available for most salt, pH, and temperature ranges. Commercially available lysozyme was further purified by a number of methods. Crystals grown with the purified lysozyme and with the unpurified lysozyme in citrate buffer were different shapes but were found to be of the same symmetry space group by precession photos. Differences were seen in the lysozyme crystals grown using different evaporation rates. At three of the four initial conditions for lysozyme crystal growth, longer evaporation times yielded better crystals. The evaporation times required to see a change in the appearance of the crystals was much longer than expected. The number of rates studied so far represent only a small fraction of the ones now available with the gas evaporation device. The technique also provides for control of both solution pH and temperature which are related to the solubilities of proteins.

Transfer RNA: a dancer between charging and mis-charging for protein biosynthesis.

PubMed

Zhou, Xiaolong; Wang, Enduo

2013-10-01

Transfer RNA plays a fundamental role in the protein biosynthesis as an adaptor molecule by functioning as a biological link between the genetic nucleotide sequence in the mRNA and the amino acid sequence in the protein. To perform its role in protein biosynthesis, it has to be accurately recognized by aminoacyl-tRNA synthetases (aaRSs) to generate aminoacyl-tRNAs (aa-tRNAs). The correct pairing between an amino acid with its cognate tRNA is crucial for translational quality control. Production and utilization of mis-charged tRNAs are usually detrimental for all the species, resulting in cellular dysfunctions. Correct aa-tRNAs formation is collectively controlled by aaRSs with distinct mechanisms and/or other trans-factors. However, in very limited instances, mis-charged tRNAs are intermediate for specific pathways or essential components for the translational machinery. Here, from the point of accuracy in tRNA charging, we review our understanding about the mechanism ensuring correct aa-tRNA generation. In addition, some unique mis-charged tRNA species necessary for the organism are also briefly described.

Afzelin ameliorates D‐galactosamine and lipopolysaccharide‐induced fulminant hepatic failure by modulating mitochondrial quality control and dynamics

PubMed Central

Lee, Sang‐Bin; Kang, Jung‐Woo; Kim, So‐Jin; Ahn, Jongmin; Kim, Jinwoong

2016-01-01

Background and Purpose Fulminant hepatic failure (FHF) is a fatal clinical syndrome that results in excessive inflammation and hepatocyte death. Mitochondrial dysfunction is considered to be a possible mechanism of FHF. Afzelin, a flavonol glycoside found in Houttuynia cordata Thunberg, has anti‐inflammatory and antioxidant properties. The present study elucidated the cytoprotective mechanisms of afzelin against D‐galactosamine (GalN)/LPS induced FHF, particularly focusing on mitochondrial quality control and dynamics. Experimental Approach Mice were administered afzelin i.p. 1 h before receiving GalN (800 mg·kg−1)/LPS (40 μg·kg−1), and they were then killed 5 h after GalN/LPS treatment. Key Results Afzelin improved the survival rate and reduced the serum levels of alanine aminotransferase and pro‐inflammatory cytokines in GalN/LPS‐treated mice. Afzelin attenuated the mitochondrial damage, as indicated by diminished mitochondrial swelling and mitochondrial glutamate dehydrogenase activity in GalN/LPS‐treated mice. Afzelin enhanced mitochondrial biogenesis, as indicated by increased levels of PPAR‐γ coactivator 1α, nuclear respiratory factor 1 and mitochondrial transcription factor A. Afzelin also decreased the level of mitophagy‐related proteins, parkin and PTEN‐induced putative kinase 1. Furthermore, while GalN/LPS significantly increased the level of fission‐related protein, dynamin‐related protein 1, and decreased the level of fusion‐related protein, mitofusin 2; these effects were attenuated by afzelin. Conclusions and Implications Our findings demonstrated that afzelin protects against GalN/LPS‐induced liver injury by enhancing mitochondrial biogenesis, suppressing excessive mitophagy and balancing mitochondrial dynamics. PMID:27861739

Inhibition of stress-inducible HSP70 impairs mitochondrial proteostasis and function.

PubMed

Leu, Julia I-Ju; Barnoud, Thibaut; Zhang, Gao; Tian, Tian; Wei, Zhi; Herlyn, Meenhard; Murphy, Maureen E; George, Donna L

2017-07-11

Protein quality control is an important component of survival for all cells. The use of proteasome inhibitors for cancer therapy derives from the fact that tumor cells generally exhibit greater levels of proteotoxic stress than do normal cells, and thus cancer cells tend to be more sensitive to proteasome inhibition. However, this approach has been limited in some cases by toxicity to normal cells. Recently, the concept of inhibiting proteostasis in organelles for cancer therapy has been advanced, in part because it is predicted to have reduced toxicity for normal cells. Here we demonstrate that a fraction of the major stress-induced chaperone HSP70 (also called HSPA1A or HSP72, but hereafter HSP70) is abundantly present in mitochondria of tumor cells, but is expressed at quite low or undetectable levels in mitochondria of most normal tissues and non-tumor cell lines. We show that treatment of tumor cells with HSP70 inhibitors causes a marked change in mitochondrial protein quality control, loss of mitochondrial membrane potential, reduced oxygen consumption rate, and loss of ATP production. We identify several nuclear-encoded mitochondrial proteins, including polyadenylate binding protein-1 (PABPC1), which exhibit decreased abundance in mitochondria following treatment with HSP70 inhibitors. We also show that targeting HSP70 function leads to reduced levels of several mitochondrial-encoded RNA species that encode components of the electron transport chain. Our data indicate that small molecule inhibitors of HSP70 represent a new class of organelle proteostasis inhibitors that impair mitochondrial function in cancer cells, and therefore constitute novel therapeutics.

Apo-bacteriophytochromes modulate bacterial photosynthesis in response to low light.

PubMed

Fixen, Kathryn R; Baker, Anna W; Stojkovic, Emina A; Beatty, J Thomas; Harwood, Caroline S

2014-01-14

Bacteriophytochromes (BphPs) are light-sensing regulatory proteins encoded by photosynthetic and nonphotosynthetic bacteria. This protein class has been characterized structurally, but its biological activities remain relatively unexplored. Two BphPs in the anoxygenic photosynthetic bacterium Rhodopseudomonas palustris, designated regulatory proteins RpBphP2 and RpBphP3, are configured as light-regulated histidine kinases, which initiate a signal transduction system that controls expression of genes for the low light harvesting 4 (LH4) antenna complex. In vitro, RpBphP2 and RpBphP3 respond to light quality by reversible photoconversion, a property that requires the light-absorbing chromophore biliverdin. In vivo, RpBphP2 and RpBphP3 are both required for the expression of the LH4 antenna complex under anaerobic conditions, but biliverdin requires oxygen for its synthesis by heme oxygenase. On further investigation, we found that the apo-bacteriophytochrome forms of RpBphP2 and RpBphP3 are necessary and sufficient to control LH4 expression in response to light intensity in conjunction with other signal transduction proteins. One possibility is that the system senses a reduced quinone pool generated when light energy is absorbed by bacteriochlorophyll. The biliverdin-bound forms of the BphPs have the additional property of being able to fine-tune LH4 expression in response to light quality. These observations support the concept that some bacteriophytochromes can function with or without a chromophore and may be involved in regulating physiological processes not directly related to light sensing.

Evaluation of salivary oxidate stress biomarkers, nitric oxide and C-reactive protein in patients with oral lichen planus and burning mouth syndrome.

PubMed

Tvarijonaviciute, Asta; Aznar-Cayuela, Cristina; Rubio, Camila P; Ceron, José J; López-Jornet, Pia

2017-05-01

The aim of this study was to evaluate oxidative stress factors and C-reactive protein in the saliva of patients with oral lichen planus (OLP) and burning mouth syndrome (BMS). This consecutive, cross-sectional study included 20 patients with OLP, 19 with burning mouth syndrome (BMS), and 31 control subjects. The oral cavity of each patient was examined and patients responded to a quality of life questionnaire (OHIP-14) and the xerostomia inventory. The following parameters were measured in whole non-stimulated saliva: trolox equivalent antioxidant capacity (TEAC); total antioxidant capacity (TAC); cupric reducing antioxidant capacity (CUPRAC); ferric reducing ability of plasma (FRAP); C-reactive protein (CRP); nitric oxide; nitrates; and nitrites. The OLP group presented statistically significant differences in reactive oxygen species (ROS) (29 600 cps) in comparison with the control group (39 679 cps) (P < 0.05). In the BMS group, ROS was 29 707 cps with significant difference in comparison with the control group (P < 0.05). Significantly higher salivary nitric oxide (145.7 μmol) and nitrite (141.0 μmol) levels were found in OLP patients in comparison with control group (P < 0.05). Increases in nitric oxide and C-reactive protein were found in the saliva of OLP patients in comparison with BMS and control patients. Further studies are required to confirm these findings. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Functionality of gliadin proteins in wheat flour tortillas.

PubMed

Mondal, Suchismita; Hays, Dirk B; Alviola, Noviola J; Mason, Richard E; Tilley, Michael; Waniska, Ralph D; Bean, Scott R; Glover, Karl D

2009-02-25

Gliadins are monomeric proteins that are encoded by the genes at the loci Gli 1 and Gli 2 present on the short arm of homologous wheat chromosomes 1 and 6, respectively. Studies have suggested that gliadins may play an important role in determining the functional properties of wheat flour. The main objective of this study was to understand the functionality of gliadins with respect to tortilla quality. The important tortilla quality attributes are diameter, opacity, and shelf stability, designated here as rollability or the ability to roll or fold the tortilla without cracking. In this study gliadin functionality in tortilla quality was studied using near-isogenic wheat lines that have deletions in either Gli A1, Gli D1, Gli A2, or Gli D2 gliadin loci. The deletion lines are designated by the same abbreviations. Dough and tortillas were prepared from the parent line used to derive these deletion lines, each individual deletion line, and a control commercial tortilla flour. Quantitative and qualitative evaluations were performed on the dough and tortillas derived from the flour from each of these lines. None of the deletions in the gliadin loci altered the shelf stability versus that found for the parent to the deletion lines or control tortilla flour. However, deletions in the Gli 2 loci, in particular Gli A2 reduced the relative proportion of alpha- and beta-gliadins with a greater cysteine amino acid content and gluten cross-link function versus the chain-terminating omega-gliadins in Gli 1, which were still present. As such, the dough and gluten matrix appeared to have greater extensibility, which improved the diameter and overall quality of the tortillas while not altering the rollability. Deletions in the Gli 1 loci had the opposite result with increased cross-linking of alpha- and beta-gliadins, polymeric protein content, and a stronger dough that decreased the diameter and overall quality of the tortillas. The data suggest that altering certain Gli 2 loci through null alleles could be a viable strategy to develop cultivars improved for the specific functionality requirements needed for the rapidly growing tortilla market.

Quality-of-Life Outcomes following Thoracolumbar and Lumbar Fusion with and without the Use of Recombinant Human Bone Morphogenetic Protein-2: Does Recombinant Human Bone Morphogenetic Protein-2 Make a Difference?

PubMed Central

Lubelski, Daniel; Alvin, Matthew D.; Torre-Healy, Andrew; Abdullah, Kalil G.; Nowacki, Amy S.; Whitmore, Robert G.; Steinmetz, Michael P.; Benzel, Edward C.; Mroz, Thomas E.

2014-01-01

Design Retrospective study. Objectives (1) To investigate the quality-of-life (QOL) outcomes in the population undergoing lumbar spine surgery with versus without recombinant human bone morphogenetic protein-2 (rhBMP-2); (2) to determine QOL outcomes for those patients who experience postoperative complications; and (3) to identify the effect of patient characteristics on postoperative QOL outcomes. Methods A retrospective review of QOL questionnaires, including the Patient Health Questionnaire-9, Patient Disability Questionnaire (PDQ), EuroQol-5D (EQ-5D), and quality of life-year (QALY), was performed for all patients who underwent thoracolumbar and lumbar fusion surgery with versus without rhBMP-2 between March 2008 and September 2010. Individual preoperative and postoperative QOL data were compared for each patient. Demographic factors and complications were reviewed. Results We identified 266 patients, including 60 with and 206 without rhBMP-2. Questionnaires were completed an average of 10.3 ± 5 months after surgery. For all measures, average scores improved postoperatively compared with preoperatively. No differences in postoperative QOL outcomes were identified between the rhBMP-2 and the control cohorts. Median annual household income was positively associated with EQ-5D and QALY. Compared with those without, patients with postoperative complications had fewer QOL improvements. Conclusions There was no difference in QOL outcomes in the rhBMP-2 compared with the control group. Socioeconomic status and postoperative complications affected QOL outcomes following surgery. The QOL questionnaires provide the clinician with information regarding the patients' self-perceived well-being and can be helpful in the selection of surgical candidates and for understanding the effectiveness of a given surgical procedure. PMID:25396105

Gastrointestinal Symptoms of Food Challenge-proven Non-IgE Cow's Milk Allergy Are Dissipated by Early School Age.

PubMed

Helin, Noora; Kolho, Kaija-Leena; Qvist, Erik; Mäkelä, Mika; Merras-Salmio, Laura

2018-04-01

The aim of this study was to evaluate the current well-being and dietary restrictions in children 6 years after food challenge-confirmed diagnosis of non-IgE cow's milk protein allergy, compared to peers with gastrointestinal symptoms but negative food challenge. This study aimed to evaluate the diagnostic process retrospectively. This is an Internet-based survey for mothers whose children underwent 6 years ago the double-blind, placebo-controlled food challenge for cow's milk (CM) because of gastrointestinal symptoms causing suspicion of non-IgE CM protein allergy. Concurrent dietary restrictions, overall well-being, medical history, and retrospective views on the food challenge were queried using a study-specific questionnaire, the Quality of life using PedsQL general score and parental stress with the Parenting Stress Index questionnaire. Mothers of 42 children (23 girls), median age of 6.7 years (range 5.7-8.6), participated in the survey, the response rate was 70%. All children now consumed cow's milk protein. The only food restrictions reported were empirical lactose-free diets in 7 children (17%). One-third of the children in both groups were presently reported to have eating-related issues such as picky eating. Quality of life was good and present parenting stress was average in both groups. The majority of the mothers (87%) felt positive or neutral about the food challenge performed in infancy. The non-IgE CM allergy with gastrointestinal symptoms diagnosed in infancy was a transient condition with good outcome. At an early school age, nearly all children have a good quality of life and a regular diet. The use of the double-blind, placebo-controlled food challenge was well-endorsed.

AbMiner: a bioinformatic resource on available monoclonal antibodies and corresponding gene identifiers for genomic, proteomic, and immunologic studies.

PubMed

Major, Sylvia M; Nishizuka, Satoshi; Morita, Daisaku; Rowland, Rick; Sunshine, Margot; Shankavaram, Uma; Washburn, Frank; Asin, Daniel; Kouros-Mehr, Hosein; Kane, David; Weinstein, John N

2006-04-06

Monoclonal antibodies are used extensively throughout the biomedical sciences for detection of antigens, either in vitro or in vivo. We, for example, have used them for quantitation of proteins on "reverse-phase" protein lysate arrays. For those studies, we quality-controlled > 600 available monoclonal antibodies and also needed to develop precise information on the genes that encode their antigens. Translation among the various protein and gene identifier types proved non-trivial because of one-to-many and many-to-one relationships. To organize the antibody, protein, and gene information, we initially developed a relational database in Filemaker for our own use. When it became apparent that the information would be useful to many other researchers faced with the need to choose or characterize antibodies, we developed it further as AbMiner, a fully relational web-based database under MySQL, programmed in Java. AbMiner is a user-friendly, web-based relational database of information on > 600 commercially available antibodies that we validated by Western blot for protein microarray studies. It includes many types of information on the antibody, the immunogen, the vendor, the antigen, and the antigen's gene. Multiple gene and protein identifier types provide links to corresponding entries in a variety of other public databases, including resources for phosphorylation-specific antibodies. AbMiner also includes our quality-control data against a pool of 60 diverse cancer cell types (the NCI-60) and also protein expression levels for the NCI-60 cells measured using our high-density "reverse-phase" protein lysate microarrays for a selection of the listed antibodies. Some other available database resources give information on antibody specificity for one or a couple of cell types. In contrast, the data in AbMiner indicate specificity with respect to the antigens in a pool of 60 diverse cell types from nine different tissues of origin. AbMiner is a relational database that provides extensive information from our own laboratory and other sources on more than 600 available antibodies and the genes that encode the antibodies' antigens. The data will be made freely available at http://discover.nci.nih.gov/abminer.

Dietary protein quality and quantity affect lactational responses to corn distillers grains: a meta-analysis.

PubMed

Hollmann, M; Allen, M S; Beede, D K

2011-04-01

Diet fermentability influences lactational responses to feeding corn distillers grains (CDG) to dairy cows. However, some measures of diet fermentability are inherently related to the concentration and characteristics of corn-based ingredients in the ration. Corn-based feeds have poor protein quality, unable to meet the essential AA requirements of lactating cows. We conducted a meta-analysis of treatment means (n=44) from the scientific literature to evaluate responses in milk yield (MY) and milk true protein concentration and yield to dietary CDG. The test variable was the difference in response between the CDG diet mean and the control diet mean (0% CDG) within experiment. Fixed variables were CDG concentration of the diet [% of dietary dry matter (DM)] and crude protein (CP) concentration and fractions of CP based on origin (corn-based versus non-corn-based feeds) of control and CDG diets. Diets with CDG ranged from 4 to 42% CDG, DM basis. Non-corn-based dietary CP averaged 6.3±3.32% of total DM. Milk yield and milk true protein yield responses to added CDG were maximized when approximately 8.5% of the total dietary DM was non-corn-based CP. Milk yield response peaked for higher-producing cows (>30.0 kg MY/cow per day) at 4.3% dietary corn-based CP, but decreased linearly for lower-producing cows (<30.0 kg MY/cow per day) as corn-based dietary CP increased. Milk true protein yield response decreased as corn-based dietary CP concentration increased but milk true protein concentration response was not decreased when CDG diets had more than 6.5% dietary non-corn-based CP. Overall, 8.5% dietary non-corn-based CP was necessary in lactation diets to maximize lactational responses to dietary CDG. The necessity of dietary non-corn-based CP to maximize milk and milk protein yields limits the amount of dietary corn-based CP, including that from CDG, which can be included in rations without overfeeding N. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Protein Quantity and Quality of Safflower Seed Improved by NP Fertilizer and Rhizobacteria (Azospirillum and Azotobacter spp.)

PubMed Central

Nosheen, Asia; Bano, Asghari; Yasmin, Humaira; Keyani, Rumana; Habib, Rabia; Shah, Syed T. A.; Naz, Rabia

2016-01-01

HIGHLIGHTS Rhizobacteria (Azotobacter spp.) have improved the quality and quantity of safflower seed protein.Protein quality was confirmed by SDS-PAGE and new bands were found in response to different combinations of rhizobacteria and lower doses of fertilizers.The PGPR application has reduced the use of fertilizers upto 50%. Protein is an essential part of the human diet. The aim of this present study was to improve the protein quality of safflower seed by the application of plant growth promoting rhizobacteria (PGPR) in combination with conventional nitrogen and phosphate (NP) fertilizers. The seeds of two safflower cultivars Thori and Saif-32, were inoculated with Azospirillum and Azotobacter and grown under field conditions. Protein content and quality was assessed by crude protein, amino acid analysis, and SDS-PAGE. Seed crude protein and amino acids (methionine, phenylalanine, and glutamic acid) showed significant improvements (55–1250%) by Azotobacter supplemented with a quarter dose of fertilizers (BTQ) at P ≤ 0.05. Additional protein bands were induced in Thori and Saif-32 by BTQ and BTH (Azotobacter supplemented with a half dose of fertilizer) respectively. The Azospirillum in combination with half dose of fertilizer (SPH) and BTQ enhanced both indole acetic acid (IAA) (90%) and gibberellic acid (GA) (23–27%) content in safflower leaf. Taken together, these data suggest that Azospirillum and Azotobacter along with significantly reduced (up to 75%) use of NP fertilizers could improve the quality and quantity of safflower seed protein. PMID:26941744

Quantitative phosphoproteomic analysis of caprine muscle with high and low meat quality.

PubMed

Liu, Manshun; Wei, Yanchao; Li, Xin; Quek, Siew Young; Zhao, Jing; Zhong, Huazhen; Zhang, Dequan; Liu, Yongfeng

2018-07-01

During the conversion of muscle to meat, protein phosphorylation can regulate various biological processes that have important effects on meat quality. To investigate the phosphorylation pattern of protein on rigor mortis, goat longissimus thoracis and external intercostals were classified into two groups (high quality and low quality), and meat quality was evaluated according to meat quality attributes (Warner-Bratzler shear force, Color, pH and drip loss). A quantitative mass spectrometry-based phosphoproteomic study was conducted to analyze the caprine muscle at 12h postmortem applying the TiO 2 -SIMAC-HILIC (TiSH) phosphopeptide enrichment strategy. A total of 2125 phosphopeptides were identified from 750 phosphoproteins. Among them, 96 proteins had differed in phosphorylation levels. The majority of these proteins are involved in glucose metabolism and muscle contraction. The differential phosphorylation level of proteins (PFK, MYL2 and HSP27) in two groups may be the crucial factors of regulating muscle rigor mortis. This study provides a comprehensive view for the phosphorylation status of caprine muscle at rigor mortis, it also gives a better understanding of the regulation of protein phosphorylation on various biological processes that affect the final meat quality attributes. Copyright © 2018. Published by Elsevier Ltd.

Spice MyPlate: Nutrition Education Focusing Upon Spices and Herbs Improved Diet Quality and Attitudes Among Urban High School Students.

PubMed

D'Adamo, Christopher R; McArdle, Patrick F; Balick, Lyssa; Peisach, Erin; Ferguson, Tenaj; Diehl, Alica; Bustad, Kendall; Bowden, Brandin; Pierce, Beverly A; Berman, Brian M

2016-05-01

To determine whether an experiential nutrition education intervention focusing on spices and herbs ("Spice MyPlate") is feasible and improves diet quality and healthy eating attitudes among an urban and predominantly African-American sample of adolescents more than standard nutrition education alone. A nonrandomized controlled trial compared standard nutrition education in U.S. Department of Agriculture MyPlate guidelines (control group) with standard nutrition education plus adjuvant Spice MyPlate curriculum (intervention group). Data were collected at baseline and after 3, 6, and 10 weeks. Study setting was two public high schools in Baltimore, Maryland. A total of 110 students in grades 9 to 12 participated. The 6-week school-based intervention conducted during health class focused on cooking using spices and herbs to eat healthier diets according to MyPlate. Dietary intake reported on 3-day food records and healthy eating attitudes questionnaires was analyzed. Differences in diet quality and healthy eating attitudes between study groups were estimated by t-tests, Wilcoxon-Mann-Whitney tests, and covariate-adjusted regression models. Spice MyPlate was feasible and there were modest but significant improvements (p ≤ .05) in the Spice MyPlate group compared with control in whole grains (31.2 g/wk) and protein foods (13.2 ounces per week) intake, and attitudes toward eating vegetables, whole grains, lean protein, and low-fat dairy. Although randomized trials are needed, experiential nutrition education focusing on spices and herbs may help urban and predominantly African-American adolescent populations eat healthier diets. © The Author(s) 2016.

Identification of two key genes controlling chill haze stability of beer in barley (Hordeum vulgare L).

PubMed

Ye, Lingzhen; Huang, Yuqing; Dai, Fei; Ning, Huajiang; Li, Chengdao; Zhou, Meixue; Zhang, Guoping

2015-06-11

In bright beer, haze formation is a serious quality problem, degrading beer quality and reducing its shelf life. The quality of barley (Hordeum vulgare L) malt, as the main raw material for beer brewing, largely affects the colloidal stability of beer. In this study, the genetic mechanism of the factors affecting beer haze stability in barley was studied. Quantitative trait loci (QTL) analysis of alcohol chill haze (ACH) in beer was carried out using a Franklin/Yerong double haploid (DH) population. One QTL, named as qACH, was detected for ACH, and it was located on the position of about 108 cM in chromosome 4H and can explain about 20 % of the phenotypic variation. Two key haze active proteins, BATI-CMb and BATI-CMd were identified by proteomics analysis. Bioinformatics analysis showed that BATI-CMb and BATI-CMd had the same position as qACH in the chromosome. It may be deduced that BATI-CMb and BATI-CMd are candidate genes for qACH, controlling colloidal stability of beer. Polymorphism comparison between Yerong and Franklin in the nucleotide and amino acid sequence of BATI-CMb and BATI-CMd detected the corresponding gene specific markers, which could be used in marker-assisted selection for malt barley breeding. We identified a novel QTL, qACH controlling chill haze of beer, and two key haze active proteins, BATI-CMb and BATI-CMd. And further analysis showed that BATI-CMb and BATI-CMd might be the candidate genes associated with beer chill haze.

Redox proteomics analysis of HNE-modified proteins in Down syndrome brain: clues for understanding the development of Alzheimer disease.

PubMed

Di Domenico, Fabio; Pupo, Gilda; Tramutola, Antonella; Giorgi, Alessandra; Schininà, Maria Eugenia; Coccia, Raffaella; Head, Elizabeth; Butterfield, D Allan; Perluigi, Marzia

2014-06-01

Down syndrome (DS) is the most common genetic cause of intellectual disability, due to partial or complete triplication of chromosome 21. DS subjects are characterized by a number of abnormalities including premature aging and development of Alzheimer disease (AD) neuropathology after approximately 40 years of age. Several studies show that oxidative stress plays a crucial role in the development of neurodegeneration in the DS population. Increased lipid peroxidation is one of the main events causing redox imbalance within cells through the formation of toxic aldehydes that easily react with DNA, lipids, and proteins. In this study we used a redox proteomics approach to identify specific targets of 4-hydroxynonenal modifications in the frontal cortex from DS cases with and without AD pathology. We suggest that a group of identified proteins followed a specific pattern of oxidation in DS vs young controls, probably indicating characteristic features of the DS phenotype; a second group of identified proteins showed increased oxidation in DS/AD vs DS, thus possibly playing a role in the development of AD. The third group of comparison, DS/AD vs old controls, identified proteins that may be considered specific markers of AD pathology. All the identified proteins are involved in important biological functions including intracellular quality control systems, cytoskeleton network, energy metabolism, and antioxidant response. Our results demonstrate that oxidative damage is an early event in DS, as well as dysfunctions of protein-degradation systems and cellular protective pathways, suggesting that DS subjects are more vulnerable to oxidative damage accumulation that might contribute to AD development. Further, considering that the majority of proteins have been already demonstrated to be oxidized in AD brain, our results strongly support similarities with AD in DS. Copyright © 2014 Elsevier Inc. All rights reserved.

The relevance and use of mouse embryo bioassays for quality control in an assisted reproductive technology program.

PubMed

Scott, L F; Sundaram, S G; Smith, S

1993-09-01

To define both the limits of a mouse embryo bioassay for quality control in an assisted reproductive technology (ART) program and the areas where it can be effectively used. Embryos at the pronuclear and two-cell stage from three different strains of mice were used to assess the effectiveness of this assay for media quality control using five different media routinely used in ART. Pronuclear and two-cell embryos from CD-1 mice were used to test the ability of a mouse embryo bioassay to control for water quality, contaminants in the culture system, and fluctuations in the environmental conditions using a medium, culture system, and scoring technique that were optimized for this strain. The mouse embryo bioassay is not effective in differentiating media appropriate for supporting human embryo development since the development of mouse embryos in vitro is strain, stage, and media related. However, CD-1 embryos were shown to be sensitive to variations in water quality, pH, temperature, incubator conditions, and contaminants in the system when grown in a protein-free medium optimized for their development. Both total blastocyst number and the cell count in the blastocysts were affected. Pronuclear embryos were more sensitive to perturbations in the culture system than two-cell embryos. A mouse embryo bioassay can be effectively used as a means of quality control of water, chemicals, and contact materials and for technique standardization and training in an assisted reproduction program. All the conditions of the test should be defined, pronuclear embryos should be used, and the end point should be fully expanded blastocysts and/or cell numbers in these blastocysts where appropriate.

Mitochondrial anchorage and fusion contribute to mitochondrial inheritance and quality control in the budding yeast Saccharomyces cerevisiae

PubMed Central

Higuchi-Sanabria, Ryo; Charalel, Joseph K.; Viana, Matheus P.; Garcia, Enrique J.; Sing, Cierra N.; Koenigsberg, Andrea; Swayne, Theresa C.; Vevea, Jason D.; Boldogh, Istvan R.; Rafelski, Susanne M.; Pon, Liza A.

2016-01-01

Higher-functioning mitochondria that are more reduced and have less ROS are anchored in the yeast bud tip by the Dsl1-family protein Mmr1p. Here we report a role for mitochondrial fusion in bud-tip anchorage of mitochondria. Fluorescence loss in photobleaching (FLIP) and network analysis experiments revealed that mitochondria in large buds are a continuous reticulum that is physically distinct from mitochondria in mother cells. FLIP studies also showed that mitochondria that enter the bud can fuse with mitochondria that are anchored in the bud tip. In addition, loss of fusion and mitochondrial DNA (mtDNA) by deletion of mitochondrial outer or inner membrane fusion proteins (Fzo1p or Mgm1p) leads to decreased accumulation of mitochondria at the bud tip and inheritance of fitter mitochondria by buds compared with cells with no mtDNA. Conversely, increasing the accumulation and anchorage of mitochondria in the bud tip by overexpression of MMR1 results in inheritance of less-fit mitochondria by buds and decreased replicative lifespan and healthspan. Thus quantity and quality of mitochondrial inheritance are ensured by two opposing processes: bud-tip anchorage by mitochondrial fusion and Mmr1p, which favors bulk inheritance; and quality control mechanisms that promote segregation of fitter mitochondria to the bud. PMID:26764088

The effect of supplemental energy, nitrogen, and protein on feed intake, digestibility, and nitrogen flux across the gut and liver in sheep fed low-quality forage.

PubMed

Ferrell, C L; Kreikemeier, K K; Freetly, H C

1999-12-01

Our objective was to determine the impact of supplemental energy, N, and protein on feed intake and N metabolism in sheep fed low-quality forage. Six Texel x Dorset wethers (16 mo, 63+/-3.1 kg) fitted with mesenteric, portal, and hepatic venous catheters were used in a Latin square design with five sampling periods. Lambs were fed chopped bromegrass hay (4.3% CP) to appetite, and a mineral mixture was given. Treatments were 1) control (no supplement), 2) energy (cornstarch, molasses, and soybean oil), 3) energy plus urea, 4) energy plus soybean meal (SBM), and 5) energy plus ruminally undegraded protein (RUP; 50:50 mixture of blood and feather meals). Supplements were fed once daily (.3% BW). Forage DMI did not differ (P = .13), but intake of total DM, N, and energy differed (P<.01) among treatments. Apparent digestibilities of DM, OM, and energy were less (P<.01) for control than for other treatments. Apparent N digestibility was least for control and energy and greatest for urea treatments (P<.05). As a result, digested DM, OM, and energy ranked from least to greatest were control, energy, urea, SBM, and RUP, respectively. Apparently digested N was 2.44, 2.24, 11.39, 9.80, and 11.25 g/d for control, energy, urea, SBM, and RUP (P<.01; SE = .10). Hour of sampling x treatment was a significant source of variation for blood concentrations of ammonia N and urea N, net ammonia N release from portal-drained viscera (PDV) and liver, and urea N release from splanchnic tissues. These results were primarily because patterns through time for the urea treatment differed from the other treatments. Net PDV release of alpha-amino N did not differ (P>.05) between control and energy treatments. Values for those treatments were about one-half of values for urea, SBM, and RUP treatments, which did not differ (P>.05). Hepatic net uptake (negative release) of alpha-amino N for control was 53% of values for the other treatments, which did not differ (P>.05). Net release of alpha-amino N from splanchnic tissues did not differ among treatments (P = .34) and did not differ from zero. The data indicate that arterial alpha-amino N concentration, hepatic alpha-amino N uptake, PDV release and hepatic uptake of ammonia N, and hepatic release of urea N were greater in energy than in control treatments. We also found that hepatic uptake of alpha-amino N was 187% of PDV release in energy-supplemented lambs. These results suggest that energy supplementation of a protein-limiting diet stimulated mobilization of body protein.

Protein source and quality in therapeutic foods affect the immune response and outcome in severe acute malnutrition

USDA-ARS?s Scientific Manuscript database

Protein is a vital component of therapeutic foods designed to treat severe acute malnutrition (SAM) in children; however there are still unknowns about the quality and quantity of the proteins to use in these foods. This review examines two recent studies investigating several different qualities an...

VoroMQA: Assessment of protein structure quality using interatomic contact areas.

PubMed

Olechnovič, Kliment; Venclovas, Česlovas

2017-06-01

In the absence of experimentally determined protein structure many biological questions can be addressed using computational structural models. However, the utility of protein structural models depends on their quality. Therefore, the estimation of the quality of predicted structures is an important problem. One of the approaches to this problem is the use of knowledge-based statistical potentials. Such methods typically rely on the statistics of distances and angles of residue-residue or atom-atom interactions collected from experimentally determined structures. Here, we present VoroMQA (Voronoi tessellation-based Model Quality Assessment), a new method for the estimation of protein structure quality. Our method combines the idea of statistical potentials with the use of interatomic contact areas instead of distances. Contact areas, derived using Voronoi tessellation of protein structure, are used to describe and seamlessly integrate both explicit interactions between protein atoms and implicit interactions of protein atoms with solvent. VoroMQA produces scores at atomic, residue, and global levels, all in the fixed range from 0 to 1. The method was tested on the CASP data and compared to several other single-model quality assessment methods. VoroMQA showed strong performance in the recognition of the native structure and in the structural model selection tests, thus demonstrating the efficacy of interatomic contact areas in estimating protein structure quality. The software implementation of VoroMQA is freely available as a standalone application and as a web server at http://bioinformatics.lt/software/voromqa. Proteins 2017; 85:1131-1145. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Protein digestibility evaluations of meat and fish substrates using laboratory, avian and ileally cannulated dog assays

USDA-ARS?s Scientific Manuscript database

Fish and meat protein serves as important protein sources in the human and companion animal diets: however, limited information is available on differences in protein quality. Pollock fillet, and salmon fillet, beef loin, pork loin and chicken breast, were evaluated for protein quality and amino aci...

Differential proteome analysis during early somatic embryogenesis in Musa spp. AAA cv. Grand Naine.

PubMed

Kumaravel, Marimuthu; Uma, Subbaraya; Backiyarani, Suthanthiram; Saraswathi, Marimuthu Somasundaram; Vaganan, Muthu Mayil; Muthusamy, Muthusamy; Sajith, Kallu Purayil

2017-01-01

Endogenous hormone secretion proteins along with stress and defense proteins play predominant role in banana embryogenesis. This study reveals the underlying molecular mechanism during transition from vegetative to embryogenic state. Banana (Musa spp.) is well known globally as a food fruit crop for millions. The requirement of quality planting material of banana is enormous. Although mass multiplication through tissue culture is in vogue, high-throughput techniques like somatic embryogenesis (SE) as a mass multiplication tool needs to be improved. Apart from clonal propagation, SE has extensive applications in genetic improvement and mutation. SE in banana is completely genome-dependent and most of the commercial cultivars exhibit recalcitrance. Thus, understanding the molecular basis of embryogenesis in Musa will help to develop strategies for mass production of quality planting material. In this study, differentially expressed proteins between embryogenic calli (EC) and non-embryogenic calli (NEC) with respect to the explant, immature male flower buds (IMFB), of cv. Grand Naine (AAA) were determined using two-dimensional gel electrophoresis (2DE). The 2DE results were validated through qRT-PCR. In total, 65 proteins were identified: 42 were highly expressed and 23 were less expressed in EC compared to NEC and IMFB. qRT-PCR analysis of five candidate proteins, upregulated in EC, were well correlated with expression at transcript level. Further analysis of proteins showed that embryogenesis in banana is associated with the control of oxidative stress. The regulation of ROS scavenging system and protection of protein structure occurred in the presence of heat shock proteins. Alongside, high accumulation of stress-related cationic peroxidase and plant growth hormone-related proteins like indole-3-pyruvate monooxygenase and adenylate isopentenyltransferase in EC revealed the association with the induction of SE.

Proteomics in biomanufacturing control: Protein dynamics of CHO-K1 cells and conditioned media during apoptosis and necrosis.

PubMed

Albrecht, Simone; Kaisermayer, Christian; Gallagher, Clair; Farrell, Amy; Lindeberg, Anna; Bones, Jonathan

2018-06-01

Cell viability has a critical impact on product quantity and quality during the biomanufacturing of therapeutic proteins. An advanced understanding of changes in the cellular and conditioned media proteomes upon cell stress and death is therefore needed for improved bioprocess control. Here, a high pH/low pH reversed phase data independent 2D-LC-MS E discovery proteomics platform was applied to study the cellular and conditioned media proteomes of CHO-K1 apoptosis and necrosis models where cell death was induced by staurosporine exposure or aeration shear in a benchtop bioreactor, respectively. Functional classification of gene ontology terms related to molecular functions, biological processes, and cellular components revealed both cell death independent and specific features. In addition, label free quantitation using the Hi3 approach resulted in a comprehensive shortlist of 23 potential cell viability marker proteins with highest abundance and a significant increase in the conditioned media upon induction of cell death, including proteins related to cellular stress response, signal mediation, cytoskeletal organization, cell differentiation, cell interaction as well as metabolic and proteolytic enzymes which are interesting candidates for translating into targeted analysis platforms for monitoring bioprocessing response and increasing process control. © 2018 Wiley Periodicals, Inc.

Strategies for analysing and improving the expression and quality of recombinant proteins made in mammalian cells.

PubMed

Jenkins, Nigel; Meleady, Paula; Tyther, Raymond; Murphy, Lisa

2009-05-06

The production of monoclonal antibodies and other recombinant proteins is one of the highest growth areas in the pharmaceutical industry. Mammalian cells are used to manufacture the majority of biotherapeutics, largely due to their ability to perform complex post-translational modifications. Although significant progress has been made recently in improving product yields and protein quality, many challenges still lie ahead to achieve consistently high yields while avoiding potentially damaging protein modifications. The present review first considers the strategies used to analyse and improve recombinant protein expression of industrial cell lines, with an emphasis on proteomic technologies. Next, cellular and environmental influences on protein production and quality are examined, and strategies for improvements in product yield and quality are reviewed. The analytical techniques required to detect these protein changes are also described, together with prospects for assay improvements.

Exploring Anti-Prion Glyco-Based and Aromatic Scaffolds: A Chemical Strategy for the Quality of Life.

PubMed

Blázquez-Sánchez, María Teresa; de Matos, Ana M; Rauter, Amélia P

2017-05-24

Prion diseases are fatal neurodegenerative disorders caused by protein misfolding and aggregation, affecting the brain progressively and consequently the quality of life. Alzheimer's is also a protein misfolding disease, causing dementia in over 40 million people worldwide. There are no therapeutics able to cure these diseases. Cellular prion protein is a high-affinity binding partner of amyloid β (Aβ) oligomers, the most toxic species in Alzheimer's pathology. These findings motivate the development of new chemicals for a better understanding of the events involved. Disease control is far from being reached by the presently known therapeutics. In this review we describe the synthesis and mode of action of molecular entities with intervention in prion diseases' biological processes and, if known, their role in Alzheimer's. A diversity of structures is covered, based on glycans, steroids and terpenes, heterocycles, polyphenols, most of them embodying aromatics and a structural complexity. These molecules may be regarded as chemical tools to foster the understanding of the complex mechanisms involved, and to encourage the scientific community towards further developments for the cure of these devastating diseases.

Free fatty acid particles in protein formulations, part 2: contribution of polysorbate raw material.

PubMed

Siska, Christine C; Pierini, Christopher J; Lau, Hollis R; Latypov, Ramil F; Fesinmeyer, R Matthew; Litowski, Jennifer R

2015-02-01

Polysorbate 20 (PS20) is a nonionic surfactant frequently used to stabilize protein biopharmaceuticals. During the development of mAb formulations containing PS20, small clouds of particles were observed in solutions stored in vials. The degree of particle formation was dependent on PS20 concentration. The particles were characterized by reversed-phase HPLC after dissolution and labeling with the fluorescent dye 1-pyrenyldiazomethane. The analysis showed that the particles consisted of free fatty acids (FFAs), with the distribution of types consistent with those found in the PS20 raw material. Protein solutions formulated with polysorbate 80, a chemically similar nonionic surfactant, showed a substantial delay in particle formation over time compared with PS20. Multiple lots of polysorbates were evaluated for FFA levels, each exhibiting differences based on polysorbate type and lot. Polysorbates purchased in more recent years show a greater distribution and quantity of FFA and also a greater propensity to form particles. This work shows that the quality control of polysorbate raw materials could play an important role in biopharmaceutical product quality. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Clp Protease and OR Directly Control the Proteostasis of Phytoene Synthase, the Crucial Enzyme for Carotenoid Biosynthesis in Arabidopsis.

PubMed

Welsch, Ralf; Zhou, Xiangjun; Yuan, Hui; Álvarez, Daniel; Sun, Tianhu; Schlossarek, Dennis; Yang, Yong; Shen, Guoxin; Zhang, Hong; Rodriguez-Concepcion, Manuel; Thannhauser, Theodore W; Li, Li

2018-01-08

Phytoene synthase (PSY) is the crucial plastidial enzyme in the carotenoid biosynthetic pathway. However, its post-translational regulation remains elusive. Likewise, Clp protease constitutes a central part of the plastid protease network, but its substrates for degradation are not well known. In this study, we report that PSY is a substrate of the Clp protease. PSY was uncovered to physically interact with various Clp protease subunits (i.e., ClpS1, ClpC1, and ClpD). High levels of PSY and several other carotenogenic enzyme proteins overaccumulate in the clpc1, clpp4, and clpr1-2 mutants. The overaccumulated PSY was found to be partially enzymatically active. Impairment of Clp activity in clpc1 results in a reduced rate of PSY protein turnover, further supporting the role of Clp protease in degrading PSY protein. On the other hand, the ORANGE (OR) protein, a major post-translational regulator of PSY with holdase chaperone activity, enhances PSY protein stability and increases the enzymatically active proportion of PSY in clpc1, counterbalancing Clp-mediated proteolysis in maintaining PSY protein homeostasis. Collectively, these findings provide novel insights into the quality control of plastid-localized proteins and establish a hitherto unidentified post-translational regulatory mechanism of carotenogenic enzymes in modulating carotenoid biosynthesis in plants. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

Uterine fluid proteins and egg quality characteristics for 2 commercial and 2 heritage laying hen lines in response to manipulation of dietary calcium and vitamin D3.

PubMed

Kaur, Ravinder; Rathgeber, Bruce M; Thompson, Kristen L; Macisaac, Janice

2013-09-01

The aim of this study was to evaluate the quality of eggs from 2 selected commercial strains of laying hens and 2 unselected lines of chickens fed diets with different combinations of Ca and vitamin D and relate it to the profile of uterine proteins and ultrastructure of the shell. A group of 4 chickens was housed in each of 24 cages. The group consisted of one representative from each of the following breeds: Lohmann LSL- Lite, Lohmann Classic-Brown, Fayoumi, and Light Sussex. Six dietary combinations of Ca and vitamin D(3) (3.35%, 2,500 IU; 4.10%, 2,500 IU (control); 4.85%, 2,500 IU; 3.35%, 200 IU; 4.10%, 200 IU; and 4.85%, 200 IU) were randomly assigned to 4 replicate cages for 2 treatment periods (26-29 and 56-59 wk of age). Data were analyzed as a split-plot design with cage as the main plot and hen as the subplot. Egg quality traits were different (P < 0.0001) between commercial and heritage breeds. Lohmann Brown had stronger shells with higher specific gravity compared with other breeds. Both commercial and heritage birds responded to a drop in vitamin D3 level by marked reduction in shell thickness. The SDS-PAGE profiles of uterine fluid samples revealed a decrease (P < 0.05) in 200-, 150-, 116-, and ≤6.5-kDa proteins, whereas proteins with molecular weight (MW) of 80, 55, 52, 45, 42, and 28 kDa increased with bird age. A 36- and 52-kDa protein band was most intense for Fayoumi compared with other breeds. Ultrastructural characteristics showed flattened and deeply etched mammillary caps for Lohmann Brown and the presence of type A and type B bodies between mammillary cones in eggshells from Fayoumi and Lohmann Lite. The negative correlation between ultrastructural characteristics, which decrease with bird age, and the 116-kDa uterine protein band could provide insight into reduced eggshell quality as hens age.

Scrambled eggs: Proteomic portraits and novel biomarkers of egg quality in zebrafish (Danio rerio)

PubMed Central

Yilmaz, Ozlem; Patinote, Amélie; Nguyen, Thao Vi; Com, Emmanuelle; Lavigne, Regis; Pineau, Charles; Sullivan, Craig V.; Bobe, Julien

2017-01-01

Egg quality is a complex biological trait and a major determinant of reproductive fitness in all animals. This study delivered the first proteomic portraits of egg quality in zebrafish, a leading biomedical model for early development. Egg batches of good and poor quality, evidenced by embryo survival for 24 h, were sampled immediately after spawning and used to create pooled or replicated sample sets whose protein extracts were subjected to different levels of fractionation before liquid chromatography and tandem mass spectrometry. Obtained spectra were searched against a zebrafish proteome database and detected proteins were annotated, categorized and quantified based on normalized spectral counts. Manually curated and automated enrichment analyses revealed poor quality eggs to be deficient of proteins involved in protein synthesis and energy and lipid metabolism, and of some vitellogenin products and lectins, and to have a surfeit of proteins involved in endo-lysosomal activities, autophagy, and apoptosis, and of some oncogene products, lectins and egg envelope proteins. Results of pathway and network analyses suggest that this aberrant proteomic profile results from failure of oocytes giving rise to poor quality eggs to properly transit through final maturation, and implicated Wnt signaling in the etiology of this defect. Quantitative comparisons of abundant proteins in good versus poor quality eggs revealed 17 candidate egg quality markers. Thus, the zebrafish egg proteome is clearly linked to embryo developmental potential, a phenomenon that begs further investigation to elucidate the root causes of poor egg quality, presently a serious and intractable problem in livestock and human reproductive medicine. PMID:29145436

Scrambled eggs: Proteomic portraits and novel biomarkers of egg quality in zebrafish (Danio rerio).

PubMed

Yilmaz, Ozlem; Patinote, Amélie; Nguyen, Thao Vi; Com, Emmanuelle; Lavigne, Regis; Pineau, Charles; Sullivan, Craig V; Bobe, Julien

2017-01-01

Egg quality is a complex biological trait and a major determinant of reproductive fitness in all animals. This study delivered the first proteomic portraits of egg quality in zebrafish, a leading biomedical model for early development. Egg batches of good and poor quality, evidenced by embryo survival for 24 h, were sampled immediately after spawning and used to create pooled or replicated sample sets whose protein extracts were subjected to different levels of fractionation before liquid chromatography and tandem mass spectrometry. Obtained spectra were searched against a zebrafish proteome database and detected proteins were annotated, categorized and quantified based on normalized spectral counts. Manually curated and automated enrichment analyses revealed poor quality eggs to be deficient of proteins involved in protein synthesis and energy and lipid metabolism, and of some vitellogenin products and lectins, and to have a surfeit of proteins involved in endo-lysosomal activities, autophagy, and apoptosis, and of some oncogene products, lectins and egg envelope proteins. Results of pathway and network analyses suggest that this aberrant proteomic profile results from failure of oocytes giving rise to poor quality eggs to properly transit through final maturation, and implicated Wnt signaling in the etiology of this defect. Quantitative comparisons of abundant proteins in good versus poor quality eggs revealed 17 candidate egg quality markers. Thus, the zebrafish egg proteome is clearly linked to embryo developmental potential, a phenomenon that begs further investigation to elucidate the root causes of poor egg quality, presently a serious and intractable problem in livestock and human reproductive medicine.

ProTSAV: A protein tertiary structure analysis and validation server.

PubMed

Singh, Ankita; Kaushik, Rahul; Mishra, Avinash; Shanker, Asheesh; Jayaram, B

2016-01-01

Quality assessment of predicted model structures of proteins is as important as the protein tertiary structure prediction. A highly efficient quality assessment of predicted model structures directs further research on function. Here we present a new server ProTSAV, capable of evaluating predicted model structures based on some popular online servers and standalone tools. ProTSAV furnishes the user with a single quality score in case of individual protein structure along with a graphical representation and ranking in case of multiple protein structure assessment. The server is validated on ~64,446 protein structures including experimental structures from RCSB and predicted model structures for CASP targets and from public decoy sets. ProTSAV succeeds in predicting quality of protein structures with a specificity of 100% and a sensitivity of 98% on experimentally solved structures and achieves a specificity of 88%and a sensitivity of 91% on predicted protein structures of CASP11 targets under 2Å.The server overcomes the limitations of any single server/method and is seen to be robust in helping in quality assessment. ProTSAV is freely available at http://www.scfbio-iitd.res.in/software/proteomics/protsav.jsp. Copyright © 2015 Elsevier B.V. All rights reserved.

Adverse effect versus quality control of the Fuenzalida-Palacios antirabies vaccine.

PubMed

Nogueira, Y L

1998-01-01

We evaluated the components of the Fuenzalida-Palacios antirabies vaccine, which is till used in most developing countries in human immunization for treatment and prophylaxis. This vaccine is prepared from newborn mouse brains at 1% concentration. Even though the vaccine is considered to have a low myelin content, it is not fully free of myelin or of other undesirable components that might trigger adverse effects after vaccination. The most severe effect is a post-vaccination neuroparalytic accident associated with Guillain-Barré syndrome. In the present study we demonstrate how the vaccines produced and distributed by different laboratories show different component patterns with different degrees of impurity and with varying protein concentrations, indicating that production processes can vary from one laboratory to another. These differences, which could be resolved using a better quality control process, may affect and impair immunization, with consequent risks and adverse effects after vaccination. We used crossed immunoelectrophoresis to evaluate and demonstrate the possibility of quality control in vaccine production, reducing the risk factors possibly involved in these immunizing products.

Effect of additives in the shelflife extension of chilled and frozen stored Indian octopus (Cistopus indicus).

PubMed

Manimaran, Uthaman; Shakila, Robinson Jeya; Shalini, Rajendran; Sivaraman, Balasubramanian; Sumathi, Ganesan; Selvaganapathi, Rajendran; Jeyasekaran, Geevarathnam

2016-02-01

In this study, the effect of commercial additives viz. cafodos and altesa employed to treat Indian octopus (Cistopus indicus) was examined during chilled and frozen storage. Shelf lives of treated and untreated octopus in ice were 6 and 8 days, respectively in ice. Treated and untreated frozen octopus had a shelf life of 40 days. Autolytic and microbiological changes were not controlled by the additives, as evidenced through rapid reduction in non-protein nitrogen (NPN) and α-amino nitrogen (α-AN) compounds; as well as accumulation of water soluble ammoniacal nitrogen and total volatile base- nitrogen (TVB-N) compounds. Loss of texture and colour were the major quality defects noticed in treated octopus as a result of enhanced protein solubility. Therefore, the additives approved for use in octopus neither enhanced the shelf life nor improved the sensory quality.

Dietary Protected Feed Supplement to Increase Milk Production and Quality of Dairy Cows

NASA Astrophysics Data System (ADS)

Pramono, A.; Handayanta, E.; Widayati, D. T.; Putro, P. P.; Kustono

2017-04-01

The efforts to improve and optimize productivity of dairy cows require sufficient availability of nutrients, especially high energy in the early period of lactation. Increasing energy intake in dairy cows can be conducted by increasing the density of energy. The research aimed to evaluate dietary protected feed supplement on milk production and quality, including: fat, protein, and lactose content of Friesian Holstein dairy cow milk. Protected feed supplement was produced from sardine fish oil, through saponification and microencapsulation protection methods. The experiment consists of two treatments i.e. P0: basal diet (control) and P1: basal diet + 3 % protected feed supplement. Each treatment was repeated 15 times. Data were analyzed by independent samples t-test analysis. Results showed that supplementation of protected sardine fish oil had no effect on lactose content, but increased milk yield production (p<0.01), milk fat content (p<0.05), and protein content (p<0.05).

Sperm nuclear protamines: A checkpoint to control sperm chromatin quality.

PubMed

Steger, Klaus; Balhorn, Rod

2018-05-23

Protamines are nuclear proteins which are specifically expressed in haploid male germ cells. Their replacement of histones and binding to DNA is followed by chromatin hypercondensation that protects DNA from negative influences by environmental factors. Mammalian sperm contain two types of protamines: PRM1 and PRM2. While the proportion of the two protamines is highly variable between different species, abnormal ratios within a species are known to be associated with male subfertility. Therefore, it is more than likely that correct protamine expression represents a kind of chromatin checkpoint during sperm development rendering protamines as suitable biomarkers for the estimation of sperm quality. This review presents an overview of our current knowledge on protamines comparing gene and protein structures between different mammalian species with particular consideration given to man, mouse and stallion. At last, recent insights into the possible role of inherited sperm histones for early embryo development are provided. © 2018 Blackwell Verlag GmbH.

The BEACH-containing protein WDR81 coordinates p62 and LC3C to promote aggrephagy.

PubMed

Liu, Xuezhao; Li, Yang; Wang, Xin; Xing, Ruxiao; Liu, Kai; Gan, Qiwen; Tang, Changyong; Gao, Zhiyang; Jian, Youli; Luo, Shouqing; Guo, Weixiang; Yang, Chonglin

2017-05-01

Autophagy-dependent clearance of ubiquitinated and aggregated proteins is critical to protein quality control, but the underlying mechanisms are not well understood. Here, we report the essential role of the BEACH (beige and Chediak-Higashi) and WD40 repeat-containing protein WDR81 in eliminating ubiquitinated proteins through autophagy. WDR81 associates with ubiquitin (Ub)-positive protein foci, and its loss causes accumulation of Ub proteins and the autophagy cargo receptor p62. WDR81 interacts with p62, facilitating recognition of Ub proteins by p62. Furthermore, WDR81 interacts with LC3C through canonical LC3-interacting regions in the BEACH domain, promoting LC3C recruitment to ubiquitinated proteins. Inactivation of LC3C or defective autophagy results in accumulation of Ub protein aggregates enriched for WDR81. In mice, WDR81 inactivation causes accumulation of p62 bodies in cortical and striatal neurons in the brain. These data suggest that WDR81 coordinates p62 and LC3C to facilitate autophagic removal of Ub proteins, and provide important insights into CAMRQ2 syndrome, a WDR81-related developmental disorder. © 2017 Liu et al.

Protein Information Resource: a community resource for expert annotation of protein data

PubMed Central

Barker, Winona C.; Garavelli, John S.; Hou, Zhenglin; Huang, Hongzhan; Ledley, Robert S.; McGarvey, Peter B.; Mewes, Hans-Werner; Orcutt, Bruce C.; Pfeiffer, Friedhelm; Tsugita, Akira; Vinayaka, C. R.; Xiao, Chunlin; Yeh, Lai-Su L.; Wu, Cathy

2001-01-01

The Protein Information Resource, in collaboration with the Munich Information Center for Protein Sequences (MIPS) and the Japan International Protein Information Database (JIPID), produces the most comprehensive and expertly annotated protein sequence database in the public domain, the PIR-International Protein Sequence Database. To provide timely and high quality annotation and promote database interoperability, the PIR-International employs rule-based and classification-driven procedures based on controlled vocabulary and standard nomenclature and includes status tags to distinguish experimentally determined from predicted protein features. The database contains about 200 000 non-redundant protein sequences, which are classified into families and superfamilies and their domains and motifs identified. Entries are extensively cross-referenced to other sequence, classification, genome, structure and activity databases. The PIR web site features search engines that use sequence similarity and database annotation to facilitate the analysis and functional identification of proteins. The PIR-Inter­national databases and search tools are accessible on the PIR web site at http://pir.georgetown.edu/ and at the MIPS web site at http://www.mips.biochem.mpg.de. The PIR-International Protein Sequence Database and other files are also available by FTP. PMID:11125041

The BEACH-containing protein WDR81 coordinates p62 and LC3C to promote aggrephagy

PubMed Central

Xing, Ruxiao; Tang, Changyong; Gao, Zhiyang

2017-01-01

Autophagy-dependent clearance of ubiquitinated and aggregated proteins is critical to protein quality control, but the underlying mechanisms are not well understood. Here, we report the essential role of the BEACH (beige and Chediak–Higashi) and WD40 repeat-containing protein WDR81 in eliminating ubiquitinated proteins through autophagy. WDR81 associates with ubiquitin (Ub)-positive protein foci, and its loss causes accumulation of Ub proteins and the autophagy cargo receptor p62. WDR81 interacts with p62, facilitating recognition of Ub proteins by p62. Furthermore, WDR81 interacts with LC3C through canonical LC3-interacting regions in the BEACH domain, promoting LC3C recruitment to ubiquitinated proteins. Inactivation of LC3C or defective autophagy results in accumulation of Ub protein aggregates enriched for WDR81. In mice, WDR81 inactivation causes accumulation of p62 bodies in cortical and striatal neurons in the brain. These data suggest that WDR81 coordinates p62 and LC3C to facilitate autophagic removal of Ub proteins, and provide important insights into CAMRQ2 syndrome, a WDR81-related developmental disorder. PMID:28404643

Optimisation of near-infrared reflectance model in measuring protein and amylose content of rice flour.

PubMed

Xie, L H; Tang, S Q; Chen, N; Luo, J; Jiao, G A; Shao, G N; Wei, X J; Hu, P S

2014-01-01

Near-infrared reflectance spectroscopy (NIRS) has been used to predict the cooking quality parameters of rice, such as the protein (PC) and amylose content (AC). Using brown and milled flours from 519 rice samples representing a wide range of grain qualities, this study was to compare the calibration models generated by different mathematical, preprocessing treatments, and combinations of different regression algorithm. A modified partial least squares model (MPLS) with the mathematic treatment "2, 8, 8, 2" (2nd order derivative computed based on 8 data points, and 8 and 2 data points in the 1st and 2nd smoothing, respectively) and inverse multiplicative scattering correction preprocessing treatment was identified as the best model for simultaneously measurement of PC and AC in brown flours. MPLS/"2, 8, 8, 2"/detrend preprocessing was identified as the best model for milled flours. The results indicated that NIRS could be useful in estimation of PC and AC of breeding lines in early generations of the breeding programs, and for the purposes of quality control in the food industry. Copyright © 2013 Elsevier Ltd. All rights reserved.

Fluorescence-based bioassays for the detection and evaluation of food materials.

PubMed

Nishi, Kentaro; Isobe, Shin-Ichiro; Zhu, Yun; Kiyama, Ryoiti

2015-10-13

We summarize here the recent progress in fluorescence-based bioassays for the detection and evaluation of food materials by focusing on fluorescent dyes used in bioassays and applications of these assays for food safety, quality and efficacy. Fluorescent dyes have been used in various bioassays, such as biosensing, cell assay, energy transfer-based assay, probing, protein/immunological assay and microarray/biochip assay. Among the arrays used in microarray/biochip assay, fluorescence-based microarrays/biochips, such as antibody/protein microarrays, bead/suspension arrays, capillary/sensor arrays, DNA microarrays/polymerase chain reaction (PCR)-based arrays, glycan/lectin arrays, immunoassay/enzyme-linked immunosorbent assay (ELISA)-based arrays, microfluidic chips and tissue arrays, have been developed and used for the assessment of allergy/poisoning/toxicity, contamination and efficacy/mechanism, and quality control/safety. DNA microarray assays have been used widely for food safety and quality as well as searches for active components. DNA microarray-based gene expression profiling may be useful for such purposes due to its advantages in the evaluation of pathway-based intracellular signaling in response to food materials.

Fluorescence-Based Bioassays for the Detection and Evaluation of Food Materials

PubMed Central

Nishi, Kentaro; Isobe, Shin-Ichiro; Zhu, Yun; Kiyama, Ryoiti

2015-01-01

We summarize here the recent progress in fluorescence-based bioassays for the detection and evaluation of food materials by focusing on fluorescent dyes used in bioassays and applications of these assays for food safety, quality and efficacy. Fluorescent dyes have been used in various bioassays, such as biosensing, cell assay, energy transfer-based assay, probing, protein/immunological assay and microarray/biochip assay. Among the arrays used in microarray/biochip assay, fluorescence-based microarrays/biochips, such as antibody/protein microarrays, bead/suspension arrays, capillary/sensor arrays, DNA microarrays/polymerase chain reaction (PCR)-based arrays, glycan/lectin arrays, immunoassay/enzyme-linked immunosorbent assay (ELISA)-based arrays, microfluidic chips and tissue arrays, have been developed and used for the assessment of allergy/poisoning/toxicity, contamination and efficacy/mechanism, and quality control/safety. DNA microarray assays have been used widely for food safety and quality as well as searches for active components. DNA microarray-based gene expression profiling may be useful for such purposes due to its advantages in the evaluation of pathway-based intracellular signaling in response to food materials. PMID:26473869

Assessment of infant formula quality and composition using Vis-NIR, MIR and Raman process analytical technologies.

PubMed

Wang, Xiao; Esquerre, Carlos; Downey, Gerard; Henihan, Lisa; O'Callaghan, Donal; O'Donnell, Colm

2018-06-01

In this study, visible and near-infrared (Vis-NIR), mid-infrared (MIR) and Raman process analytical technologies were investigated for assessment of infant formula quality and compositional parameters namely preheat temperature, storage temperature, storage time, fluorescence of advanced Maillard products and soluble tryptophan (FAST) index, soluble protein, fat and surface free fat (SFF) content. PLS-DA models developed using spectral data with appropriate data pre-treatment and significant variables selected using Martens' uncertainty test had good accuracy for the discrimination of preheat temperature (92.3-100%) and storage temperature (91.7-100%). The best PLS regression models developed yielded values for the ratio of prediction error to deviation (RPD) of 3.6-6.1, 2.1-2.7, 1.7-2.9, 1.6-2.6 and 2.5-3.0 for storage time, FAST index, soluble protein, fat and SFF content prediction respectively. Vis-NIR, MIR and Raman were demonstrated to be potential PAT tools for process control and quality assurance applications in infant formula and dairy ingredient manufacture. Copyright © 2018 Elsevier B.V. All rights reserved.

Effect of drying process assisted by high-pressure impregnation on protein quality and digestibility in red abalone (Haliotis rufescens).

PubMed

Cepero-Betancourt, Yamira; Oliva-Moresco, Patricio; Pasten-Contreras, Alexis; Tabilo-Munizaga, Gipsy; Pérez-Won, Mario; Moreno-Osorio, Luis; Lemus-Mondaca, Roberto

2017-10-01

Abalone (Haliotis spp.) is an exotic seafood product recognized as a protein source of high biological value. Traditional methods used to preserve foods such as drying technology can affect their nutritional quality (protein quality and digestibility). A 28-day rat feeding study was conducted to evaluate the effects of the drying process assisted by high-pressure impregnation (HPI) (350, 450, and 500 MPa × 5 min) on chemical proximate and amino acid compositions and nutritional parameters, such as protein efficiency ratio (PER), true digestibility (TD), net protein ratio, and protein digestibility corrected amino acid score (PDCAAS) of dried abalone. The HPI-assisted drying process ensured excellent protein quality based on PER values, regardless of the pressure level. At 350 and 500 MPa, the HPI-assisted drying process had no negative effect on TD and PDCAAS then, based on nutritional parameters analysed, we recommend HPI-assisted drying process at 350 MPa × 5 min as the best process condition to dry abalone. Variations in nutritional parameters compared to casein protein were observed; nevertheless, the high protein quality and digestibility of HPI-assisted dried abalones were maintained to satisfy the metabolic demands of human beings.

Multiplexed MRM-based assays for the quantitation of proteins in mouse plasma and heart tissue.

PubMed

Percy, Andrew J; Michaud, Sarah A; Jardim, Armando; Sinclair, Nicholas J; Zhang, Suping; Mohammed, Yassene; Palmer, Andrea L; Hardie, Darryl B; Yang, Juncong; LeBlanc, Andre M; Borchers, Christoph H

2017-04-01

The mouse is the most commonly used laboratory animal, with more than 14 million mice being used for research each year in North America alone. The number and diversity of mouse models is increasing rapidly through genetic engineering strategies, but detailed characterization of these models is still challenging because most phenotypic information is derived from time-consuming histological and biochemical analyses. To expand the biochemists' toolkit, we generated a set of targeted proteomic assays for mouse plasma and heart tissue, utilizing bottom-up LC/MRM-MS with isotope-labeled peptides as internal standards. Protein quantitation was performed using reverse standard curves, with LC-MS platform and curve performance evaluated by quality control standards. The assays comprising the final panel (101 peptides for 81 proteins in plasma; 227 peptides for 159 proteins in heart tissue) have been rigorously developed under a fit-for-purpose approach and utilize stable-isotope labeled peptides for every analyte to provide high-quality, precise relative quantitation. In addition, the peptides have been tested to be interference-free and the assay is highly multiplexed, with reproducibly determined protein concentrations spanning >4 orders of magnitude. The developed assays have been used in a small pilot study to demonstrate their application to molecular phenotyping or biomarker discovery/verification studies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Valorization of Proteins from Co- and By-Products from the Fish and Meat Industry.

PubMed

Aspevik, Tone; Oterhals, Åge; Rønning, Sissel Beate; Altintzoglou, Themistoklis; Wubshet, Sileshi Gizachew; Gildberg, Asbjørn; Afseth, Nils Kristian; Whitaker, Ragnhild Dragøy; Lindberg, Diana

2017-06-01

Large volumes of protein-rich residual raw materials, such as heads, bones, carcasses, blood, skin, viscera, hooves and feathers, are created as a result of processing of animals from fisheries, aquaculture, livestock and poultry sectors. These residuals contain proteins and other essential nutrients with potentially bioactive properties, eligible for recycling and upgrading for higher-value products, e.g. for human, pet food and feed purposes. Here, we aim to cover all the important aspects of achieving optimal utilization of proteins in such residual raw materials, identifying those eligible for human consumption as co-products and for feed applications as by-products. Strict legislation regulates the utilization of various animal-based co- and by-products, representing a major hurdle if not addressed properly. Thorough understanding and optimization of all parts of the production chain, including conservation and processing, are important prerequisites for successful upgrading and industrial implementation of such products. This review includes industrially applied technologies such as freezing/cooling, acid preservation, salting, rendering and protein hydrolysis. In this regard, it is important to achieve stable production and quality through all the steps in the manufacturing chain, preferably supported by at- or online quality control points in the actual processing step. If aiming for the human market, knowledge of consumer trends and awareness are important for production and successful introduction of new products and ingredients.

Heat Shock Proteins: A Review of the Molecular Chaperones for Plant Immunity.

PubMed

Park, Chang-Jin; Seo, Young-Su

2015-12-01

As sessile organisms, plants are exposed to persistently changing stresses and have to be able to interpret and respond to them. The stresses, drought, salinity, chemicals, cold and hot temperatures, and various pathogen attacks have interconnected effects on plants, resulting in the disruption of protein homeostasis. Maintenance of proteins in their functional native conformations and preventing aggregation of non-native proteins are important for cell survival under stress. Heat shock proteins (HSPs) functioning as molecular chaperones are the key components responsible for protein folding, assembly, translocation, and degradation under stress conditions and in many normal cellular processes. Plants respond to pathogen invasion using two different innate immune responses mediated by pattern recognition receptors (PRRs) or resistance (R) proteins. HSPs play an indispensable role as molecular chaperones in the quality control of plasma membrane-resident PRRs and intracellular R proteins against potential invaders. Here, we specifically discuss the functional involvement of cytosolic and endoplasmic reticulum (ER) HSPs/chaperones in plant immunity to obtain an integrated understanding of the immune responses in plant cells.

C-Terminal End-Directed Protein Elimination by CRL2 Ubiquitin Ligases.

PubMed

Lin, Hsiu-Chuan; Yeh, Chi-Wei; Chen, Yen-Fu; Lee, Ting-Ting; Hsieh, Pei-Yun; Rusnac, Domnita V; Lin, Sung-Ya; Elledge, Stephen J; Zheng, Ning; Yen, Hsueh-Chi S

2018-05-17

The proteolysis-assisted protein quality control system guards the proteome from potentially detrimental aberrant proteins. How miscellaneous defective proteins are specifically eliminated and which molecular characteristics direct them for removal are fundamental questions. We reveal a mechanism, DesCEND (destruction via C-end degrons), by which CRL2 ubiquitin ligase uses interchangeable substrate receptors to recognize the unusual C termini of abnormal proteins (i.e., C-end degrons). C-end degrons are mostly less than ten residues in length and comprise a few indispensable residues along with some rather degenerate ones. The C-terminal end position is essential for C-end degron function. Truncated selenoproteins generated by translation errors and the USP1 N-terminal fragment from post-translational cleavage are eliminated by DesCEND. DesCEND also targets full-length proteins with naturally occurring C-end degrons. The C-end degron in DesCEND echoes the N-end degron in the N-end rule pathway, highlighting the dominance of protein "ends" as indicators for protein elimination. Copyright © 2018 Elsevier Inc. All rights reserved.

Altered Escherichia coli membrane protein assembly machinery allows proper membrane assembly of eukaryotic protein vitamin K epoxide reductase.

PubMed

Hatahet, Feras; Blazyk, Jessica L; Martineau, Eugenie; Mandela, Eric; Zhao, Yongxin; Campbell, Robert E; Beckwith, Jonathan; Boyd, Dana

2015-12-08

Functional overexpression of polytopic membrane proteins, particularly when in a foreign host, is often a challenging task. Factors that negatively affect such processes are poorly understood. Using the mammalian membrane protein vitamin K epoxide reductase (VKORc1) as a reporter, we describe a genetic selection approach allowing the isolation of Escherichia coli mutants capable of functionally expressing this blood-coagulation enzyme. The isolated mutants map to components of membrane protein assembly and quality control proteins YidC and HslV. We show that changes in the VKORc1 sequence and in the YidC hydrophilic groove along with the inactivation of HslV promote VKORc1 activity and dramatically increase its expression level. We hypothesize that such changes correct for mismatches in the membrane topogenic signals between E. coli and eukaryotic cells guiding proper membrane integration. Furthermore, the obtained mutants allow the study of VKORc1 reaction mechanisms, inhibition by warfarin, and the high-throughput screening for potential anticoagulants.

Altered Escherichia coli membrane protein assembly machinery allows proper membrane assembly of eukaryotic protein vitamin K epoxide reductase

PubMed Central

Hatahet, Feras; Blazyk, Jessica L.; Martineau, Eugenie; Mandela, Eric; Zhao, Yongxin; Campbell, Robert E.; Beckwith, Jonathan; Boyd, Dana

2015-01-01

Functional overexpression of polytopic membrane proteins, particularly when in a foreign host, is often a challenging task. Factors that negatively affect such processes are poorly understood. Using the mammalian membrane protein vitamin K epoxide reductase (VKORc1) as a reporter, we describe a genetic selection approach allowing the isolation of Escherichia coli mutants capable of functionally expressing this blood-coagulation enzyme. The isolated mutants map to components of membrane protein assembly and quality control proteins YidC and HslV. We show that changes in the VKORc1 sequence and in the YidC hydrophilic groove along with the inactivation of HslV promote VKORc1 activity and dramatically increase its expression level. We hypothesize that such changes correct for mismatches in the membrane topogenic signals between E. coli and eukaryotic cells guiding proper membrane integration. Furthermore, the obtained mutants allow the study of VKORc1 reaction mechanisms, inhibition by warfarin, and the high-throughput screening for potential anticoagulants. PMID:26598701

A compact disk-like centrifugal microfluidic system for high-throughput nanoliter-scale protein crystallization screening.

PubMed

Li, Gang; Chen, Qiang; Li, Junjun; Hu, Xiaojian; Zhao, Jianlong

2010-06-01

A centrifuge-based microfluidic system has been developed that enables automated high-throughput and low-volume protein crystallizations. In this system, protein solution was automatically and accurately metered and dispensed into nanoliter-sized multiple reaction chambers, and it was mixed with various types of precipitants using a combination of capillary effect and centrifugal force. It has the advantages of simple fabrication, easy operation, and extremely low waste. To demonstrate the feasibility of this system, we constructed a chip containing 24 units and used it to perform lysozyme and cyan fluorescent protein (CyPet) crystallization trials. The results demonstrate that high-quality crystals can be grown and harvested from such a nanoliter-volume microfluidic system. Compared to other microfluidic technologies for protein crystallization, this microfluidic system allows zero waste, simple structure and convenient operation, which suggests that our microfluidic disk can be applied not only to protein crystallization, but also to the miniaturization of various biochemical reactions requiring precise nanoscale control.

Interplay of PDZ and protease domain of DegP ensures efficient elimination of misfolded proteins

PubMed Central

Krojer, Tobias; Pangerl, Karen; Kurt, Juliane; Sawa, Justyna; Stingl, Christoph; Mechtler, Karl; Huber, Robert; Ehrmann, Michael; Clausen, Tim

2008-01-01

Aberrant proteins represent an extreme hazard to cells. Therefore, molecular chaperones and proteases have to carry out protein quality control in each cellular compartment. In contrast to the ATP-dependent cytosolic proteases and chaperones, the molecular mechanisms of extracytosolic factors are largely unknown. To address this question, we studied the protease function of DegP, the central housekeeping protein in the bacterial envelope. Our data reveal that DegP processively degrades misfolded proteins into peptides of defined size by employing a molecular ruler comprised of the PDZ1 domain and the proteolytic site. Furthermore, peptide binding to the PDZ domain transforms the resting protease into its active state. This allosteric activation mechanism ensures the regulated and rapid elimination of misfolded proteins upon folding stress. In comparison to the cytosolic proteases, the regulatory features of DegP are established by entirely different mechanisms reflecting the convergent evolution of an extracytosolic housekeeping protease. PMID:18505836

Characterization of Proteins in Filtrate from Biodegradation of Crop Residue

NASA Technical Reports Server (NTRS)

Horton, Wileatha; Trotman, A. A.

1997-01-01

Biodegradation of plant biomass is a feasible path for transformation of crop residue and recycling of nutrients for crop growth. The need to model the effects of factors associated with recycling of plant biomass resulting from hydroponic sweet potato production has led to investigation of natural soil isolates with the capacity for starch hydrolysis. This study sought to use nondenaturing gel electrophoresis to characterize the proteins present in filtered effluent from bioreactors seeded with starch hydrolyzing bacterial culture used in the biodegradation of senesced sweet potato biomass. The study determined the relative molecular weight of proteins in sampled effluent and the protein banding pattern was characterized. The protein profiles of effluent were similar for samples taken from independent runs under similar conditions of starch hydrolysis. The method can be used as a quality control tool for confirmation of starch hydrolysis of crop biomass. In addition, this method will allow monitoring for presence of contaminants within the system-protein profiles indicative of new enzymes in the bioreactors.

Hsp42 is required for sequestration of protein aggregates into deposition sites in Saccharomyces cerevisiae

PubMed Central

Specht, Sebastian; Miller, Stephanie B.M.

2011-01-01

The aggregation of proteins inside cells is an organized process with cytoprotective function. In Saccharomyces cerevisiae, aggregating proteins are spatially sequestered to either juxtanuclear or peripheral sites, which target distinct quality control pathways for refolding and degradation. The cellular machinery driving the sequestration of misfolded proteins to these sites is unknown. In this paper, we show that one of the two small heat shock proteins of yeast, Hsp42, is essential for the formation of peripheral aggregates during physiological heat stress. Hsp42 preferentially localizes to peripheral aggregates but is largely absent from juxtanuclear aggregates, which still form in hsp42Δ cells. Transferring the amino-terminal domain of Hsp42 to Hsp26, which does not participate in aggregate sorting, enables Hsp26 to replace Hsp42 function. Our data suggest that Hsp42 acts via its amino-terminal domain to coaggregate with misfolded proteins and perhaps link such complexes to further sorting factors. PMID:22065637

Selective Targeting of Proteins within Secretory Pathway for Endoplasmic Reticulum-associated Degradation

PubMed Central

Vecchi, Lara; Petris, Gianluca; Bestagno, Marco; Burrone, Oscar R.

2012-01-01

The endoplasmic reticulum-associated degradation (ERAD) is a cellular quality control mechanism to dispose of misfolded proteins of the secretory pathway via proteasomal degradation. SEL1L is an ER-resident protein that participates in identification of misfolded molecules as ERAD substrates, therefore inducing their ER-to-cytosol retrotranslocation and degradation. We have developed a novel class of fusion proteins, termed degradins, composed of a fragment of SEL1L fused to a target-specific binding moiety located on the luminal side of the ER. The target-binding moiety can be a ligand of the target or derived from specific mAbs. Here, we describe the ability of degradins with two different recognition moieties to promote degradation of a model target. Degradins recognize the target protein within the ER both in secretory and membrane-bound forms, inducing their degradation following retrotranslocation to the cytosol. Thus, degradins represent an effective technique to knock-out proteins within the secretory pathway with high specificity. PMID:22523070

Antenatal dietary education and supplementation to increase energy and protein intake.

PubMed

Ota, Erika; Hori, Hiroyuki; Mori, Rintaro; Tobe-Gai, Ruoyan; Farrar, Diane

2015-06-02

Gestational weight gain is positively associated with fetal growth, and observational studies of food supplementation in pregnancy have reported increases in gestational weight gain and fetal growth. To assess the effects of education during pregnancy to increase energy and protein intake, or of actual energy and protein supplementation, on energy and protein intake, and the effect on maternal and infant health outcomes. We searched the Cochrane Pregnancy and Childbirth Group's Trials Register (31 January 2015), reference lists of retrieved studies and contacted researchers in the field. Randomised controlled trials of dietary education to increase energy and protein intake, or of actual energy and protein supplementation, during pregnancy. Two review authors independently assessed trials for inclusion and assessed risk of bias. Two review authors independently extracted data and checked for accuracy. Extracted data were supplemented by additional information from the trialists we contacted. We examined 149 reports corresponding to 65 trials. Of these trials, 17 were included, 46 were excluded, and two are ongoing. Overall, 17 trials involving 9030 women were included. For this update, we assessed methodological quality of the included trials using the standard Cochrane criteria (risk of bias) and the GRADE approach. The overall risk of bias was unclear. Nutritional education (five trials, 1090 women) Women given nutritional education had a lower relative risk of having a preterm birth (two trials, 449 women) (risk ratio (RR) 0.46, 95% CI 0.21 to 0.98, low-quality evidence), and low birthweight (one trial, 300 women) (RR 0.04, 95% CI 0.01 to 0.14). Head circumference at birth was increased in one trial (389 women) (mean difference (MD) 0.99 cm, 95% CI 0.43 to 1.55), while birthweight was significantly increased among undernourished women in two trials (320 women) (MD 489.76 g, 95% CI 427.93 to 551.59, low-quality evidence), but did not significantly increase for adequately nourished women (MD 15.00, 95% CI -76.30 to 106.30, one trial, 406 women). Protein intake increased significantly (three trials, 632 women) (protein intake: MD +6.99 g/day, 95% CI 3.02 to 10.97). No significant differences were observed on any other outcomes such as neonatal death (RR 1.28, 95% CI 0.35 to 4.72, one trial, 448 women, low-quality evidence), stillbirth (RR 0.37, 95% CI 0.07 to 1.90, one trial, 431 women, low-quality evidence), small-for-gestational age (RR 0.97, 95% CI 0.45 to 2.11, one trial, 404 women, low-quality evidence) and total gestational weight gain (MD -0.41, 95% CI -4.41 to 3.59, two trials, 233 women). There were no data on perinatal death. Balanced energy and protein supplementation (12 trials, 6705 women)Risk of stillbirth was significantly reduced for women given balanced energy and protein supplementation (RR 0.60, 95% CI 0.39 to 0.94, five trials, 3408 women, moderate-quality evidence), and the mean birthweight was significantly increased (random-effects MD +40.96 g, 95% CI 4.66 to 77.26, Tau² = 1744, I² = 44%, 11 trials, 5385 women, moderate-quality evidence). There was also a significant reduction in the risk of small-for-gestational age (RR 0.79, 95% CI 0.69 to 0.90, I² = 16%, seven trials, 4408 women, moderate-quality evidence). No significant effect was detected for preterm birth (RR 0.96, 95% CI 0.80 to 1.16, five trials, 3384 women, moderate-quality evidence) or neonatal death (RR 0.68, 95% CI 0.43 to 1.07, five trials, 3381 women, low-quality evidence). Weekly gestational weight gain was not significantly increased (MD 18.63, 95% CI -1.81 to 39.07, nine trials, 2391 women, very low quality evidence). There were no data reported on perinatal death and low birthweight. High-protein supplementation (one trial, 1051 women)High-protein supplementation (one trial, 505 women), was associated with a significantly increased risk of small-for-gestational age babies (RR 1.58, 95% CI 1.03 to 2.41, moderate-quality evidence). There was no significant effect for stillbirth (RR 0.81, 95% CI 0.31 to 2.15, one trial, 529 women), neonatal death (RR 2.78, 95% CI 0.75 to 10.36, one trial, 529 women), preterm birth (RR 1.14, 95% CI 0.83 to 1.56, one trial, 505 women), birthweight (MD -73.00, 95% CI -171.26 to 25.26, one trial, 504 women) and weekly gestational weight gain (MD 4.50, 95% CI -33.55 to 42.55, one trial, 486 women, low-quality evidence). No data were reported on perinatal death. Isocaloric protein supplementation (two trials, 184 women)Isocaloric protein supplementation (two trials, 184 women) had no significant effect on birthweight (MD 108.25, 95% CI -220.89 to 437.40) and weekly gestational weight gain (MD 110.45, 95% CI -82.87 to 303.76, very low-quality evidence). No data reported on perinatal mortality, stillbirth, neonatal death, small-for-gestational age, and preterm birth. This review provides encouraging evidence that antenatal nutritional education with the aim of increasing energy and protein intake in the general obstetric population appears to be effective in reducing the risk of preterm birth, low birthweight, increasing head circumference at birth, increasing birthweight among undernourished women, and increasing protein intake. There was no evidence of benefit or adverse effect for any other outcome reported.Balanced energy and protein supplementation seems to improve fetal growth, and may reduce the risk of stillbirth and infants born small-for-gestational age. High-protein supplementation does not seem to be beneficial and may be harmful to the fetus. Balanced-protein supplementation alone had no significant effects on perinatal outcomes.The results of this review should be interpreted with caution. The risk of bias was either unclear or high for at least one category examined in several of the included trials, and the quality of the evidence was low for several important outcomes. Also, as the anthropometric characteristics of the general obstetric population is changing, those developing interventions aimed at altering energy and protein intake should ensure that only those women likely to benefit are included. Large, well-designed randomised trials are needed to assess the effects of increasing energy and protein intake during pregnancy in women whose intake is below recommended levels.

Tailoring recombinant protein quality by rational media design.

PubMed

Brühlmann, David; Jordan, Martin; Hemberger, Jürgen; Sauer, Markus; Stettler, Matthieu; Broly, Hervé

2015-01-01

Clinical efficacy and safety of recombinant proteins are closely associated with their structural characteristics. The major quality attributes comprise glycosylation, charge variants (oxidation, deamidation, and C- & N-terminal modifications), aggregates, low-molecular-weight species (LMW), and misincorporation of amino acids in the protein backbone. Cell culture media design has a great potential to modulate these quality attributes due to the vital role of medium in mammalian cell culture. The purpose of this review is to provide an overview of the way both classical cell culture medium components and novel supplements affect the quality attributes of recombinant therapeutic proteins expressed in mammalian hosts, allowing rational and high-throughput optimization of mammalian cell culture media. A selection of specific and/or potent inhibitors and activators of oligosaccharide processing as well as components affecting multiple quality attributes are presented. Extensive research efforts in this field show the feasibility of quality engineering through media design, allowing to significantly modulate the protein function. © 2015 American Institute of Chemical Engineers.

Potential of the Lectin/Inhibitor Isolated from Crataeva tapia Bark (CrataBL) for Controlling Callosobruchus maculatus Larva Development.

PubMed

Nunes, Natalia N S; Ferreira, Rodrigo S; Silva-Lucca, Rosemeire A; de Sá, Leonardo F R; de Oliveira, Antônia Elenir A; Correia, Maria Tereza dos S; Paiva, Patrícia Maria G; Wlodawer, Alexander; Oliva, Maria Luiza V

2015-12-09

Callosobruchus maculatus is an important predator of cowpeas. Due to infestation during storage, this insect affects the quality of seed and crop yield. This study aimed to investigate the effects of CrataBL, a multifunction protein isolated from Crataeva tapia bark, on C. maculatus larva development. The protein, which is stable even in extreme pH conditions, showed toxic activity, reducing the larval mass 45 and 70% at concentrations of 0.25 and 1.0% (w/w), respectively. Acting as an inhibitor, CrataBL decreased by 39% the activity of cysteine proteinases from larval gut. Conversely, the activity of serine proteinases was increased about 8-fold. The toxic properties of CrataBL may also be attributed to its capacity of binding to glycoproteins or glycosaminoglycans. Such binding interferes with larval metabolism, because CrataBL-FITC was found in the fat body, Malpighian tubules, and feces of larvae. These results demonstrate the potential of this protein for controlling larva development.

Functional Genomics of Allergen Gene Families in Fruits

PubMed Central

Maghuly, Fatemeh; Marzban, Gorji; Laimer, Margit

2009-01-01

Fruit consumption is encouraged for health reasons; however, fruits may harbour a series of allergenic proteins that may cause discomfort or even represent serious threats to certain individuals. Thus, the identification and characterization of allergens in fruits requires novel approaches involving genomic and proteomic tools. Since avoidance of fruits also negatively affects the quality of patients’ lives, biotechnological interventions are ongoing to produce low allergenic fruits by down regulating specific genes. In this respect, the control of proteins associated with allergenicity could be achieved by fine tuning the spatial and temporal expression of the relevant genes. PMID:22253972

Can Solution Supersaturation Affect Protein Crystal Quality?

NASA Technical Reports Server (NTRS)

Gorti, Sridhar

2013-01-01

The formation of large protein crystals of "high quality" is considered a characteristic manifestation of microgravity. The physical processes that predict the formation of large, high quality protein crystals in the microgravity environment of space are considered rooted in the existence of a "depletion zone" in the vicinity of crystal. Namely, it is considered reasonable that crystal quality suffers in earth-grown crystals as a result of the incorporation of large aggregates, micro-crystals and/or large molecular weight "impurities", processes which are aided by density driven convective flow or mixing at the crystal-liquid interface. Sedimentation and density driven convection produce unfavorable solution conditions in the vicinity of the crystal surface, which promotes rapid crystal growth to the detriment of crystal size and quality. In this effort, we shall further present the hypothesis that the solution supersaturatoin at the crystal surface determines the growth mechanism, or mode, by which protein crystals grow. It is further hypothesized that protein crystal quality is affected by the mechanism or mode of crystal growth. Hence the formation of a depletion zone in microgravity environment is beneficial due to inhibition of impurity incorporatoin as well as preventing a kinetic roughening transition. It should be noted that for many proteins the magnitude of neither protein crystal growth rates nor solution supersaturation are predictors of a kinetic roughening transition. That is, the kinetic roughening transition supersaturation must be dtermined for each individual protein.

A step towards long-wavelength protein crystallography: subjecting protein crystals to a vacuum

PubMed Central

Panjikar, Santosh; Thomsen, Lars; O’Donnell, Kane Michael; Riboldi-Tunnicliffe, Alan

2015-01-01

Using the UHV experimental endstation on the soft X-ray beamline at the Australian Synchrotron, lysozyme and proteinase K crystals have been exposed to a vacuum of 10−5 mbar, prior to flash-cooling in a bath of liquid nitrogen. Subsequent data collection on the MX2 beamline at the Australian Synchrotron demonstrated that, for lysozyme and proteinase K, it is possible to subject these mounted crystals to a vacuum pressure of 10−5 mbar without destroying the crystal lattice. Despite the lower data quality of the vacuum-pumped crystals compared with control crystals, it is demonstrated that the protein crystals can survive in a vacuum under suitable conditions. PMID:26089765

Analysis of glycoprotein processing in the endoplasmic reticulum using synthetic oligosaccharides.

PubMed

Ito, Yukishige; Takeda, Yoichi

2012-01-01

Protein quality control (QC) in the endoplasmic reticulum (ER) comprises many steps, including folding and transport of nascent proteins as well as degradation of misfolded proteins. Recent studies have revealed that high-mannose-type glycans play a pivotal role in the QC process. To gain knowledge about the molecular basis of this process with well-defined homogeneous compounds, we achieved a convergent synthesis of high-mannose-type glycans and their functionalized derivatives. We focused on analyses of UDP-Glc: glycoprotein glucosyltransferase (UGGT) and ER Glucosidase II, which play crucial roles in glycoprotein QC; however, their specificities remain unclear. In addition, we established an in vitro assay system mimicking the in vivo condition which is highly crowded because of the presence of various biomacromolecules.

Application of the quality by design approach to the drug substance manufacturing process of an Fc fusion protein: towards a global multi-step design space.

PubMed

Eon-duval, Alex; Valax, Pascal; Solacroup, Thomas; Broly, Hervé; Gleixner, Ralf; Strat, Claire L E; Sutter, James

2012-10-01

The article describes how Quality by Design principles can be applied to the drug substance manufacturing process of an Fc fusion protein. First, the quality attributes of the product were evaluated for their potential impact on safety and efficacy using risk management tools. Similarly, process parameters that have a potential impact on critical quality attributes (CQAs) were also identified through a risk assessment. Critical process parameters were then evaluated for their impact on CQAs, individually and in interaction with each other, using multivariate design of experiment techniques during the process characterisation phase. The global multi-step Design Space, defining operational limits for the entire drug substance manufacturing process so as to ensure that the drug substance quality targets are met, was devised using predictive statistical models developed during the characterisation study. The validity of the global multi-step Design Space was then confirmed by performing the entire process, from cell bank thawing to final drug substance, at its limits during the robustness study: the quality of the final drug substance produced under different conditions was verified against predefined targets. An adaptive strategy was devised whereby the Design Space can be adjusted to the quality of the input material to ensure reliable drug substance quality. Finally, all the data obtained during the process described above, together with data generated during additional validation studies as well as manufacturing data, were used to define the control strategy for the drug substance manufacturing process using a risk assessment methodology. Copyright © 2012 Wiley-Liss, Inc.

Strategies for the structural analysis of multi-protein complexes: lessons from the 3D-Repertoire project.

PubMed

Collinet, B; Friberg, A; Brooks, M A; van den Elzen, T; Henriot, V; Dziembowski, A; Graille, M; Durand, D; Leulliot, N; Saint André, C; Lazar, N; Sattler, M; Séraphin, B; van Tilbeurgh, H

2011-08-01

Structural studies of multi-protein complexes, whether by X-ray diffraction, scattering, NMR spectroscopy or electron microscopy, require stringent quality control of the component samples. The inability to produce 'keystone' subunits in a soluble and correctly folded form is a serious impediment to the reconstitution of the complexes. Co-expression of the components offers a valuable alternative to the expression of single proteins as a route to obtain sufficient amounts of the sample of interest. Even in cases where milligram-scale quantities of purified complex of interest become available, there is still no guarantee that good quality crystals can be obtained. At this step, protein engineering of one or more components of the complex is frequently required to improve solubility, yield or the ability to crystallize the sample. Subsequent characterization of these constructs may be performed by solution techniques such as Small Angle X-ray Scattering and Nuclear Magnetic Resonance to identify 'well behaved' complexes. Herein, we recount our experiences gained at protein production and complex assembly during the European 3D Repertoire project (3DR). The goal of this consortium was to obtain structural information on multi-protein complexes from yeast by combining crystallography, electron microscopy, NMR and in silico modeling methods. We present here representative set case studies of complexes that were produced and analyzed within the 3DR project. Our experience provides useful insight into strategies that are more generally applicable for structural analysis of protein complexes. Copyright © 2011 Elsevier Inc. All rights reserved.

In vitro bioassay for reactive toxicity towards proteins implemented for water quality monitoring.

PubMed

Tang, Janet Y M; Glenn, Eva; Thoen, Hanne; Escher, Beate I

2012-03-01

Reactive organic chemicals comprise a large number of compounds with a variety of reactive moieties. While most assays for reactive toxicity focus on DNA damage, reactivity towards proteins can also lead to irreparable damage, but reactivity towards proteins is typically not included in any test battery for water quality assessment. Glutathione (GSH) is a small tripeptide whose cysteine moiety can serve as a model for nucleophilic sites on proteins. GSH is also an important indicator of detoxification processes and the redox status of cells and due to its protective role, depletion of GSH ultimately leads to adverse effects. A bioassay based on genetically modified Escherichia coli strains was used to quantify the specific reactivity towards the protein-like biological nucelophile GSH. The significance of GSH for detoxification was assessed by comparing the growth inhibition induced by reference chemicals or water samples in a GSH-deficient strain to its fully functional parent strain. The GSH deficient strain showed the same sensitivity as the GSH proficient strain to non-reactive and DNA damaging chemicals, but was more sensitive to chemicals that attack cysteine in proteins. The difference in effect concentrations for 50% inhibition of growth assessed as biomass increase (EC(50)) between the two strains indicates the relevance of GSH conjugation as a detoxification step as well as direct reactivity with cysteine-containing proteins. Seven reference compounds serving as positive and negative controls were investigated. The E. coli strain that lacks GSH was four times more sensitive towards the positive control Sea-Nine, while negative controls benzo[a]pyrene, 2-aminoanthracene, phenol, t-butylhydroquinone, methyl methane sulfonate and 4-nitroquinoline oxide showed equal effect concentrations in both strains. Water samples collected across an indirect potable reuse scheme representing the complete water cycle from sewage to drinking water in South East Queensland, Australia were used to evaluate the applicability of the E. coli assay for reactive toxicity in water samples. While the EC(50) values of the GSH+ strain showed similar trends as in other biological endpoints over the various treatment chains, the specific response indicative of protein damage was only observed in samples that had undergone chlorination as a disinfection process. High natural organic matter or other matrix components disturbed the bioassay so much that we recommend it for future routine testing only in tertiary treated water or drinking water. This journal is © The Royal Society of Chemistry 2012

Effects of progressive resistance training combined with a protein-enriched lean red meat diet on health-related quality of life in elderly women: secondary analysis of a 4-month cluster randomised controlled trial.

PubMed

Torres, Susan J; Robinson, Sian; Orellana, Liliana; O'Connell, Stella L; Grimes, Carley A; Mundell, Niamh L; Dunstan, David W; Nowson, Caryl A; Daly, Robin M

2017-06-01

Resistance training (RT) and increased dietary protein are recommended to attenuate age-related muscle loss in the elderly. This study examined the effect of a lean red meat protein-enriched diet combined with progressive resistance training (RT+Meat) on health-related quality of life (HR-QoL) in elderly women. In this 4-month cluster randomised controlled trial, 100 women aged 60-90 years (mean 73 years) from self-care retirement villages participated in RT twice a week and were allocated either 160 g/d (cooked) lean red meat consumed across 2 meals/d, 6 d/week or ≥1 serving/d (25-30 g) carbohydrates (control group, CRT). HR-QoL (SF-36 Health Survey questionnaire), lower limb maximum muscle strength and lean tissue mass (LTM) (dual-energy X-ray absorptiometry) were assessed at baseline and 4 months. In all, ninety-one women (91 %) completed the study (RT+Meat (n 48); CRT (n 43)). Mean protein intake was greater in RT+Meat than CRT throughout the study (1·3 (sd 0·3) v. 1·1 (sd 0·3) g/kg per d, P<0·05). Exercise compliance (74 %) was not different between groups. After 4 months there was a significant net benefit in the RT+Meat compared with CRT group for overall HR-QoL and the physical component summary (PCS) score (P<0·01), but there were no changes in either group in the mental component summary (MCS) score. Changes in lower limb muscle strength, but not LTM, were positively associated with changes in overall HR-QoL (muscle strength, β: 2·2 (95 % CI 0·1, 4·3), P<0·05). In conclusion, a combination of RT and increased dietary protein led to greater net benefits in overall HR-QoL in elderly women compared with RT alone, which was because of greater improvements in PCS rather than MCS.

Massive integration of diverse protein quality assessment methods to improve template based modeling in CASP11.

PubMed

Cao, Renzhi; Bhattacharya, Debswapna; Adhikari, Badri; Li, Jilong; Cheng, Jianlin

2016-09-01

Model evaluation and selection is an important step and a big challenge in template-based protein structure prediction. Individual model quality assessment methods designed for recognizing some specific properties of protein structures often fail to consistently select good models from a model pool because of their limitations. Therefore, combining multiple complimentary quality assessment methods is useful for improving model ranking and consequently tertiary structure prediction. Here, we report the performance and analysis of our human tertiary structure predictor (MULTICOM) based on the massive integration of 14 diverse complementary quality assessment methods that was successfully benchmarked in the 11th Critical Assessment of Techniques of Protein Structure prediction (CASP11). The predictions of MULTICOM for 39 template-based domains were rigorously assessed by six scoring metrics covering global topology of Cα trace, local all-atom fitness, side chain quality, and physical reasonableness of the model. The results show that the massive integration of complementary, diverse single-model and multi-model quality assessment methods can effectively leverage the strength of single-model methods in distinguishing quality variation among similar good models and the advantage of multi-model quality assessment methods of identifying reasonable average-quality models. The overall excellent performance of the MULTICOM predictor demonstrates that integrating a large number of model quality assessment methods in conjunction with model clustering is a useful approach to improve the accuracy, diversity, and consequently robustness of template-based protein structure prediction. Proteins 2016; 84(Suppl 1):247-259. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

The Stanford Automated Mounter: Enabling High-Throughput Protein Crystal Screening at SSRL

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

Smith, C.A.; Cohen, A.E.

2009-05-26

The macromolecular crystallography experiment lends itself perfectly to high-throughput technologies. The initial steps including the expression, purification, and crystallization of protein crystals, along with some of the later steps involving data processing and structure determination have all been automated to the point where some of the last remaining bottlenecks in the process have been crystal mounting, crystal screening, and data collection. At the Stanford Synchrotron Radiation Laboratory, a National User Facility that provides extremely brilliant X-ray photon beams for use in materials science, environmental science, and structural biology research, the incorporation of advanced robotics has enabled crystals to be screenedmore » in a true high-throughput fashion, thus dramatically accelerating the final steps. Up to 288 frozen crystals can be mounted by the beamline robot (the Stanford Auto-Mounting System) and screened for diffraction quality in a matter of hours without intervention. The best quality crystals can then be remounted for the collection of complete X-ray diffraction data sets. Furthermore, the entire screening and data collection experiment can be controlled from the experimenter's home laboratory by means of advanced software tools that enable network-based control of the highly automated beamlines.« less

Effect of processing on nutritive values of milk protein.

PubMed

Borad, Sanket G; Kumar, Anuj; Singh, Ashish K

2017-11-22

Milk is an essential source of nutritionally excellent quality protein in human, particularly in vegan diet. Before consumption, milk is invariably processed depending upon final product requirement. This processing may alter the nutritive value of protein in a significant manner. The processing operations like thermal treatment, chemical treatment, biochemical processing, physical treatments, nonconventional treatments, etc. may exert positive or negative influence on nutritional quality of milk proteins. On one side, processing enhances the nutritive and therapeutic values of protein while on other side intermediate or end products generated during protein reactions may cause toxicity and/or antigenicity upon consumption at elevated level. The review discusses the changes occurring in nutritive quality of milk proteins under the influence of various processing operations.

The effects of sex, tissue type, and dietary components on stable isotope discrimination factors (Δ13C and Δ15N) in mammalian omnivores.

PubMed

Kurle, Carolyn M; Koch, Paul L; Tershy, Bernie R; Croll, Donald A

2014-01-01

We tested the effects of sex, tissue, and diet on stable isotope discrimination factors (Δ(13)C and Δ(15)N) for six tissues from rats fed four diets with varied C and N sources, but comparable protein quality and quantity. The Δ(13)C and Δ(15)N values ranged from 1.7-4.1‰ and 0.4-4.3‰, respectively. Females had higher Δ(15)N values than males because males grew larger, whereas Δ(13)C values did not differ between sexes. Differences in Δ(13)C values among tissue types increased with increasing variability in dietary carbon sources. The Δ(15)N values increased with increasing dietary δ(15)N values for all tissues except liver and serum, which have fast stable isotope turnover times, and differences in Δ(15)N values among tissue types decreased with increasing dietary animal protein. Our results demonstrate that variability in dietary sources can affect Δ(13)C values, protein source affects Δ(15)N values even when protein quality and quantity are controlled, and the isotope turnover rate of a tissue can influence the degree to which diet affects Δ(15)N values.

Increased biomass and quality and reduced heavy metal accumulation of edible tissues of vegetables in the presence of Cd-tolerant and immobilizing Bacillus megaterium H3.

PubMed

Wang, Qi; Zhang, Wen-Ji; He, Lin-Yan; Sheng, Xia-Fang

2018-02-01

A Cd-resistant and immobilizing Bacillus megaterium H3 was characterized for its impact on the biomass and quality and heavy metal uptake of edible tissues of two vegetables (Brassica campestris L. var. Aijiaohuang and Brassica rapa L. var. Shanghaiqing) grown in heavy metal-polluted soil. The impact of strain H3 on the soil quality was also evaluated. The increase in the edible tissue biomass and the contents of soluble proteins and vitamin C of the vegetables inoculated with strain H3 ranged from 18% to 33%, 17% to 31%, and 15% to 19%, respectively, compared with the controls. Strain H3 significantly decreased the edible tissue Cd and Pb contents of the two greens (41-80%), DTPA-extractable Cd content (35-47%) of the rhizosphere soils, and Cd and Pb translocation factors (25-56%) of the greens compared with the controls. Moreover, strain H3 significantly increased the organic matter content (17-21%) and invertase activity (13-14%) of the rhizosphere soils compared with the controls. Our results demonstrated the increased edible tissue biomass and quality, decreased Cd and Pb uptake of the edible tissues, and improved soil quality in the presence of strain H3. The results also suggested an effective bacterial-enhanced technique for decreased metal uptake of greens and improved vegetable and soil qualities in the metal-contaminated soils. Copyright © 2017 Elsevier Inc. All rights reserved.

Comprehensive proteomic analysis of developing protein bodies in maize (Zea mays) endosperm provides novel insights into its biogenesis.

PubMed

Wang, Guifeng; Wang, Gang; Wang, Jiajia; Du, Yulong; Yao, Dongsheng; Shuai, Bilian; Han, Liang; Tang, Yuanping; Song, Rentao

2016-12-01

Prolamins, the major cereal seed storage proteins, are sequestered and accumulated in the lumen of the endoplasmic reticulum (ER), and are directly assembled into protein bodies (PBs). The content and composition of prolamins are the key determinants for protein quality and texture-related traits of the grain. Concomitantly, the PB-inducing fusion system provides an efficient target to produce therapeutic and industrial products in plants. However, the proteome of the native PB and the detailed mechanisms underlying its formation still need to be determined. We developed a method to isolate highly purified and intact PBs from developing maize endosperm and conducted proteomic analysis of intact PBs of zein, a class of prolamine protein found in maize. We thus identified 1756 proteins, which fall into five major categories: metabolic pathways, response to stimulus, transport, development, and growth, as well as regulation. By comparing the proteomes of crude and enriched extractions of PBs, we found substantial evidence for the following conclusions: (i) ribosomes, ER membranes, and the cytoskeleton are tightly associated with zein PBs, which form the peripheral border; (ii) zein RNAs are probably transported and localized to the PB-ER subdomain; and (iii) ER chaperones are essential for zein folding, quality control, and assembly into PBs. We futher confirmed that OPAQUE1 (O1) cannot directly interact with FLOURY1 (FL1) in yeast, suggesting that the interaction between myosins XI and DUF593-containing proteins is isoform-specific. This study provides a proteomic roadmap for dissecting zein PB biogenesis and reveals an unexpected diversity and complexity of proteins in PBs. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.